Thrombin Receptor Occupancy Modulates Aggregation Efficiency and Platelet Surface Expression of vWF and Thrombospondin at Low Thrombin Concentrations

Summary

Previous studies evaluating requirements for occupancy of thrombin receptors in normal platelet secretion and aggregation, using the thrombin antagonists hirudin and PPACK (D-Phe-Pro-Arg-chloromethylke-tone), have suggested that at low thrombin activating concentrations (0.025–0.13 U/ml), occupancy was required only in the first 45–60 s following activation. In our study, we differentiate between thrombin receptor occupancy requirements for surface expression of secreted adhesive proteins, for activation of GPIIb-IIIa receptors, and for aggregation of washed platelets (WP) in laminar shear flow. Platelets activated with 0.05 U/ml thrombin for 10 min to allow maximal secretion (hereafter referred to as “pre-activated platelets”), then sheared, showed a 50–70% decrease in platelet counts after 60 s of shear. Treatment of pre-activated platelets with hirudin or PPACK produced a 65% reduction of capture efficiencies, α_G (reflecting experimental/theoretical initial rates of aggregation), as well as a 30–40% decrease in the surface expression of von Willebrand factor (vWF) and thrombospondin (TSP). However, α-granule membrane P-selectin expression and numbers of activated GPIIb-IIIa receptors were comparable for treated and non-treated platelets. No significant difference in any of the parameters tested was observed when platelets were similarly pre-activated with 0.2 U/ml thrombin, due to treatment with thrombin antagonists. Binding of soluble FITC-vWF (GRGDSP-sensitive) to pre-activated, thrombin antagonist treated platelets, was greatly reduced (≥80%). Soluble Fg was shown to bind to antagonist-treated pre-activated platelets, but could not significantly enhance platelet aggregation. Although occupancy of thrombin receptors by catalytically active thrombin is required transiently for secretion and activation of platelets, there is a further requirement for thrombin occupancy at low thrombin concentrations, for optimizing initial rates of platelet aggregation, surface expression of vWF and TSP, and activated GPIIb-IIIa ligand recognition.

• References

• 1 Naski MC, Fenton JW I I, Maraganore JM, Olson ST, Shafer JA. The COOH-terminal domain of hirudin. An exosite-directed competitive inhibitor of the action of alpha thrombin on fibrinogen. J Biol Chem 1990; 265: 13484-9.
• 2 Rydel TJ, Tulinsky A, Bode W, Huber R, Roitsch C, Fenton I JW. I. Refined structure of the hirudin-thrombin complex. J Mol Biol 1991; 221: 583-601.
• 3 Holmsen H, Dangelmaier CA, Rongved S. Tight coupling of thrombin-induced acid hydrolase secretion and phosphatidate synthesis to receptor occupancy in human platelets. Biochem J 1984; 222: 157-67.
• 4 Jandrot-Perrus M, Didry D, Guillin MC, Nurden A. Cross-linking of α- and γ-thrombin to distinct binding sites on human platelets. Eur J Biochem 1988; 174: 359-67.
• 5 Yamamoto N, Greco NJ, Barnard MR, Tanque K, Yamazaki H, Jamieson GA, Michelson AD. Glycoprotein Ib (GPIb)-dependent and GPIb-independent pathways of thrombin-induced platelet activation. Blood 1991; 77:: 1740-8.
• 6 Topol EJ, Fuster V, Harrington RA, Califf RM, Klieman NS, Kereiakes DJ, Cohen M, Chapekis A, Gold HK, Tannenbaum MA, Rao AK, Debowey D, Schwartz D, Henis M, Chesebro JH. Recombinant hirudin for unstable angina pectoris. A multicentre randomised angiographic trial. Circulation 1994; 89: 1557-66.
• 7 Eriksson BI, Ekman S, Kälebo P, Zachrisson B, Bach D, Close P. Prevention of deep-vein thrombosis after total hip replacement: direct thrombin inhibition with recombinant hirudin CGP 39393. Lancet 1996; 347: 635-9.
• 8 van den Bos AA, Deckers JW, Heyndrickx GR, Laarman G-J, Suryapranata H, Zijlstra F, Close P, Rijnierse JJMM, Báller HR, Serruys PW. Safety and efficacy of recombinant hirudin (CGP 39393) versus heparin in patients with unstable angina undergoing coronary angioplasty. Circulation 1993; 88: 2058-66.
• 9 Global Use of Strategies to Open Occluded Arteries (GUSTO) IIa Investigators Randomized trial of intravenous heparin versus recombinant hirudin for acute coronary syndromes. Circulation 1994; 90:: 1631-7.
• 10 Holmsen H, Dangelmaier CA, Holmsen HK.. Thrombin-induced platelet responses differ in requirement for receptor occupancy; evidence for tight coupling of occupancy and compartmentalized phosphatidic acid formation. J Biol Chem 1981; 256:: 9393-6.
• 11 Greco NJ, Tenner Jr. TE, Tandon NN, Jamieson GA. PPACK-Thrombin inhibits thrombin-induced platelet aggregation and cytoplasmic acidification but does not inhibit platelet shape change. Blood 1990; 75: 1983-90.
• 12 Greco NJ, Jones GD, Tandon NN, Kornhauser R, Jackson B, Jamieson GA. Differentiation of the two forms of GPIb functioning as receptors for alpha-thrombin and von Willebrand factor: Ca²⁺ responses of protease-treated human platelets activated with alpha thrombin and the tethered ligand peptide. Biochem 1996; 35: 915-21.
• 13 Plow EF, Pierschbacher MD, Ruoslahti E, Marguerie GA, Ginsberg MH. The effect of Arg-Gly-Asp-containing peptides on fibrinogen and von Willebrand factor binding to platelets. Proc Nat Acad Sci USA 1985; 82: 8057-61.
• 14 Kettner C, Shaw E. D-Phe-Pro-ArgCH2CL- A selective affinity label for thrombin. Thromb Res 1979; 14: 969-73.
• 15 Schor K, Darius H, Matzky R, Ohlendort R. The antiplatelet and cardiovascular actions of a new carbaxylic derivative (ZK 36374)- equipotent to PGI2 in vitro. Arch Pharm 1981; 316: 252-5.
• 16 Coller BS, Peerschke I E, Scudder LE, Sullivan CA. Studies with a murine monoclonal antibody that abolishes ristocetin-induced binding of von Willebrand factor to platelets; additional evidence in support of GPIb as a platelet receptor for von Willebrand factor. Blood 1983; 61: 99-110.
• 17 Shattil SJ, Cunningham M, Hoaxie JA. Detection of activation platelets in whole blood using activation-dependent monoclonal antibodies and flow cytometry. Blood 1987; 70: 307-15.
• 18 Goto S, Salomon DR, Ikeda Y, Ruggeri ZM. Characterization of the unique mechanism mediating the shear-dependent binding of soluble von Wille-brand factor to platelets. J Biol Chem 1995; 270: 23352-61.
• 19 Legrand C, Dubernard V, Kieffer N, Nurden AT.. Use of a monoclonal antibody to measure the surface expression of thrombospondin following platelet activation. Eur J Biochem 1988; 171:: 393-9.
• 20 Shattil SJ, Hoxie JA, Cunningham M, Brass L. Changes in the platelet membrane glycoprotein IIb-IIIa complex during platelet activation. J Biol Chem 1985; 260: 11107-14.
• 21 Kasirer-Friede A, Frojmovic MM. Ristocetin and thrombin-induced platelet aggregation at physiological shear rates: Differential roles for GPIb and GPIIb-IIIa receptor. Thromb Haemost 1998; 80: 428-36.
• 22 Ramsamooj P, Doellgast GJ, Hantgan RR. Inhibition of fibrin(ogen) binding to stimulated platelets by a monoclonal antibody specific for a conformational determinant of GPIIIa. Thromb Res 1990; 58: 577-92.
• 23 Goldsmith HL, Frojmovic MM, Braovac S, McIntosh F, Wong T. Adenosine diphosphate-induced aggregation of human platelets in flow through tubes: III. Shear and extrinsic fibrinogen-dependent effects. Thromb Haemost 1994; 71: 78-90.
• 24 Tang SS, Frojmovic MM. The effects of pCO2 and pH on platelet shape change and aggregation for human and rabbit platelet-rich plasma. Thromb Res 1977; 10: 135-45.
• 25 George JN, Torres MM. Thrombin decreases von Willebrand factor binding to platelet Glycoprotein Ib. Blood 1988; 71: 1253-9.
• 26 Legrand C, Dubernard V, Nurden AT. Studies on the mechanism of expression of secreted fibrinogen on the surface of activated platelets. Blood 1989; 73: 1226-34.
• 27 Huang EM, Detweiler TC. Thrombin-induced phosphoinositide hydrolysis in platelets. Biochem J 1987; 242: 11-8.
• 28 Prasa D, Svendsen L, Stürzebecher J. The ability of thrombin inhibitors to reduce the thrombin activity generated in plasma on extrinsic and intrinsic activation. Thomb Haemost 1997; 77: 498-503.
• 29 Jackman MP, Parry MA, Hofsteenge J, Stone SR.. Intrinsic fluorescence changes and rapid kinetics of the reaction of thrombin with hirudin. J Biol Chem 1992; 267:: 15375-83.
• 30 Frojmovic MM, Mooney RF, Wong T. Dynamics of platelet glycoprotein IIb-IIIa receptor expression and fibrinogen binding. I. Quantal activation of platelet subpopulations varies with adenosine diphosphate concentration. Biophys J 1994; 67: 2060-8.
• 31 Xia A, Frojmovic MM. Aggregation efficiency of activated normal or fixed platelets in a simple shear field: Effect of shear and fibrinogen occupancy. Biophys J 1994; 66: 2190-201.
• 32 Xia Z, Wong T, Liu Q, Kasirer-Friede A, Brown E, Frojmovic MM. Optimally functional fluorescein isothiocyanate-labelled fibrinogen for quantitative studies of binding to activated platelets and platelet aggregation. Br J Haematol 1996; 93: 204-14.
• 33 Van de Ven TGM.. Two colloidal particles subjected to an external field. In Colloidal Hydrodynamics Academic Press; London: pp 384-91. 1989
• 34 Frojmovic MM, Kasirer-Friede A, Goldsmith HL, Brown EA. Surface-secreted von Willebrand factor mediates aggregation of ADP-activated platelets at moderate shear stress: facilitated by GPIb but controlled by GPIIb-IIIa. Thromb Haemost 1997; 77: 568-76.
• 35 Born GVR, Hume M. Effects of the numbers and sizes of platelet aggregates on the optical density of plasma. Nature 1967; 215: 1027-9.
• 36 Frojmovic MM, Milton JG, Duchastel A. Microscopic measurements of platelet aggregation reveal a low ADP-dependent process distinct from turbidimetrically measured aggregation. J Lab Clin Med 1983; 101: 964-76.
• 37 Ikeda Y, Handa M, Kawano K, Kamata T, Murata M, Araki Y, Anbo H, Kawai Y, Watanabe K, Itagaki I, Sakai K, Ruggeri Z. The role of von Wille-brand factor and fibrinogen in platelet aggregation under varying shear stress. J Clin Invest 1991; 87: 1234-40.
• 38 Frojmovic MM, Mooney RF, Wong T. Dynamics of platelet glycoprotein IIb-IIIa receptor expression and fibrinogen binding. II. Quantal activation parallels platelet capture in stir-associated microaggregation. Biophys J 1994; 67: 2069-75.
• 39 Leung LLK. Role of thrombospondin in platelet aggregation. J Clin Invest 1984; 74: 1764-72.
• 40 Rabhi-Sabile S, Thibert V, Legrand C. Thrombospondin peptides inhibit the secretion-dependent phase of platelet aggregation. Blood Coag Fibrinolys 1996; 7: 237-40.
• 41 Nakatani S, Hato T, Minamoto Y, Fujita S. Differential inhibition of fibrinogen binding to agonist-and RGD peptide-activated states of GPIIb-IIIa by an anti-GPIIIa monoclonal antibody. Thromb Haemost 1996; 76: 1030-7.
• 42 Nurden P, Humbert M, Piotrowicz RS, Bihour C, Poujol C, Nurden AT, Kunicki TJ. Distribution of ligand-occupied αIIbβ₃ in resting and activated human platelets determined by expression of a novel class of ligand-induced binding site recognized by monoclonal antibody AP-6. Blood 1996; 88: 887-99.
• 43 Amrani DL, Newman PJ, Meh D, Mosesson MW. The role of fibrinogen Aα chains in ADP-induced platelet aggregation in the presence of fibrinogen molecules containing γ = chains. Blood 1988; 72: 919-24.
• 44 Liu Q, Matsueda G, Brown E, Frojmovic MM. The AGDV residues on the gamma chain carboxyl terminus of the platelet-bound fibrinogen are needed for platelet aggregation. Biochimica Biophysica Acta 1997; 1343: 316-26.
• 45 D’Souza SE, Ginsburg MH, Matsueda GR, Plow EF. A discrete sequence in a platelet integrin is involved in ligand recognition. Nature 1991; 350: 66-8.
• 46 D’Souza SE, Ginsberg MH, Burke TA, Lam SC, Plow EF. Localization of an Arg-Gly-Asp recognition site within an integrin adhesion receptor. Science 1988; 242: 91-3.
• 47 Giesberts AN, van Willigen G, Lapetina EG, Akkerman JN.. Regulation of platelet glycoprotein IIb/IIIa (integrin α_IIbβ₃) function via the thrombin receptor. Biochem J 309:: 613-620. 1995
• 48 van Willigen G, Hers I, Gorter G, Akkerman JWN. Exposure of ligand-binding sites on integrin α_IIbβ₃ by phosphorylation of the β₃ subunit. Biochem J 1996; 314: 769-79.
• 49 Newman PJ, McEver RP, Doers MP, Kunicki TJ. Synergistic action of two murine monoclonal antibodies that inhibit ADP-induced platelet aggregation without blocking fibrinogen binding. Blood 1987; 69: 668-76.
• 50 Peerschke I E, Zucker MB. Fibrinogen receptor exposure and aggregation of human blood platelets produced by ADP and chilling. Blood 1981; 57: 663-9.
• 51 Wencel-Drake JD, Boudignon-Proudhon C, Dieter MG, Criss AB, Parise LV. Internalization of bound fibrinogen modulates platelet aggregation. Blood 1996; 87: 602-12.