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• W2109421983 abstract "Thrombin plays a central role in normal and abnormal hemostatic processes. It is assumed that α-thrombin activates platelets by hydrolyzing the protease-activated receptor (PAR)-1, thereby exposing a new N-terminal sequence, a tethered ligand, which initiates a cascade of molecular reactions leading to thrombus formation. This process involves cross-linking of adjacent platelets mediated by the interaction of activated glycoprotein (GP) IIb/IIIa with distinct amino acid sequences, LGGAKQAGDV and/or RGD, at each end of dimeric fibrinogen molecules. We demonstrate here the existence of a second α-thrombin-induced platelet-activating pathway, dependent on GP Ib, which does not require hydrolysis of a substrate receptor, utilizes polymerizing fibrin instead of fibrinogen, and can be inhibited by the Fab fragment of the monoclonal antibody LJIb-10 bound to the GP Ib thrombin-binding site or by the cobra venom metalloproteinase, mocarhagin, that hydrolyzes the extracellular portion of GP Ib. This alternative α-thrombin pathway is observed when PAR-1 or GP IIb/IIIa is inhibited. The recognition sites involved in the cross-linking of polymerizing fibrin and surface integrins via the GP Ib pathway are different from those associated with fibrinogen. This pathway is insensitive to RGDS and anti-GP IIb/IIIa antibodies but reactive with a mutant fibrinogen, γ407, with a deletion of the γ-chain sequence, AGDV. The reaction is not due to simple trapping of platelets by the fibrin clot, since ligand binding, signal transduction, and second messenger formation are required. The GP Ib pathway is accompanied by mobilization of internal calcium and the platelet release reaction. This latter aspect is not observed with ristocetin-induced GP Ib-von Willebrand factor agglutination nor with GP Ib-von Willebrand factor-polymerizing fibrin trapping of platelets. Human platelets also respond to γ-thrombin, an autoproteolytic product of α-thrombin, through PAR-4. Co-activation of the GP Ib, PAR-1, and PAR-4 pathways elicit synergistic responses. The presence of the GP Ib pathway may explain why anti-α-thrombin/anti-platelet regimens fail to completely abrogate thrombosis/restenosis in the cardiac patient. Thrombin plays a central role in normal and abnormal hemostatic processes. It is assumed that α-thrombin activates platelets by hydrolyzing the protease-activated receptor (PAR)-1, thereby exposing a new N-terminal sequence, a tethered ligand, which initiates a cascade of molecular reactions leading to thrombus formation. This process involves cross-linking of adjacent platelets mediated by the interaction of activated glycoprotein (GP) IIb/IIIa with distinct amino acid sequences, LGGAKQAGDV and/or RGD, at each end of dimeric fibrinogen molecules. We demonstrate here the existence of a second α-thrombin-induced platelet-activating pathway, dependent on GP Ib, which does not require hydrolysis of a substrate receptor, utilizes polymerizing fibrin instead of fibrinogen, and can be inhibited by the Fab fragment of the monoclonal antibody LJIb-10 bound to the GP Ib thrombin-binding site or by the cobra venom metalloproteinase, mocarhagin, that hydrolyzes the extracellular portion of GP Ib. This alternative α-thrombin pathway is observed when PAR-1 or GP IIb/IIIa is inhibited. The recognition sites involved in the cross-linking of polymerizing fibrin and surface integrins via the GP Ib pathway are different from those associated with fibrinogen. This pathway is insensitive to RGDS and anti-GP IIb/IIIa antibodies but reactive with a mutant fibrinogen, γ407, with a deletion of the γ-chain sequence, AGDV. The reaction is not due to simple trapping of platelets by the fibrin clot, since ligand binding, signal transduction, and second messenger formation are required. The GP Ib pathway is accompanied by mobilization of internal calcium and the platelet release reaction. This latter aspect is not observed with ristocetin-induced GP Ib-von Willebrand factor agglutination nor with GP Ib-von Willebrand factor-polymerizing fibrin trapping of platelets. Human platelets also respond to γ-thrombin, an autoproteolytic product of α-thrombin, through PAR-4. Co-activation of the GP Ib, PAR-1, and PAR-4 pathways elicit synergistic responses. The presence of the GP Ib pathway may explain why anti-α-thrombin/anti-platelet regimens fail to completely abrogate thrombosis/restenosis in the cardiac patient. glycoprotein protease-activated receptor von Willebrand factor 4-[2-aminoethyl]-benzene sulfonyl fluoride platelet-rich plasma thrombin receptor-activating peptide Despite advances in anti-platelet and anti-thrombotic treatment regimens, cardiovascular diseases remain the leading cause of death in the United States (1Schafer A.I. Am. J. Med. 1996; 101: 199-209Abstract Full Text PDF PubMed Scopus (200) Google Scholar). Clinically employed anti-platelet and anti-thrombotic agents include heparin, aspirin (2Rigel D.F. Olson R.W. Lappe R.W. Circulation Res. 1993; 72: 1091-1102Crossref PubMed Scopus (40) Google Scholar), integrilin (3Curley G.P. Blum H. Humphries M.J. CMLS Cell. Mol. Life Sci. 1999; 52: 427-441Crossref Scopus (108) Google Scholar), and anti-GP1 IIb/IIIa antibodies (c7E3 Fab, abciximab, or, ReoPro) (4Coller B.S. Thromb. Haemostastasis. 1997; 78: 730-735Crossref PubMed Scopus (144) Google Scholar, 5Coller B.S. J. Clin. Invest. 1997; 99: 1467-1471Crossref PubMed Scopus (189) Google Scholar). α-Thrombin, generated at the site of vessel injury, is generally assumed to catalyze the hydrolysis of an N-terminal peptide from the human platelet seven-transmembrane thrombin receptor, protease-activated receptor 1 (PAR-1), which initiates a cascade of molecular reactions leading to thrombus formation. Thrombin-induced activation of PAR-1, as for other agonist-activated platelet receptors, results in an outside-in signal transduced process followed by the alteration of the surface integrin, GP IIb/IIIa, by an inside-out signal (6Schwartz M.A. Schaller M.D. Ginsberg M.H. Annu. Rev. Cell Biol. 1995; 11: 549-599Crossref Scopus (1474) Google Scholar). The conformational change of GP IIb/IIIa leads to the Ca2+-dependent binding of the bifunctional fibrinogen molecule (7Bodary S.C. Napier M.A. McLean J.W. J. Biol. Chem. 1989; 264: 18859-18862Abstract Full Text PDF PubMed Google Scholar). The fibrinogen-GP IIb/IIIa binding sites recognize RGDX sequences on the fibrinogen α-chains and an LGGAKQAGDV sequence on the γ-chains (8Bennett J.S. Shattil S.J. Power J.W. Gartner T.K. J. Biol. Chem. 1988; 263: 12948-12953Abstract Full Text PDF PubMed Google Scholar). Potential competing peptides of RGDS and peptides including the γ sequence LGGAKQAGDV were found to be effective antagonists of platelet aggregation (9Hawiger J. Kloczewiak M. Bednarek M.A. Timmons S. Biochemistry. 1989; 28: 2909-2914Crossref PubMed Scopus (97) Google Scholar). Anti-GP IIb/IIIa antibodies such as c7E3 Fab (4Coller B.S. Thromb. Haemostastasis. 1997; 78: 730-735Crossref PubMed Scopus (144) Google Scholar, 5Coller B.S. J. Clin. Invest. 1997; 99: 1467-1471Crossref PubMed Scopus (189) Google Scholar) and LJ-CP8 (10Niiya K. Hodson E. Bader R. Byers-Ward V. Koziol J.A. Plow E.F. Ruggeri Z.M. Blood. 1987; 70: 475-483Crossref PubMed Google Scholar) are also potent inhibitors of fibrinogen binding to this glycoprotein complex in activated platelets. Early studies of the cellular thrombin receptor indicated that more than one species exist in platelets (11Greco N.J. Jamieson G.A. Proc. Soc. Exp. Biol. Med. 1991; 198: 792-799Crossref PubMed Scopus (49) Google Scholar,12Harmon J.T. Jamieson G.A. J. Biol. Chem. 1986; 261: 15928-15933Abstract Full Text PDF PubMed Google Scholar). Many questions related to the identity and mechanism(s) of action of the platelet thrombin receptor(s) were resolved with the cloning and sequencing of PAR-1 (13Vu T-K.H. Hung D.T. Wheaton V.I. Coughlin S.R. Cell. 1991; 64: 1057-1068Abstract Full Text PDF PubMed Scopus (2680) Google Scholar). Human platelets appear to respond to PAR-1 and a second minor receptor PAR-4 (14Kahn M.L. Zheng Y.-W. Huang W. Bigornia V. Zheng D. Moff S. Farese R.V. Tam C. Coughlin S.R. Nature. 1998; 394: 690-694Crossref PubMed Scopus (882) Google Scholar, 15Xu W.-F. Anderson H. Whitmore T.E. Presnell S.R. Yee D.P. Ching A. Gilbert T. Davie E.W. Foster D.C. Proc. Natl. Acad. Sci. 1998; 95: 6642-6646Crossref PubMed Scopus (759) Google Scholar, 16Covic L. Gresser A.L. Kuliopulos A. Biochemistry. 2000; 39: 5458-5467Crossref PubMed Scopus (261) Google Scholar), while the recently cloned PAR-3 (17Ishihara H. Connolly A.J. Zang D. Kahn M.L. Zheng Y.-W. Timmons C. Tram T. Coughlin S.R. Nature. 1997; 386: 502-506Crossref PubMed Scopus (804) Google Scholar) is either absent or present in only trace amounts. Mouse platelets, on the other hand, respond to α-thrombin primarily through PAR-3 and, secondarily, PAR-4, with no involvement of PAR-1 (14Kahn M.L. Zheng Y.-W. Huang W. Bigornia V. Zheng D. Moff S. Farese R.V. Tam C. Coughlin S.R. Nature. 1998; 394: 690-694Crossref PubMed Scopus (882) Google Scholar). Other important issues still remain unresolved with regard to the PARs. Another platelet membrane protein, GP Ib, may also function, in part, as a thrombin receptor (11Greco N.J. Jamieson G.A. Proc. Soc. Exp. Biol. Med. 1991; 198: 792-799Crossref PubMed Scopus (49) Google Scholar, 12Harmon J.T. Jamieson G.A. J. Biol. Chem. 1986; 261: 15928-15933Abstract Full Text PDF PubMed Google Scholar, 18Clemetson K.J. Thromb. Haemostasis. 1995; 74: 111-116Crossref PubMed Scopus (73) Google Scholar, 19Greco N.J. Jones G.D. Tandon N.N. Kornhauser R. Jackson B. Jamieson G.A. Biochemistry. 1996; 35: 915-992Crossref PubMed Scopus (46) Google Scholar, 20Okumura T. Hasitz M. Jamieson G.A. J. Biol. Chem. 1978; 253: 3435-3443Abstract Full Text PDF PubMed Google Scholar, 21Greco N.J. Tandon N.N. Jones G.D. Kornhauser R. Jackson B. Yamamoto N. Tanoue K. Jamieson G.A. Biochemistry. 1996; 35: 906-914Crossref PubMed Scopus (69) Google Scholar). A major role of GP Ib, complexed with GP IX, is the specific interaction with subendothelium-bound von Willebrand factor (vWF) under high shear rates to facilitate platelet adhesion to injured vascular walls (22Ruggeri Z.M. Semin. Hematol. 1994; 31: 229-239PubMed Google Scholar). The expression on the plasma membrane of the vWF receptor, GP Ib, requires the stable expression of GP Ibβ, GP Ibα, and GP IX (23Lopez J.A. Leung B. Reynold C.C. Li C.Q. Fox J.E.B. J. Biol. Chem. 1991; 267: 12851-12859Abstract Full Text PDF Google Scholar). The GP Ib-IX complex associates with the cytoskeletal actin-binding protein via the cytoplasmic domain of GP Ibα (24Andrews R.K. Fox J.E.B. J. Biol. Chem. 1992; 267: 18605-18611Abstract Full Text PDF PubMed Google Scholar, 25Cunningham J.G. Meyer S.C. Fox J.E.B. J. Biol. Chem. 1996; 271: 11581-11587Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). This GP Ibα-actin-binding protein association is initiated by the binding of vWF to GP Ib and appears to be linked to vWF-induced transmembrane signaling (25Cunningham J.G. Meyer S.C. Fox J.E.B. J. Biol. Chem. 1996; 271: 11581-11587Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Signal transduction appears to be regulated, at least in part, by one form of the 14-3-3 ζ protein (26Du X. Fox J.E.B. Pei S. J. Biol. Chem. 1996; 271: 7362-7367Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar) and its association with the GP Ib-IX-V complex (27Andrews R.K. Harris S.J. McNally T. Berndt M.C. Biochemistry. 1998; 37: 638-647Crossref PubMed Scopus (120) Google Scholar). The GP Ib receptor also possesses a thrombin binding site that may respond to lower concentrations of thrombin than required to activate the PARs (11Greco N.J. Jamieson G.A. Proc. Soc. Exp. Biol. Med. 1991; 198: 792-799Crossref PubMed Scopus (49) Google Scholar, 12Harmon J.T. Jamieson G.A. J. Biol. Chem. 1986; 261: 15928-15933Abstract Full Text PDF PubMed Google Scholar). The GP Ib-thrombin complex may serve to prime the activation of PAR-1 as the thrombin levels rise (11Greco N.J. Jamieson G.A. Proc. Soc. Exp. Biol. Med. 1991; 198: 792-799Crossref PubMed Scopus (49) Google Scholar, 18Clemetson K.J. Thromb. Haemostasis. 1995; 74: 111-116Crossref PubMed Scopus (73) Google Scholar). The physiologic roles of the three purported platelet thrombin receptors have yet to be clearly defined. While an in vitro functional role of PAR-1 has been demonstrated for α-thrombin-induced platelet aggregation (13Vu T-K.H. Hung D.T. Wheaton V.I. Coughlin S.R. Cell. 1991; 64: 1057-1068Abstract Full Text PDF PubMed Scopus (2680) Google Scholar), no comparable response has ever been described for PAR-4 or GP Ib with a natural thrombin agonist. We demonstrate here the existence of two thrombin receptors on human platelets that respond to α-thrombin. One is the PAR-1 receptor, and the second is GP Ib. Unlike the activation of platelets via PAR-1, activation by the GP Ib pathway does not require thrombin hydrolysis of the substrate receptor, utilizes polymerizing fibrin instead of fibrinogen, and is inhibited by the Fab fragment of the monoclonal antibody LJIb-10 that specifically binds to the GP Ib thrombin-binding site (28Handa M. Titani K. Holland L.Z. Roberts J.R. Ruggeri Z.M. J. Biol. Chem. 1986; 261: 12579-12585Abstract Full Text PDF PubMed Google Scholar). This alternative pathway is readily observed in the presence of PAR-1 or GP IIb/IIIa inhibitors. Human platelets also respond to γ-thrombin, the autoproteolytic product of α-thrombin, through activation of a second protease-activated receptor, PAR-4. Co-activation of the GP Ib, PAR-1, and PAR-4 pathways elicits synergistic responses. The α-thrombin was initially obtained from Ortho Diagnostic Systems (Raritan NJ), as Fibrindex. Production of Fibrindex was discontinued, and α-thrombin was subsequently obtained from Chronolog Corp. (Havertown, PA). The two products at 0.05–0.1 units/ml gave identical results. The γ-thrombin was obtained from Hematologic Technologies. The thrombin receptor-activating peptide for PAR-1 (TRAP-1), SFLLRNP, was synthesized by TANA Laboratories (Houston, TX), and peptides for PAR-4, GYPGQV (TRAP-4) and AYPGKF (TRAP-4A), were supplied from the Schering-Plough stocks. The anti-PAR-1 drug, SCH203099, was also supplied by Schering-Plough. Human fibrinogen, RGDS, GPRP-amide, and the water-soluble serine protease inhibitor, 4-[2-aminoethyl]-benzene sulfonyl fluoride (AEBSF) were purchased from Sigma. The thrombin substrate CBS 34.47 came from Diagnostica Stago-American Bioproducts. Fura-2 AM was purchased from Molecular Probes, Inc. (Eugene, OR). The ristocetin and Chrono-lume (luciferin-luciferase) were obtained from Chronolog Corp. Anti-PAR-1 antibodies were kindly supplied by Drs. Greco and Jamieson (polyclonal antibody that recognizes the sequence LLRNPNDKYEPF) and Dr. Brass (monoclonal antibody ATAP-2). The anti-GP IIb/IIIa antibody c7E3 Fab was a kind gift from Dr. Coller. All other anti-GP Ib and anti-GP IIb/IIIa antibodies employed along with the recombinant fibrinogens were from our laboratories. The cobra metalloproteinase, mocarhagin, from Naja mocambique mocambique was kindly supplied by Dr. Berndt (Baker Medical Research Institute, Victoria, Australia). The concentrations and conditions employed with all reagents are described throughout. Blood was drawn by venipuncture into plastic tubes that contained 110 volume of 3.8‥ citrate and platelet-rich plasma (PRP) prepared as previously described (29Soslau G. Giles J. Thromb. Res. 1982; 26: 443-455Abstract Full Text PDF PubMed Scopus (46) Google Scholar). Blood samples were obtained from healthy graduate student donors who were medication-free and signed informed consent forms approved by the institutional human studies committee. Washed platelets were prepared from the PRP as previously described (30Basheer A.R. El-Asmar M.F. Soslau G. Biochim. Biophys. Acta. 1995; 1250: 97-109Crossref PubMed Scopus (16) Google Scholar). Briefly, PRP was diluted with 3 volumes of 100 mm citrate buffer (pH 6.0) plus 1–2 volumes of Hepes-Tyrode buffer, pH 7.4, final volume 50 ml; pelleted; and resuspended in Hepes-Tyrode buffer (136 mm NaCl, 2.7 mm KCl, 3.3 mm NaH2PO4,10 mm MgCl2, 3.8 mm Hepes, pH 7.4) with 1 mg/ml dextrose plus 1 mg/ml bovine serum albumin at 2–3 × 105/μl or at a l0× normal concentration of 2–3 × 106/μl. Platelet aggregations were performed on a dual channel Chronolog lumiaggregometer (Chronolog Corp.) as previously described (31Soslau G. El-Asmar M.F. Parker J. Biochem. Biophys Res. Commun. 1988; 15: 909-916Crossref Scopus (25) Google Scholar). Aggregations were conducted with 480 μl of washed platelets or a 50-μl sample of the concentrated platelets added to 430 μl of Hepes-Tyrode buffer with a final platelet count of 2–3 × 105/μl. Agonists and inhibitors were added as detailed throughout with the concentration of the reagents noted. In some experiments, the α-thrombin plus fibrinogen was added to the platelets, while in others the order of addition was reversed. This reversal of order allowed for the generation of polymerizing fibrin prior to the addition of the platelets. The mobilization of internal stores of calcium, [Ca2+] i , was monitored with a Hitachi F-2000 fluorescence spectrophotometer as previously reported (32Soslau G. McKenzie R.J. Brodsky I. Devlin T.M. Biochim. Biophys. Acta. 1995; 1268: 73-80Crossref PubMed Scopus (26) Google Scholar) in the presence of extracellular EGTA to chelate extracellular Ca2+. Platelets, as PRP, were preloaded with 1 μm Fura-2/AM for 45–60 min and then washed and resuspended in Hepes-Tyrode buffer as described above, at a 10× concentration. Samples were incubated, at room temperature, with or without 10 μm SCH203099 for 1 h prior to analysis. A 50-μl sample of platelets was added to 430 μl of Hepes-Tyrode buffer in a special quartz microcuvette with a 4.5-mm path length with stirring at 37 °C. Agonists were added through an injection port at the levels described. Excitation wavelengths were 340 and 380 nm, and emission was measured at 505 nm. Calibration and conversion of raw data were performed exactly as reported (32Soslau G. McKenzie R.J. Brodsky I. Devlin T.M. Biochim. Biophys. Acta. 1995; 1268: 73-80Crossref PubMed Scopus (26) Google Scholar). The platelet release reaction was monitored simultaneously, in some experiments, with aggregation as previously reported (33Soslau G. Parker J. Thromb. Res. 1992; 66: 15-21Abstract Full Text PDF PubMed Scopus (10) Google Scholar). The release of dense granule ATP from aggregating platelets was detected as light emission in the Chronolog lumiaggregometer produced by the reaction of ATP with luciferin catalyzed by luciferase (Chrono-lume). Platelets were washed in cold phosphate-buffered saline containing 0.1‥ (w/v) bovine serum albumin and 15 mm NaN3 (Buffer) for 5 min at 1200 × g. Pellets were resuspended and washed once, and the pellet was resuspended in 1 ml of room temperature Buffer. Platelets were incubated with 20 μl of antibody at 20 μg/ml for 40 min at 4 °C, washed once in Buffer, and incubated with 20 μl of secondary fluorescein isothiocyanate-conjugated goat anti-mouse antibody (1:40 dilution) for 30 min at 4 °C. Then platelets were washed as above, resuspended in 1 ml of count solution, and analyzed for fluorescence in a FACSort flow cytometer (Becton Dickinson, San Jose, CA) using the Lysis II software program. An acquisition in a live gate containing the platelets and excluding red blood cells and debris was performed. Platelet aggregations were performed as described above and monitored in the aggregometer. A 300-μl sample of Trump's EM fixative (1‥ glutaraldehyde plus 4‥ formaldehyde) was added to the 500-μl aggregated platelet sample and incubated for 5 min. Samples were then pelleted at 4000 rpm for 5 min in a microcentrifuge, and the pellet was fixed with Trump's EM fixative. Samples were postfixed with OsO4 and embedded in epon, and sections were stained with uranyl acetate and lead citrate. The experiments described here indicate that an α-thrombin-GP Ib interaction may induce a distinct pathway of platelet aggregation and, along with PAR-1 and PAR-4, may be functionally relevant. α-Thrombin (0.05–0.1 units/ml) and the peptide SFLLRNP, the PAR-1 TRAP-1, induced platelet aggregation with similar kinetics (Fig.1, A (a) andB (a)). An equivalent amount of γ-thrombin (10–20 nm, comparable with the activity of 0.05–0.1 units/ml α-thrombin) induced platelet aggregation with distinctly slower kinetics but a similar end point (Fig. 1 C(a)), as did the PAR-4 thrombin receptor-activating peptide (TRAP-4), GYPGQV (Fig. 1 D (a)). Platelets that had been preincubated with an anti-PAR-1 antibody (either a polyclonal antibody that recognizes the sequence LLRNPNDKYEPF or the monoclonal antibody ATAP-2 (34Brass L.F. Vassallo Jr R.R. Belmonte E. Ahuja M. Cichowski K. Hoxie J.A. J. Biol. Chem. 1992; 267: 13795-13798Abstract Full Text PDF PubMed Google Scholar); only results with the latter are shown) or treated with a chemically defined PAR-1 inhibitor, SCH203099 (35Ahn H-S. Arik L. Boykow G. Burnett D.A. Caplen M.A. Czarniecki M. Domalski M.S. Foster C. Manna M. Stamford A.W. Wu Y. Bioorganic Med. Chem. Lett. 1999; 9: 2073-2078Crossref PubMed Scopus (56) Google Scholar), had a delayed aggregation profile relative to controls when stimulated with α-thrombin but ultimately reached a similar level of aggregation (Fig. 1, A (e) and E (a), respectively). SCH203099 at 5–10 μm did not inhibit platelet activation induced by 1 mm TRAP-4 or 10–30 nm γ-thrombin (Fig. 1, G and H); however, TRAP-1-induced aggregation was completely inhibited but not platelet shape change (Fig. 1 F). Total inhibition of platelet aggregation was maintained for greater than 15 min, at which time monitoring ceased. Platelets treated with SCH203099 (7.5 μm) plus ATAP-2 (50 μg/ml) have the PAR-1 receptor blocked at two levels: at the tethered ligand, blocking hydrolysis by α-thrombin, and at the PAR-1 receptor site for the tethered ligand or synthetic TRAP-1. These platelets still responded to α-thrombin after a delay period (Fig. 1 E (e)), suggesting the presence of another receptor. The complete inhibition of platelet aggregation upon the addition of the Fab fragment of the anti-GP Ib antibody, LJIb-10, (binds to the GP Ib thrombin binding site) to the SCH203099 plus ATAP-2-treated platelets indicates that GP Ib is the second α-thrombin receptor (Fig. 1 E (g)). The potential presence of three distinct thrombin receptors on human platelets could be defined with combinations of inhibitors and PAR-4 desensitization. Platelets preincubated for 1 h with 350–700 μm TRAP-4, under nonstirring conditions, could not be activated by subsequent additions of 10 nm γ-thrombin or TRAP-4 at mm concentrations (Fig. 1, C(b) and D (b)). Identical results were obtained with the more reactive PAR-4-activating peptide, AYPGKF (TRAP-4A) (36Faruqi T.R. Weiss E.J. Shapiro M.J. Huang W. Coughlin S.R. J. Biol. Chem. 2000; 275: 19728-19734Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). Control platelets aggregated optimally with 100 μm TRAP-4A. Platelets incubated for 20 min with suboptimal concentrations of TRAP-4A (30 μm) were totally unreactive with 100 μm TRAP-4A or 10 nmγ-thrombin but were fully aggregated upon the addition of 0.1 units/ml α-thrombin (data not shown). Furthermore, control platelets and platelets treated with SCH203099 plus ATAP-2 that were preincubated with TRAP-4 still aggregated upon the addition of α-thrombin (Fig. 1,A (b) and E (f), respectively). This evidence indicates that TRAP-4 and γ-thrombin activate PAR-4, while α-thrombin activates PAR-1 and a third receptor. Further evidence for the presence of a thrombin-GP Ib platelet aggregation pathway comes from studies with the cobra venom metalloproteinase, mocarhagin, which has been shown to hydrolyze the extracellular portion of GP Ib that contains the vWF and thrombin binding domains (37Ward C.M. Andrews R.K. Smith A.I. Berndt M.C. Biochemistry. 1996; 35: 4929-4938Crossref PubMed Scopus (182) Google Scholar). Concentrated platelet samples were incubated with 5–20 μg/ml mocarhagin for 60–90 min at 37 °C in the absence or presence of SCH203099 and/or Fab LJ Ib-10 (Fab-10). Mocarhagin (20 μg/ml for 90 min) alone did not alter the slope or extent of platelet aggregation induced by the PAR-1 or PAR-4 agonists, TRAP-1 or γ-thrombin, respectively (Fig.2 A). However, in a dose- and time-dependent fashion, mocarhagin significantly inhibited α-thrombin-induced aggregation of platelets simultaneously incubated with the PAR-1 inhibitor, SCH203099. Fig. 2 B is representative of three different experiments where 10 μmSCH203099 plus 20 μg/ml mocarhagin inhibited α-thrombin-induced platelet aggregation ∼100‥ for the first 3 min with the eventual slow aggregation phase occurring at the 3–6-min point after the addition of α-thrombin. The delayed aggregation appears to be due to residual intact GP Ib molecules on the platelet surface as demonstrated by fluorescence-activated cell sorting analysis (Fig. 2 C). The combined addition of SCH203099 plus Fab-10 and mocarhagin essentially abrogated any delayed aggregation phase in the first 7 min (Fig. 2 B). We reasoned that GP Ib may be the third thrombin receptor that, along with PAR-1, is involved in the α-thrombin-induced activation of platelets. In this regard, we hypothesized that the 1–2-min delay in aggregation observed with platelets treated with anti-PAR-1 antibody or SCH203099 may correspond to the time required for the generation of polymerizing fibrin, which could then participate in platelet aggregation in a manner different from fibrinogen, as previously reported (38 39). This alternative α-thrombin pathway would normally be obscured by the action of the rapidly acting PAR-1 pathway. We further hypothesized that α-thrombin-induced aggregation via the PAR-1 pathway, as depicted in Fig. 1 A, is entirely, or predominantly, dependent upon fibrinogen-platelet interactions. Initial studies with TRAP-1-induced platelet aggregation demonstrated that the PAR-1 pathway was blocked by RGDS but little affected by GPRP-amide (Sigma), an inhibitor of fibrin polymerization (Fig. 1,B (c) and B (d), respectively). Thus, we tested the hypothesis that an alternate thrombin-induced pathway is associated with platelet aggregation mediated by polymerizing fibrin in lieu of native fibrinogen. Washed platelets (50 μl), as a 10× concentrate, were added to 0.05–0.1 units/ml α-thrombin plus fibrinogen preincubated for 2 min (polymerizing fibrin) in 430 μl of buffer. The kinetics of aggregation with thrombin plus polymerizing fibrin (Fig.3 B (a)) was essentially the same as that seen with thrombin plus fibrinogen added simultaneously to platelets (Fig. 3 A (a)). Aggregation in the presence of polymerizing fibrin was little affected by RGDS (Fig. 3 D (a)). In contrast, the rapid onset α-thrombin-induced PAR-1 pathway, seen in Fig. 3 A(a), was inhibited by the fibrinogen-competing peptide RGDS for the first few minutes, a time sufficient for the generation of polymerizing fibrin (Fig. 3 C (a)), after which aggregation ensued via the GP Ib pathway, overriding the RGDS inhibition. The addition of GPRP along with RGDS completely blocked platelet aggregation (Fig. 3, C (b) andD (b)), while the addition of GPRP alone had little effect on α-thrombin-induced aggregation (Fig. 3 A(b)). A recombinant mutant fibrinogen (γ407), lacking the AGDV sequence in the γ-chain required for GP IIb/IIIa-fibrinogen interactions (40Rooney M.M. Parise L.V. Lord S.T. J. Biol. Chem. 1996; 271: 8553-8555Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 41Smith R.A. Mosesson M.W. Rooney M.M. Lord S.T. Daniels A.U. Gartner T.K. J. Biol. Chem. 1997; 272: 22080-22085Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar), still supported α-thrombin-induced platelet aggregation with normal kinetics, as did recombinant wild type fibrinogen (data not shown). Aggregation via the PAR-1 pathway was again initially blocked by the addition of RGDS when γ407 replaced normal fibrinogen, until γ407 presumably began to polymerize (Fig. 3, compare E (a) and C (a)). Residual adhering/endogenous fibrinogen cannot account for the observed aggregation, since none occurred in the presence of RGDS without added fibrinogen (Fig. 3 E (c)). Aggregation occurred with polymerizing γ407 via the GP Ib pathway even in the presence of RGDS (Fig. 3 F) although at a reduced level. RGDS completely blocked aggregation induced by U46619 and ADP, two agonists that cannot generate polymerizing fibrin (Fig. 3, Gand H, without (a) versus with (b) RGDS) like α-thrombin. When the thrombin inhibitor, hirudin, was added to platelets (1–2 units/ml) followed by the addition of thrombin plus fibrinogen, aggregation was completely inhibited (Fig. 5 I). If this inhibitor was added 2 min after fibrinogen was preincubated with thrombin and then platelets were added 30 s later, hirudin continued to prevent aggregation completely (Fig. 3 B (d)), indicating that polymerizing fibrin alone could not account for the observed aggregations as platelet “trapping.” Electron micrographic analysis was conducted with control platelets aggregated by α-thrombin (Fig.4 A) and with 10 μm SCH203099-treated platelets added to polymerizing fibrin (fibrinogen plus α-thrombin preincubated for 2 min) (Fig.4 B) as described in experiments above. The platelet aggregates of both samples are indistinguishable and indicate true platelet aggregation of the SCH203099-treated platelets in the presence of polymerizing fibrin as opposed to platelet trapping.Figure 4Electron microscopic analysis of aggregated control and SCH203099-treated platelets. Platelet aggregation was induced by the addition of α-thrombin p" @default.
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