Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.5 (thrombin)
33,306 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The lectin-like domain of P-selectin, an adhesive receptor (also known as PADGEM, GMP-140 or CD62) is implicated in platelet or endothelial cell interactions with leukocytes. The aim of this study was to characterize the lectin-like domain of rat P-selectin by the use of synthetic peptides. The lectin and EGF-like domains of rat P-selectin were cloned in our laboratory and shown to present very strong homologies to its human counterpart. Peptides corresponding with the lectin-like domain of P-selectin were tested for their ability to inhibit thrombin-activated platelets rosetting to neutrophils. Peptides 23-30 (A) and 76-90 (C), but not peptide 51-61 (B), inhibited thrombin activated rat platelets interactions with rat neutrophils (A = 33%, C = 46%, P < 0.05). Using a combination of peptides (A+B = 35%, P = 0.008 and A+C = 62%, P < 0.001), we observe different degrees of inhibition of platelets binding to neutrophils. The IC50 of peptides A+C was 0.11 mM. LYP-20, an anti-human P-selectin monoclonal antibody, was also observed to inhibit thrombin-activated rat platelets binding to rat neutrophils in a very significant manner (57% of inhibition, P < 0.001). Moreover, heparin inhibited thrombin-stimulated platelet/neutrophils rosetting (36% of inhibition, P < 0.01). These results show the importance of two sites (23-30 and 76-90) on the lectin-like domain of P-selectin in mediating platelet-neutrophil interactions in rats. Such peptides may be potent in vivo inhibitors of cell-cell interactions involving P-selectin.
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PMID:Two sites (23-30, 76-90) on rat P-selectin mediate thrombin activated platelet-neutrophil interactions. 753 Jan 56

1. P-selectin (PADGEM, GMP140, or CD62) a member of lectin-like adhesive proteins is expressed on the surface of activated degranulated canine platelets and is the calcium-dependent receptor for leukocyte adhesion. 2. The electrophoretic mobility of P-selectin, by Western blot analysis and immunoprecipitation from radiolabeled membranes of canine and human platelets, was similar or identical and immunocytochemical studies localized P-selectin in internal vesicles similar to the alpha granule localization in human platelets. 3. Two antibodies to human P-selectin KC4.1 and AC1.2 crossreacted with canine platelets whose surface binding, in response to agonists thrombin, calcium ionophore (A23187), phorbol esters and ADP, was similar. 4. Anti-P-selectin antibodies in conjunction with crossreacting anti-GPIIb/IIIa antibodies (A2A9, 7E3, RUU-PL7F12) enables the analysis of activated platelets, platelet-derived microparticles and platelet-leukocyte interactions in canine models by flow cytometry.
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PMID:Characterization of canine platelet P-selectin (CD 62) and its utility in flow cytometry platelet studies. 768 39

Treatment of human platelets with the lectin Concanavalin A (Con A) resulted in the tyrosine phosphorylation of several proteins with molecular masses 65, 80, 85, 95, 120, 135, and 150 kDa. These proteins were divided in two groups: the first group included the 65-, 85-, 95-, and 120-kDa bands, which were tyrosine phosphorylated also in thrombin-stimulated platelets; the second group (80-, 135-, and 150-kDa bands) included proteins whose tyrosine phosphorylation was exclusively promoted by Con A, but not by thrombin. Members of the second group were rapidly dephosphorylated when the lectin was displaced from the cell surface by methyl alpha-D-mannopyranoside. Pretreatment of intact platelets with the prostacyclin analog iloprost, inhibited Con A-induced tyrosine phosphorylation of the first group of proteins, but had no effect on the tyrosine phosphorylation of the proteins of the second group. Succinyl-Con A, a dimeric derivative of the lectin, which binds to the platelet surface but does not promote clustering of the receptor, did not induce tyrosine phosphorylation of the second group of proteins, although phosphorylation of some members of the first group was observed. Our results demonstrate the presence of two different mechanisms leading to protein-tyrosine phosphorylation in Con A-stimulated platelets, and identify a new signal transduction pathway, promoted by the clustering of membrane glycoproteins, that produces tyrosine phosphorylation of specific substrates. This new pathway may be activated by platelet interaction with multivalent ligands, such as adhesive proteins, during adhesion, spreading, and aggregation.
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PMID:Dual mechanism of protein-tyrosine phosphorylation in concanavalin A-stimulated platelets. 772 57

A new glycoprotein Ib (GPIb) antagonist, agkicetin, was purified from the venom of Agkistrodon acutus and characterized. It is a disulfide-linked heterodimer consisting subunits of 15 and 14 kDa. The subunits are homologous to each other and to other snake venom proteins of the C-type (Ca(2+)-dependent) lectin superfamily. Agkicetin behaved as a potent antagonist of von Willebrand Factor (vWF)-induced platelet agglutination (IC50 = 12.5 nM) and bound specifically to GPIb of fixed platelets with high affinity (Kd = 38 nM). It did not bind coagulation factor IX and thrombin. Monoclonal antibody against epitope on the N-terminal domain of GPIb competed the binding of agkicetin with platelets. Reduced and alkylated agkicetin lost most of its inhibitory efficacy toward vWF-induced platelet agglutination.
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PMID:Functional and sequence characterization of agkicetin, a new glycoprotein Ib antagonist isolated from Agkistrodon acutus venom. offf2p4. 775 23

Galactose-specific lectin from Viscum album (VAA) was found to induce aggregation of human platelets in a dose- and sugar-dependent manner. Small nonaggregating concentrations of VAA primed the response of platelets to known aggregants (ADP, arachidonic acid, thrombin, ristocetin, and A23187). VAA-induced platelet aggregation was completely reversible by addition of the sugar inhibitor lactose and the platelets from disrupted aggregates maintained the response to other aggregants. The lectin-induced aggregation of washed platelets was more resistant to metabolic inhibitors than thrombin- or arachidonic acid-dependent cell interaction. In contrast to the related galactose-specific lectin from Ricinus communis and the soy bean agglutinin, the lectin did not aggregate liposomes prepared from total platelet lipids, indicating different affinities of aggregation-mediating lectins to platelet glycolipids.
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PMID:Galactose-specific lectin from Viscum album as a mediator of aggregation and priming of human platelets. 776 7

The synthesis of phosphatidylinositol 3',4'-bisphosphate (PtdIns(3,4)P2) in 32P-labeled human platelets induced by the tetrameric lectin concanavalin A and the physiological agonist thrombin were compared. Like thrombin, concanavalin A stimulated a time-dependent accumulation of PtdIns(3,4)P2, which reached maximal levels after 5 min of stimulation. However, while synthesis of PtdIns(3,4)P2 induced by thrombin was dependent on platelet aggregation, the production of PtdIns(3,4)P2 induced by concanavalin A was unchanged when aggregation was prevented by the omission of stirring or when fibrinogen binding to platelets was inhibited by the tetrapeptide RGDS. Accumulation of PtdIns(3,4)P2 was not observed in platelets stimulated with succinyl-concanavalin A, a dimeric derivative of the lectin that binds to the same receptors on the platelet surface but does not promote clustering of membrane glycoproteins. The synthesis of PtdIns(3,4)P2 induced by concanavalin A was also independent of the membrane glycoprotein IIb-IIIa, as normal accumulation of this lipid was observed in platelets from two patients affected by Glanzmann thrombasthenia. In contrast, thrombin showed a strongly reduced ability to stimulate PtdIns(3,4)P2 production in thrombasthenic platelets. Although concanavalin A was able to induce association of the regulatory subunit of the phosphatidylinositol 3-kinase with tyrosine-phosphorylated proteins, the tyrosine kinase inhibitor tyrphostin AG-213 did not inhibit the lectin-induced synthesis of PtdIns(3,4)P2. These results demonstrate the existence of a novel mechanism of PtdIns(3,4)P2 synthesis in human platelets, which is independent of glycoprotein IIb-IIIa and aggregation, but requires clustering of membrane glycoproteins. As clustering events occur during platelet aggregation promoted by physiological agonists, this new mechanism may also be involved in the aggregation-dependent production of PtdIns(3,4)P2 in thrombin-stimulated platelets.
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PMID:Evidence for a glycoprotein IIb-IIIa- and aggregation-independent mechanism of phosphatidylinositol 3',4'-bisphosphate synthesis in human platelets. 776 14

Stimulation of human platelets with concanavalin A resulted in a significant increase in the concentration of cytoplasmic free Ca2+. This effect was due to two different processes: Ca2+ mobilization from internal stores and Ca2+ influx from the extracellular medium. Kinetic analysis revealed that the release of Ca2+ from internal storage sites occurred sooner than the opening of plasma membrane Ca2+ channels. The ability of concanavalin A to induce a sustained increase in cytoplasmic Ca2+ concentration was antagonized and reversed by methyl alpha-D-mannopyranoside, demonstrating that it was promoted by the interaction of the lectin with cell surface glycoproteins. Succinyl-concanavalin A, a dimeric derivative of the lectin, that does not promote patching/capping of the receptor, was able to bind to the platelet surface, and antagonized the effects of native concanavalin A. In addition, succinyl-concanavalin A, per se, was unable to induce Ca2+ mobilization in human platelets. Therefore, the action of the native concanavalin A was mediated by receptor clustering events. Concanavalin A mobilized Ca2+ from the same internal stores from which Ca2+ was mobilized in response to strong platelet agonists, such as thrombin and arachidonic acid. However, while thrombin was ineffective in inducing Ca2+ release after stimulation of platelets with ConA, ConA was able to cause a full discharge of Ca2+ from internal stores even in platelets previously stimulated with thrombin. These results demonstrate for the first time that the clustering of specific membrane glycoproteins can trigger platelet activation. The physiological implications during platelet aggregation are discussed.
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PMID:Intracellular calcium mobilization is triggered by clustering of membrane glycoproteins in concanavalin A-stimulated platelets. 827 48

The lectin wheat germ agglutinin (WGA) elicited a prompt and sharp increase in intracellular Ca2+ concentration in human platelets. The WGA-induced Ca2+ mobilization was markedly inhibited by a protein kinase inhibitor staurosporine, whereas Ca2+ mobilization by receptor-mediated agonists, including thrombin, platelet-activating factor, and arginine-vasopressin, was not. In contrast, the lectin-induced Ca2+ mobilization was resistant to cyclic AMP inhibition, compared with that induced by receptor-mediated agonists. These findings indicate that the mechanism of intracellular Ca2+ mobilization, or possibly phospholipase C activation, induced by WGA is different from that induced by receptor-mediated agonists in human platelets.
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PMID:Wheat germ agglutinin-induced intracellular calcium mobilization in human platelets: suppression by staurosporine and resistance to cyclic AMP inhibition. 838 40

A new coagulation factor IX/factor X-binding protein (IX/X-bp) from Echis carinatus leucogaster venom has been purified and designated ECLV IX/X-bp. ECLV IX/X-bp binds factor IX and X in a Ca(2+)-dependent manner and is devoid of thrombin-inhibitory and platelet-aggregating activities. The apparent dissociation constants (Kd) for binding of ECLV IX/X-bp to factor IX and factor X are 6.6 and 125 nM, respectively. Upon the addition of Mg2+, the required Ca2+ concentration for optimal binding of ECLV IX/X-bp to factor IX and factor X was prominently reduced. Mg2+ also increases the affinity of factor X for the venom protein. Direct binding of IX/X-bp to factor IX and X could also be detected by far-Western blotting, and results of the experiment ruled out the lectin-like mechanism of ECLV IX/X-bp. The complete amino acid sequence and the disulfide pattern of ECLV IX/X-bp was deduced by enzymatic hydrolysis and automated sequencing of the S-pyridylethylated protein. The venom protein is a heterodimer with one subunit of 131 amino acid residues and another of 125 residues. Both subunits are homologous to each other and to other snake venom proteins of the C-type lectin superfamily.
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PMID:Functional and sequence characterization of coagulation factor IX/factor X-binding protein from the venom of Echis carinatus leucogaster. 861 13

Stimulation of platelets by the extracellular matrix protein collagen leads to activation of a tyrosine kinase-dependent mechanism resulting in secretion and aggregation. Tyrosine phosphorylation of the tyrosine kinase Syk and phospholipase Cgamma2 are early events in collagen-induced activation. We recently proposed that collagen-signaling in platelets involves a receptor or a receptor-associated protein containing an immunoreceptor tyrosine-based activation motif (ITAM) enabling interaction with Syk. In this report we show that collagen stimulation of platelets causes rapid tyrosine phosphorylation of the ITAM containing Fc receptor gamma-chain and that this is precipitated by the tandem Src homology 2 (SH2) domains of Syk expressed as a fusion protein. In addition we demonstrate an association between the Fc receptor gamma-chain with endogenous Syk in collagen-stimulated platelets. The Fc receptor gamma-chain undergoes tyrosine phosphorylation in platelets stimulated by a collagen-related peptide which does not bind the integrin alpha2beta1 and by the lectin wheat germ agglutinin. In contrast, cross-linking of the platelet low affinity receptor for immune complexes, FcgammaRIIA, or stimulation by thrombin does not induce phosphorylation of the Fc receptor gamma-chain. The present results provide a molecular basis for collagen activation of platelets which is independent of the integrin alpha2beta1 and involves phosphorylation of the Fc receptor gamma-chain, its association with Syk and subsequent phosphorylation of phospholipase Cgamma2. Collagen is the first example of a nonimmune receptor stimulus to signal through a pathway closely related to signaling by immune receptors.
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PMID:Tyrosine phosphorylation of the Fc receptor gamma-chain in collagen-stimulated platelets. 866 60


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