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I'm reading on hormones and the book talks about how peptide or amine hormones are easily broken down by proteases present in the blood plasma. This has led me to question the interactions between these proteases and the blood proteins of the blood (such as albumins and globulins). Does having proteases in the blood mean the blood proteins are constantly broken down? How do the blood proteins get anything done then? And what good do binding proteins do in protecting hormones during vascular transit when the binding proteins are also susceptible to blood proteases?
Edit: I've been requested to provide some quotes and references.
Because water-soluble hormones can dissolve in blood, many circulate as free hormones, meaning that most of them dissolve directly into the blood and are delivered to their target tissue without binding to a binding protein.
The book goes on to say
Water-soluble hormones, such as proteins, peptides, and amino acid derivatives, have relatively short half-lives because they are rapidly degraded by enzymes, called proteases, within the bloodstream. The kidneys then remove the hormone breakdown products from the blood.
These quotes were from Seeley's Anatomy and Physiology, 10th edition.
Platelets and proteases
Circulating platelets are essential for the formation of blood clots. Studies of mice reveal more about the proteins involved in activating platelets, with implications for understanding strokes and heart attacks.
How do animals stop bleeding after an injury? By the time humans evolved, a common solution lay in rapidly generating a complex clot made up of blood-derived proteins (fibrin) and cells (platelets). Working together, fibrin and platelets create a plug that prevents bleeding long enough for healing to occur. The downside is that clots can also form in blood vessels scarred by cholesterol-laden plaques (atherosclerosis), interrupting blood flow and leading to strokes and heart attacks.
Writing on page 74 of this issue, Sambrano and colleagues 1 investigate one of the key unanswered questions about clotting: what is the role in mice of a protein called protease-activated receptor-4 (PAR4)? Their results confirm the need for this protein in clotting, but the implications go beyond this, touching on the fine-tuning of platelet activation and intriguing differences between mice and humans. There may also be implications for developing drugs to prevent cardiovascular problems.
One of the earliest steps in the formation of a blood clot is the localized generation of the active enzyme thrombin from its circulating inactive precursor, prothrombin. Thrombin cleaves fibrinogen in blood plasma, creating monomers of fibrin that then polymerize into a mesh across a wound in a blood-vessel wall. Thrombin also activates platelets 2 . A study published a decade ago 3 established that human platelets express on their surface protease-activated receptor-1 (PAR1), a signalling protein that is a substrate for thrombin. By cleaving PAR1, thrombin initiates signalling within platelets, causing them to stick to each other. The basic structure of PAR1 places it within a large family of cell-surface receptors that work through intracellular switches called G proteins.
With PAR1 identified, the mystery of how a plasma protease could activate platelets seemed to have been solved. But before long, questions began to accumulate. If PAR1 is indeed the universal thrombin receptor, why did deleting its gene in mice have no effect on platelet activation by thrombin? How can cleavage of a single type of receptor activate more than one signalling pathway within platelets, and how could these responses be modulated over time and over the wide range of thrombin concentrations that platelets are likely to encounter? What, if any, is the role of other thrombin-binding proteins found on the platelet surface?
Answers to at least some of these questions are now known. As it turns out, there are three other members of the PAR family 4 : PAR2, PAR3 and PAR4. Like PAR1, two of these receptors — PAR3 and PAR4 — are activated by thrombin PAR2 is not. Human platelets express PAR1 and PAR4, whereas mouse platelets express PAR3 and PAR4 but not PAR1. This, of course, explains why the loss of PAR1 had no effect on platelet function in mice.
Thrombin produces a variety of responses in platelets because the PAR receptors are coupled to more than one type of G protein. Graded responses to a range of thrombin concentrations can be explained in part by a relationship between the amount of thrombin present and the number of PAR proteins cleaved per second, and in part by differences among PAR family members in the concentration of thrombin required for cleavage. So, for example, PAR1 is a better substrate for thrombin than PAR4. When both are present, cleavage of PAR1 presumably precedes cleavage of PAR4 as thrombin accumulates (Fig. 1a) 5,6 . On the other hand, once PAR4 is cleaved, it appears to signal for longer 7 .
Communication between the protein-cleaving enzyme thrombin (scissors) and members of the PAR protein family sets off signalling pathways that activate the blood cells. a, In humans, platelets express PAR1 and PAR4. These are coupled to intracellular signalling pathways through molecular switches from the Gq, G12 and Gi protein families. When thrombin removes the amino termini of PAR1 and PAR4, several signalling pathways (coloured arrows) are activated, one result of which is the secretion of ADP. By binding to its receptor, P2Y12, ADP activates additional Gi-mediated pathways. In the absence of wounding, platelet activation is counteracted by signalling from prostaglandin I2. Cleavage of PAR1 by thrombin appears to be enabled by thrombin binding to glycoprotein Ibα. b, Mouse platelets express PAR3 and PAR4. PAR4 seems to be solely responsible for signalling 1 , whereas PAR3 enables cleavage of PAR4, allowing it to respond to lower concentrations of thrombin.
The fact that human platelets express PAR1 and PAR4 whereas mouse platelets express PAR3 and PAR4 seems to be an example of divergent solutions to a common problem — how to induce a variety of responses to thrombin. Although mouse PAR3 is a good substrate for thrombin, it signals poorly, if at all. In other words, it has for some reason lost the ability to activate G proteins, while retaining the ability to be cleaved by thrombin. Nonetheless, when the gene encoding PAR3 was deleted from mice, platelets responded considerably less well to thrombin 5 . So it was suggested that PAR4 is the main mediator of mouse responses to thrombin, and that PAR3 has a supportive role, enabling the cleavage of PAR4 at low thrombin concentrations. If so, then one would predict that deleting PAR4 would abolish thrombin signalling in mouse platelets.
Sambrano et al. 1 have now deleted the PAR4 gene in mice, and find that platelets are indeed no longer activated by thrombin. The results show the requirement for PAR4 and support the predicted helper role for PAR3 (Fig. 1b). For this awkward arrangement to have arisen, mouse PAR3 not only had to lose its intrinsic ability to signal when cleaved it also had to 'acquire' the ability to aid PAR4 activation. One might speculate that these differences between mouse and human platelets are not mere happenstance, but are appropriate for other reasons. That remains to be seen.
As the authors show, platelet responses to thrombin were completely abolished in mice lacking both copies of the PAR4 gene. Mice with one copy of the gene showed a modest impairment in clot formation. Although this impairment did not reach statistical significance, there seems to be a trend here: mouse platelets may require that most, if not all, of their thrombin receptors are fully functional. If this is true of human platelets, too, then inherited variations in the structure of PAR family members that result in a reduction in signalling might translate into differences between people in their risk of developing strokes and heart attacks. So far, no function-altering variations in PAR1 and PAR4 have been reported, but one has been described 8 for PAR2.
Are the PAR proteins sufficient to account for all of the responses of platelets to thrombin? Given the new results 1 and the fact that simultaneous blockade of PAR1 and PAR4 abolishes the ability of human platelets to respond to thrombin 9 , the answer would appear to be 'yes'. But recent studies 10,11 have reinvigorated a debate about the role of the glycoprotein Ib/IX/V multiprotein complex as a binding site and substrate for thrombin. It is still too early to predict the outcome of that debate. Nonetheless, the results from Sambrano et al. provide a conclusive demonstration of the essential role of PAR4 in the activation of platelets by thrombin and, by extension, of the need for thrombin itself. Their work also re-emphasizes the possibility that drugs that block PAR1, PAR4 or both might be useful in treating a variety of clotting disorders in humans.
What is Protease? (with pictures)
A protease is a member of a very large group of enzymes that have a variety of functions in the body. A primary one is as a digestive enzyme to process protein. Without protease, the body would not be able to digest the protein in food. Other types of proteases are involved in the regulation of cellular events, such as blood clotting. These are also called proteolytic enzymes or proteinases.
Proteins are long chains of amino acids that are held together by peptide bonds. Small fragments of proteins are known as peptides, and larger fragments are referred to as polypeptides. Enzymes that break down peptides are called peptidases.
Proteases are types of proteins that accelerate the degradation of others. They differ in the manner in which they carry out this activity. Exopeptidases cleave off terminal amino acids and nibble away at proteins. They break down peptide bonds to release amino acids. In contrast, endopeptidases act within the protein, and also cleave peptide bonds, producing polypeptides as the result of their activities.
There are several classes of proteases, depending on the type of amino acid at the site where the reaction occurs, and any additional molecule needed for activity. For instance, many proteins require a metal atom to be active. They are known as metalloproteinases. Other proteases have an amino acid known as serine at their active site, and are known as serine proteases.
The initial studies of proteases, in human physiology, were done to discern their role in digestion in the gastrointestinal system. The goal of enzymatic digestion is to break larger molecules into smaller ones. Several proteases work in concert with peptidases to degrade the proteins in foods to small peptides and amino acids. Such small molecules can be absorbed by the intestinal cells and used as fuel or to build new protein molecules.
One thing all of these digestive proteases have in common is that they are synthesized as larger, inactive forms to prevent the tissue that contains them from enzymatic damage. Such precursors are known as zymogens. Another feature they share is that they are all endopeptidases, although they vary in their preference for which part of proteins they will cleave. This substrate specificity is based on the location of specific amino acids in the target proteins.
The stomach contains the digestive protease pepsin, which is stimulated by the stomach’s hydrochloric acid. Pepsin breaks the proteins into polypeptides, which travel to the intestine. In this location, they are broken into even smaller pieces by the additional digestive proteases trypsin and chymotrypsin. All of these enzymes are serine proteases.
Other types of protease act to regulate the activity of other proteins. By cleaving a specific site on a protein, they can either turn them on or off. This can be part of a mechanism for signaling a physiological change. Another function of proteases is to help in the processing of proteins that are produced in larger forms, such as the amyloid precursor protein. Other proteases degrade proteins that are no longer needed for cellular function.
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ADAMTS proteases in cardiovascular physiology and disease
The a disintegrin-like and metalloproteinase with thrombospondin motif (ADAMTS) family comprises 19 proteases that regulate the structure and function of extracellular proteins in the extracellular matrix and blood. The best characterized cardiovascular role is that of ADAMTS-13 in blood. Moderately low ADAMTS-13 levels increase the risk of ischeamic stroke and very low levels (less than 10%) can cause thrombotic thrombocytopenic purpura (TTP). Recombinant ADAMTS-13 is currently in clinical trials for treatment of TTP. Recently, new cardiovascular roles for ADAMTS proteases have been discovered. Several ADAMTS family members are important in the development of blood vessels and the heart, especially the valves. A number of studies have also investigated the potential role of ADAMTS-1, -4 and -5 in cardiovascular disease. They cleave proteoglycans such as versican, which represent major structural components of the arteries. ADAMTS-7 and -8 are attracting considerable interest owing to their implication in atherosclerosis and pulmonary arterial hypertension, respectively. Mutations in the ADAMTS19 gene cause progressive heart valve disease and missense variants in ADAMTS6 are associated with cardiac conduction. In this review, we discuss in detail the evidence for these and other cardiovascular roles of ADAMTS family members, their proteolytic substrates and the potential molecular mechanisms involved.
The extracellular matrix (ECM) guides the formation of cardiovascular tissues during embryogenesis and supports it throughout adulthood by providing structural support, guidance of cell behaviour and sequestering of growth factors. In large arteries and blood vessels, collagen and elastic fibres provide essential structural support to prevent rupture. A healthy artery consists of three layers (tunicae): tunica adventitia, media and intima (figure 1). The intima is in contact with the vessel lumen and consists of endothelial cells (ECs) attached to a basal membrane rich in collagen IV, laminin, nidogen and heparan sulfate (HS) proteoglycans (PGs) (syndecan, perlecan). The tunica media is made up of vascular smooth muscle cells (VSMC), which express chondroitin-sulfate (CS)/dermatan-sulfate (DS)-PGs (CSPGs) (such a versican and aggrecan) and elastin. There are fenestrated sheets of elastin called lamellae between which there are collagen fibres, thin layers of PG-rich ECM and VSMCs. Elastin, which is distensible and has a low tensile strength, functions as an elastic reservoir and distributes stress evenly throughout the wall and onto collagen fibres . The adventitia is the outermost layer of a blood vessel and consists of a collagen-rich ECM secreted by fibroblasts that prevents vascular rupture at very high pressures. The adventitia is also a reservoir of stem cells and plays an essential role in regulating the functions of cell populations in the tunica intima and media . Various PGs are present in the different layers. In the tunica media, they contribute to the viscoelastic properties of the vessel wall. This function of PGs is mediated by their glycosaminoglycan (GAG) chains. Owing to their high negative charge, these GAGs attract counter-ions and water into the tissue [3,4]. Moreover, CSPGs such as versican and aggrecan are essential components of cardiac valve leaflets and, in particular, the middle ECM layer, the spongiosa, while elastin and collagens predominate in the ventricularis/atrialis and fibrosa layers, respectively [5,6] (figure 2). The ratios of PGs and the interspersed collagens/elastic fibres provide a means of balance between stiffness and flexibility of the cardiovascular tissues . For this reason, remodelling of the vascular ECM by proteases secreted by both ECs and VSMCs is crucial to establish the mechanical properties of these tissues. Moreover, ECM degradation is required for migration and proliferation of VSMCs, as well as infiltration of inflammatory cells under pathological conditions. Among the proteases involved in this dynamic action, members of the matrix metalloproteinases (MMP) family have been extensively investigated owing to their ability to cleave the elastic ECM components such as collagens (MMP-1, -8, -13, -14) and elastin (MMP-12). In addition, proteases of the related family of a disintegrin and metalloproteinases (ADAMs) exert a fundamental role in the vascular ECM owing to their ability to selectively cleave the ectodomain of membrane proteins (shedding). The role of these metalloproteinase families in cardiovascular disorders has been exhaustively reviewed elsewhere [8,9].
Figure 1. Involvement of ADAMTS proteases in cardiovascular physiology and disease. Atherosclerosis and haemostasis: decreased proteoglycanase activity (a1) is associated with proteoglycan accumulation, increased low-density lipoprotein (LDL) internalization (a2) and foam cell formation (a3), which eventually leads to formation of an atherosclerotic plaque. A thrombus can form on top of a plaque if collagen is exposed to the blood. ADAMTS-13 regulates the capacity of von Willebrand Factor (VWF) to recruit platelets to the site of collagen exposure and initiate thrombus formation. In the aorta, reduced proteoglycanase activity (a1) causes proteoglycan accumulation, increased osmotic pressure, reduced viability of vascular smooth muscle cells (VSMC) and disruption of mechanosensing (b). BM, basal membrane EC, endothelial cell VWF, von Willebrand factor.
Figure 2. Extracellular matrix (ECM) organization in cardiac valves. (a) The ECM composition in a mature valve is shown. The formation of the ventricularis and the fibrosa during development is a complex and tightly regulated molecular process in which ADAMTS proteases play an essential role. (b) Dysfunctional ADAMTS activity can lead to a certain degree of disorganized ECM, altered valve shape and leakage of the valve. Disorganized ECM can present as an abundance of proteoglycans, which can be owing to insufficient proteoglycanase activity or possibly owing to incorrect assembly of the ECM in the ventricularis (e.g. fibrillin-1 microfibrils/elastin). ADAMTS-1, -5 and -9 have been implicated in cardiac valve development by mouse studies and ADAMTS-19 by a human genetic disease.
This review focuses on a closely related metalloprotease family, the a disintegrin-like and metalloproteinase with thrombospondin motif (ADAMTS) and their (patho)physiological role in heart and blood vessels. In humans, the ADAMTS family comprises 19 secreted metalloproteinases as well as 7 ADAMTS-like proteins devoid of catalytic activity . They share a common domain composition consisting of a signal peptide, a prodomain, a metalloproteinase catalytic domain (Mp, absent in the ADAMTS-like proteins), followed by non-catalytic ancillary domains such as a disintegrin-like (Dis) domain, a central thrombospondin-type I motif (TSR), a cysteine-rich (CR) domain, a spacer (Sp) domain, and, with the exception of ADAMTS-4, a various number of TSRs at the C-terminus (table 1). Some members have additional C-terminal domains, such as a mucin domain (present in ADAMTS-7 and ADAMTS-12), a Gon-1 like domain (in ADAMTS-20 and ADAMTS-9) and a PLAC (protease and lacunin) domain (in ADAMTS-2, -3, -6, -10, -12, 14, -16, -17, -18 and -19). Moreover, ADAMTS-13 is unique among ADAMTS proteases since it presents two CUB [complement subcomponent C1r/C1s/embryonic sea urchin protein Uegf (urchin epidermal growth factor)/bone morphogenic protein 1] domains [11,12]. A common feature of the family is the presence of a zinc-binding motif in the Mp domain, containing the consensus sequence H EXX H XXGXX H , in which the three underlined histidine residues coordinate a zinc atom, which together with a glutamate residue, exerts a catalytic role. This motif is followed C-terminally by a methionine residue that constitutes a structural turn (Met-turn) conserved within the metzincin family of metallopeptidases (comprising MMPs, ADAMs, ADAMTSs and astacins) .
Table 1. Summary of the cardiovascular roles, proteolytic functions and domain organization of the ADAMTS family. For the cardiovascular roles and diseases, the numbers in brackets indicate specific ADAMTS family members. CAD, coronary artery disease PAH, pulmonary arterial hypertension TAAD, thoracic aortic aneurysms and dissections TTP, thrombotic thrombocytopenic purpura VSMC, vascular smooth muscle cells VWF, von Willebrand factor WMS, Weill-Marchesani syndrome. The ADAMTS protein domains are abbreviated as follows: S, signal peptide Pro, prodomain Mp, metalloproteinase domain Dis, disintegrin-like domain CR, cysteine-rich domain Sp, spacer domain Muc, mucin-like domain CUB, complement subcomponent C1r/C1s/embryonic sea urchin protein Uegf (urchin epidermal growth factor)/bone morphogenic protein 1 PL, protease and lacunin domain GON, gon-1 like domain.
ADAMTS proteases are generally activated following proteolytic removal of the N-terminal prodomain by subtilisin-type proprotein convertases (e.g. Furin, PCSK6) . With the exception of ADAMTS-13, which circulates in blood, the other ADAMTS family members appear to function in the ECM. The secreted activated enzymes are mainly regulated through inhibition by tissue inhibitors of metalloproteinases (TIMPs)  and endocytosis .
Distinct ADAMTS subfamilies can be defined on the basis of sequence homology (table 1). They likely evolved through a process of gene duplication resulting in either neo-functionalization or sub-functionalization  and some still share a common substrate repertoire.
ADAMTS-1, -4, -5, -8 and -15 are characterized by their ability to cleave PGs and are therefore collectively named ‘proteoglycanases'. Although more distantly related, ADAMTS-9 and -20 also exhibit this distinct proteolytic activity.
ADAMTS-2 is a well-characterized procollagen N-propeptidase that cleaves the N-terminal propeptides of type I, II and III collagen . ADAMTS-3 and ADAMTS-14 show a high degree of homology with ADAMTS-2 and can cleave pro-collagen in vitro. ADAMTS-3 is essential for lymphangiogenesis through activation of vascular endothelial growth factor (VEGF)-C [19,20].
The related pairs ADAMTS7/12, ADAMTS6/10, ADAMTS16/18 and ADAMTS17/19 are not well characterised . ADAMTS10 and ADAMTS17 mutations give rise to Weill-Marchesani syndrome (WMS)-like spectrum and have been functionally associated with the assembly of fibrillin microfibrils . ADAMTS-6 has also been shown to promote fibrillin-1 microfibril formation .
ADAMTS-13, by far the best characterized family member, regulates the function of von Willebrand Factor (VWF) in primary haemostasis [23,24].
Whereas non-cardiovascular roles of the different ADAMTS family members have been discussed in recent excellent reviews [10,14], here, we will discuss in detail the involvement of ADAMTS proteases in cardiovascular biology and disease.
2. Proteoglycans and proteoglycanases in cardiovascular physiology and disease
Several ADAMTS family members specifically cleave PGs. Because ‘The biology of a protease is really the biology of its substrates' , we will briefly discuss the role of PGs in the cardiovascular system. In blood vessels, PGs are mainly expressed by ECs and VSMCs in the tunica intima and media, respectively, where they regulate the biophysical properties of the ECM [3,19]. Moreover, through their interactions with ECM proteins, growth factors and chemokines, PGs regulate a variety of processes such as cell signalling, proliferation, migration and apoptosis . Increased accumulation of PGs is a feature of atherosclerosis [26,27] and aneurysms , as well as hereditary diseases such as pediatric aortic valve disease and adult myxomatous mitral valves .
PGs are classed according to the predominant GAG covalently attached to serine residues in their protein core, heparin/heparan sulfate (HS) PGs and chondroitin-sulfate/dermatan sulfate (CS/DS) PGs. CS-GAGs contain D-glucuronic acid and N-acetyl-D-galactosamine, whereas in DS-GAGs, the D-glucuronic acid is epimerized into L-iduronic acid. HS-GAGs contain D-glucuronic acid or L-iduronic acid alternating with N-acetyl-D-glucosamine. GAGs are not only important to generate a Donnan osmotic pressure in the vascular tissue owing to their negative charges  but also to mediate lipoprotein uptake from the circulation.
Sub-intimal accumulation of lipid-rich and inflammatory deposits (plaques) in medium and large arteries is a hallmark of atherosclerosis (figure 1a). The enlargement of plaques hampers the normal blood flow, leading to organ ischemia and tissue necrosis. Plaque rupture with subsequent thrombus formation can cause vascular occlusion leading to potentially fatal cardiovascular events such as myocardial infarction and stroke. PGs such as versican and aggrecan also tend to accumulate in thoracic aortic aneurysms and dissections (TAAD) [28,30,31] and during normal ageing of the aorta , resulting in an increased osmotic pressure that is disruptive to the ECM (figure 1b). Moreover, PG accumulation can disrupt mechanosensing by VSMCs and create stress-risers in the aortic wall that may predispose to or propagate a dissection .
HSPGs such as perlecan and the four transmembrane syndecans are synthesized mainly by ECs in the intima and inhibit pro-atherogenic processes such as lipoprotein retention, infiltration of inflammatory cells, proliferation of VSMCs and thrombosis [34,35]. Since HSPGs are not major ADAMTS substrates, they will not be discussed in detail here. We will instead focus on CSPGs.
CSPGs are mainly expressed by VSMCs in the tunica media . In contrast with HSPGs, CSPGs may initiate atherosclerotic processes by enhancing both deposition and internalization of low-density lipoprotein (LDL) particles that have penetrated into the arterial wall after transcytosis or endothelial dysfunction [27,36] (figure 1a). CSPGs can form high-affinity complexes with LDL particles, which are then internalized more efficiently by VSMCs and infiltrated macrophages than native LDLs [37,38]. Once internalized, LDLs enhance intracellular cholesteryl ester synthesis and subsequent foam formation . The interaction between lipoproteins and CSPGs involves an ionic bond between basic amino acids in apoprotein B (apoB) and negatively charged sulfate groups on the GAGs . Moreover, the longer the CS chains, the higher the affinity for LDL . To support the notion that the direct binding of LDL particles to CSPGs is a key step in atherogenesis, transgenic mice expressing LDL particles where positively charged amino acids in apoB had been replaced with neutral ones exhibited significantly less atherosclerotic lesions than mice expressing wild-type apoB . CSPGs comprise large aggregating PGs such as versican and aggrecan and small leucine-rich PGs (SLRPs) such as biglycan and decorin. Owing to their ability to bind both hyaluronan, the only non-sulfated GAG, and lectins, large aggregating PGs are also called hyalectans . Hyalectans have a similar structure, comprising two globular domains at the N- and C-terminus, named G1 and G3, respectively, and a central GAG domain containing attachment sites for GAG chains. The G1 domain binds to hyaluronan, whereas the G3 domain contains the lectin-binding region (figure 1a).
Versican is the main hyalectan in the vasculature, where it plays a role in developmental and repair processes such as cell adhesion, proliferation and migration, ECM assembly and inflammation [25,26,44]. It is present in 5 isoforms (V0-V4), generated by alternative splicing within the central GAG-rich region. Expression of versican is essential for normal development of heart and blood vessels [45,46].Versican is involved in various aspects of vascular lesion development and is present in atherosclerotic plaques, restenotic lesions, lesions arising during graft repair and aneurysmal lesions .Versican levels increase dramatically in atherosclerosis [27,47], suggesting that its accumulation may in part be responsible for increased LDL deposition/internalization in the vessel wall (figure 1a).
Aggrecan, a major CSPG in cartilage, has been recently identified in human aortas [28,30,31,48–53] and, together with versican, it has been shown to accumulate in the aortas of patients with TAAD, resulting in an increased osmotic pressure that is disruptive to the ECM  (figure 1b). Here, it is important to note that aggrecan has an order of magnitude more CS than versican , and therefore more potential to exert osmotic pressure. In addition to CS, aggrecan also contains keratan sulfate (KS) GAGs (where the D-galactose replaces hexuronic acid) clustered into a KS-rich region .
The protein core of SLRPs such as biglycan is small (40–60 kDa) and characterized by the presence of 11–12 leucine-rich tandem repeats and the attachment of 1–2 CS/DS GAG chains . The leucine-rich tandem repeats bind to collagens, thus regulating collagen fibril formation [56–58].
Biglycan is one of the major PGs found in human atherosclerotic lesions [59–62]. Like aggrecan and versican, biglycan binds to LDL particles, although with reduced affinity owing to the lower number of GAG chains . Bgn knockout mice did not show reduced LDL retention in arterial wall, most likely owing to compensation from other CSPGs . However, overexpression of biglycan in mice increased arterial retention of apoB lipoproteins and promoted atherosclerosis [62,65]. Biglycan may also exert an anti-atherosclerotic function. In mice genetically susceptible to develop atherosclerotic lesions (already bearing deletion of either apolipoprotein-E, ApoE, or LDL-receptor, LDLR), abolishing biglycan expression increased macrophage-mediated plaque inflammation . Biglycan is also considered as an early initiator of aortic stenosis lesion, contributing to wall thickening . Furthermore, biglycan has been implicated in the formation of TAAD, since Bgn/LDLR double knockout mice showed increased incidence of TAAD  and loss of function mutations in Bgn results in syndromic early-onset TAAD in humans . Bgn knockout mice also showed spontaneous abdominal aortic aneurysms (AAA) . Since biglycan deficiency impairs the formation of collagen fibres in the aortic wall and contributes to the breakdown of elastic fibres [67,70], this will affect the ability of the vessel to sustain tension forces.
Physiological levels of PGs are regulated by the proteoglycanase activity of several ADAMTS family members. ADAMTS-1, -4, -5, -8, -9, -15 and -20 have been shown to cleave, albeit to a different extent, both versican [71–75] and aggrecan . The only ADAMTS cleavage of versican V1 isoform described to date occurs at the Glu441↓442Ala bond within the βGAG domain [71–75]. The equivalent cleavage sites in the αGAG region in the V0 and V2 isoforms have been identified as Glu1428↓1429Ala  and Glu405↓406Gln, respectively . Aggrecan is cleaved by ADAMTS proteases at multiple sites , although the cleavage event most detrimental for its function occurs at the Glu392↓Ala393 bond (Uniprot P16112 numbering) in the region between the G1 and G2 domains . Importantly, ADAMTS-mediated cleavage of both aggrecan and versican has been shown to release bioactive fragments. The G1-DPEAAE 441 versican V1 cleavage fragment, named versikine, is involved in a variety of biological processes such as immune signalling  and apoptosis , while the G1-NIVSFE 405 V2 cleavage fragment has been identified as a hyaluronan-binding protein previously identified in human brain . A 32-amino acid long aggrecan fragment generated by MMP-mediated cleavage at Asn360↓Phe361 and ADAMTS-mediated cleavage at Glu392↓Ala393 has been shown to interact with toll-like receptor-2 and excite nociceptive neurons in chondrocytes [82,83]. Following ADAMTS-cleavage at Glu392↓Ala393, the 393 ARGS neopeptide can diffuse from the ECM into plasma, urine and synovial fluid [84,85]. Among the aforementioned ADAMTS proteases, ADAMTS-4 and -5 show the strongest proteolytic activity against both aggrecan [86,87] and versican  moreover, they have also been shown to cleave the SLRP biglycan at Asn186↓187Cys [86,89]. This proteoglycanase activity exerts important functions in vascular biology and may contribute to cardiovascular diseases such as atherosclerosis and aneurysms, as outlined in the following sections.
2.2.1. ADAMTS-1: friend (in TAAD) or foe (in atherosclerosis)?
ADAMTS-1 was the first member of the family to be described . Early murine knockout models showed the involvement of this enzyme in fertilization and the development of the urogenital system [91–94], but it was soon recognized that ADAMTS-1 plays an important cardiovascular role. The substrate repertoire of ADAMTS-1 appears to extend beyond the CSPGs aggrecan  and versican . Reported in vitro substrates include nidogen-1 and -2 [96,97], semaphorin 3C , Tissue factor pathway inhibitor (TFPI)-2 , insulin growth factor binding protein (IGFBP)-2 , syndecan-4  and thrombospondins 1 and 2 [98,102]. ADAMTS-1 versicanase activity in vitro is considerably weaker than that of either ADAMTS-4 or ADAMTS-5  but appears biologically important in certain developmental processes such folliculogenesis . Mouse studies suggest that ADAMTS-1 versicanase activity within the endocardial cushion contributes to valve maturation and myocardial trabeculation [103,104]. However, ADAMTS-1 proteolysis of non-PG substrates or non-proteolytic activities may be partially responsible for these phenotypes.
In the blood vessels, ADAMTS-1, like ADAMTS-4 and -5, is expressed by ECs, VSMCs and invading macrophages [105–108]. ADAMTS-1 expression was upregulated in human atherosclerotic lesions  and its versicanase activity may contribute to plaque instability [71,105]. Transgenic mice overexpressing Adamts1 on an ApoE −/− background were investigated by Jönsson-Rylander et al. , but measurements of atherosclerotic lesion formation were not reported. However, carotid artery ligation in these mice showed a significant increase in neo-intima formation, suggesting an effect of ADAMTS-1 on VSMC migration/proliferation (table 2). In support of this, two miRNAs targeting ADAMTS1, miR-265b-3p and miR-362–3p, appeared to inhibit VSMC proliferation and migration [123,124].
Table 2. Adamts knockout mice and overexpression in vivo. AB, aortic banding-induced cardiac hypertrophy BAV, bicuspid aortic valve AngII, angiotensin II PAH, pulmonary arterial hypertension PPS, pentosan polysulfate TAAD, thoracic aortic aneurysms and dissections. Genetic manipulation is in mice if not otherwise specified.
It has also been reported that angiotensin II (Ang II) and other stimuli associated with vascular remodelling induce the expression of ADAMTS-1 in aorta . Since Adamts1 knockout mice have elevated perinatal mortality , heterozygous Adamts1 +/− mice were tested in a TAAD model . The Adamts1 +/− genotype exacerbated aortic aneurisms and lethal aortic dissections induced by treatment with the hypertensive factor Ang II. Administration of Ang II induced TAAD in nearly 80% of the Adamts1 +/− mice and lethal aortic dissections in nearly 50% of these mice, compared with nearly 10 and 8% in wild-type mice . This phenotype resembles that of Fbn1 C1039G/+ , a mouse model of Marfan syndrome (MFS) characterized by a low incidence of aortic dissections and ruptures compared to other MFS mouse models . In these ‘Marfan mice’, the introduced Fbn1 mutation C1039G disrupts the microfibril scaffold, which has complex knock-on effects (including decreased ADAMTS-1 protein levels) owing to the many mechanistic complexities of the fibrillin microfibril niche and its roles in elastic fibre formation, growth factor signalling and VSMC biology [127,128]. However, in human aortic aneurisms, ADAMTS-1 levels are either unchanged or increased [30,129,130].
In conclusion, these findings suggest that ADAMTS-1 may play a detrimental role in the aetiology of atherosclerosis, whereas further studies are required to establish its involvement in the development of aortopathies.
2.2.2. ADAMTS-4, a potential therapeutic target in atherosclerosis and TAAD
ADAMTS-4 cleaves CSPGs such as versican and aggrecan [87,88], brevican  and SLRPs such as fibromodulin [86,87] and biglycan [86,89] as well as non-PG substrates such as cartilage oligomeric matrix protein (COMP) .
Although ADAMTS-4, like all other ADAMTS family members, with the exception of ADAMTS-13, is predominantly bound to the ECM [86,87], it may diffuse into plasma following cardiovascular damage. Elevated plasma levels of ADAMTS-4 have been consistently found in patients affected by coronary artery disease (CAD) [129,133–135], atherosclerosis [106,136–138] and TAAD [112,129]. Some of these studies associated elevated ADAMTS-4 plasma levels with increased severity of CAD [134,136] and plaque destabilization [137,138]. Increased ADAMTS-4 levels were also found in macrophage-rich areas of human atherosclerotic plaques and unstable coronary plaques [106,138]. Importantly, these findings in humans are in agreement with those obtained from various mouse models (table 2). ADAMTS-4 levels were shown to increase in the atherosclerotic plaques and plasma of ApoE knockout [111,138] and LDLR −/− ApoB 100/100  double knockout mice as atherosclerosis progressed. Moreover, genetic deletion of Adamts4 in an ApoE knockout background produced a milder atherosclerotic phenotype, with increased plaque stability compared to their littermates . ApoE/Adamts4 double knockout mice also showed reduced cleavage of both versican and aggrecan in arteries, with no compensation shown by other proteoglycanases such as ADAMTS-1 and −5. This was associated with reduced lipid deposition and macrophage infiltration but increased VSMC proliferation . Moreover, in the absence of ADAMTS-4, increased macrophage apoptosis and decreased levels of proinflammatory cytokines were observed . These data suggest that the activity of ADAMTS-4 is associated with more unstable atherosclerotic plaques. Therefore, therapeutic inhibition of ADAMTS-4 may be beneficial at late stages of atherosclerotic development.
Deletion of Adamts4 was shown to significantly reduce aortic diameter enlargement, aneurysm formation, dissection and aortic rupture in a mouse model of sporadic TAAD induced by a high-fat diet and AgII infusion . These phenotypes were associated with decreased macrophage infiltration, VSMC apoptosis and versican degradation . Expression of Adamts4 is increased upon Ang II treatment and injection of miR-126a-5p, a miRNA targeting Adamts4, has been recently shown to reduce aortic dilation and versican degradation as well as increase survival in these mice . Severe downregulation of this miRNA may be one of the mechanisms responsible for the observed upregulation of Adamts4 expression in the Ang II model .
In vitro, ADAMTS4 knockdown has been shown to reduce macrophage infiltration  and VSMC apoptosis , two processes that are critical for the development of aortic aneurysms , and plaque rapture . In both cases, the versicanase activity of ADAMTS-4 may play a role. Full-length versican is endowed with adhesive properties and its cleavage by ADAMTS proteoglycanases facilitates the migration of immune cells [80,142]. Moreover, versikine, the ADAMTS-generated versican cleavage fragment, can promote apoptosis , thus antagonising the anti-apoptotic effect of full-length versican [143,144]. ADAMTS-4 may also be directly involved in apoptosis of VSMCs after translocation to the nucleus and cleavage of poly ADP ribose polymerase-1 (PARP-1), a key molecule in DNA repair and cell survival . Full-length PARP-1 promotes cell survival, whereas cleaved PARP-1 can induce apoptosis . Finally, ADAMTS-4 has been shown to exert pro-apoptotic effects independently of its catalytic activity , suggesting that ADAMTS-4 can induce apoptosis through different mechanisms.
Interestingly, ADAMTS-4 expression was increased in the myocardium of rats subjected to hypertension and addition of pentosan polysulfate inhibited both Adamts4 expression and versican cleavage and ameliorated myocardial function .
These clinical observations and in vivo data from mouse models of disease point towards multiple roles of ADAMTS-4 in diseased cardiovascular tissues. Whether therapeutic inhibition of ADAMTS-4 could slow the progression of vascular disease in particular warrants further investigation.
2.2.3. ADAMTS-5 regulates cardiovascular proteoglycan levels
ADAMTS-5 has been extensively studied in the context of aggrecan degradation in cartilage and is a validated target for the treatment of osteoarthritis . More recently, a cardiovascular role has emerged for ADAMTS-5, which has been recently reviewed  and will be briefly summarized here.
In vitro, ADAMTS-5 is a more potent proteoglycanase than ADAMTS-1 and -4 [87,88] and the absence of ADAMTS-5 in vivo causes accumulation of cardiovascular PGs (table 2). For example, Adamts5 knockout mice showed severe anomalies in the pulmonary valve cusps owing to decreased versican cleavage and subsequent versican and aggrecan accumulation [53,113,114] similarly, they exhibited dilation of thoracic aorta with accumulation of aggrecan and biglycan . It would be interesting to compare this phenotype with that of knock-in mice expressing ADAMTS cleavage-resistant versican (i.e. mutated at the Glu1428↓1429Ala site), called V1R mice, but unfortunately their cardiovascular phenotype has not been described . While the majority of V1R mice were viable and fertile, some of them die of organ haemorrahage after backcrossing .
ADAMTS-5 was found to be markedly reduced in aortas of ApoE knockout mice, which spontaneously developed atherosclerotic lesions, resulting in accumulation of versican and biglycan . Recombinant ADAMTS-5 reduced the LDL-binding ability of biglycan and released LDL particles from human aortic lesions , thus suggesting a role for this enzyme in regulating PG-mediated lipoprotein retention (figure 1a). ADAMTS-5 is expressed in both VSMCs [151,152] and macrophages , two cell types that also express versican [151,153–155] and TIMP-3 [156–158], the major inhibitor of ADAMTS-4 and -5 . Once a plaque is formed, its stability is associated with high expression levels of TIMP-3 [158,159] and versican . Therefore, atherosclerotic plaque development is impacted by an imbalance between the expression of proteoglycanases, proteoglycanase inhibitors and PGs, where each protein may exert a beneficial/detrimental role at different stages of the process.
In studies of TAAD, the mRNA levels of ADAMTS-5 are found to be decreased [30,161] and this has been recently confirmed at the protein level, both in plasma and in aortic biopsies . Mouse models of TAAD may help to clarify the role of ADAMTS-5 in this disease. In the Ang II model, Adamts5 knockout mice showed increased aortic dilation, suggesting that ADAMTS-5 plays a non-redundant role in maintaining the viscoelastic properties of aortic ECM . Loss of ADAMTS-5 was associated with increased protein levels of versican and TGF-β , a crucial player in the development of TAAD . At the same time, low-density lipoprotein receptor-related protein-1 (LRP-1) expression was downregulated , a phenomenon that has itself been shown to exacerbate aortic dilation . Remarkably, in this model, an increase in ADAMTS-1 protein levels did not compensate for the absence of ADAMTS-5, since versican cleavage was severely diminished . This may be explained by the higher intrinsic versicanase activity of ADAMTS-5 .
Taken together, these data suggest that the proteolytic activity of ADAMTS-5 is essential in regulating the levels of cardiovascular PGs and that disturbances could affect the disease process in atherosclerosis and TAAD. As a consequence, any treatment for osteoarthritis should aim to spare ADAMTS-5 activity in the blood vessels to avoid imbalance in PG levels .
2.2.4. ADAMTS-8, a contributor to pulmonary arterial hypertension
The enzymatic properties of ADAMTS-8, including its substrate repertoire, have not been extensively investigated. The only reported substrate is aggrecan, which was cleaved in vitro, but at an extremely high enzyme/substrate ratio . Notwithstanding the homology of ADAMTS-8 with ADAMTS-1, -4 and -5, these data cast doubt on the inclusion of ADAMTS-8 in the proteoglycanase subfamily. ADAMTS8 has been identified as a tumour suppressor gene in several types of cancers  and single nucleotide polymorphisms (SNPs) in the ADAMTS8 locus have been associated with hypertension in a genome-wide association study (GWAS) . At the protein level, ADAMTS-8 is highly expressed in the lung and the heart [119,166] and, together with ADAMTS-1, -4 and -5, has been detected within human carotid lesions and advanced coronary atherosclerotic plaques [106,107]. ADAMTS-8 expression was increased in the lungs of patients with pulmonary arterial hypertension (PAH) and in mouse/rat models of PAH . In a model of hypoxia-induced PAH, mice bearing a targeted deletion of Adamts8 in pulmonary arterial smooth muscle cells (Adamts8 ΔSM22α ) showed decreased right ventricular systolic pressure and right ventricular hypertrophy compared with wild-type mice, suggesting a crucial role for ADAMTS-8 in the development of PAH . Addition of recombinant ADAMTS-8 to pulmonary artery ECs seemed to exert a pro-inflammatory and pro-apoptotic role , similar to its effects on nasopharyngeal carcinoma cell lines . These data suggest potential similarities between the function of ADAMTS-8 in PAH and in cancer. Since ADAMTS-8 is also expressed in the heart, a conditional knockout model with cardiomyocyte-specific deletion of Adamts8 (Adamts8 ΔαMHC ) was generated . These mice showed decreased cardiac hypertrophy, fibrosis and right ventricular dysfunction in response to hypoxia.
Taken together, these results implicate ADAMTS-8 in the development of PAH and potentially other cardiovascular phenotypes, but further studies are necessary to characterize the ADAMTS-8 substrate repertoire, mechanism of action and regulation.
2.2.5. ADAMTS-9 in heart development
Evolutionarily, ADAMTS-9 appears to be the oldest family member, based on its high homology to nematode and fruit fly proteases, named Gon-1 and Adamts-A, respectively [72,168–170]. It is also the largest member of the ADAMTS family, comprising 14 TSR repeats and one Gon-1-like domain at the C-terminus (table 1).
Adamts9 knockout mice did not survive past 7.5 days of gestation for unknown reasons, but possibly owing to an important role of ADAMTS-9 in the formation and function of primary cilia [171,172]. Heterozygous knockout mice showed a variable penetrance of cardiac anomalies involving the myocardium, mitral valves, aortic valves and proximal aorta, associated with excess versican , suggesting that this enzyme is involved in development of the heart (table 2).
In a study of gene expression associated with AAA rupture, aortic tissues from emergency repair of ruptured AAA were compared to tissue from elective surgery. This identified a set of 5 fibroblast-expressed genes exclusively upregulated in AAA, including ADAMTS9 .
3. ADAMTS-7 in coronary artery disease
ADAMTS-7 is a potential therapeutic target in atherosclerosis and associated diseases such as CAD. The evidence for a detrimental role has accumulated over the past decade and includes 1) reduced atherosclerosis upon ablation of the Adamts7 gene in mice (table 2) 2) GWAS that show an association of ADAMTS7 SNPs with CAD and 3) immunohistochemical detection of ADAMTS-7 in human atherosclerotic plaques.
Knockout of Adamts7 on an atherosclerotic background reduced total atherosclerotic lesion area in the aorta of Adamts7 −/− /ApoE −/− mice by 62% (male) and 54% (female) compared to littermate controls. In Adamts7 −/− /Ldlr −/− mice, the reductions were 37% and 52%, respectively . These findings suggest that pharmacological inhibition of ADAMTS-7 activity could slow down the progression of atherosclerosis. Adamts7 −/− mice also showed an altered response of VSMCs to arterial wire injury [116,117]. They showed greatly reduced neointima formation upon vascular injury, which had been seen previously in rats upon Adamts7 knockdown with siRNA in a balloon injury model . Using the same model, the opposite effect was seen when ADAMTS-7 was overexpressed . These effects of ADAMTS-7 on VSMC in vivo agree with findings in vitro [116–118,174].
GWAS have shown that SNPs in the ADAMTS7 locus are associated with CAD . The SNP that is thought to cause the association is rs3825807, of which the G allele is associated with a reduced risk of CAD. It causes a serine to proline substitution in position 214 of the prodomain of ADAMTS-7. The proline is located near recognition motifs of subtilisin-like proprotein convertases such as furin and PCSK6, which activate ADAMTS-7 by proteolytic removal of the inhibitory prodomain. The proline was shown to hamper prodomain removal, which consequently reduces the activation of ADAMTS-7  and potentially mediates the reduced CAD risk associated with the G allele. VSMCs of the G/G genotype for rs3825807 also migrated less in vitro compared to the A/A genotype.
Immunohistochemistry of human atherosclerotic plaques identified ADAMTS-7 protein [174,175]. In carotid plaques, ADAMTS-7 levels were increased in patients with cerebrovascular symptoms compared to patients without these symptoms and high levels also correlated with increased risk of post-operative cardiovascular events .
Several in vitro substrates of ADAMTS-7 have been reported in the literature, but clear links with atherosclerosis and VSMC behaviour have not been established [117,176,177]. The mass spectrometry-based method terminal amine isotopic labelling of substates (TAILS) was used to identify potential ECM substrates, which identified latent TGF-β binding protein 4 (LTBP4) as a substrate . LTBP4 is a component of microfibrils/elastic fibres in the lung and large blood vessels and is also co-expressed with ADAMTS-7 in the heart [179,180]. It binds to several ECM proteins, including fibrillin-1, fibulin-4 and fibulin-5, which are essential for the formation of elastic fibres in large blood vessels [179,181,182]. Proteolysis of LTBP4 by ADAMTS-7 likely affects this process, but this remains to be investigated in vivo. The significance of LTBP4 proteolysis by ADAMTS-7 for atherosclerosis is also currently unclear. However, a recent report showed that the LTBP4 gene was differentially expressed between plaques from symptomatic and asymptomatic patients , suggesting LTBP4 may affect the composition of atherosclerotic plaques.
Whereas several other ADAMTS family members are regulated by the endogenous metalloprotease inhibitor TIMP-3, ADAMTS-7 is more susceptible to inhibition by TIMP-4, which inhibited ADAMTS-7 efficiently at low nanomolar concentrations . Both TIMP-4 and ADAMTS-7 have a restricted tissue distribution with particularly abundant expression in adult cardiovascular tissues [116,184].
In summary, ADAMTS-7 is a potential therapeutic target in CAD and related diseases resulting from atherosclerosis, but more research is needed to validate it as a target and allow a better understanding of the molecular mechanisms involved.
4. ADAMTS-13, thrombotic thrombocytopenic purpura and stroke
ADAMTS-13 circulates in blood at a concentration of approximately 6 nM, where it trims newly secreted VWF multimers that would otherwise be too thrombogenic  (figure 1a). Very low (less than 10%) ADAMTS-13 activity causes the disease TTP and moderately low ADAMTS-13 activity is associated with ischaemic stroke, which is also a feature of TTP. In TTP, the VWF multimers that are too long spontaneously aggregate platelets in the absence of endothelial damage . TTP is a rare, life-threatening condition that most commonly presents in previously healthy young to middle-aged adults, with an annual incidence of 6 per million in the UK [187,188]. They show severe microangiopathic hemolytic anemia (MAHA), severe thrombocytopenia and end-organ damage. The organs most commonly affected are the heart, brain, kidneys and gastrointestinal tract . The MAHA is the result of the microthombi that occlude the small vessels and damage the red blood cells, whereas the severe thrombocytopenia is caused by consumption of the platelets that are caught up in the microthrombi. The end-organ damage results from the occlusion of the small vessels that oxygenate the organs . In a small minority of TTP patients, ADAMTS-13 activity is low because of inherited mutations in the coding region of the ADAMTS13 gene . The most common form of TTP is immune-mediated TTP (iTTP), involving autoantibodies against ADAMTS-13, which rapidly clear the enzyme from the circulation and inhibit its activity by preventing binding to its substrate VWF . Autoantibodies that target the N-terminal domains up to the Sp domain can be inhibitory, in line with the discovery of several exosites in these domains [190–195]. Treatment of iTTP currently consists of plasma exchange (PEX) to remove pathogenic autoantibodies and provide ADAMTS-13, in conjunction with Rituximab to suppress autoantibody production [186,196]. PEX is critical and lifesaving when patients present at the hospital in an emergency. More recently, PEX is also supplemented with Caplacizumab, a construct consisting of two fused single-domain antibodies that both target the same epitope in the VWF A1 domain and reduce platelet aggregation . Recombinant ADAMTS-13 is currently undergoing a Phase 2 clinical trial, in which it is used to supplement PEX in iTTP patients with the aim to speed up recovery and reduce the use of PEX (ClinicalTrials.gov: NCT03922308). Whereas most antithrombotic agents carry a risk of bleeding, this is unlikely for recombinant ADAMTS-13 because of the unique mechanism by which its activity is regulated. It is not regulated by a physiological inhibitor (e.g. TIMPs, ), but by the limited and conditional availability of VWF exosites and scissile bonds. These are normally buried inside the globular VWF A2 domain and only exposed when the A2 domain unfolds upon elevated shear stress in the circulation [199–201]. Ultra long VWF undergoes elevated shear stress when it exits the endothelium and enters the circulation, causing proteolysis by ADAMTS-13, which reduces the size of the multimer and thereby reduces the tensile force exerted upon the molecule, preventing further unfolding and cleavage of VWF A2 domains [199,202]. VWF is the only reported substrate of ADAMTS-13. The specificity of the protease is conferred by exosites in several ADAMTS-13 domains that bind complementary exosites in the VWF A2 domain C-terminal to the scissile bond [193,194,203–205]. In addition, subsites in the Mp domain accommodate VWF side chains either side of the scissile bond and add to the overall specificity of the enzyme [204,206,207].
Whereas ADAMTS-13 activity levels less than 10% can cause TTP, low levels that fall within the normal range (lowest quartile) are associated with increased risk of developing ischemic stroke [208–210]. In patients who present with acute ischemic brain injury, the ratio of VWF antigen levels to ADAMTS-13 activity (VWF:Ag/ADAMTS-13Ac) predicts mortality and is associated with impact on brain function in survivors . Also, in TTP patients who have recovered and are in remission, lower ADAMTS13 activity levels after recovery are associated with stroke . In animal models of ischemic stroke, the administration of recombinant ADAMTS-13 after a stroke appears to be beneficial [213,214].
5. Other ADAMTS family members
5.1. The procollagenase ADAMTS-2 in myocardial repair
ADAMTS-2 is the major enzyme cleaving the N-propeptide from type I procollagen, thus allowing the assembly of collagen trimers into fibrils/fibres . Although this has primarily been studied in the skin, it may also occur in other tissues. For example, the analysis of hearts from cardiomyopathic patients showed upregulation of ADAMTS2, which may reflect the important role of collagen in myocardial repair and scarring. The collagen that is deposited to ‘repair’ myocardial damage requires prodomain removal by ADAMTS-2 for collagen fibres to form. Increased collagen expression is likely to require increased ADAMTS-2 expression. This may also explain the altered Adamts2 expression in mice treated with isoproterenol, which induces cardiac myopathy and hypertrophy  (table 2). In Adamts2 null mice, the detrimental effects of pressure overload on the heart were enhanced , possibly owing to disturbed repair mechanisms involving collagen.
5.2. ADAMTS-6 in heart development and QRS duration
ADAMTS6 mRNA has been detected in the mouse heart, specifically in the outflow tract, valves, atria and the ventricular myocardium . The physiological function of ADAMTS-6 is not known but in vitro studies suggest that it may be linked to that of fibrillin-1 microfibrils and focal adhesions . Importantly, ADAMTS-6 has been implicated in cardiac biology by a GWAS, which found that two ADAMTS6 missense variants (S90 L and R603 W) were associated with the duration of the QRS interval of the electrocardiogram , which reflects cardiac ventricular depolarization. A prolonged QRS is a predictor of mortality both in the general population and in patients affected by cardiovascular disease [217–219]. The missense variants S90 L in the prodomain and R603 W in the first TSP-1-like domain both severely impair protein secretion . Puzzingly, S90 L was associated with longer QRS and R603 W with shorter QRS duration. In people of European descent, approximately 1/500 is heterozygous for the S90 L variant, whereas R603 W is rare in individuals of European ancestry but is common in people of African decent, where approximately 1/70 is heterozygous (https://gnomad.broadinstitute.org/). Both variants may, however, be pathogenic in a homozygous state and/or function as risk factors for cardiac disease in heterozygous form. A cardiac role for ADAMTS-6 is also in line with the fatal congenital heart defects of mice homozygous for a null mutation in Adamts6, which die pre/peri-natally . Their embryonic heart defects comprise the double outlet right ventricle, an atrioventricular septal defect and ventricular hypertrophy. Interestingly, mice hemizygous for the null mutation are viable and do not show structural heart defects but their ventricles express low levels of connexin-43 protein, the main myocardial gap junction protein in mouse and human heart. The reduced levels of connexin-43 appear to have a post-transcriptional cause, as the mRNA levels of the corresponding gene (Gja1) were unaffected . In summary, these findings suggest that ADAMTS-6 may play a role in development of the heart and in regulating gap junction-mediated ventricular depolarization.
5.3. ADAMTS-10 and cardiovascular manifestations of WMS
Mutations in ADAMTS10 cause an autosomal recessive form of WMS [220–222]. WMS is a rare inherited disorder of the connective tissues characterized by short stature, brachydactyly, joint stiffness, broad skull, heart defects and a variety of eye abnormalities [222,223]. Three distinct ADAMTS10 mutations were identified in two consanguineous families and in one sporadic WMS case, including one nonsense mutation and two splice mutations . Among the clinical features of WMS patients bearing ADAMTS10 mutations were aortic and pulmonary stenosis with dysplastic valves and hypertrophic obstructive cardiomyopathy . Weil-Marchesani syndrome is also caused by mutations in fibrillin-1 (FBN1) and LTBP2, implicating ADAMTS-10 in fibrillin-1 microfibril biology . Fibrillin-1 microfibrils are ECM assemblies that are essential for the formation of the elastic fibres in blood vessels, lungs, skin, ligaments and other elastic tissues. They also regulate the bioavailability of growth factors of the TGF-β and bone morphogenetic protein (BMP) superfamily . ADAMTS-10 was subsequently confirmed to regulate fibrillin microfibril function, and to bind fibrillin-1 at two sites that coincide with the fibrillin-1 mutations in WMS . It also co-localizes with fibrillin-1 in tissues and accelerates microfibril assembly in fibroblast cultures . Ocular features in WMS caused by ADAMTS10 mutations may be owing to reduced fibrillin-2 cleavage . Mutations in other genes involved in fibrilin microfibril biology cause the related disorders WMS-like syndrome (ADAMTS17) and geleophysic dysplasia (LTBP3, ADAMTSL2, Fibrillin 1) .
5.4. ADAMTS-16, a potential regulator of blood pressure
The function of ADAMTS-16 is currently unknown, with contradictory reports on its possible involvement in male fertility and sex determination [229,230]. So far, the only described substrate is fibronectin . ADAMTS-16 has been identified as a quantitative trait gene (QTG) for blood pressure in humans [232,233] and rats . To investigate the cardiovascular function of ADAMTS-16, Adamts16 knockout rats have been generated  (table 2). These rats show lower systolic blood pressure, decreased arterial stiffness and thickness of the tunica media compared with wild-type rats, suggesting an involvement of ADAMTS-16 in regulating haemodynamics . Moreover, Adamts16 knockout rats survived longer, although they exhibited renal anomalies . More data are needed to ascertain a possible role of ADAMTS-16 in blood pressure regulation.
5.5. ADAMTS-19 in progressive heart valve disease
A recent study of early-onset valvular disease identified mutations in ADAMTS19 by whole exome sequencing. Four patients in two consanguineous families carried homozygous mutations in ADAMTS19 causing a large multi exon deletion in one family and a truncated ADAMTS-19 protein in the other family . To confirm causality, Adamts19 knockout mice were generated (table 2). Of the homozygous Adamts19 knockout mice, 38% showed aortic valve regurgitation and/or aortic stenosis at three months of age, confirming an important role of ADAMTS-19 in aortic valve physiology. Expression analysis of lacZ in Adamts19 knockout mice showed strong localized expression of lacZ by valvular interstitial cells in all four valves around E14.5 and expression by these cells until adulthood. Ultrastructural analysis of the ECM suggested alterations in ECM organization, including PG accumulation. The observation of PG accumulation raises the question whether this is secondary to disturbed valve physiology/cellular function or reduced PG turnover by ADAMTS-19.
6. Targeting ADAMTS therapeutically
ADAMTS inhibitors could potentially be used therapeutically to reduce enzyme activity that contributes to or aggravates cardiovascular disease (e.g. for ADAMTS-7). However, it has proven challenging to develop therapeutic metalloprotease inhibitors with sufficient selectivity to prevent side effects caused by cross-inhibition of related metalloproteinases . Selective inhibitors can be either monoclonal antibodies  or small molecules, with the latter having the distinct advantage that they can be administered orally. Selective small molecule inhibitors can be designed on the basis of the available tridimensional structure of the target enzyme or identified by high throughput screening (HTS) of large compound libraries. To screen small molecule libraries for their inhibitory potential, purified enzyme and a high throughput activity assay are needed. Typically, high throughput activity assays for proteases involve Förster resonance energy transfer (FRET) technology, where proteolysis of a small peptide generates a fluorescent signal . For ADAMTS-7 we have developed such an activity assay using a small LTBP4 peptide as an efficient substrate and are currently converting this for HTS of small molecule libraries (manuscript in preparation)
On the other hand, where the activity of an ADAMTS family member has proven to be beneficial in a certain pathological context, so-called enhancers or activators may be envisaged. For example, monoclonal antibodies able to increase the catalytic activity of ADAMTS-13 have been reported . Another innovative approach to increase ADAMTS activity may involve interfering with LRP-1-mediated endocytosis. We have recently reported a monoclonal antibody that is able to bind to ADAMTS-5 and block its binding to LRP-1 without interfering with its proteoglycanase activity , resulting in accumulation of active ADAMTS-5 in the extracellular milieu. Such an antibody may be used to rescue ADAMTS-5 proteoglycanase activity in mouse models of atherosclerosis and TAAD (table 2).
ADAMTS family members fulfil multiple distinct roles in cardiovascular tissues (table 1). Several of them are important for cardiac valve embryogenesis and homeostasis (figure 2), but in general the multitude of physiological processes affected by ADAMTS proteases reflects the many functions of the ECM, such as regulating cell behaviour and sequestering of a wide range of cellular growth factors. Much has been learned about ADAMTS proteases from animal studies, but caution should be exercised when extrapolating mouse data to human disease in the absence of clinical studies. In this regard, a conclusion should be supported by high-quality data collected in vitro, in vivo and ex vivo but so far this goal has been achieved just for few family members. Nevertheless, exciting new findings are published every year, incrementally elucidating the physiological roles of this fascinating family of proteases.
The protease data
The proteases in P. falciparum were predicted using a comparative genomics approach and a support vector machine (SVM)-based, supervised machine learning approach [1-3]. The classification and annotation were according to the MEROPS protease nomenclature, which is based on intrinsic evolutionary and structural relationships .
Network data and analysis
The complete set of protein-protein associations for P. falciparum was extracted from the downloaded STRING database  each association between a pair of proteins has a confidence score (S) ranging from 0.15 to 0.999 that was inferred from the evidence used to establish the association, such as homology transfer, KEGG pathway assignments, conserved chromosome synteny, phylogenetic co-occurence, and literature co-occurence . This set of associations was visualized in Cytoscape  and converted to an undirected weighted graph, where there is a single edge between any pair of proteins and the S value is used as the weight. The network was characterized using NetworkAnalyzer  and significant modules were detected using MINE  and MCODE . The default values were used for all three plugins. The set of proteins directly associated with the 77 proteases in the association set were screened using BiNGO  to determine if any categories of proteins, as identified by their Gene Ontology terms, were over-represented. The hypergeometric test was used with the Benjamini and Hochberg false discovery date correction. A significance level of 0.05 was selected.
The omics data mining
We downloaded the P. falciparum genomic sequence and annotation data , transcriptomic microarray data [6-8], mass-spectrometry proteomic data [9-12], and protein-protein interactome  data for network associated proteins from PlasmoDB, the Plasmodium Genome resource center (http://www.plasmodb.org) . Conserved domains/motifs in P. falciparum sequences were identified by searching InterPro . Multiple alignments were obtained using the ClustalX program  and T-coffee , followed by manual inspection and editing. Phylogenetic trees were inferred by the neighbor-joining, maximum-parsimony and maximum-likelihood methods, using MEGA5 .
Proteases are currently classified into six groups:
- Serine proteases
- Threonine proteases
- Cysteine proteases
- Aspartic acid proteases
- Glutamic acid proteases
The threonine and glutamic acid proteases were not described until 1995 and 2004, respectively. The mechanism used to cleave a peptide bond involves making an amino acid residue that has the cysteine and threonine (peptidases) or a water molecule (aspartic acid, metallo- and glutamic acid peptidases) nucleophilic so that it can attack the peptide carbonyl group. One way to make a nucleophile is by a catalytic triad, where a histidine residue is used to activate serine, cysteine, or threonine as a nucleophile.
Proteases in the blood - Biology
In his 1996 book, Darwin's Black Box, Michael Behe argued that the vertebrate blood clotting cascade was "Irreducibly Complex." What Prof. Behe means by this is that each and every element of the complex cascade of enzymes and cofactors must be in place for blood clotting to work. Since, according to Behe, an irreducibly complex system cannot be produced by Darwinian natural selection, it must have been produced by something else. It must have been designed .
After the publication of Behe's book, a number of scientists were quick to point out that Behe was mistaken in many of his assertions about the blood clotting cascade. The work of Russell Doolittle and many other scientists has shown quite clearly that the system evolved by a process of gene duplications from serine proteases that once were digestive enzymes. Not surprisingly, Behe asserts that the many criticisms of his work are incorrect. In August, 2000 he placed an essay on the internet:
In defense of the Irreducible Complexity of the Blood Clotting Cascade.
A Response to Russell Doolittle, Ken Miller, and Keith Robison.
A Response to Behe's Attempted Defense
As I wrote in my 1999 book Finding Darwin's God , Russell Doolittle's pioneering work on protein evolution has indeed shown that the blood clotting cascade could, and indeed was, produced by Darwinian evolution. Dr. Behe's defense of his position to the contrary requires him to explain why my description of the system's evolution (pp. 152-161) is not valid. In his web-published defense he writes that my description is "a just-so story that doesn't deal with any of the difficulties the evolution of such an intricate system would face."
Curiously, Behe all but ignores my description of the evolution of blood clotting in the lobster, and provides no rebuttal to my scenario for its evolution (based, of course, on Doolittle's research work). I described the lobster system in my book for two reasons: (1) It, like the vertebrate system is (by Behe's standards) irreducibly complex, and (2) It is a simpler system whose step-by-step evolution is relatively easy to account for.
Behe quite properly notes that my own description of the evolution of the vertebrate clotting system is rather brief. a decision, alas, of my Editor at Harper-Collins (the book's publisher). Nonetheless, I still have my original draft of this edited-out section, which the reader may wish to consult. (Click here for my ideas on the evolution of the vertebrate clotting system). However, which elements of my description, besides its sketchiness, does he take issue with?
Behe asserts that the targeting of a protease, a digestive enzyme, to the bloodstream is a "potentially deadly situation," and tells the readers of his web document that we can tell how deadly this might be by looking at situations "where regulatory proteins are missing from modern organisms." In other words, Behe wants us to look at what happens when the highly-regulated current versions of clotting proteases are missing their regulatory factors. Despite this bluster, however, Behe has no evidence that the mistargeting of an inactive protease to the bloodstream would cause harm. Indeed, the recent discovery that antifreeze protein genes in fish arose from exactly such a mistargeting of proteases into the bloodstream (Chen, L., DeVries, A. L. & Cheng, C.- H. C. Proc. Natl Acad. Sci. USA 94, 3811­3816 (1997) and Chen, L., DeVries, A. L. & Cheng, C.-H. C. Proc. Natl Acad. Sci. USA 94, 3817­3822 (1997)) suggests that exactly the opposite is true.
Having made unsupported claims about the "danger" of such a mutation, Behe says that it would be difficult to see what "advantage" this would present to the organism. The answer, of course, is that it would provide a slight improvement in the organism's ability to clot blood - and that's the point. The clotting system doesn't have to work full-blast right away. In a primitive vertebrate with a low-pressure circulatory system, a very slight improvement in clotting would be advantageous, and would be favored by natural selection.
Behe then wonders how the circulating protease could become localized at the site of a clot, as if this were an insurmountable difficulty. It's not. As I suggested in my original draft on the evolution of clotting, a well-understood process called exon shuffling could have placed an "EGF domain" onto the protease sequence, and the "problem" that Behe puzzles over is solved in a flash.
Finally, Behe emphasizes that the real problem is not to generate a clot - it is to "regulate" that clot by means of an inhibitor of the protease so that it doesn't become destructive. But that's not a problem for evolution, either. As usual, Behe envisions a clotting protease that is just as powerful as the fully-evolved proteases in modern vertebrates. However, remember that this is the same guy who fretted a moment or two ago that the protease would not be strong enough to clot effectively. He wants to have it both ways. The answer to his objection is just what I wrote in the draft:
" . a primitive clotting system, adequate for an animal with low blood pressure and minimal blood flow, doesn't have the clotting capacity to present this kind of a threat. But just as soon as the occasional clot becomes large enough to present health risks, natural selection would favor the evolution of systems to keep clot formation in check. And where would these systems come from? From pre-existing proteins, of course, duplicated and modified. The tissues of the body produce a protein known as alpha-1-antitrypsin which binds to the active site of serine proteases found in tissues and keeps them in check. So, just as soon as clotting systems became strong enough, gene duplication would have presented natural selection with a working protease inhibitor that could then evolve into antithrombin , a similar inhibitor that today blocks the action of the primary fibrinogen-cleaving protease, thrombin."
In short, none of the points raised by Behe are adequate to explain why the vertebrate clotting system could not have evolved. Furthermore, as Doolittle's work has shown clearly, the hypothesis of evolution makes testable predictions with respect to the DNA sequences of clotting proteins, and these predictions have turned out to be correct time and time again.
Why has Behe's "Biochemical Challenge to Evolution" met with so little support within the scientific community? I would suggest that the reason is simple. His hypothesis is wrong. The complex biochemical systems of living organisms, including the vertebrate clotting cascade, are fully understandable in terms of Darwinian evolution.
Kenneth R. Miller
Professor of Biology
Providence, RI 02912
PARs and proteases—cooperation in cancer progression
TF is a membrane glycoprotein present on subendothelial cells that initiates blood coagulation. The disruption of endothelium exposes TF to coagulation factors present in the bloodstream. TF binds to FVII and causes its activation (FVIIa). The TF/FVIIa complex may further activate FX (FXa), which together with its cofactor FVa, generates thrombin (FIIa) from prothrombin by proteolytic conversion. Thrombin initiates coagulation by platelet activation and fibrin conversion from fibrinogen, resulting in effective blood clotting .
TF is also the most prominent procoagulant of cancer cells and is a determinant of tumor progression . TF has been discovered on the surface of distinct malignant cells, tumor vasculature, and tumor microenvironment: stem cells, macrophages, ECs, and myofibroblasts [9, 10, 40, reviewed in 98, 99]. It is also widely recognized that TF expression correlates with greater invasiveness and higher clinical stage of the malignant disease and is associated with poor overall prognosis [reviewed in 40, 97]. Tumor cells endogenously express TF constitutively, or they induce production of TF in their surroundings by producing soluble substances capable of triggering monocytes and ECs to express it . TF expression is associated with carcinogenic events during oncogenic transformation, as there exists mounting evidence that mutations of proto-oncogenes and tumor suppressor genes influence its expression . In colorectal cancer, the K-ras and p53 mutations eliciting the MAPK and PI3-mediated signaling pathways result in enhanced expression of TF . In lung cancer, similar observations were made for PTEN and p53 mutations . TF expression has also been shown to be modulated in other cancers by constitutively active mutant forms of epidermal growth factor receptor (EGFRvIII) in glioma and vulva cells, as well as Src family kinases, TGF-β production, and hypoxia .
There are numerous mechanisms by which TF impacts cancer biology. First, upon activation by factors VII and X and creating complexes with them (TF/VIIa, TF/Xa, TF/VIIa/Xa), TF promotes PAR-1- and PAR-2-mediated signaling responsible for the proliferative response of cancer cells [38, 97, 102]. In addition, TF may directly signal via its cytoplasmic tail through Rac1 and p38 and cytoskeletal remodeling . Furthermore, an alternatively spliced isoform of TF (asTF) also affects tumor growth independently of VIIa and PARs cleavage, through the activation of integrins α6β1 and αVβ3 on ECs to promote angiogenesis [97, 104]. Human asTF promoted tumor growth and angiogenesis in pancreatic cancer  but was inactive in the coagulant-dependent mechanism of metastasis in a breast cancer model .
In experimental and clinical models, cancer cells expressing TF had greater tendency to metastasize compared to TF-deficient cells . TF effects on metastasis may be mediated via mechanisms that are either dependent on or independent of coagulation activation, i.e., through TF signaling function. Tissue factor likely promotes proliferative and infiltrative potential rather than adhesive properties of metastatic cells [30, 68, 107]. There is also evidence that TF plays a role in tumor cell intravasation, which is the first step in dissemination of malignant cells .
Although TF may promote both PAR-1 and PAR-2 activation, it seems that TF or TF/FVIIa complex typically triggers PAR-2 but not PAR-1 signaling in cancer cells [38, 67–69, 99, 102, 109, 110]. In breast cancer experimental models, inhibition of tumor growth and angiogenesis was observed after blocking the signaling function of TF but not its coagulation activity, and after inhibition of PAR-2, but not PAR-1 activity [109, 110]. A similar phenotype was observed in glioblastoma (GBM), which is the most aggressive primary brain tumor characterized by intense neovascularization, EC hyperplasia and hypercoagulation . Experiments with GBM cell lines determined that there was expression of PAR-1 and PAR-2 in these cells as well as in vascular vessel walls within the invasive area of brain tumors [31, 72, 73]. However, only stimulation of the PAR-2 pathway led to increased secretion of VEGF and IL-8 suggesting that PAR-2/MAPK/ERK1/2, but not PAR-1/PI3K/Akt, signaling regulates angiogenesis in GBM. It is noteworthy that in GBM cells there is a correlation between TF and PAR-2 expression . There is also evidence that hypoxia upregulates PAR-2 expression in brain tumors. There is an approximately 2.5-fold increase in PAR-2 expression in hypoxic vs. normoxic microvascular ECs of GBM, resulting in HB-EGF upregulation and a proangiogenic phenotype . Poole et al. have recently demonstrated that PAR-2, which is a central factor in neurogenic inflammation and pain, sustains inflammation through a novel TRP channel-coupling mechanism. By generating bioactive lipids such as 5′,6′-EET and 12(S)-HETE, the proinflammatory effects of PAR-2 are sustained through TRPV4-dependent Ca 2+ signals . This may prove extremely relevant in this context as TRPV4 has been shown to impact angiogenesis at multiple levels [113, 114]. Finally, EGFR-induced signaling in glioma cells stimulates expression of TF, FVII, and PAR-2, thereby increasing TF/VIIa-mediated PAR-2 activation in cancer cells , and cancer cells may secrete aFVII that can act alone to activate PAR-2 .
TF/VIIa-mediated PAR-2 activation results in a transient increase in Ca 2+ levels and triggers intracellular signaling that is dependent on the MAPK family (p44/42, p38, JNK), PI3, Src-like kinases, Jak/STAT, Rho GTPases, Rac1, and Cdc42 pathways [40, 80, 102]. In addition, elevated levels of proangiogenic proteins, such as VEGF, Cyr61, VEGF-C, CTGF, CXCL1, IL8, and immune modulators, such as GM-CSF (or CSF2) and M-CSF (or CSF1), have been observed [38, 68, 73, 97]. The efficacy of TF/VIIa/PAR-2-mediated activation of angiogenic mediators is greater than that induced by PAR-1 signaling . TF-triggered PAR-2 signaling also results in increased MMP-9 expression, which positively correlates with the invasiveness of MCF-7 breast tumor cells  and may be linked to MMP-9 response to arachidonic acid metabolism . It was reported that TF/FVIIa/PAR-2 interactions are critical for MDA-MB-231 breast cancer cell migration and invasion toward NIH-3T3 fibroblast-conditioned medium . Therefore, TF/VIIa-induced PAR-2 activation facilitates proliferation and survival as well as metastatic potential of cancer cells [50, 68, 70].
In breast cancer cells, PAR-2 activation may also be induced by FXa as well as by TF/FXa or TF/FVIIa/FXa complexes. Subsequent MAPK phosphorylation or Erk1/2 activation then stimulates cancer cell migration and invasion [67, 68].
In tumors with high levels of TF (prothrombotic state), the predominant metastatic mechanism results from the coagulation activity of TF instead of its inherent signaling capacity . The procoagulant activity of TF leads to thrombin generation, platelet activation, and platelet-dependent protection from natural killer cells as well as fibrin formation and monocyte/macrophage recruitment, all of which influence angiogenic and metastatic properties of the tumor [6, 97, 106, 118]. Studies of Yokota et al.  provided new insight into thrombin-mediated TF-dependent metastasis based on a hyperthrombotic mouse model with thrombomodulin deficiency (TM Pro mice). TF-dependent, but contact-pathway-independent, breast cancer metastases were associated with hyperactivity of platelets and formation of platelet-leukocyte aggregates. Genetic deletion of platelet glycoprotein Ibα (GPIbα) and leukocyte CD11b excluded these receptors from platelet-dependent metastases. In addition, blockade of both host and tumor PAR-1 significantly decreased tumor cell metastatic potential. Similar results were obtained in melanoma models, thus confirming the contribution PAR-1 to melanoma and breast metastases .
Generation of thrombin (IIa) is the central step in blood coagulation. As mentioned above, thrombin cleaves fibrinogen to yield fibrin and activates blood platelets resulting in the formation of an effective blood plug after vessel injury. However, enzymatically active thrombin is also detected in various types of surgically removed malignant tumors (e.g., small cell lung cancer, renal, ovarian, laryngeal, pancreatic, and gastric cancer, as well as melanoma) [11, 98, 119, 120].
The presence of TF on tumor cells contributes to thrombin generation in the tumor microenvironment independently of blood coagulation. Multiple thrombin targets (e.g., blood platelets and EC activation, fibrin generation) contribute to cancer progression by providing matrix for new vessels and metastatic tumor cell colonies [118, 121, 122]. The first reports of a novel role for thrombin in tumor cell metastases were published in the early 1990s [123–128]. When incubated with W256 carcinoma cells, α-thrombin produced a 50–300 % increase in adhesion to rat aortic endothelial cells and fibronectin [123–127]. Thrombin precursors and analogues including prothrombin, prothrombin-1, mesyl-thrombin, exo-site-thrombin, DFP-thrombin, and nitro-thrombin imitated the effect of α-thrombin [123–127]. Interestingly, α-thrombin coupled with its inhibitors, namely hirudin or antithrombin III-heparin complex, was not as effective at enhancing tumor cell adhesion as the native form of the enzyme [123–127]. The data indicate a new mechanism of thrombin interaction in tumor cell metastasis that is nonproteolytic. Moreover, mice transplanted with human ovarian cancer cells (SKOV3) demonstrated elevated tumor size and decreased survival rate when treated with thrombin . Whether thrombin signaling works synergistically with the arachidonate metabolizing pathways that stimulate ovarian cancer growth remains to be determined . In addition to its pivotal role in the coagulation pathway, thrombin is regarded as the main PAR-1 and PAR-4 activator. Thus, many cellular responses, including the ones observed in cancer cells such as cytoskeletal rearrangement , are thrombin-dependent. The evidence for a crucial role of TF-dependent thrombin generation and thrombin-mediated platelet PAR-4 activation in cancer progression and metastasis comes from studies performed on genetically modified mice. Stromal and tumor cells are involved in multiple steps of tumorigenesis, including proliferation, angiogenesis, invasion, and survival. Those animals depleted of platelets, PAR-4, or fibrinogen were protected from metastasis [118, 121, 122]. Treatment of melanoma B16a cells with α-thrombin resulted in a significantly increased number of metastatic lung colonies [123–127]. Prothrombin, ɣ-thrombin, and mouse thrombin, but not nitro-thrombin, were able to mimic the α-thrombin effect of enhancing lung colonization potential of tumor cells [123–127]. Administering thrombin intravenously with colon cancer cells (CT26) and melanoma cells (B16a) increased murine pulmonary metastases 4- to 413-fold . The metastatic potential was diminished by hirudin, a specific inhibitor of thrombin [4, 122, 132].
Thrombin/PAR-1 in fibroblasts
During the coagulation process the conversion of prothrombin to thrombin and its subsequent activity leads to cleavage of fibrinogen to form fibrin. Fibrin deposits in the tumor microenvironment are the store of thrombin that is released upon degradation of fibrin by plasmin . The in vitro experiments provided evidence that stromal cells of malignant tumors, such as fibroblasts express elevated PAR-1 and PAR-2 compared to benign lesions or normal tissues where such expression is not observed . Chronic PAR-1 mediated signaling in NIH-3T3 fibroblasts can cause growth transformation . Reportedly, PAR-1 expression in the microenvironment drives progression and induces chemoresistance of pancreatic cancer  by regulating monocyte migration and fibroblast-dependent chemokine production.
Thrombin/PAR-1 in endothelial cells
Endothelial cells are another target of thrombin/PARs interactions. Thrombin-mediated PAR-1 activation regulates inflammatory pathways that are also implicated in cancer progression. Increased lipid production and expression of PAF, IL-1, IL-6, IL-8, TNF-α and adhesive molecules (E-selectin, P-selectin, intracellular adhesion molecule-1 and vascular cell adhesion molecule-1, integrins) promotes EC proliferation, platelet recruitment, and malignant cell attachment [4, 5, 128, 135–138]. Inhibition of PAR-1 activity inhibits EC growth by increasing the sub-G0/G1 fraction, thereby reducing the percentage of cells in S-phase . Moreover, thrombin/PAR-1 activation regulates barrier function between ECs by modulating adherens junctions (AJ) . The increase in endothelial barrier permeability in response to thrombin/PAR-associated actions results from VE-cadherin, p120, and β-catenin modification via protein kinase C-dependent signaling [138, 140]. The dysfunction in the endothelial barrier generates a temporary proangiogenic matrix that is the basis for the activation of the thrombin/PAR/IP3/Ca 2+ /MAPK cascade and subsequent cellular responses . Upregulation of angiogenic factors such as VEGF, VEGFR2, and angiopoietin-2 via the thrombin/PAR-dependent pathway together with enhanced barrier permeability of ECs results in the induction of angiogenesis and cancer dissemination [4, 141].
The integrin αvβ3 is found mainly on blood vessel cells and plays an essential role in angiogenesis. Localization of αvβ3 is altered in response to proinflammatory eicosanoid metabolites such as 12(S)-HETE leading to EC retraction and disruption in barrier function [142–144]. The expression of integrin αvβ3 is regulated by thrombin-mediated PAR-1 activity. Thrombin activation of PARs also leads to increased expression of gelatinases that degrade collagen IV and increase vessel permeability to promote endothelial and cancer cell migration and invasion .
In ECs, thrombin can directly cleave PAR-1, which is thought to lead to a proinflammatory phenotype, or it can do so indirectly after it activates an intermediate protease called protein C (activated protein C (APC)) that then acts on PAR-1. However, when the GLA-domain of APC is in complex with its cognate receptor, EPCR, and thrombomodulin (TM), the signaling specificity of PAR-1 is altered to an anti-inflammatory or protective phenotype. Thus, in ECs, modulation of coagulation protease signaling specificity through PAR-1 depends on whether thrombin is acting directly on PAR-1, or indirectly, through APC, and whether APC is bound to EPCR . In ECs, PAR-1 can be acted on by both thrombin and activated protein C (APC) to affect opposite outcomes, but this is thought to depend on whether the latter protease is in complex with EPCR.
The APC/EPCR/PAR-1 pathway induces motility, proliferation of ECs, and angiogenesis via vascular-protective signaling and tube formation to promote cancer cell dissemination [146, 147]. Moreover, the EC-associated modulator of hemostasis, TM, also strongly influences metastatic potential associated with thrombin procoagulant function .
Recent reports have linked PAR-2 and TRPV4 activation, where TRPV4 is known to enhance EC proliferation and arachidonic acid-mediated tumor EC migration [112, 113].
Thrombin/PARs in platelets
Human platelets express two types of thrombin-triggered PARs, namely the high-affinity PAR-1 and low-affinity PAR-4. Both receptors activate pleiotropic cellular effects via coupling to protein Gαq and Gα13, which leads to the activation of phospholipase Cβ, hydrolysis of phosphoinositides, and increased cytoplasmic calcium concentration, resulting in activation of integrin αIIbβ3, and platelet aggregation [5, 6, 91, 149]. Initial reports describing the dual PARs system in human platelets explained this phenomenon by the fact that PAR-1 and PAR-4 interact with different concentrations of activator and thus may tune to thrombin signaling more efficiently . Additional studies revealed that PAR-4 functions differently than PAR-1, in that thrombin-induced cleavage of PAR-4 results in much longer activation of Gαq. This leads to a sustained Ca 2+ response, which prolongs secondary signaling, compared to PAR-1, which is crucial for the late phase of platelet aggregation . At low thrombin concentrations, PAR-1 may act as a cofactor of PAR-4. There is also thrombin-mediated mitogenic PAR activity derived from platelets as well as for ECs and myocytes of vessels [97, 120]. Platelets coated with thrombin survive longer, which gives cancer cells opportunity to adhere and invade further [4, 120, 151]. Moreover, tumor cells coated by platelets are protected from natural killer cell-mediated elimination .
The aggregation of platelets and resultant fibrin generation is accompanied by increased expression of adhesive proteins (glycoprotein GPIIb/IIIa, von Willebrand factor, P-selectin, fibronectin) in platelets that have undergone thrombin stimulation . These adhesive proteins enable malignant cells to form complexes with fibrin thrombus and blood platelets in vascular spaces in melanoma and epithelial cancers [reviewed in 4]. These complexes enhance cancer cell survival and metastatic potential. Thrombin treatment of platelets promoted melanoma cell adhesion to platelets, which increased lung metastasis .
In addition to platelet aggregation, thrombin-mediated PAR-1 and PAR-4 cleavage induces selective release of platelet proangiogenic and mitogenic regulators (PDGF, VEGF, and angiopoietin-1) that facilitate migration of endothelial progenitor cells and new capillary net formation, which is a pivotal step to metastases . Compared to healthy subjects, platelets from breast cancer patients produce much higher levels of VEGF in response to thrombin stimulation . Thrombin induced this effect through PAR-1 activation, while PAR-4 stimulation resulted in secretion of endostatin, an antiangiogenic factor .
Thrombin/PAR-1 in cancer cells
Thrombin can elicit a signaling response via direct interaction with PAR-1 present on tumor cells [4, 14, 41, 48]. In vitro studies with various cancer cell lines showed correlation between overexpression of PAR-1 in cancer cells and greater invasiveness and development of distant metastases [14, 17, 18, 41–44, 52, 94, 154]. Moreover, in patients with lung, gastric, or breast cancer, PAR-1 expression was an independent, unfavorable prognostic factor in terms of overall survival, while in prostate cancer patients, it turned out to be a prognostic factor for local recurrence [17, 18, reviewed in 155]. Decreased expression of PAR-1 was associated with reduced invasiveness of cancer cells .
PAR-1 expression has been confirmed in melanoma, breast, lung, esophageal, gastric, colon, prostate, pancreatic, liver, ovarian, endometrial, and head and neck cancers (Table 1) [17, 38, 43–45, 78, 79, reviewed in 155–157]. Intriguingly, although PAR-1 is expressed in normal hematopoietic stem cells, its expression is markedly diminished in acute myeloid leukemia . The cellular effect induced by PAR-1 depends on the concentration of agonist such that low concentration of thrombin (less than 3 nM) stimulates cancer cell proliferation and tumor growth, while high thrombin levels lead to apoptosis . Most cellular effects are triggered via long-lasting activation of second messengers ERK1/2. However, multiple intracellular signaling pathways may be implicated in thrombin/PAR-1 activation (described below) [118, 160].
Apoptosis, proliferation, migration, and invasion
In murine models of benign tumors, PAR-1 activation results in tumor growth and invasion by silencing proapoptotic genes . However, in epithelial cancers and melanoma cells thrombin-mediated PAR-1 activation triggers prosurvival pathways [5, 50, 75, 77, 154, 161]. Overexpression and activation of PAR-1 in nonmetastatic melanoma cell lines stimulates the Akt/PKB signaling pathway, leading to a decrease in Bim and Bax expression, as well as cleaved caspase-3 and caspase-9 levels. Inhibition of PAR-1 activity decreased tumor growth during in vivo experiments, confirming apoptosis-related effects elicited by this receptor .
In numerous cancers, the response to thrombin-induced PAR-1 activation increases cell proliferation, as well as motility and migration in Matrigel barrier assays [45, 46, 50, 77]. In Hep3B liver carcinoma cells, PAR-1 and PAR-4 activate common promigratory signaling pathways via activation of the receptor tyrosine kinases Met, PDGFR, and ROS kinase, as well as the inactivation of the protein tyrosine phosphatase, PTP1B . In nasopharyngeal cancer, thrombin-induced PAR-1 activation leads to increased expression of MMP-2 and MMP-9, which are closely associated with tumor metastasis as they can degrade the extracellular matrix and disrupt the basement membrane [43, 60].
Increased expression of integrins
Integrins are transmembrane proteins that mediate the interactions between ECs and extracellular matrix that are vital for successful angiogenesis [41, 42, 120]. There is substantial evidence that enhanced expression of adhesion proteins due to thrombin-mediated PAR activity results in increased metastatic potential of cancer cells [4, 41–43]. PAR-1 increases the invasive properties of tumor cells primarily by promoting adhesion to extracellular matrix components. Several cancer cell lines (e.g., lung and melanoma) exhibit increased adhesion to platelets as well as aortic and capillary ECs after thrombin/PAR-1 stimulation [4, 14, 41, 42, 130]. PAR-1-driven adhesion to extracellular matrix components occurs via three mechanisms: (1) phosphorylation of focal adhesion kinase and paxillin, and induction of focal contact complexes, (2) mobilization of integrins on the cell surface without altering their level of expression, and (3) specific recruitment of integrin αvβ5 to focal contact sites . Interaction of cancer cells with integrin αvβ5 and cytoskeletal reorganization facilitates cell migration, invasion, and metastatic development in lung cancer and melanoma [43, 163, 164]. Moreover, the application of anti-αvβ5 antibodies specifically attenuates this PAR-1-induced invasion . Expression of integrin αIIbβ3 and P-selectin in response to PAR-1 may lead to attachment of melanoma cells to ECs and platelets and in this way also increase metastatic potential of cancer cells [14, 41, 42, 120]. Increased expression of αIIbβ3 protein was reported in several malignant tumors [4, 14, 41, 42, 165, 166].
The development of new blood vessels, angiogenesis (angio—vessel, genesis—creation) is the pivotal process for tumor growth and progression [167, 168]. Small blood vessels provide cancer cells with oxygen and nutrients and remove metabolic waste products. It is assumed that malignant tumors cannot grow above 2–3 mm 3 without vasculature . Murine embryogenesis and cancer studies demonstrated that PAR-1 expression is necessary for angiogenesis as half the animal embryos deprived of PAR-1 died due to insufficient vasculature development, while activation of PAR-1 signaling prevented cancer cell death . In melanoma and breast cancer cells PAR-1 expression correlates with increased VEGF levels, and stimulation of angiogenesis and tumor growth . There is also correlation between thrombin and VEGF expression in glioma cells suggestive of a possible autocrine mechanism of regulation of angiogenesis in brain tumors.
Thrombin-mediated cleavage of PARs in cancer, blood cells, and vessel wall cells results in activation of transcription of many proangiogenic genes such as VEGF and its receptor (VEGFR), TF, MMP-2, angiopoetin-2 (Ang-2), basic fibroblast growth factor (bFGF), MAP, and PI3 kinases [120, 142, 170–173]. Based on in vitro studies, VEGF stemming from platelets and cancer cells may be secreted within minutes of activation . Moreover, thrombin-mediated PAR activation induces production of reactive oxygen species (ROS) via increased expression of hypoxia induced factor-1 (HIF-1) . HIF-1 activates VEGF gene transcription, and its expression is responsive to arachidonic acid metabolites .
PAR-1 and PAR-4 signaling after platelet activation leads to synthesis and release of thromboxane (TXA2) and 12-hydroxyeicosatetraenoic acid (12(S)-HETE) [6, 142, 143, 175–181]. These are metabolic end products of cyclooxygenase (COX-1) and lipoxygenase (12-LOX) activity on arachidonic acid and are important mediators of thrombus formation, vascular tone, and angiogenesis through their action on specific receptors (TPα, GPR31) and transcriptional regulation of factors such as VEGF and HIF1α [144, 174–186]. Arachidonic acid is released as a substrate for these enzymes from the cell membrane by cytosolic phospholipase A2 (cPLA2a) that responds to signaling from PAR-1 and PAR-4 differentially depending on whether it is coupled to the COX-1 pathway or the 12-LOX pathway . Thrombin activation of PAR-1 and PAR-4 also leads to the formation of esterified eicosanoids at the same rate as the release of free acids. However, HETE esterified to phosphatidylethanolamine after this reaction gets presented to the cell exterior instead of recycling in the interior substrate pool and has unique functions in that context .
Another potentially important phenomenon in cancer metastasis, at least in part regulated by thrombin, is epithelial–mesenchymal transition (EMT) . The mechanism, with its reverse process, a mesenchymal–epithelial transition (MET), enhances the ability of solid cancers to disseminate and colonize distant sites . Malignant tumors composed of moderately differentiated cells can also contain regions of poor differentiation. These cells may detach from the tumor mass and invade the adjacent stroma after undergoing an EMT-like event. They lose expression of epithelial differentiation markers and gain the capacity to express mesenchymal and “stemness” markers. These cells also contribute to migrating circulating stem cells (CSCs) that disseminate and give rise to metastases. During EMT, some characteristics of differentiated epithelium (e.g., apico-basal polarity and cell–cell adhesions) are replaced with mesenchymal traits—rear to-front polarity, capacity for individual cell migration, and invasion of basal lamina and blood vessels . To effectively colonize new sites, such cells must also be capable of undergoing the reverse MET process to re-differentiate and re-establish the organization of cells .
Experimental studies on gastric cancer cell lines revealed that thrombin-mediated PAR-1 activation leads to reprogramming of gene expression by stimulation of transcription factors like SNAIL1 that is known to drive EMT in the embryo . Moreover, in epithelial cancers (e.g., gastric and breast), the thrombin/PAR-1 complex leads to alteration in basement membrane components (increased expression of fibronectin, Wnt and β-catenin, decreased expression of E-cadherin) as well as cytoskeletal proteins (myosin IIA and filamin B), which collectively regulate EMT involved in malignant tumor progression [45, 46, 75, 77, 94, 189].
MMPs are zinc-dependent proteases secreted by both tumor and host cells. It is widely recognized that MMPs are involved in cancer progression and metastasis by facilitating tumor cell invasion through the basement membrane and stromal tissue [39, 157, 190]. Coexpression of MMPs and PARs is associated with high invasiveness (deeper infiltration of tumor, lymphovascular invasion, more frequent occurrence of lymph node metastases, more advanced clinical stage of the disease) and poor survival in several malignant tumors, e.g., breast, gastric, esophageal, gallbladder, hepatocellular, lung, and ovarian cancers [18, reviewed in 39, 157, 189].
In addition, studies with breast, gallbladder and ovarian cancer cell lines have shown that MMPs (MMP-1, MMP-9, MMP-13, MMP-14) may activate PARs signaling, especially by cleavage of PAR-1 (majority of tumors) or PAR-2 (lung cancer) [15, 39, 52, 71]. Moreover, it was determined that senescent fibroblasts enhance early skin carcinogenic events via MMP-1-mediated PAR-1 activation . Of the MMPs tested, MMP-1 presents the strongest positive correlation with cell migration and invasiveness. The blockade of MMP-1-mediated PAR-1 activity in xenograft models of advanced peritoneal ovarian cancer results in the inhibition of angiogenesis and metastasis .
Activation of platelet PAR-1 by MMP-1 can also lead to Rho-GTP as well as MAPK signal activation, thereby promoting platelet aggregation as well as increasing platelet motility and cell proliferation . The ProMMP-1 zymogen is converted to MMP-1 on the platelet surface after contact with collagen fibrils. Blockade of MMP-1/PAR-1 signaling greatly inhibits thrombosis in animals, demonstrating that the collagen/MMP-1/PAR-1 pathway is an activator of platelet signaling events independent of thrombin. As PARs stimulate the expression and release of 12(S)-HETE that upregulates MMP9 , there appears to be a precedent for bi-directional regulation of MMPs and PARs signaling.
Trypsin is another serine protease that activates PAR-2 in cancer cells. The concentration of trypsin is increased in patients with gastric, colon, pancreatic, and ovarian cancer [54, 155, 192]. Increased expression of PAR-2 and its influence on cancer cell proliferation was defined in gastric, esophageal, colorectal, pancreatic, oral squamous, liver, cholangiocarcinoma, lung, breast, and ovarian cancers, as well as in melanoma and brain tumors (Table 3) [53, 54, 56, 57, 60, 61, 64, 65, 71, 155, 193–195]. PAR-2 may be highly expressed in stroma-rich tumor regions also. Studies by Shi et al. have demonstrated intriguing dual roles for stromal PAR-2 in pancreatic cancer development, namely that PAR-2 potentiated primary tumor growth but diminished lymphangiogenesis and subsequent lymph node metastasis . The findings defined PAR-2 as a negative regulator of lymphangiogenesis in pancreatic cancer. In contrast, the expression of PAR-2 correlated with the depth of wall invasion, liver metastasis, as well as lymphatic and venous infiltration in gastric cancer patients . Patients with PAR-2-positive tumors had significantly poorer prognosis than those with expression-negative tumors.
In vitro studies with epithelial cancers have shown that PAR-2, like PAR-1, exerts mitogenic activity [46, 53, 54, 56–61, 64, 71, 155, 193–195]. Trypsin and PAR-2 activating peptide, SLIGKV, significantly increased gelatinolytic activity of MMP-2, as well as ERK/AP-1, MEK1/2, and MAPK signaling to promote cancer cell proliferation, migration, and metastasis [53, 57, 58, 60–62, 195]. The increased activity of MMP-2 suggests that PAR-2 may be implicated in cancer invasion by the MMP/EGFR/MAPK/ERK1/2 pathway . PAR-2 may also activate Ca 2+ channels to promote prostaglandin E2 release resulting in EGFR-stimulated cell proliferation . Employing a migration assay through Matrigel barrier, it was determined that the Met receptor tyrosine kinase transactivation by PAR-2 is involved in hepatocellular and cholangiocarcinoma cell invasion .
The influence of inflammation in cancer is undeniable. There are interesting connections between the nervous system and regulation of inflammation, where the vagus nerve participates in a systemic feedback loop that also involves PARs [reviewed in 196]. Recent studies determined that the PAR-1 isoform on vagal C-fibers in mouse lungs could evoke an action potential in response to thrombin, trypsin, or the PAR-1-activating peptide TFLLR-NH(2) . The TRPV channels that induce pain and inflammation are also regulated by the PARs and their downstream proinflammatory bioactive lipid mediators such as 12(S)-HETE [198–204]. While we mostly associate neurogenic inflammation with nociception, it should be noted that tumor cells can migrate via a perineural route, which may speak to the proinflammatory PARs-bioactive lipid gradients along the nerves serving as metastasis beacons [205–207]. Similarly, neurogenic mechanisms have been described that relate PARs activation to extravasation of plasma and that depend on bioactive lipid mediators [112, 208]. Biopsies around the Bartholin gland of women with vestibulodynia reveal more intraepithelial nerve endings than healthy individuals and increased release of inflammatory mediators that lead to C-nerve fiber sensitization and increased proliferation . Because of this neurogenic inflammation, these patients typically experience recalcitrant yeast infections that can lead to epithelial hyperplasia and cancer .
Microbiome, PARs, cancer
PARs have been implicated in many host–microbe interactions that in time may prove relevant to deciphering the role of microbiome in cancer onset and progression as well as other diseases with roots in infectious inflammatory processes [211–217]. Microbial insult by Streptococcus pneumoniae is known to stimulate host-derived proteases so as to activate PARs . Porphyromonas gingivalis can activate PARs on oral epithelial cells to upregulate IL-6 , and the bacterium was recently demonstrated to stimulate PAR-2 resulting in MMP9 expression and promotion of oral squamous cell carcinoma . Both Streptococcus pyogenes and Staphylococcus aureus on the skin produce proteases that fuel the activation of PARs on keratinocytes leading to inflammation . Microbes themselves produce numerous proteases that aid in microbial dissemination by overcoming some of the same logistical processes that metastasizing cancer cells must circumvent to spread [222–225]. The interplay between microbiome and host to affect changes in tissue and hematologic microenvironment are actively being investigated [226, 227]. Bacterial proteases can cleave PARs to modulate inflammation and have been studied for their potential to compromise host barrier function . To that end, it is conceivable that circulating or metastasizing cells from tumors or stem niches could take advantage of such changes. Recently, there is also evidence for microbial protease activation of a novel TLR in a mechanism similar to PAR activation .
As food for thought, the gut microbiome has received a lot of attention in relation to disease and well-being [230–233]. Therefore, it is noteworthy in the climate of genetically modified foods that are either bred or engineered that bountiful yields of certain grains in the crop industry rely on serpin expression [233, 234], which may have implications for PAR regulation in the gut [235–239].
Results of theoretical studies presented above suggest that PARs and PARs-associated signaling may be used as a possible therapeutic target, either alone or in combination with other modalities, such as chemotherapy, antiangiogenic agents, and proapoptotic drugs. A PAR-directed approach is appealing since it targets both the tumor and its microenvironment. In vitro and in vivo studies provide evidence that inhibition of PAR-associated signaling results in reduced tumor growth, invasiveness, and metastasis [5, 41, 42, 240]. There are functional (inhibitors of proteases) and pharmacological (inhibitors of tethered ligand or cleavage site of PAR) PAR-associated signaling antagonists [19, 20]. Clinical benefit may be provided by direct blockade of PAR-1 or PAR-2 on tumor cells, inhibition of PAR-1 on platelets, fibroblasts, and ECs (ATAP2, WEDE15, SCH530348, SCH79797, vorapaxar), as well as administering inhibitors of thrombin (hirudin, argatroban), TF (TFPI, mAb-10H10), MMPs, and other serine protease inhbitors (serpins) [4, 5, 16, 22, 24, 87, 95, 96, 241, 242]. Although experimental trypsin inhibition is feasible, it seems that trypsin as a target for clinical therapy is unlikely to be successful due to its universal distribution . The blockade of proteins expressed in response to PAR-elicited signaling, e.g., anti-αvβ5 antibodies, EGFR, Erb, Erk, MEK inhibitors, as well as agents interfering with PAR RNA (short hairpin RNA (shRNA)), also have therapeutic potential [24, 61].
The inhibition of related activities that are not associated directly with cancer-promoting effects of PARs may also benefit cancer patients. There are intriguing findings from an animal model that thrombin-mediated PAR-1 and PAR-2 activation plays a role in the pathogenesis of acute side effects of radiotherapy, e.g., enteritis, where PAR-mediated signaling activates inflammatory, mitogenic, and proliferative processes in cells of the gut after radiotherapy. PAR-1 inhibitors decreased intensity of acute, immediate-early side effects (enteritis), but did not affect late-onset side effects [243–245]. The pathogenesis of late adverse effects is presumed to be PAR-independent. Moreover, PAR-2 antagonists potentiate analgesic effects of systemic morphine in a rat model of bone cancer pain .
Although results from experimental models are promising, inhibition of PAR activity on both normal and tumor cells may cause side effects, such as hemorrhage, so that PAR-tailored drug discovery is a great challenge. Clinical trials are still limited and so far directed to patients with diseases other than cancer. PAR-1 antagonists, such as vorapaxar and atopaxar, have been assessed in clinical trials in patients with acute coronary syndrome, cerebral infarction, and atherosclerosis [24, 247].
However, insight into the molecular basis of breast cancer and melanoma provides new potential targets for anticancer drug discovery tailored to PAR-dependent signaling.
There is growing evidence that PARs, mainly PAR-1 and PAR-2, are strong mediators of cell invasion in epithelial cancers [68, 77]. Breast cancer cells may express both PAR-1 and PAR-2 [66, 68, 77], and their role in breast carcinoma is the most widely studied. PAR-1 is not expressed in normal breast epithelium, dysplasia, or adenoma but is upregulated in carcinoma in situ (low expression) and is highly expressed in invasive breast carcinoma cell lines [47, 77, 154]. Experimental studies on breast cancer have shown that PAR-1 is activated by thrombin, MMPs and TF, while PAR-2 is activated by coagulation factors VIIa, Xa, or their complexes with TF [16, 52, 66, 68, 77]. There are also observations that PAR-1 and PAR-2 act as a functional unit in this tumor type . Silencing PAR-2 by shRNA attenuates thrombin-mediated PAR-1 activation, leading to reduced colony formation and decreased cell invasion .
PAR activity mediates breast cancer cell migration through Matrigel (a reconstituted basement membrane), facilitates cell chemokinesis through the Gαi/c-Src/JNK/paxillin signaling pathway, activates Akt-dependent survival pathways, and correlates with the level of invasiveness and metastatic potential of numerous cancer cell lines [66, 77, 154, 163]. PARs also regulate EMT processes in breast cancer tumors, which facilitates cell proliferation (in situ carcinoma), encroachment of basement membrane, matrix degradation, and local infiltration (invasive cancer). Furthermore, PAR interactions with integrins, formation of focal contact complexes, and cytoskeleton reorganization enable distant dissemination via intravasation and extravasation (via lymphatic or blood vessels). Finally, the MET process, and interactions with blood and ECs, facilitates metastases formation (disseminated cancer) . Inhibition of PAR activation in highly metastatic MDA-435 breast cancer cells reduced cell invasion . Administering an MMP-1 inhibitor and P1pal-7 (inhibitor of cell viability mediated by Akt signaling) attenuates Akt activity, significantly promoting apoptosis in breast tumor xenografts and inhibiting metastasis to the lungs by up to 88 % .
There is evidence from in vivo studies for PAR-mediated breast cancer progression . PAR-1 expression was essential for tumor growth and invasion in mammary xenografts via thrombin-mediated interaction with EGFR- and ErbB or by the fibroblast-derived MMP-1-mediated Ca 2+ pathway [32, 52]. Persistent transactivation of EGFR and ErbB2/Her2 by the thrombin-cleaved PAR-1 pathway has been demonstrated in invasive breast carcinoma, but not in normal mammary epithelial cells [32, 94]. There is evidence that Gαi/o, metalloprotease activity and release of HB-EGF (heparin-binding EGF) ligand are critical for transactivation of EGFR. Finally, EGFR and ErbB2/Her2 signaling triggered by PARs results in prolonged Erk-1/2 activation leading to breast carcinoma cell invasion. These results indicate potential therapeutic benefit of inhibitors of thrombin, EGFR, ErbB and Erk kinases in metastatic breast cancer patients.
In epithelial cancers, the predominant mechanism leading to metastatic dissemination is EMT. In melanoma, the transition of a lesion from the noninvasive radial growth phase (RGP) to the invasive and metastasis-competent vertical growth phase (VGP) is a major step in tumor progression, and PAR expression is implicated in the RGP-VGP transition process . Melanoma cells express both PAR-1 and PAR-2 [50, 250]. The PAR-2 role in melanoma metastasis was not previously appreciated, but the newest findings have shown its dual role in melanoma [187, 194]. In a murine model of spontaneous metastatic B16 melanoma, PAR-2 contributed to the limitation of local cancer progression in one area, while enhancing distant metastatic spread. Numerous reports document the role of PAR-1 signaling in the prometastatic phenotype of melanoma cells [4, 5, 21]. Experimental studies on melanoma cell lines demonstrated that PAR-1-elicited signaling activates adhesive, invasive, antiapoptotic, and angiogenic factors to promote melanoma metastasis [4, 5, 21, 251]. Additional proof for the role of PAR-1 in melanoma dissemination is the fact that it is highly expressed both in metastatic melanoma cell lines and in metastatic lesions in comparison to primary nevi and normal skin [21, 250]. Moreover, melanoma cells isolated from lesions giving rise to metastases in patients had higher PAR-1 mRNA and protein expression, as compared to those obtained from lesions that did not develop metastatic disease . Motility and migration of melanoma cells is also regulated by thrombin-mediated PAR-1 activation [50, 252]. Thrombin, whose generation is TF-dependent (procoagulant expressed in melanoma cells), is the predominant PAR-1 activator [21, 107]. However, there is also evidence that MMP-1-mediated PARs activation exists in melanoma cells [5, 249]. Both MMP-1 and PAR-1 are highly expressed by VGP melanomas. MMP-1 is thought to facilitate melanoma invasion by degrading type I collagen within the skin, while PAR-1 activation leads to increased activation of growth factors: FGFR-2 and IGF-1 [5, 249].
Experiments with the B16F10 murine metastasis model of melanoma demonstrated that cells transfected with PAR-1 exhibited substantially higher pulmonary metastasis potential than those deprived of PAR-1 signaling [4, 48]. PAR-1 promoted metastatic melanoma by regulating the tumor suppressor Maspin and the gap junction protein Connexin 43. Villares et al.  determined that Connexin 43 facilitates interaction between malignant cells and ECs, and maspin expression is decreased in metastatic melanoma cells, where there is an inverse correlation between PAR-1 and Maspin expression . PAR-1 also promotes expression of melanoma cell adhesion molecule MCAM/MUC18 (MUC18), which is a key marker of melanoma metastasis. It is of interest that PAR-1 activity increases expression of platelet-activating factor receptor (PAFR) and its ligand, and so not only promotes platelet aggregation but also enhances MUC18 levels. This is extremely relevant to the metastatic process as it was demonstrated that the PAR1/PAFR/MUC18 pathway mediates melanoma cell adhesion to microvascular ECs, transendothelial migration and metastatic retention in the lungs .
PAR-1 silencing and thrombin inhibition affects the ability of metastatic melanoma cell lines to disseminate [21, 22, 251]. Inhibition of PAR activity by 80 % through the use of lentiviral shRNA decreases lung metastatic potential of PAR-1 overexpressing melanoma cell lines . PAR-1 silencing also inhibits expression of the adhesive protein MUC18, which attenuates the metastatic phenotype of melanoma cells .
To reduce the toxic immune responses of viral therapy, PAR-1 small interfering RNA (siRNA) incorporated into neutral liposomes (1,2-dioleoyl-sn-glycero-3-phosphatidylcholine, DOPC) was used in experiments on melanoma models. There was a significant decline in tumor growth, weight, and formation of metastatic lung colonies in mice treated with the PAR-1 siRNA-DOPC . siRNA delivery also resulted in a decline in VEGF, IL-8, and MMP-2 expression levels, and decreased blood vessel density. In another study, the reduction of PAR-1 expression by siRNA and the inhibition of PAR-1 function by the specific antagonist SCH79797 significantly decreased melanoma cell motility and invasiveness to the extent of the non-metastatic and low PAR-1 expressing cells . A specific thrombin inhibitor, argatroban, also decreases migration and bone metastatic potential of B16BL6 melanoma cells .
These findings suggest that PAR-1-dependent stimulation of tumor growth and metastasis is regulated by invasive, adhesive and proangiogenic factors and that PAR-1 could be a potential therapeutic target for metastatic melanoma patients.
Tumor cell invasion and metastasis involves complex interactions between mesenchymal cells and extracellular matrix as well as blood components and ECs. The coagulation proteases, matrix metalloproteases and serine proteases interact with PARs, thus promoting multiple activities leading to cancer progression. Further studies are necessary to convert theoretical knowledge into practical value.
How Serpins Work
The serpins inhibit the action of their respective serine protease by mimicking the three-dimensional structure of the normal substrate of the protease.
- The serine protease binds the serpin instead of its normal substrate. This alone would block any further activity by the protease. But the serpin has another trick to play.
- The protease makes a cut in the serpin leading to
- the formation of a covalent bond linking the two molecules
- a massive allosteric change in the tertiary structure of the serpin
- which moves the attached protease to a site where it can be destroyed.
Borrelia burgdorferi, Host-Derived Proteases, and the Blood-Brain BarrierFIG. 1 . Human BMEC in vitro express P-glycoprotein and tight junctional proteins. (A) Confluent and subconfluent human BMEC monolayers were lifted with EDTA. The cells were fixed with 2% paraformaldehyde in PBS for 30 min, permeabilized with PBS containing 0.01% Triton X-100, and quenched with 10 mM glycine-PBS. After blocking with 10% normal goat serum, the cells were incubated with fluorescein isothiocyanate-labeled mouse anti-P-glycoprotein monoclonal antibody and rabbit anti-factor VIII-Rag polyclonal antibody, followed by secondary antibody coupled to phycoerythrin. Analysis was done by using a FACScan and the CellQuest software (Becton Dickinson, San Jose, Calif.). The percentages of gated human BMEC positive for both factor VIII-Rag and P-glycoprotein are shown. (B) Human BMEC monolayers were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and stained with anti-human ZO-1 monoclonal antibody. The presence of ZO-1 was visualized with Alexa 488-conjugated secondary antibody. Differential interference contrast (left panel) and fluorescence (right panel) images of human BMEC showed that ZO-1 is expressed at the cell junctions. Magnification, ×400. FIG. 2 . In vitro model for B. burgdodrferi 297 crossing the human BBB. A total of 10 7 B. burgdodrferi 297 (Bb) cells were incubated overnight with human BMEC grown to confluence in Transwell inserts. (A) Dark-field microscopy was used to determine the number of spirochetes that crossed the monolayers. The data from two independent experiments with triplicate determinations are expressed as the percentages of B. burgdorferi (means ± standard errors of the means) that crossed relative to the total number of spirochetes in control wells without inserts. (Inset) Comparison of B. burgdorferi crossing with human BMEC (bar +) or without human BMEC (bar −). (B) TEER expressed as means ± standard errors of the means for the experiments shown in panel A. Cont, control. FIG. 3 . B. burgdorferi differentially crosses human BMEC and HUVEC (EA.hy926) monolayers. A total of 10 6 B. burgdodrferi 297 (Bb) cells were incubated overnight with human BMEC or HUVEC (EAhy.926) grown to confluence in Transwell inserts. (A) Dark-field microscopy was used to determine the number of spirochetes that crossed human BMEC or HUVEC monolayers. (B) Real-time PCR based on the single-copy B. burgdorferi sensu stricto fla gene (50) was used to determine the number of spirochetes that crossed human BMEC or HUVEC monolayers in the experiments whose results are shown in panel A. The data from triplicate determinations are expressed as the percentages of B. burgdorferi (means ± standard errors of the means) that crossed relative to the total number of spirochetes in control wells without inserts. (C) TEER measured as mean resistance (means ± standard errors of the means) for the experiments whose results are shown in panels A and B as described in Materials and Methods. The P values (as determined by a paired Student's t test) for the TEER changes in the 5- and 18-h HUVEC samples were 0.019 and 0.017, respectively (indicated by asterisks). Cont, control. FIG. 4 . Plasminogen activation by cocultures of B. burgdorferi and human BMEC. Human BMEC were grown on 96-well plates until they reached confluence (approximately 10 5 cells). The medium was exchanged with DMEM—F-12 medium, and the next day increasing amounts of B. burgdorferi N40 (0, 10 3 , 10 4 , 10 5 cells) were added. (A) Plasminogen activation by human BMEC in response to spirochete addition. Twenty-four hours after spirochete addition the supernatants from the cultures were collected and incubated with 1 μg of human plasminogen per ml and 5 mM Spectrozyme PL. The increase in absorbance at 405 nm was directly proportional to the amount of plasmin in a given sample. The error bars indicate the standard deviations based on six experiments performed in duplicate. (B) Binding of exogenous urokinase-type plasminogen activator to human BMEC after spirochete addition. The cultures were washed three times with PBS prior to addition of 125 U of human recombinant uPA per ml. After a 60-min incubation, the cultures were again washed three times with PBS. After washing, 200 μl of PBS containing 1 μg of plasminogen per ml was added together with 5 mM Spectrozyme PL. The increase in the absorbance at 405 nm was determined after 3 h. Note the increased ability of B. burgdorferi N40 to stimulate human BMEC binding to uPA and activation of plasminogen compared to control cultures incubated with spirochetes. The error bars indicate the standard deviations based on three experiments performed in duplicate. FIG. 5 . Role of proteases in B. burgdorferi (Bb) transmigration across human BMEC. Serum-reduced conditions were used to examine the role of host proteases in spirochete transmigration across human BMEC as described in Materials and Methods and the legend to Fig. 4. The traversal of human BMEC by B. burgdorferi N40 was examined in the presence of 1 μg of plasminogen per ml. The traversal of human BMEC by B. burgdorferi N40 with 50 nM BB-94 alone, with 200 μM EACA alone, with both BB-94 and EACA, or with 40 μg of α2-antiplasmin per ml is shown. The results are the averages of two experiments performed in duplicate. The error bars indicate the standard deviations. FIG. 6 . MMP-1 expression in response to B. burgdorferi. A total of 10 5 human BMEC were inoculated with increasing amounts of B. burgdorferi N40. After 24 h, cell culture supernatants were harvested and tested by immunoblot analysis with the antibody raised against MMP-1. Note the increased levels of MMP-1 with the increased numbers of B. burgdorferi cells.
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