Platelet activation via PAR4 is involved in the initiation of thrombin generation and in clot elasticity development

Summary

Thrombin is a pivotal enzyme formed in the coagulation cascade and an important and potent platelet activator. The two pro-tease-activated thrombin receptors on human platelets are denoted PARI and PAR4. The physiological relevance of PAR4 is still unclear, as both aggregation and secretion can be accomplished by PARI activation alone. In the present study we have investigated the role of PARs in platelet activation, blood coagulation, clot elasticity and fibrinolysis. Flow cytometry, free oscillation rheometry and thrombin generation measurements were used to analyze blood or platelet-rich plasma from healthy individuals. Maximum PARI activation with the peptide SFLLRN gave fewer fibrinogen-binding platelets with lower mean fluorescent intensity than maximum PAR4 activation with AYPGKF. Inhibition of any of the receptors prolonged clotting times. However, PARI is more important for fibrinolysis; inhibition of this receptor prolonged all the steps in the fibrinolytic process. Clot elasticity decreased significantly when the PAR4 receptor was inhibited. In the thrombin generation measurements, PAR4 inhibition delayed the thrombin generation start and peak, but did not affect the total amount of thrombin generated. PAR I inhibition had no significant impact on thrombin generation. We found that PAR4 is most likely activated by low concentrations of thrombin during the initial phase of thrombin generation and is of importance to the clotting time. Furthermore, we suggest that the PAR4 receptor may have a physiological role in the stabilisation of the coagulum.

Keywords

Blood coagulation - clot elasticity - fibrinolysis - PAR receptors

• References

• 1 Monroe DM, Hoffman M. What does it take to make the perfect clot?. Arterioscler Thromb Vasc Biol 2006; 26: 41-48.
• 2 Klages B, Brandt U, Simon MI. et al. Activation of G12/G13 results in shape change and Rho/Rho-kinasemediated myosin light chain phosphorylation in mouse platelets. J Cell Biol 1999; 144: 745-754.
• 3 Vassallo Jr RR, Kieber-Emmons T, Cichowski K. et al. Structure-function relationships in the activation of platelet thrombin receptors by receptor-derived peptides. J Biol Chem 1992; 267: 6081-6085.
• 4 Xu WF, Andersen H, Whitmore TE. et al. Cloning and characterization of human protease-activated receptor 4. Proc Natl Acad Sci USA 1998; 95: 6642-6646.
• 5 Vu TK, Wheaton VI, Hung DT. et al. Domains specifying thrombin-receptor interaction. Nature 1991; 353: 674-677.
• 6 Gerszten RE, Chen J, Ishii M. et al. Specificity of the thrombin receptor for agonist peptide is defined by its extracellular surface. Nature 1994; 368: 648-651.
• 7 Chen J, Ishii M, Wang L. et al. Thrombin receptor activation. Confirmation of the intramolecular tethered liganding hypothesis and discovery of an alternative intermolecular liganding mode. J Biol Chem 1994; 269: 16041-16045.
• 8 Vu TK, Hung DT, Wheaton VI. et al. M cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell 1991; 64: 1057-1068.
• 9 Faruqi TR, Weiss EJ, Shapiro MJ. et al. Structurefunction analysis of protease-activated receptor 4 tethered ligand peptides. Determinants of specificity and utility in assays of receptor function. J Biol Chem 2000; 275: 19728-19734.
• 10 Kahn ML, Nakanishi-Matsui M, Shapiro MJ. et al. Protease-activated receptors 1 and 4 mediate activation of human platelets by thrombin. J Clin Invest 1999; 103: 879-887.
• 11 Covic L, Gresser AL, Kuliopulos A. Biphasic kinetics of activation and signaling for PAR1 and PAR4 thrombin receptors in platelets. Biochemistry 2000; 39: 5458-5467.
• 12 Vanhoorelbeke K, Ulrichts H, Romijn RA. et al. The GPIbalpha-thrombin interaction: far from crystal clear. Trends Mol Med 2004; 10: 33-39.
• 13 Nylander S, Mattsson C. Thrombin-induced platelet activation and its inhibition by anticoagulants with different modes of action. Blood Coagul Fibrinolysis 2003; 14: 159-167.
• 14 Seeley S, Covic L, Jacques SL. et al. Structural basis for thrombin activation of a protease-activated receptor: inhibition of intramolecular liganding. Chem Biol 2003; 10: 1033-1041.
• 15 Kinlough-Rathbone RL, Perry DW, Guccione MA. et al. Degranulation of human platelets by the thrombin receptor peptide SFLLRN: comparison with degranulation by thrombin. Thromb Haemost 1993; 70: 1019-1023.
• 16 Sambrano GR, Huang W, Faruqi T. et al. Cathepsin G activates protease-activated receptor-4 in human platelets. J Biol Chem 2000; 275: 6819-6823.
• 17 Covic L, Singh C, Smith H. et al. Role of the PAR4 thrombin receptor in stabilizing platelet-platelet aggregates as revealed by a patient with Hermansky-Pudlak syndrome. Thromb Haemost 2002; 87: 722-727.
• 18 Bohlin L. Method of measuring rheological properties and rheometer for carrying out the method, in PCT (Patent Cooperation Treaty) publication number WO94/08222. 1994, Aktiebolaget Medicinsk Reologi Lund: United States
• 19 Bohlin L. Bearing device, in PCT (Patent Cooperation Treaty) publication number WO98/54475. 1998, Global Hemostasis Institute
• 20 Ranby M, Ramstrom S, Svensson PO. et al. Clotting time by free oscillation rheometry and visual inspection and a viscoelastic description of the clotting phenomenon. Scand J Clin Lab Invest 2003; 63: 397-406.
• 21 Ramstrom S, Ranby M, Lindahl T. Effects of inhibition of P2Y1 and P2Y12 on whole blood clotting, coagulum elasticity and fibrinolysis resistance studied with free oscillation rheometry. Thromb Res 2003; 109: 315-322.
• 22 Lindahl TL, Festin R, Larsson A. Studies of fibrinogen binding to platelets by flow cytometry: an improved method for studies of platelet activation. Thromb Haemost 1992; 68: 221-225.
• 23 Akita EM, Nakai S. Comparison of four purification methods for the production of immunoglobulins from eggs laid by hens immunized with an enterotoxigenic E. coli strain. J Immunol Methods 1993; 160: 207-214.
• 24 Brass LF, Pizarro S, Ahuja M. et al. Changes in the structure and function of the human thrombin receptor during receptor activation, internalization, and recycling. J Biol Chem 1994; 269: 2943-2952.
• 25 Molino M, Bainton DF, Hoxie JA. et al. Thrombin receptors on human platelets. Initial localization and subsequent redistribution during platelet activation. J Biol Chem 1997; 272: 6011-6017.
• 26 Molino M, Blanchard N, Belmonte E. et al. Proteolysis of the human platelet and endothelial cell thrombin receptor by neutrophil-derived cathepsin G. J Biol Chem 1995; 270: 11168-11175.
• 27 Hemker HC, Al Dieri R, Beguin S. Thrombin generation assays: accruing clinical relevance. Curr Opin Hematol 2004; 11: 170-175.
• 28 Andersen H, Greenberg DL, Fujikawa K. et al. Protease- activated receptor 1 is the primary mediator of thrombin-stimulated platelet procoagulant activity. Proc Natl Acad Sci USA 1999; 96: 11189-11193.
• 29 Devine DV, Carter CJ. Profibrinolytic and antifibrinolytic effects of platelets. Coron Artery Dis 1995; 6: 915-922.
• 30 Korbut R, Gryglewski RJ. Platelets in fibrinolytic system. J Physiol Pharmacol 1995; 46: 409-418.
• 31 Lenich C, Liu JN, Gurewich V. Thrombin stimulation of platelets induces plasminogen activation mediated by endogenous urokinase-type plasminogen activator. Blood 1997; 90: 3579-3586.
• 32 Bajzar L. Thrombin activatable fibrinolysis inhibitor and an antifibrinolytic pathway. Arterioscler Thromb Vasc Biol 2000; 20: 2511-2518.
• 33 Glanzmann E. Hereditäre hämorragische Thrombasthenie. Ein Beitrag zur Pathologie der Blutplättchen. Jahr Kinderh 1918; 88: 113-141.
• 34 Blombäck B, Carlsson K, Fatah K. et al. Fibrin in human plasma: gel architectures governed by rate and nature of fibrinogen activation. Thromb Res 1994; 75: 521-538.
• 35 Collet JP, Park D, Lesty C. et al. Influence of fibrin network conformation and fibrin fiber diameter on fibrinolysis speed: dynamic and structural approaches by confocal microscopy. Arterioscler Thromb Vasc Biol 2000; 20: 1354-1361.
• 36 Tynngård N, Lindahl T, Ramström S. et al. Effects of different blood components on clot retraction analysed by measuring elasticity with a free oscillating rheometer. Platelets 2006; 17: 545-554.
• 37 Harper MT, Sage SO. Actin polymerisation regulates thrombin-evoked Ca(2+) signalling after activation of PAR-4 but not PAR-1 in human platelets. Platelets 2006; 17: 134-142.