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• W2035721901 startingPage "1997" @default.
• W2035721901 abstract "The roles of the G-protein-linked thrombin receptor and platelet glycoprotein Ib (GPIb) as α-thrombin-binding sites on platelets remain controversial. α-Thrombin has been proposed to bind to both GPIb and the hirudin-like domain of the G-protein-linked receptor (from which it cleaves the NH2-terminal extracellular domain to release a 41-mer peptide (TR-(1-41), where TR is α-thrombin receptor)) to initiate platelet activation. Using affinity-purified rabbit anti-human TR-(1-41) IgG and immunoblotting, we demonstrated TR-(1-41) release from platelets suspended in Tyrode's buffer containing 2 mM CaCl2 and incubated with ≥0.5 nMα-thrombin for 10-60 s at 37°C. As quantified by enzyme-linked immunosorbent assay, 0.32-0.59 nM TR-(1-41) was released from washed platelets (5 x 1011 platelets/liter) after their incubation with 10 nMα-thrombin for 10 s. Parallel binding of α-thrombin to and activation of the platelets were confirmed by flow cytometry. A monoclonal antibody against the hirudin-like domain of the G-protein-linked receptor abrogated α-thrombin binding to platelets, cleavage of TR-(1-41), and platelet activation by ≤1.0 nM (but not 10 nM) α-thrombin. Proteolysis of platelet GPIb with Serratia marcescens protease or O-sialoglycoprotein endopeptidase had no effect on α-thrombin binding to platelets or their subsequent activation. In contrast, chymotrypsin, which cleaves both GPIb and the G-protein-linked receptor, abrogated α-thrombin binding to platelets, TR-(1-41) release, and platelet activation. Furthermore, monoclonal antibodies directed against the reported α-thrombin-binding site on GPIb inhibited neither α-thrombin binding to nor activation of the platelets. Thus, α-thrombin binds to and cleaves the G-protein-linked receptor when it activates platelets, and GPIb does not appear to serve as an important binding site when α-thrombin activates platelets. The roles of the G-protein-linked thrombin receptor and platelet glycoprotein Ib (GPIb) as α-thrombin-binding sites on platelets remain controversial. α-Thrombin has been proposed to bind to both GPIb and the hirudin-like domain of the G-protein-linked receptor (from which it cleaves the NH2-terminal extracellular domain to release a 41-mer peptide (TR-(1-41), where TR is α-thrombin receptor)) to initiate platelet activation. Using affinity-purified rabbit anti-human TR-(1-41) IgG and immunoblotting, we demonstrated TR-(1-41) release from platelets suspended in Tyrode's buffer containing 2 mM CaCl2 and incubated with ≥0.5 nMα-thrombin for 10-60 s at 37°C. As quantified by enzyme-linked immunosorbent assay, 0.32-0.59 nM TR-(1-41) was released from washed platelets (5 x 1011 platelets/liter) after their incubation with 10 nMα-thrombin for 10 s. Parallel binding of α-thrombin to and activation of the platelets were confirmed by flow cytometry. A monoclonal antibody against the hirudin-like domain of the G-protein-linked receptor abrogated α-thrombin binding to platelets, cleavage of TR-(1-41), and platelet activation by ≤1.0 nM (but not 10 nM) α-thrombin. Proteolysis of platelet GPIb with Serratia marcescens protease or O-sialoglycoprotein endopeptidase had no effect on α-thrombin binding to platelets or their subsequent activation. In contrast, chymotrypsin, which cleaves both GPIb and the G-protein-linked receptor, abrogated α-thrombin binding to platelets, TR-(1-41) release, and platelet activation. Furthermore, monoclonal antibodies directed against the reported α-thrombin-binding site on GPIb inhibited neither α-thrombin binding to nor activation of the platelets. Thus, α-thrombin binds to and cleaves the G-protein-linked receptor when it activates platelets, and GPIb does not appear to serve as an important binding site when α-thrombin activates platelets. INTRODUCTIONBinding of α-thrombin to platelets precedes platelet activation by this enzyme and two platelet membrane glycoproteins have been identified as thrombin-binding sites (1Jamieson G.A. Okumura T. J. Clin. Invest. 1978; 61: 861-864Crossref PubMed Scopus (124) Google Scholar, 2Okumura T. Hasitz M. Jamieson G.A. J. Biol. Chem. 1978; 253: 3435-3443Abstract Full Text PDF PubMed Google Scholar, 3Harmon J.T. Jamieson G.A. Biochemistry. 1988; 27: 2151-2157Crossref PubMed Scopus (42) Google Scholar, 4Harmon J.T. Jamieson G.A. J. Biol. Chem. 1986; 261: 15928-15933Abstract Full Text PDF PubMed Google Scholar, 5Lopez J.A. Blood Coagul. & Fibrinolysis. 1994; 5: 97-119Crossref PubMed Scopus (291) Google Scholar, 6Jandrot-Perrus M. Didry D. Guillin M.C. Nurden A.T. Eur. J. Biochem. 1988; 174: 359-367Crossref PubMed Scopus (38) Google Scholar, 7Yamamoto N. Greco N.J. Barnard M.R. Tanoue K. Yamazaki H. Jamieson G.A. Michelson A.D. Blood. 1991; 77: 1740-1748Crossref PubMed Google Scholar, 8Ruggeri Z.M. Prog. Hemostasis Thromb. 1991; 10: 35-68PubMed Google Scholar, 9Vu T.K.H. Hung D.T. Wheaton V.I. Coughlin S.R. Cell. 1991; 64: 1057-1068Abstract Full Text PDF PubMed Scopus (2649) Google Scholar, 10Rasmussen U.B. Vouret-Craviari V. Jallat S. Schlesinger Y. Pages G. Pavirani A. Lecocq J.P. Pouyssegur J. Van Obberghen-Schilling E. FEBS Lett. 1991; 288: 123-128Crossref PubMed Scopus (422) Google Scholar, 11De Marco L. Mazzucato M. Masotti A. Fenton II, J.W. Ruggeri Z.M. J. Biol. Chem. 1991; 266: 23776-23783Abstract Full Text PDF PubMed Google Scholar, 12De Marco L. Mazzucato M. Masotti A. Ruggeri Z.M. J. Biol. Chem. 1994; 269: 6478-6484Abstract Full Text PDF PubMed Google Scholar). Based on the results of studies estimating α-125I-thrombin binding to platelets, ∼50 high-affinity sites (Kd∼ 1 nM) involving GPIb 1The abbreviations used are: GPglycoproteinTBSTris-buffered salineELISAenzyme-linked immunosorbent assayPBSphosphate-buffered saline. and ∼2000 GPIb-independent binding sites with moderate affinity (Kd∼ 10 nM) for α-thrombin on platelets have been reported (3Harmon J.T. Jamieson G.A. Biochemistry. 1988; 27: 2151-2157Crossref PubMed Scopus (42) Google Scholar, 4Harmon J.T. Jamieson G.A. J. Biol. Chem. 1986; 261: 15928-15933Abstract Full Text PDF PubMed Google Scholar, 7Yamamoto N. Greco N.J. Barnard M.R. Tanoue K. Yamazaki H. Jamieson G.A. Michelson A.D. Blood. 1991; 77: 1740-1748Crossref PubMed Google Scholar). GPIb is a disulfide-linked, two-chain protein consisting of a heavy (α) chain (Mr 140,000) and a light (β) chain (Mr 24,000). Distinct sites on GPIb for α-thrombin and von Willebrand factor binding are located within the Mr 45,000 NH2-terminal domain of GPIbα (5Lopez J.A. Blood Coagul. & Fibrinolysis. 1994; 5: 97-119Crossref PubMed Scopus (291) Google Scholar, 7Yamamoto N. Greco N.J. Barnard M.R. Tanoue K. Yamazaki H. Jamieson G.A. Michelson A.D. Blood. 1991; 77: 1740-1748Crossref PubMed Google Scholar, 8Ruggeri Z.M. Prog. Hemostasis Thromb. 1991; 10: 35-68PubMed Google Scholar, 11De Marco L. Mazzucato M. Masotti A. Fenton II, J.W. Ruggeri Z.M. J. Biol. Chem. 1991; 266: 23776-23783Abstract Full Text PDF PubMed Google Scholar, 12De Marco L. Mazzucato M. Masotti A. Ruggeri Z.M. J. Biol. Chem. 1994; 269: 6478-6484Abstract Full Text PDF PubMed Google Scholar). Support for GPIb as a high-affinity binding site for α-thrombin arises from observations that Bernard-Soulier platelets (congenitally deficient in platelet GPIb) are poorly activable by α-thrombin (1Jamieson G.A. Okumura T. J. Clin. Invest. 1978; 61: 861-864Crossref PubMed Scopus (124) Google Scholar). Additionally, cleavage of GPIb by chymotrypsin, elastase, or Serratia marcescens protease impairs the responses of platelets to subnanomolar (but not higher) concentrations of α-thrombin (13Cooper H.A. Bette W.P. White II, G.C. Wagner R.H. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 1433-1438Crossref PubMed Scopus (27) Google Scholar, 14Wicki A.N. Clemetson K.J. Eur. J. Biochem. 1985; 153: 1-11Crossref PubMed Scopus (95) Google Scholar, 15Tam S.W. Fenton II, J.W. Detwiler T.C. J. Biol. Chem. 1980; 255: 6626-6632Abstract Full Text PDF PubMed Google Scholar, 16McGowan E.B. Detwiler T.C. J. Biol. Chem. 1986; 261: 739-746Abstract Full Text PDF PubMed Google Scholar). Furthermore, monoclonal antibodies recognizing epitopes in the Mr 45,000 NH2-terminal domain of GPIbα inhibit the responses of platelets to ≤1.0 nMα-thrombin (17Brass L.F. Thromb. Haemostasis. 1995; 74: 499-505Crossref PubMed Scopus (26) Google Scholar, 18Katagiri Y. Hayashi Y. Yamamoto K. Tanoue K. Kosaki G. Yamazaki H. Thromb. Haemostasis. 1990; 63: 122-126Crossref PubMed Scopus (78) Google Scholar, 19Mazurov A.V. Vinogradov D.V. Vlasik T.N. Thromb. Res. 1991; 62: 673-684Abstract Full Text PDF PubMed Scopus (48) Google Scholar).Another α-thrombin receptor on platelets, a member of the superfamily of G-protein-linked receptors and also found on endothelial cells, smooth muscle cells, and fibroblasts, has been cloned (9Vu T.K.H. Hung D.T. Wheaton V.I. Coughlin S.R. Cell. 1991; 64: 1057-1068Abstract Full Text PDF PubMed Scopus (2649) Google Scholar, 17Brass L.F. Thromb. Haemostasis. 1995; 74: 499-505Crossref PubMed Scopus (26) Google Scholar, 20Vu T.K.H. Wheaton V.I. Hung D.T. Charo I. Coughlin S.R. Nature. 1991; 353: 674-677Crossref PubMed Scopus (467) Google Scholar, 21Coughlin S.R. Vu T.K.H. Hung D.T. Wheaton V.I. J. Clin. Invest. 1991; 89: 351-355Crossref Scopus (243) Google Scholar, 22Coughlin S.R. Vu T.K.H. Hung D.T. Wheaton V.I. Semin. Thromb. Hemostasis. 1992; 18: 161-166Crossref PubMed Scopus (30) Google Scholar). α-Thrombin binds to and cleaves this receptor at Arg-41/Ser-42, releasing a 41-mer activation peptide (called TR-(1-41) in this study, where TR is α-thrombin receptor) and exposing a new NH2-terminal domain, which then binds to an undefined part of the same receptor to activate the platelets (9Vu T.K.H. Hung D.T. Wheaton V.I. Coughlin S.R. Cell. 1991; 64: 1057-1068Abstract Full Text PDF PubMed Scopus (2649) Google Scholar, 20Vu T.K.H. Wheaton V.I. Hung D.T. Charo I. Coughlin S.R. Nature. 1991; 353: 674-677Crossref PubMed Scopus (467) Google Scholar, 21Coughlin S.R. Vu T.K.H. Hung D.T. Wheaton V.I. J. Clin. Invest. 1991; 89: 351-355Crossref Scopus (243) Google Scholar). Whether interactions of α-thrombin with this G-protein-linked thrombin receptor, GPIb, or both are required for platelet activation by α-thrombin remains an unresolved question. Some investigators consider the G-protein-linked α-thrombin receptor to be the moderate-affinity binding site since there are 1700 copies of this α-thrombin receptor/platelet (23Gralnick H.R. Williams S. McKeown L.P. Hansmann K. Fenton J.W. II Krutzsch H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6334-6338Crossref PubMed Scopus (64) Google Scholar, 24Gralnick H.R. Williams S. McKeown L. Hansmann K. Vail M. Krutzsch H. Blood. 1992; 80: 265aGoogle Scholar), and Bernard-Soulier platelets have normal numbers of this receptor (24Gralnick H.R. Williams S. McKeown L. Hansmann K. Vail M. Krutzsch H. Blood. 1992; 80: 265aGoogle Scholar). However, monoclonal antibodies that bind to the hirudin-like domain of the G-protein-linked thrombin receptor abrogate the responses of platelets to ≤1.0 nMα-thrombin (25Hung D.T. Vu T.K.H. Wheaton V.I. Ishii K. Coughlin S.R. J. Clin. Invest. 1992; 89: 1350-1353Crossref PubMed Scopus (165) Google Scholar, 26Hung D.T. Vu T.K.H. Wheaton V.I. Charo I.F. Nelken N.A. Esmon N. Esmon C.T. Coughlin S.R. J. Clin. Invest. 1992; 89: 444-450Crossref PubMed Scopus (38) Google Scholar, 27Brass 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, 28Liu L. Freedman J. Hornstein A. Fenton II, J.W. Ofosu F.A. Br. J. Haematol. 1994; 88: 592-600Crossref PubMed Scopus (21) Google Scholar). This level of α-thrombin would be expected to bind preferentially to its high-affinity binding sites on platelets. It is possible that GPIb, by initiating α-thrombin binding to platelets, could localize α-thrombin to sites on platelets where the cleavage of the G-protein-linked α-thrombin receptor would be facilitated to cause platelet activation (19Mazurov A.V. Vinogradov D.V. Vlasik T.N. Thromb. Res. 1991; 62: 673-684Abstract Full Text PDF PubMed Scopus (48) Google Scholar, 23Gralnick H.R. Williams S. McKeown L.P. Hansmann K. Fenton J.W. II Krutzsch H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6334-6338Crossref PubMed Scopus (64) Google Scholar).This study examined whether the cleavage of the G-protein-linked thrombin receptor necessarily occurs when platelets are activated with 0.5, 1.0, and 10 nMα-thrombin. Affinity-purified polyclonal antibodies against the 41-mer activation peptide (TR-(1-41)) released from the G-protein-linked thrombin receptor by α-thrombin were used to detect cleavage of this receptor and release of the 41-mer activation peptide from platelets incubated with α-thrombin. Binding of α-thrombin to and activation of the same platelets were assessed by flow cytometry (28Liu L. Freedman J. Hornstein A. Fenton II, J.W. Ofosu F.A. Br. J. Haematol. 1994; 88: 592-600Crossref PubMed Scopus (21) Google Scholar). Whether α-thrombin binding to GPIb is a prerequisite for platelet activation by α-thrombin was also explored by cleaving GPIb from platelets with three proteases known to cleave this platelet glycoprotein (13Cooper H.A. Bette W.P. White II, G.C. Wagner R.H. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 1433-1438Crossref PubMed Scopus (27) Google Scholar–16McGowan E.B. Detwiler T.C. J. Biol. Chem. 1986; 261: 739-746Abstract Full Text PDF PubMed Google Scholar) and by using a panel of monoclonal anti-GPIb antibodies previously reported to inhibit α-thrombin binding to platelets (11De Marco L. Mazzucato M. Masotti A. Fenton II, J.W. Ruggeri Z.M. J. Biol. Chem. 1991; 266: 23776-23783Abstract Full Text PDF PubMed Google Scholar, 17Brass L.F. Thromb. Haemostasis. 1995; 74: 499-505Crossref PubMed Scopus (26) Google Scholar, 18Katagiri Y. Hayashi Y. Yamamoto K. Tanoue K. Kosaki G. Yamazaki H. Thromb. Haemostasis. 1990; 63: 122-126Crossref PubMed Scopus (78) Google Scholar).DISCUSSIONPlatelets have ∼25,000 copies of GPIb, the platelet glycoprotein proposed to provide ∼50 high-affinity binding sites for α-thrombin (Kd∼ 1 nM) since platelets of Bernard-Soulier patients (and thus congenitally deficient in GPIb) aggregate slowly, but demonstrate normal dense body release in response to subnanomolar α-thrombin. Additionally, cleavage of GPIb or occupancy of GPIb by some monoclonal anti-GPIb antibodies inhibits platelet aggregation and release by ≤1.0 nMα-thrombin, but not by 10 nMα-thrombin (1Jamieson G.A. Okumura T. J. Clin. Invest. 1978; 61: 861-864Crossref PubMed Scopus (124) Google Scholar, 5Lopez J.A. Blood Coagul. & Fibrinolysis. 1994; 5: 97-119Crossref PubMed Scopus (291) Google Scholar, 7Yamamoto N. Greco N.J. Barnard M.R. Tanoue K. Yamazaki H. Jamieson G.A. Michelson A.D. Blood. 1991; 77: 1740-1748Crossref PubMed Google Scholar, 8Ruggeri Z.M. Prog. Hemostasis Thromb. 1991; 10: 35-68PubMed Google Scholar, 11De Marco L. Mazzucato M. Masotti A. Fenton II, J.W. Ruggeri Z.M. J. Biol. Chem. 1991; 266: 23776-23783Abstract Full Text PDF PubMed Google Scholar, 12De Marco L. Mazzucato M. Masotti A. Ruggeri Z.M. J. Biol. Chem. 1994; 269: 6478-6484Abstract Full Text PDF PubMed Google Scholar–16McGowan E.B. Detwiler T.C. J. Biol. Chem. 1986; 261: 739-746Abstract Full Text PDF PubMed Google Scholar). A G-protein-linked thrombin receptor on platelets to which α-thrombin binds (probably via the hirudin-like domain of this receptor) and cleaves off the first 41 amino acid residues (called TR-(1-41) in this study) has been described (9Vu T.K.H. Hung D.T. Wheaton V.I. Coughlin S.R. Cell. 1991; 64: 1057-1068Abstract Full Text PDF PubMed Scopus (2649) Google Scholar, 10Rasmussen U.B. Vouret-Craviari V. Jallat S. Schlesinger Y. Pages G. Pavirani A. Lecocq J.P. Pouyssegur J. Van Obberghen-Schilling E. FEBS Lett. 1991; 288: 123-128Crossref PubMed Scopus (422) Google Scholar, 19Mazurov A.V. Vinogradov D.V. Vlasik T.N. Thromb. Res. 1991; 62: 673-684Abstract Full Text PDF PubMed Scopus (48) Google Scholar–21Coughlin S.R. Vu T.K.H. Hung D.T. Wheaton V.I. J. Clin. Invest. 1991; 89: 351-355Crossref Scopus (243) Google Scholar). There are ∼1700 copies of this receptor/platelet (27Brass 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), and some investigators have assigned the moderate-affinity α-thrombin-binding sites (Kd∼ 10 nM) on platelets to this receptor (11De Marco L. Mazzucato M. Masotti A. Fenton II, J.W. Ruggeri Z.M. J. Biol. Chem. 1991; 266: 23776-23783Abstract Full Text PDF PubMed Google Scholar, 23Gralnick H.R. Williams S. McKeown L.P. Hansmann K. Fenton J.W. II Krutzsch H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6334-6338Crossref PubMed Scopus (64) Google Scholar, 24Gralnick H.R. Williams S. McKeown L. Hansmann K. Vail M. Krutzsch H. Blood. 1992; 80: 265aGoogle Scholar). Antibodies against the hirudin-like domain of this G-protein-linked receptor inhibit the responsiveness of platelets to α-thrombin (27Brass 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, 28Liu L. Freedman J. Hornstein A. Fenton II, J.W. Ofosu F.A. Br. J. Haematol. 1994; 88: 592-600Crossref PubMed Scopus (21) Google Scholar, 33Norton K.J. Scarborough R.M. Kutok J.L. Escobedo M.A. Nannizzi L. Coller B.S. Blood. 1993; 82: 2125-2136Crossref PubMed Google Scholar). Thus, the primary site on platelets to which α-thrombin binds to initiate platelet activation remains unclear.In this study, cleavage of platelet TR-(1-41) by α-thrombin was directly monitored, as were α-thrombin binding to platelets and the subsequent activation of the same platelets. No attempt was made in this study to quantify the number of α-thrombin molecules/platelet or the concentrations of markers of platelet activation that became expressed on activated platelets. Rather, the percentages of platelets that rapidly bound α-thrombin and subsequently expressed surface P-selection, CD63, and the activated conformer of GPIIb-IIIa for each concentration of the enzyme were quantified. We have presented data demonstrating the parallel binding of α-thrombin to platelets, cleavage and release of TR-(1-41) from the platelets, and activation of the same platelets with each concentration of α-thrombin. There was a similar (∼1:1) relationship between the binding of α-thrombin to platelets and the expression of each of the three markers of platelet activation within 60 s of α-thrombin addition. This study also confirmed the observation by Norton et al. (33Norton K.J. Scarborough R.M. Kutok J.L. Escobedo M.A. Nannizzi L. Coller B.S. Blood. 1993; 82: 2125-2136Crossref PubMed Google Scholar) that α-thrombin releases TR-(1-41) from platelets. It is unclear why 1.0 nMα-thrombin did not release TR-(1-41) as effectively as 10 nMα-thrombin when both concentrations of the enzyme activated ≥75% of the washed platelets (Fig. 1, Fig. 2, Fig. 3). We eliminated the possibility that this α-thrombin receptor became inaccessible to α-thrombin following the exposure of platelets to suboptimal concentrations of α-thrombin. Specifically, we demonstrated that platelets preincubated with 0.5 or 1 nMα-thrombin responded appropriately to a subsequent addition of α-thrombin. Thus, the fraction of the thrombin receptor not previously occupied by suboptimal concentrations of α-thrombin remained accessible to added α-thrombin. Since ∼2.0 nM TR-(1-41) could be theoretically released from platelets (27Brass 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), the fact that 10 nMα-thrombin fully activated the platelets but released only ≤0.6 nM TR-(1-41) suggests that complete cleavage of the receptor is not required for maximum platelet activation. Nonetheless, partial cleavage of this α-thrombin receptor is required to initiate platelet activation since abrogation of thrombin-mediated cleavage of this receptor by ATAP-138 also abrogated platelet activation.A likely reason for the failure of α-thrombin to quantitatively cleave all available TR-(1-41) from platelets may reside in the ability of α-thrombin to induce endocytosis of this receptor, as demonstrated for two megaloblastic cell lines, namely human erythroleukemia cells and Children's Hospital Research Foundation cell line 288 (34Hoxie J.A. Ahuja M. Belmonte E. Pizarro S. Parton R. Brass L.F. J. Biol. Chem. 1993; 268: 13756-13763Abstract Full Text PDF PubMed Google Scholar–36Brass L.F. Pizarro S. Ahuja M. Belmonte E. Blanchard N. Stadel J.M. Hoxie J.A. J. Biol. Chem. 1994; 269: 2943-2952Abstract Full Text PDF PubMed Google Scholar). This failure of up to 10 nMα-thrombin to fully cleave the G-protein-linked thrombin receptor and to release TR-(1-41) from platelets parallels the effects α-thrombin has on fibrinogen and fibrin has on the enzymatic activity of α-thrombin. Similar to the release of TR-(1-41), α-thrombin cleaves fibrinogen in a dose-dependent manner, with fibrinopeptide A release proceeding to the maximum extent achievable with each α-thrombin concentration within 60 s (37Weitz J.I. Hudoba M. Massel D. Maraganore J. Hirsh J. J. Clin. Invest. 1990; 86: 385-391Crossref PubMed Scopus (1077) Google Scholar). α-Thrombin binding to fibrin also clearly impairs the ability of this enzyme to release fibrinopeptide A from fibrinogen (37Weitz J.I. Hudoba M. Massel D. Maraganore J. Hirsh J. J. Clin. Invest. 1990; 86: 385-391Crossref PubMed Scopus (1077) Google Scholar). Binding of α-thrombin to the cleaved receptor (which then becomes phosphorylated (17Brass L.F. Thromb. Haemostasis. 1995; 74: 499-505Crossref PubMed Scopus (26) Google Scholar, 38Lau L.F. Pumiglia K. Cole Y.P. Feinstein M.B. Biochem. J. 1994; 303: 391-400Crossref PubMed Scopus (66) Google Scholar)) may similarly impair the ability of the bound enzyme to cleave nearby receptors. Continued tight binding of α-thrombin to this site may be important, and one study has reported that continued occupancy of the G-protein-linked receptor by α-thrombin is required to propagate tyrosine phosphorylation. Specifically, Lau et al. (38Lau L.F. Pumiglia K. Cole Y.P. Feinstein M.B. Biochem. J. 1994; 303: 391-400Crossref PubMed Scopus (66) Google Scholar) have reported that addition of hirudin to platelets preincubated with α-thrombin for 60 s does not deaggregate the platelets, but inhibits specific tyrosine phosphorylation and simultaneously accelerates specific tyrosine dephosphorylation. Occupancy of this receptor by α-thrombin at the hirudin-like domain of the receptor is clearly crucial for platelet activation since ATAP-138 abrogates the binding of 0.5 or 1 nMα-thrombin to platelets, release of TR-(1-41) from the platelets, and activation of the platelets. As previously reported by Brass et al. (27Brass 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), we found that 10 nMα-thrombin binds to and activates washed platelets in the presence of a saturating concentration ATAP-138.The high-affinity binding sites for α-thrombin on GPIb are reportedly located within the Mr 45,000 NH2-terminal domain of GPIbα (3Harmon J.T. Jamieson G.A. Biochemistry. 1988; 27: 2151-2157Crossref PubMed Scopus (42) Google Scholar, 5Lopez J.A. Blood Coagul. & Fibrinolysis. 1994; 5: 97-119Crossref PubMed Scopus (291) Google Scholar, 7Yamamoto N. Greco N.J. Barnard M.R. Tanoue K. Yamazaki H. Jamieson G.A. Michelson A.D. Blood. 1991; 77: 1740-1748Crossref PubMed Google Scholar, 18Katagiri Y. Hayashi Y. Yamamoto K. Tanoue K. Kosaki G. Yamazaki H. Thromb. Haemostasis. 1990; 63: 122-126Crossref PubMed Scopus (78) Google Scholar, 39De Marco L. Mazzucato M. Fabris F. De Roia D. Cosser P. Girolami A. Vincente V. Ruggeri Z.M. J. Clin. Invest. 1990; 86: 25-31Crossref PubMed Scopus (57) Google Scholar–42Greco N.T. Jones G.D. Tandon N.N. Kornhauser R. Jackson B. Jamieson G.A. Biochemistry. 1996; 35: 915-921Crossref PubMed Scopus (45) Google Scholar), and removal of GPIb from platelets by chymotrypsin, S. marcescens protease, or elastase yields platelets with a lower sensitivity to ≤1.0 nMα-thrombin (13Cooper H.A. Bette W.P. White II, G.C. Wagner R.H. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 1433-1438Crossref PubMed Scopus (27) Google Scholar–16McGowan E.B. Detwiler T.C. J. Biol. Chem. 1986; 261: 739-746Abstract Full Text PDF PubMed Google Scholar). This study has demonstrated that platelets with this putative high-affinity α-thrombin-binding domain on GPIb removed (by protease digestion) bound normally to α-thrombin. In further experiments, two monoclonal antibodies against this putative high-affinity α-thrombin-binding domain on GPIb (TM60 and LJ-1B10) that inhibit the responses of platelets to ≤1 nMα-thrombin (7Yamamoto N. Greco N.J. Barnard M.R. Tanoue K. Yamazaki H. Jamieson G.A. Michelson A.D. Blood. 1991; 77: 1740-1748Crossref PubMed Google Scholar, 18Katagiri Y. Hayashi Y. Yamamoto K. Tanoue K. Kosaki G. Yamazaki H. Thromb. Haemostasis. 1990; 63: 122-126Crossref PubMed Scopus (78) Google Scholar, 39De Marco L. Mazzucato M. Fabris F. De Roia D. Cosser P. Girolami A. Vincente V. Ruggeri Z.M. J. Clin. Invest. 1990; 86: 25-31Crossref PubMed Scopus (57) Google Scholar, 40Ware J. Russell S.R. Marchese P. Murata W. Mazzucato M. De Marco L. Ruggeri Z.M. J. Clin. Invest. 1993; 92: 1213-1220Crossref PubMed Scopus (113) Google Scholar, 41Greco N.T. Tandon N.N. Jones G.D. Kornhauser R. Jackson B. Yamamoto N. Tonoue K. Jamieson G.A. Biochemistry. 1996; 35: 906-914Crossref PubMed Scopus (68) Google Scholar, 42Greco N.T. Jones G.D. Tandon N.N. Kornhauser R. Jackson B. Jamieson G.A. Biochemistry. 1996; 35: 915-921Crossref PubMed Scopus (45) Google Scholar) were used in another attempt to prevent α-thrombin binding to platelets via GPIb. In the presence of 2 mM CaCl2, α-thrombin bound normally to and activated platelets that had been preincubated with either monoclonal anti-GPIb antibody.Therefore, we conclude that GPIb does not normally participate in the initial interactions of α-thrombin with platelets and that cleavage(s) by chymotrypsin additional to GPIb abrogate the responsiveness of platelets to α-thrombin. Chymotrypsin cleaves the G-protein thrombin-linked receptor at a point distal to Arg-41/Ser-42 (43Vouret-Craviari V. Grall D. Chambard J.-C. Rasmussen U.B. Pouyssegur J. Van Obberghen-Schilling E. J. Biol. Chem. 1995; 270: 8367-8372Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 44Bouton M.-C. Jandrot-Perrus M. Moog S. Cazenave J.-P. Guillin M.-C. Lanza F. Biochem. J. 1995; 305: 635-641Crossref PubMed Scopus (34) Google Scholar). This cleavage may explain why only <30 pM TR-(1-41) was detected by the ELISA for TR-(1-41). Using a chimeric fusion protein consisting of glutathione S-transferase and residues 25-97 corresponding to the NH2-terminal extracellular domain of the G-protein-linked thrombin as the substrate, Bouton et al. (44Bouton M.-C. Jandrot-Perrus M. Moog S. Cazenave J.-P. Guillin M.-C. Lanza F. Biochem. J. 1995; 305: 635-641Crossref PubMed Scopus (34) Google Scholar) reported that the glycocalicin portion of GPIb did not alter the kinetics describing the cleavage of this fusion protein by α-thrombin, whereas fibrinogen fragment E, thrombomodulin, and hirudin fragment 54-65 did. These results suggest minimal rapid binding interactions between α-thrombin and the extracellular domain of GPIb when the enzyme normally cleaves the G-protein-linked thrombin receptor.There are three reasons why we could not ascribe a critical role to GPIb for mediating α-thrombin binding to platelets in the time required for α-thrombin to optimally activate platelets. (i) We used 10-60-s incubations to demonstrate optimal binding of α-thrombin to platelets, compared with ≥30-min incubations used in some of the previous studies (7Yamamoto N. Greco N.J. Barnard M.R. Tanoue K. Yamazaki H. Jamieson G.A. Michelson A.D. Blood. 1991; 77: 1740-1748Crossref PubMed Google Scholar, 18Katagiri Y. Hayashi Y. Yamamoto K. Tanoue K. Kosaki G. Yamazaki H. Thromb. Haemostasis. 1990; 63: 122-126Crossref PubMed Scopus (78) Google Scholar, 24Gralnick H.R. Williams S. McKeown L. Hansmann K. Vail M. Krutzsch H. Blood. 1992; 80: 265aGoogle Scholar). The incubation times of 10 and 60 s were chosen as activation of platelets by α-thrombin proceeds to the maximum extent achievable with each concentration of thrombin in ≤60 s (28Liu L. Freedman J. Hornstein A. Fenton II, J.W. Ofosu F.A. Br. J. Haematol. 1994; 88: 592-600Crossref PubMed Scopus (21) Google Scholar). This choice was also justified by the demonstration of decreased binding of α-thrombin to platelets after the enzyme was incubated with platelets for 30 min (compared with 10 s), as shown in Table I. (ii) The platelets used in this study were fixed with 10 g/liter paraformaldehyde after their incubation with α-thrombin to immobilize the enzyme on platelets. Fixation of the platelets also inactivated α-thrombin and halted further platelet reactions resulting from α-thrombin binding to the platelets. Fixation does not alter the binding of α-thrombin to platelets (40Ware J. Russell S.R. Marchese P. Murata W. Mazzucato M. De Marco L. Ruggeri Z.M. J. Clin. Invest. 1993; 92: 1213-1220Crossref PubMed Scopus (113) Google Scholar). (iii) We also estimated α-thrombin binding to platelets resuspended in CaCl2-containing media, while the previous studies were without addition of this salt. CaCl2 enhances the binding of α-thrombin to platelets and stabilizes the expression of P-selectin on the activated platelets (28Liu L. Freedman J. Hornstein A. Fenton II, J.W. Ofosu F.A. Br. J. Haematol. 1994; 88: 592-600Crossref PubMed Scopus (21) Google Scholar), as was confirmed in this study. Additionally, two monoclonal anti-GPIb antibodies (LJ-IB10 and TM60) inhibited α-thrombin binding to washed platelets and their activation, but only in the absence of added CaCl2 (Table I). Inhibition of α-thrombin binding to platelets by these two monoclonal anti-GPIb antibodies (in the absence of Ca2+) has been reported by many other investigators (11De Marco L. Mazzucato M. Masotti A. Fenton II, J.W. Ruggeri Z.M. J. Biol. Chem. 1991; 266: 23776-23783Abstract Full Text PDF PubMed Google Scholar, 12De Marco L. Mazzucato M. Masotti A. Ruggeri Z.M. J. Biol. Chem. 1994; 269: 6478-6484Abstract Full Text PDF PubMed Google Scholar, 39De Marco L. Mazzucato M. Fabris F. De Roia D. Cosser P. Girolami A. Vincente V. Ruggeri Z.M. J. Clin. Invest. 1990; 86: 25-31Crossref PubMed Scopus (57) Google Scholar–42Greco N.T. Jones G.D. Tandon N.N. Kornhauser R. Jackson B. Jamieson G.A. Biochemistry. 1996; 35: 915-921Crossref PubMed Scopus (45) Google Scholar).Previous reports have hypothesized that GPIb and the G-protein-linked thrombin receptor form a functional complex on platelets. Specifically, interactions of α-thrombin with GPIb localize α-thrombin to sites that facilitate cleavage of nearby G-protein-linked thrombin receptors during the activation process (18Katagiri Y. Hayashi Y. Yamamoto K. Tanoue K. Kosaki G. Yamazaki H. Thromb. Haemostasis. 1990; 63: 122-126Crossref PubMed Scopus (78) Google Scholar, 23Gralnick H.R. Williams S. McKeown L.P. Hansmann K. Fenton J.W. II Krutzsch H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6334-6338Crossref PubMed Scopus (64) Google Scholar). Our results do not support significant interactions between α-thrombin and GPIb to effect α-thrombin binding to platelets, in the presence of Ca2+, to initiate platelet activation. The G-protein-linked thrombin receptor appears to be the primary site to which α-thrombin binds to initiate platelet activation. Our observations, however, do not exclude GPIb modulating additional signaling events, including changes in extracellular Ca2+ and aggregation resulting from α-thrombin binding to the platelets (12De Marco L. Mazzucato M. Masotti A. Ruggeri Z.M. J. Biol. Chem. 1994; 269: 6478-6484Abstract Full Text PDF PubMed Google Scholar, 40Ware J. Russell S.R. Marchese P. Murata W. Mazzucato M. De Marco L. Ruggeri Z.M. J. Clin. Invest. 1993; 92: 1213-1220Crossref PubMed Scopus (113) Google Scholar, 41Greco N.T. Tandon N.N. Jones G.D. Kornhauser R. Jackson B. Yamamoto N. Tonoue K. Jamieson G.A. Biochemistry. 1996; 35: 906-914Crossref PubMed Scopus (68) Google Scholar). INTRODUCTIONBinding of α-thrombin to platelets precedes platelet activation by this enzyme and two platelet membrane glycoproteins have been identified as thrombin-binding sites (1Jamieson G.A. Okumura T. J. Clin. Invest. 1978; 61: 861-864Crossref PubMed Scopus (124) Google Scholar, 2Okumura T. Hasitz M. Jamieson G.A. J. Biol. Chem. 1978; 253: 3435-3443Abstract Full Text PDF PubMed Google Scholar, 3Harmon J.T. Jamieson G.A. Biochemistry. 1988; 27: 2151-2157Crossref PubMed Scopus (42) Google Scholar, 4Harmon J.T. Jamieson G.A. J. Biol. Chem. 1986; 261: 15928-15933Abstract Full Text PDF PubMed Google Scholar, 5Lopez J.A. Blood Coagul. & Fibrinolysis. 1994; 5: 97-119Crossref PubMed Scopus (291) Google Scholar, 6Jandrot-Perrus M. Didry D. Guillin M.C. Nurden A.T. Eur. J. Biochem. 1988; 174: 359-367Crossref PubMed Scopus (38) Google Scholar, 7Yamamoto N. Greco N.J. Barnard M.R. Tanoue K. Yamazaki H. Jamieson G.A. Michelson A.D. Blood. 1991; 77: 1740-1748Crossref PubMed Google Scholar, 8Ruggeri Z.M. Prog. Hemostasis Thromb. 1991; 10: 35-68PubMed Google Scholar, 9Vu T.K.H. Hung D.T. Wheaton V.I. Coughlin S.R. Cell. 1991; 64: 1057-1068Abstract Full Text PDF PubMed Scopus (2649) Google Scholar, 10Rasmussen U.B. Vouret-Craviari V. Jallat S. Schlesinger Y. Pages G. Pavirani A. Lecocq J.P. Pouyssegur J. Van Obberghen-Schilling E. FEBS Lett. 1991; 288: 123-128Crossref PubMed Scopus (422) Google Scholar, 11De Marco L. Mazzucato M. Masotti A. Fenton II, J.W. Ruggeri Z.M. J. Biol. Chem. 1991; 266: 23776-23783Abstract Full Text PDF PubMed Google Scholar, 12De Marco L. Mazzucato M. Masotti A. Ruggeri Z.M. J. Biol. Chem. 1994; 269: 6478-6484Abstract Full Text PDF PubMed Google Scholar). Based on the results of studies estimating α-125I-thrombin binding to platelets, ∼50 high-affinity sites (Kd∼ 1 nM) involving GPIb 1The abbreviations used are: GPglycoproteinTBSTris-buffered salineELISAenzyme-linked immunosorbent assayPBSphosphate-buffered saline. and ∼2000 GPIb-independent binding sites with moderate affinity (Kd∼ 10 nM) for α-thrombin on platelets have been reported (3Harmon J.T. Jamieson G.A. Biochemistry. 1988; 27: 2151-2157Crossref PubMed Scopus (42) Google Scholar, 4Harmon J.T. Jamieson G.A. J. Biol. Chem. 1986; 261: 15928-15933Abstract Full Text PDF PubMed Google Scholar, 7Yamamoto N. Greco N.J. Barnard M.R. Tanoue K. Yamazaki H. Jamieson G.A. Michelson A.D. Blood. 1991; 77: 1740-1748Crossref PubMed Google Scholar). GPIb is a disulfide-linked, two-chain protein consisting of a heavy (α) chain (Mr 140,000) and a light (β) chain (Mr 24,000). Distinct sites on GPIb for α-thrombin and von Willebrand factor binding are located within the Mr 45,000 NH2-terminal domain of GPIbα (5Lopez J.A. Blood Coagul. & Fibrinolysis. 1994; 5: 97-119Crossref PubMed Scopus (291) Google Scholar, 7Yamamoto N. Greco N.J. Barnard M.R. Tanoue K. Yamazaki H. Jamieson G.A. Michelson A.D. Blood. 1991; 77: 1740-1748Crossref PubMed Google Scholar, 8Ruggeri Z.M. Prog. Hemostasis Thromb. 1991; 10: 35-68PubMed Google Scholar, 11De Marco L. Mazzucato M. Masotti A. Fenton II, J.W. Ruggeri Z.M. J. Biol. Chem. 1991; 266: 23776-23783Abstract Full Text PDF PubMed Google Scholar, 12De Marco L. Mazzucato M. Masotti A. Ruggeri Z.M. J. Biol. Chem. 1994; 269: 6478-6484Abstract Full Text PDF PubMed Google Scholar). Support for GPIb as a high-affinity binding site for α-thrombin arises from observations that Bernard-Soulier platelets (congenitally deficient in platelet GPIb) are poorly activable by α-thrombin (1Jamieson G.A. Okumura T. J. Clin. Invest. 1978; 61: 861-864Crossref PubMed Scopus (124) Google Scholar). Additionally, cleavage of GPIb by chymotrypsin, elastase, or Serratia marcescens protease impairs the responses of platelets to subnanomolar (but not higher) concentrations of α-thrombin (13Cooper H.A. Bette W.P. White II, G.C. Wagner R.H. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 1433-1438Crossref PubMed Scopus (27) Google Scholar, 14Wicki A.N. Clemetson K.J. Eur. J. Biochem. 1985; 153: 1-11Crossref PubMed Scopus (95) Google Scholar, 15Tam S.W. Fenton II, J.W. Detwiler T.C. J. Biol. Chem. 1980; 255: 6626-6632Abstract Full Text PDF PubMed Google Scholar, 16McGowan E.B. Detwiler T.C. J. Biol. Chem. 1986; 261: 739-746Abstract Full Text PDF PubMed Google Scholar). Furthermore, monoclonal antibodies recognizing epitopes in the Mr 45,000 NH2-terminal domain of GPIbα inhibit the responses of platelets to ≤1.0 nMα-thrombin (17Brass L.F. Thromb. Haemostasis. 1995; 74: 499-505Crossref PubMed Scopus (26) Google Scholar, 18Katagiri Y. Hayashi Y. Yamamoto K. Tanoue K. Kosaki G. Yamazaki H. Thromb. Haemostasis. 1990; 63: 122-126Crossref PubMed Scopus (78) Google Scholar, 19Mazurov A.V. Vinogradov D.V. Vlasik T.N. Thromb. Res. 1991; 62: 673-684Abstract Full Text PDF PubMed Scopus (48) Google Scholar).Another α-thrombin receptor on platelets, a member of the superfamily of G-protein-linked receptors and also found on endothelial cells, smooth muscle cells, and fibroblasts, has been cloned (9Vu T.K.H. Hung D.T. Wheaton V.I. Coughlin S.R. Cell. 1991; 64: 1057-1068Abstract Full Text PDF PubMed Scopus (2649) Google Scholar, 17Brass L.F. Thromb. Haemostasis. 1995; 74: 499-505Crossref PubMed Scopus (26) Google Scholar, 20Vu T.K.H. Wheaton V.I. Hung D.T. Charo I. Coughlin S.R. Nature. 1991; 353: 674-677Crossref PubMed Scopus (467) Google Scholar, 21Coughlin S.R. Vu T.K.H. Hung D.T. Wheaton V.I. J. Clin. Invest. 1991; 89: 351-355Crossref Scopus (243) Google Scholar, 22Coughlin S.R. Vu T.K.H. Hung D.T. Wheaton V.I. Semin. Thromb. Hemostasis. 1992; 18: 161-166Crossref PubMed Scopus (30) Google Scholar). α-Thrombin binds to and cleaves this receptor at Arg-41/Ser-42, releasing a 41-mer activation peptide (called TR-(1-41) in this study, where TR is α-thrombin receptor) and exposing a new NH2-terminal domain, which then binds to an undefined part of the same receptor to activate the platelets (9Vu T.K.H. Hung D.T. Wheaton V.I. Coughlin S.R. Cell. 1991; 64: 1057-1068Abstract Full Text PDF PubMed Scopus (2649) Google Scholar, 20Vu T.K.H. Wheaton V.I. Hung D.T. Charo I. Coughlin S.R. Nature. 1991; 353: 674-677Crossref PubMed Scopus (467) Google Scholar, 21Coughlin S.R. Vu T.K.H. Hung D.T. Wheaton V.I. J. Clin. Invest. 1991; 89: 351-355Crossref Scopus (243) Google Scholar). Whether interactions of α-thrombin with this G-protein-linked thrombin receptor, GPIb, or both are required for platelet activation by α-thrombin remains an unresolved question. Some investigators consider the G-protein-linked α-thrombin receptor to be the moderate-affinity binding site since there are 1700 copies of this α-thrombin receptor/platelet (23Gralnick H.R. Williams S. McKeown L.P. Hansmann K. Fenton J.W. II Krutzsch H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6334-6338Crossref PubMed Scopus (64) Google Scholar, 24Gralnick H.R. Williams S. McKeown L. Hansmann K. Vail M. Krutzsch H. Blood. 1992; 80: 265aGoogle Scholar), and Bernard-Soulier platelets have normal numbers of this receptor (24Gralnick H.R. Williams S. McKeown L. Hansmann K. Vail M. Krutzsch H. Blood. 1992; 80: 265aGoogle Scholar). However, monoclonal antibodies that bind to the hirudin-like domain of the G-protein-linked thrombin receptor abrogate the responses of platelets to ≤1.0 nMα-thrombin (25Hung D.T. Vu T.K.H. Wheaton V.I. Ishii K. Coughlin S.R. J. Clin. Invest. 1992; 89: 1350-1353Crossref PubMed Scopus (165) Google Scholar, 26Hung D.T. Vu T.K.H. Wheaton V.I. Charo I.F. Nelken N.A. Esmon N. Esmon C.T. Coughlin S.R. J. Clin. Invest. 1992; 89: 444-450Crossref PubMed Scopus (38) Google Scholar, 27Brass 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, 28Liu L. Freedman J. Hornstein A. Fenton II, J.W. Ofosu F.A. Br. J. Haematol. 1994; 88: 592-600Crossref PubMed Scopus (21) Google Scholar). This level of α-thrombin would be expected to bind preferentially to its high-affinity binding sites on platelets. It is possible that GPIb, by initiating α-thrombin binding to platelets, could localize α-thrombin to sites on platelets where the cleavage of the G-protein-linked α-thrombin receptor would be facilitated to cause platelet activation (19Mazurov A.V. Vinogradov D.V. Vlasik T.N. Thromb. Res. 1991; 62: 673-684Abstract Full Text PDF PubMed Scopus (48) Google Scholar, 23Gralnick H.R. Williams S. McKeown L.P. Hansmann K. Fenton J.W. II Krutzsch H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6334-6338Crossref PubMed Scopus (64) Google Scholar).This study examined whether the cleavage of the G-protein-linked thrombin receptor necessarily occurs when platelets are activated with 0.5, 1.0, and 10 nMα-thrombin. Affinity-purified polyclonal antibodies against the 41-mer activation peptide (TR-(1-41)) released from the G-protein-linked thrombin receptor by α-thrombin were used to detect cleavage of this receptor and release of the 41-mer activation peptide from platelets incubated with α-thrombin. Binding of α-thrombin to and activation of the same platelets were assessed by flow cytometry (28Liu L. Freedman J. Hornstein A. Fenton II, J.W. Ofosu F.A. Br. J. Haematol. 1994; 88: 592-600Crossref PubMed Scopus (21) Google Scholar). Whether α-thrombin binding to GPIb is a prerequisite for platelet activation by α-thrombin was also explored by cleaving GPIb from platelets with three proteases known to cleave this platelet glycoprotein (13Cooper H.A. Bette W.P. White II, G.C. Wagner R.H. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 1433-1438Crossref PubMed Scopus (27) Google Scholar–16McGowan E.B. Detwiler T.C. J. Biol. Chem. 1986; 261: 739-746Abstract Full Text PDF PubMed Google Scholar) and by using a panel of monoclonal anti-GPIb antibodies previously reported to inhibit α-thrombin binding to platelets (11De Marco L. Mazzucato M. Masotti A. Fenton II, J.W. Ruggeri Z.M. J. Biol. Chem. 1991; 266: 23776-23783Abstract Full Text PDF PubMed Google Scholar, 17Brass L.F. Thromb. Haemostasis. 1995; 74: 499-505Crossref PubMed Scopus (26) Google Scholar, 18Katagiri Y. Hayashi Y. Yamamoto K. Tanoue K. Kosaki G. Yamazaki H. Thromb. Haemostasis. 1990; 63: 122-126Crossref PubMed Scopus (78) Google Scholar)." @default.
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• W2035721901 title "Binding of Thrombin to the G-protein-linked Receptor, and Not to Glycoprotein Ib, Precedes Thrombin-mediated Platelet Activation" @default.
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