Protease‐activated receptor 4 is more important than protease‐activated receptor 1 for the thrombin‐induced procoagulant effect on platelets

Abstract

Thrombin is the most important enzyme in coagulation and also a potent activator of platelets. It activates platelets via N‐terminal cleavage of protease‐activated receptors (PARs) 1 and 4. Although structurally similar, both PARs have distinct and complementary roles; for example, PAR1 is more sensitive than PAR4 to low concentrations of thrombin 1.Kahn M.L. Zheng Y.W. Huang W. Bigornia V. Zeng D. Moff S. Farese Jr, R.V. Tam C. Coughlin S.R. A dual thrombin receptor system for platelet activation.Nature. 1998; 394: 690-4Crossref PubMed Scopus (868) Google Scholar, whereas PAR4 gives rise to a more prolonged calcium mobilization after activation compared with a transient ‘spike’ by PAR1 2.Shapiro M.J. Weiss E.J. Faruqi T.R. Coughlin S.R. Protease‐activated receptors 1 and 4 are shut off with distinct kinetics after activation by thrombin.J Biol Chem. 2000; 275: 25216-21Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar. Moreover, PAR4 is more important for clot lysis resistance 3.Vretenbrant K. Ramstrom S. Bjerke M. Lindahl T.L. Platelet activation via PAR4 is involved in the initiation of thrombin generation and in clot elasticity development.Thromb Haemost. 2007; 97: 417-24Crossref PubMed Scopus (30) Google Scholar. After activation, a subpopulation of the platelets become procoagulant by allowing the formation of coagulation factor complexes on their surface. They do not participate in aggregation but facilitate generation of even more thrombin and, consequently, amplification of the response 4.Mazepa M. Hoffman M. Monroe D. Superactivated platelets: thrombus regulators, thrombin generators, and potential clinical targets.Arterioscler Thromb Vasc Biol. 2013; 33: 1747-52Crossref PubMed Scopus (57) Google Scholar. Experimental data suggest that collagen exposed during vessel damage in combination with thrombin is the most potent trigger for formation of procoagulant platelets 5.Bevers E.M. Comfurius P. van Rijn J.L. Hemker H.C. Zwaal R.F. Generation of prothrombin‐converting activity and the exposure of phosphatidylserine at the outer surface of platelets.Eur J Biochem. 1982; 122: 429-36Crossref PubMed Scopus (398) Google Scholar, but the signaling processes regulating the formation of the aggregatory and procoagulant platelet subpopulations are still not known in detail. The article by Hamilton and co‐workers in this issue of JTH 6.French S.L. Arthur J.F. Lee H. Nesbitt W.S. Andrews R.K. Gardiner E.E. Hamilton J.R. Inhibition of protease‐activated receptor 4 impairs platelet procoagulant activity during thrombus formation in human blood.J Thromb Haemost. 2016; 14: 1642-54Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar shows for the first time that activation of PAR4 is crucial for the thrombin‐induced activation of platelets in an in vitro thrombus model; that is, platelets adhered on a collagen surface in a flow chamber system. Two tools were essential for this study, a rabbit polyclonal antibody directed towards the cleavage site for thrombin at the N‐terminal of PAR4 and a fluorescence resonance energy transfer‐based thrombin activity sensor linked to an anti‐CD41a antibody, to prevent washing out by the flowing blood, which was invented by Welsh et al. 7.Welsh J.D. Colace T.V. Muthard R.W. Stalker T.J. Brass L.F. Diamond S.L. Platelet‐targeting sensor reveals thrombin gradients within blood clots forming in microfluidic assays and in mouse.J Thromb Haemost. 2012; 10: 2344-53Crossref PubMed Scopus (75) Google Scholar. The anti‐PAR4 antibody only attenuated the peak of the thrombin‐induced cytosolic calcium but abolished the sustained elevation of cytosolic calcium; thus PAR4 was responsible for the sustained elevation, in accordance with previous studies 8.Covic L. Gresser A.L. Kuliopulos A. 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Sanchez Centellas D, Wallstedt M, Ramstrom S, Grenegard M, Lindahl TL. Thrombin‐induced platelet activation via PAR4: pivotal role for exosite II.Thromb Haemost. 2014; 112: 558-65Crossref PubMed Scopus (11) Google Scholar. Presumably this sustained elevation is essential for the creation of the negatively charged surface and the procoagulant activity, which is supported by the known very strong effect in this direction by the calcium ionophore A23187 (see Fig. 4G in 6.French S.L. Arthur J.F. Lee H. Nesbitt W.S. Andrews R.K. Gardiner E.E. Hamilton J.R. Inhibition of protease‐activated receptor 4 impairs platelet procoagulant activity during thrombus formation in human blood.J Thromb Haemost. 2016; 14: 1642-54Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar and 13.Ramström S. Ranby M. Lindahl T.L. Platelet phosphatidylserine exposure and procoagulant activity in clotting whole blood ‐ different effects of collagen, TRAP and calcium ionophore A23187.Thromb Haemost. 2003; 89: 132-41Crossref PubMed Scopus (30) Google Scholar). The relative importance of PAR1 and PAR4 for different aspects of platelet activation is far from clear; published studies have shown various, even contradicting, results. To be able to answer questions regarding the relative importance of PAR1 and PAR4 in the platelet procoagulant response, we need to study dose‐responses to increasing concentrations of the specific receptor activating peptides. Such data are rare in previous publications, but studies by, for example, Duvernay et al. and Vretenbrant et al. 3.Vretenbrant K. Ramstrom S. Bjerke M. Lindahl T.L. Platelet activation via PAR4 is involved in the initiation of thrombin generation and in clot elasticity development.Thromb Haemost. 2007; 97: 417-24Crossref PubMed Scopus (30) Google Scholar, 14.Duvernay M. Young S. Gailani D. Schoenecker J. Hamm H.E. Protease‐activated receptor (PAR) 1 and PAR4 differentially regulate factor V expression from human platelets.Mol Pharmacol. 2013; 83: 781-92Crossref PubMed Scopus (47) Google Scholar report a higher platelet activation with the most efficient PAR4‐activating peptide, AYPGKF 15.Faruqi T.R. Weiss E.J. Shapiro M.J. Huang W. Coughlin S.R. Structure‐function analysis of protease‐activated receptor 4 tethered ligand peptides. Determinants of specificity and utility in assays of receptor function.J Biol Chem. 2000; 275: 19728-34Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar, as compared with the most potent PAR1‐activating peptide, SFLLRN 16.Hui K.Y. Jakubowski J.A. Wyss V.L. Angleton E.L. Minimal sequence requirement of thrombin receptor agonist peptide.Biochem Biophys Res Commun. 1992; 184: 790-6Crossref PubMed Scopus (83) Google Scholar. However, it should be kept in mind that the PAR4‐activating peptides reach their maximum effects at concentrations 10‐fold higher than the PAR1‐activating peptides, which means that potential unspecific effects caused by the presence of the peptides themselves have to be considered as potential confounding factors. A different option would be to use specific inhibitors for the receptors. Such inhibitors are widely available for PAR1, but as stated by Hamilton and co‐workers 6.French S.L. Arthur J.F. Lee H. Nesbitt W.S. Andrews R.K. Gardiner E.E. Hamilton J.R. Inhibition of protease‐activated receptor 4 impairs platelet procoagulant activity during thrombus formation in human blood.J Thromb Haemost. 2016; 14: 1642-54Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, for PAR4 the situation is more problematic. Pepducins, that is, cell membrane‐penetrating palmitylated peptides targeting specific intracellular loops of PAR1 or PAR4, have been claimed to be specific inhibitors and used in a number of studies 17.Covic L. Gresser A.L. Talavera J. Swift S. Kuliopulos A. Activation and inhibition of G protein‐coupled receptors by cell‐penetrating membrane‐tethered peptides.Proc Natl Acad Sci U S A. 2002; 99: 643-8Crossref PubMed Scopus (275) Google Scholar. In our experience, there is less effect in whole blood, probably because pepducins will also enter the more numerous red blood cells (T. L. Lindahl, L. Faxälv, N. Boknäs, A. Macwan and S. Ramström, unpublished data). More important for research use, the specificity has been seriously questioned 18.Stampfuss J.J. Schror K. Weber A.A. Inhibition of platelet thromboxane receptor function by a thrombin receptor‐targeted pepducin.Nat Med. 2003; 9: 1447Crossref PubMed Scopus (18) Google Scholar. In contrast to these objections, there is one very recent phase 1 study on a pepducin targeting PAR1 19.Gurbel P.A. Bliden K.P. Turner S.E. Tantry U.S. Gesheff M.G. Barr T.P. Covic L. Kuliopulos A. Cell‐Penetrating Pepducin Therapy Targeting PAR1 in Subjects With Coronary Artery Disease.Arterioscler Thromb Vasc Biol. 2016; 36: 189-97Crossref PubMed Scopus (78) Google Scholar, which was preceded by a promising report on a baboon thrombosis model 20.Zhang P. Gruber A. Kasuda S. Kimmelstiel C. O'Callaghan K. Cox D.H. Bohm A. Baleja J.D. Covic L. Kuliopulos A. Suppression of arterial thrombosis without affecting hemostatic parameters with a cell‐penetrating PAR1 pepducin.Circulation. 2012; 126: 83-91Crossref PubMed Scopus (65) Google Scholar. Low‐molecular‐weight inhibitors of PAR4 have not been used to inhibit procoagulant effects by thrombin, and are anyway weak. For trans‐cinnamoyl‐YPGKF‐NH2 there is one study in rodents, but mice and rats lack PAR1 21.Hollenberg M.D. Saifeddine M. Proteinase‐activated receptor 4 (PAR4): activation and inhibition of rat platelet aggregation by PAR4‐derived peptides.Can J Physiol Pharmacol. 2001; 79: 439-42Crossref PubMed Scopus (66) Google Scholar. In our hands it was too inefficient in human platelets. YD‐3 ([1‐benzyl‐3‐[ethoxycarbonylphenyl]‐indazole]) inhibits activation by AYPGKF 22.Wu C.C. Hwang T.L. Liao C.H. Kuo S.C. Lee F.Y. Lee C.Y. Teng C.M. Selective inhibition of protease‐activated receptor 4‐dependent platelet activation by YD‐3.Thromb Haemost. 2002; 87: 1026-33Crossref PubMed Scopus (79) Google Scholar, 23.Hosokawa K. Ohnishi T. Miura N. Sameshima H. Koide T. Tanaka K.A. Maruyama I. Antithrombotic effects of PAR1 and PAR4 antagonists evaluated under flow and static conditions.Thromb Res. 2014; 133: 66-72Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 24.Edelstein L.C. Simon L.M. Lindsay C.R. Kong X. Teruel‐Montoya R. Tourdot B.E. Chen E.S. Ma L. Coughlin S. Nieman M. Holinstat M. Shaw C.A. Bray P.F. Common variants in the human platelet PAR4 thrombin receptor alter platelet function and differ by race.Blood. 2014; 124: 3450-8Crossref PubMed Scopus (78) Google Scholar but showed only a weak effect on thrombin 25.Wu C.C. Wang W.Y. Wei C.K. Teng C.M. Combined blockade of thrombin anion binding exosite‐1 and PAR4 produces synergistic antiplatelet effect in human platelets.Thromb Haemost. 2011; 105: 88-95Crossref PubMed Scopus (12) Google Scholar and no effect on tissue factor‐induced coagulation in a flow chamber system, not even on top of the PAR1‐inhibitor SCH70797, and had no effect on thrombin generation 23.Hosokawa K. Ohnishi T. Miura N. Sameshima H. Koide T. Tanaka K.A. Maruyama I. Antithrombotic effects of PAR1 and PAR4 antagonists evaluated under flow and static conditions.Thromb Res. 2014; 133: 66-72Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar. Thus, so far the only undisputed specific, reasonable and efficient tools to inhibit thrombin activation of PAR4 are antibodies directed towards the N‐terminal 3.Vretenbrant K. Ramstrom S. Bjerke M. Lindahl T.L. Platelet activation via PAR4 is involved in the initiation of thrombin generation and in clot elasticity development.Thromb Haemost. 2007; 97: 417-24Crossref PubMed Scopus (30) Google Scholar, 6.French S.L. Arthur J.F. Lee H. Nesbitt W.S. Andrews R.K. Gardiner E.E. Hamilton J.R. Inhibition of protease‐activated receptor 4 impairs platelet procoagulant activity during thrombus formation in human blood.J Thromb Haemost. 2016; 14: 1642-54Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 26.Kahn M.L. Nakanishi‐Matsui M. Shapiro M.J. Ishihara H. Coughlin S.R. Protease‐activated receptors 1 and 4 mediate activation of human platelets by thrombin.J Clin Investig. 1999; 103: 879-87Crossref PubMed Scopus (694) Google Scholar. Unfortunately, to the best of our knowledge no PAR4 inhibitor is yet commercially available. What needs to be kept in mind is that thrombin or PAR‐activating peptides alone are not very efficient inducers of platelet procoagulant responses. Studies by Bevers et al. in 1982 5.Bevers E.M. Comfurius P. van Rijn J.L. Hemker H.C. Zwaal R.F. Generation of prothrombin‐converting activity and the exposure of phosphatidylserine at the outer surface of platelets.Eur J Biochem. 1982; 122: 429-36Crossref PubMed Scopus (398) Google Scholar clearly identified that collagen is more potent, but that the combination of collagen and thrombin greatly potentiated the capability of platelets to support thrombin formation 5.Bevers E.M. Comfurius P. van Rijn J.L. Hemker H.C. Zwaal R.F. Generation of prothrombin‐converting activity and the exposure of phosphatidylserine at the outer surface of platelets.Eur J Biochem. 1982; 122: 429-36Crossref PubMed Scopus (398) Google Scholar. The small percentage of phosphatidylserine (PS)‐positive platelets reported in this paper are well in accordance with other studies 14.Duvernay M. Young S. Gailani D. Schoenecker J. Hamm H.E. Protease‐activated receptor (PAR) 1 and PAR4 differentially regulate factor V expression from human platelets.Mol Pharmacol. 2013; 83: 781-92Crossref PubMed Scopus (47) Google Scholar, 27.Keuren J.F. Wielders S.J. Ulrichts H. Hackeng T. Heemskerk J.W. Deckmyn H. Bevers E.M. Lindhout T. Synergistic effect of thrombin on collagen‐induced platelet procoagulant activity is mediated through protease‐activated receptor‐1.Arterioscler Thromb Vasc Biol. 2005; 25: 1499-505Crossref PubMed Scopus (74) Google Scholar, 28.Panteleev M.A. Ananyeva N.M. Greco N.J. Ataullakhanov F.I. Saenko E.L. Two subpopulations of thrombin‐activated platelets differ in their binding of the components of the intrinsic factor X‐activating complex.J Thromb Haemost. 2005; 3: 2545-53Crossref PubMed Scopus (58) Google Scholar, where only a small percentage of platelets turn PS positive even in response to high concentrations of thrombin, although it should be noticed that PS exposure determined by annexin V binding could vary considerably as a result of variations in experimental conditions 29.Ramstrom S. O'Neill S. Dunne E. Kenny D. Annexin V binding to platelets is agonist, time and temperature dependent.Platelets. 2010; 21: 289-96Crossref PubMed Scopus (20) Google Scholar. The important contribution by collagen is also evident in this paper, as the combination of thrombin and collagen‐related peptide in Fig. 4G results in approximately 40% PS‐positive platelets, as compared with the less than 15% generated by thrombin alone in Fig. 4C and the approximately 7% generated by the PAR4 peptide in Fig. 4D. Nevertheless, it is very interesting and important that this paper shows that the inhibition of PAR4 reduced thrombus formation by approximately 50%, as it has been previously shown that the relation between platelet PS‐exposure and blood coagulation is not clear‐cut 13.Ramström S. Ranby M. Lindahl T.L. Platelet phosphatidylserine exposure and procoagulant activity in clotting whole blood ‐ different effects of collagen, TRAP and calcium ionophore A23187.Thromb Haemost. 2003; 89: 132-41Crossref PubMed Scopus (30) Google Scholar; this could indicate that thrombin pathways make a contribution with other factors that are important for the procoagulant response. However, it needs to be kept in mind that in the thrombus formation experiments in the study by Hamilton's group 6.French S.L. Arthur J.F. Lee H. Nesbitt W.S. Andrews R.K. Gardiner E.E. Hamilton J.R. Inhibition of protease‐activated receptor 4 impairs platelet procoagulant activity during thrombus formation in human blood.J Thromb Haemost. 2016; 14: 1642-54Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, a collagen‐coated surface is also present, which means that the thrombus build‐up also includes platelet exposure to collagen. In conclusion, the results in this and other papers suggest that PAR4 inhibition is a potential candidate for new, antithrombotic drugs, maybe even with potential for reduced risk of bleeding 3.Vretenbrant K. Ramstrom S. Bjerke M. Lindahl T.L. Platelet activation via PAR4 is involved in the initiation of thrombin generation and in clot elasticity development.Thromb Haemost. 2007; 97: 417-24Crossref PubMed Scopus (30) Google Scholar, 6.French S.L. Arthur J.F. Lee H. Nesbitt W.S. Andrews R.K. Gardiner E.E. Hamilton J.R. Inhibition of protease‐activated receptor 4 impairs platelet procoagulant activity during thrombus formation in human blood.J Thromb Haemost. 2016; 14: 1642-54Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 12.Boknas N. Faxalv L. Sanchez Centellas D, Wallstedt M, Ramstrom S, Grenegard M, Lindahl TL. Thrombin‐induced platelet activation via PAR4: pivotal role for exosite II.Thromb Haemost. 2014; 112: 558-65Crossref PubMed Scopus (11) Google Scholar, 17.Covic L. Gresser A.L. Talavera J. Swift S. Kuliopulos A. Activation and inhibition of G protein‐coupled receptors by cell‐penetrating membrane‐tethered peptides.Proc Natl Acad Sci U S A. 2002; 99: 643-8Crossref PubMed Scopus (275) Google Scholar. Indeed, it will be interesting and important to follow the development in this field. The knowledge gained through more complex studies, enabled by specific and efficient inhibitors such as the antibody developed by the Hamilton group, will tease out the relative importance of different activation pathways and mechanisms contributing to the platelet procoagulant response and thus pave the way for efficient but safer strategies to prevent thrombosis. T. L. Lindahl wrote the first draft of the manuscript. All authors read and discussed the relevant scientific articles, revised the manuscript and approved the final version of the manuscript. T. Lindahl reports support from Diapensia HB during the conduct of the study.