Identification of the thrombin receptor on human platelets by chemical crosslinking.

Takamatsu, Junki; Horne, McDonald K.; Gralnick, Harvey R.

doi:10.1172/jci112313

Cited by 71 publications

(19 citation statements)

References 28 publications

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“…The amount of a-thrombin bound to platelets corresponded to 18 2 4.5 % of the '251-a-thrombin added. As previously reported by our group [24] and others [31] and illustrated in Fig. 6B, bound '251-a-thrombin was either non-covalently associated to platelets, as indicated by the 36.5-kDa band, or associated with several covalent complexes.…”

Section: Resultssupporting

confidence: 80%

Late‐fibrin(ogen) fragment E modulates human α‐thrombin specificity

Bouton

Jandrot‐Perrus

Bezeaud

et al. 1993

European Journal of Biochemistry

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Fibrinogen contains at least two independent sites having demonstrable affinity for a-thrombin. One of these two sites, located in the fibrin E domain, binds to structures within the anion-binding exosite of a-thrombin. Taking advantage of its solubility, we have used late-fibrin(ogen) fragment E in competition experiments to examine its effect on a-thrombin specificity. We show that fragment E modulates a-thrombin enzymic activity towards small synthetic substrates, suggesting that fibrinthrombin interaction might induce subtle changes in the conformation near the catalytic center of the enzyme. In addition, fragment E behaved as a competitive inhibitor of a-thrombin-catalyzed fibrinopeptide-A cleavage (K, = 5.2 2 1.3 pM), indicating that a-thrombin interaction with the fibrin moiety of fibrinogen makes a major contribution to the efficacy of fibrinogen hydrolysis. Fragment E inhibited a-thrombin-induced serotonin release by platelets (concentration required to obtain 50% inhibition, IC,, = 10 pM) and a-thrombin binding to GPIb. Fragment E competitively inhibited athrombin binding to thrombomodulin (K, = 18.3 20.8 pM) but did not inhibit protein-C activation in the absence of thrombomodulin. The data are consistent with the proposal that fibrin, platelet GPIb and thrombomodulin bind to overlapping, but probably non-identical sites, while protein C binds to an independent site on a-thrombin.The serine protease a-thrombin plays a critical role in hemostasis [l]. In addition to its proteolytic action on fibrinogen, a-thrombin also activates factor V, factor VII, factor VIII, factor XI, factor XI11 and, in the presence of thrombomodulin, protein C. Moreover, a-thrombin interacts with receptors or ligands on the surface of most of the cells to elicit various responses [2].The structures of a-thrombin required for binding to other macromolecules have received considerable attention. Thrombin specificity can be attributed not only to the catalytic triad and surrounding regions, but also to exosite(s) remote from the catalytic site [3, 41. Consistent information has been obtained by characterization of proteolytic derivatives of thrombin, which have indicated that a positively charged region located some distance from the catalytic site, named the anion-binding exosite, is involved in the recognition of fibrin(ogen), protein C, thrombomodulin and platelets [5 -81. Crystallographic studies of a-thrombin complexed with its specific inhibitor, hirudin, have confirmed the existence of multiple binding sites both close to and distant from the catalytic site, and have shown that the anion-binding exosite makes numerous interactions with the C-terminal tail of hirudin [9, lo]. Fibrin(ogen), protein C, thrombomodulin and platelet GPIb all appear to compete with the C-terminal do- Enzymes. Thrombin (EC 3.4.21 3. main of hirudin for binding to the anion binding exosite [8, 11 -151. However, the study of recombinant mutant thrombins with single amino-acid substitutions [16] has indicated that the binding sites for fibrin(ogen),...

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Section: Resultssupporting

confidence: 80%

Late‐fibrin(ogen) fragment E modulates human α‐thrombin specificity

Bouton

Jandrot‐Perrus

Bezeaud

et al. 1993

European Journal of Biochemistry

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“…Neomycin most probably alters the interaction of thrombin with the platelet surface. This interaction is complex and involves at least two glycoproteins, glycoprotein Ib and V. Glycoprotein Ib might serve as the binding site for thrombin [21]; glycoprotein V is proteolytically cleaved by thrombin [22,23]. Studies using cy-and y-thrombin provide evidence for two types of thrombin receptors and coupling mechanisms [24].…”

Section: Effects Of Neomycin On Washed Plateletsmentioning

confidence: 99%

Neomycin inhibits inositol phosphate formation in human platelets stimulated by thrombin but not other agonists

Siess

Lapetina

1986

FEBS Letters

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Neomycin (0.1-l mM) added to human platelet-rich plasma or washed platelets prelabeled with [3H]inositol inhibits aggregation, ATP secretion (ID,, 0.2 mM) and formation of [3H]inositol mono-, bis-and trisphosphate (ID,, 060.8 mM) in response to thrombin (0.25 U/ml). The production of inositol phosphates in response to other platelet agonists (vasopressin, platelet activating factor, prostaglandin endoperoxide analogs and collagen) is not inhibited by neomycin, even at a concentration of 2 mM. At this concentration neomycin reduces the secretion of ATP stimulated by these agents (by up to 50%). The results indicate that neomycin has multiple effects on platelets that are unrelated to a specific inhibition of inositol phospholipid degradation by phospholipase C. Low concentrations (0. l-l mM) of neomycin might selectively inhibit the interaction of thrombin with the platelet surface, and high concentrations (> 2 mM) might unspecifically reduce platelet secretion in response to various platelet agonists. Neomycin Platelet aggregation Platelet release reaction

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“…The platelet surface membrane contains an estimated 26,000-The glycocalicin fragment contains the GPIb receptor site for von Willebrand factor (7) and thrombin (4,5).…”

Section: Introductionmentioning

confidence: 99%

Platelet storage results in a redistribution of glycoprotein Ib molecules. Evidence for a large intraplatelet pool of glycoprotein Ib.

Michelson¹,

Adelman²,

Barnard³

et al. 1988

J. Clin. Invest.

110

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Platelet membrane glycoprotein (GP) Ib contains receptors for von Willebrand factor and thrombin. Its proteolytic fragment, glycocalicin, circulates in normal plasma. In this study, storage of platelet concentrates for 5 d resulted in a 221% increase in plasma glycocalicin (1.3 times the total amount of glycocalicin present on the surface of all platelets), an 8% overall increase in platelet surface GPIb, and the appearance of a surface GPIb-negative subpopulation of platelets. Total platelet GPIb content of fresh washed platelets, determined by gel electrophoresis and immunoassay of Triton X-100 lysates, averaged 159,740 molecules per platelet. There were 36,360 surface GPIb molecules per platelet, determined by immunoassay of the supernatant of fresh washed platelets whose surface GPIb had been completely plasmin-cleaved. In sumnpary, these studies provide evidence for (a) a redistribution of GPIb molecules with platelet storage, and (b) a large intraplatelet pool of GPIb (approximately threefold larger than the platelet surface pool of GPIb).

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Identification of the thrombin receptor on human platelets by chemical crosslinking.

Cited by 71 publications

References 28 publications

Late‐fibrin(ogen) fragment E modulates human α‐thrombin specificity

Late‐fibrin(ogen) fragment E modulates human α‐thrombin specificity

Neomycin inhibits inositol phosphate formation in human platelets stimulated by thrombin but not other agonists

Platelet storage results in a redistribution of glycoprotein Ib molecules. Evidence for a large intraplatelet pool of glycoprotein Ib.

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