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)

We have investigated mechanisms involved in integrin-mediated signal transduction in platelets by examining integrin-dependent phosphorylation and activation of a newly identified protein tyrosine kinase, pp125FAK (FAK, focal adhesion kinase). This kinase was previously shown to be localized in focal adhesions in fibroblasts, and to be phosphorylated on tyrosine in normal and Src-transformed fibroblasts. We show that thrombin and collagen activation of platelets causes an induction of tyrosine phosphorylation of pp125FAK and that pp125FAK molecules isolated from activated platelets display enhanced levels of phosphorylation in immune-complex kinase assays. pp125FAK was not phosphorylated on tyrosine after thrombin or collagen treatment of Glanzmann's thrombasthenic platelets deficient in the fibrinogen receptor GPIIb-IIIa, or of platelets pretreated with an inhibitory monoclonal antibody to GP IIb-IIIa. Fibrinogen binding to GP IIb-IIIa was not sufficient to induce pp125FAK phosphorylation because pp125FAK was not phosphorylated on tyrosine in thrombin-treated platelets that were not allowed to aggregate. These results indicate that tyrosine phosphorylation of pp125FAK is dependent on platelet aggregation mediated by fibrinogen binding to the integrin receptor GP IIb-IIIa. The induction of tyrosine phosphorylation of pp125FAK was inhibited in thrombin- and collagen-treated platelets preincubated with cytochalasin D, which prevents actin polymerization following activation. Under all of these conditions, there was a strong correlation between the induction of tyrosine phosphorylation of pp125FAK in vivo and stimulation of the phosphorylation of pp125FAK in vitro in immune-complex kinase assays. This study provides the first genetic evidence that tyrosine phosphorylation of pp125FAK is dependent on integrin-mediated events, and demonstrates that there is a strong correlation between tyrosine phosphorylation of pp125FAK in platelets, and the activation of pp125FAK-associated phosphorylating activity in vitro.
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PMID:Integrin-dependent phosphorylation and activation of the protein tyrosine kinase pp125FAK in platelets. 138 45

G619, a 4-OH-isophthalic acid derivative, was studied for its capacity to inhibit platelet aggregation. G619 dose-dependently inhibited U46619, collagen, ADP, PAF, thrombin and epinephrine-induced platelet aggregation in vitro. The IC50 values for inhibition of U46619-induced human and rabbit platelet aggregation were 39 and 43 microM, respectively. G619, at 100 microM, inhibited high concentration collagen (10 micrograms/ml)-induced aggregation of rabbit platelets pretreated with indomethacin and increased the level of cAMP in washed rabbit platelets by 30% (p less than 0.01 vs basal). However, G619, did not inhibit fibrinogen binding to GPIIb/IIIa receptor, phosphodiesterase, U46619-induced contractile responses on canine saphenous vein or rabbit aorta, calcium-induced vasoconstriction and thrombin or PAF-induced elevation of [Ca++]i in platelets in vitro. In vivo, the U46619-induced maximal thrombocytopenia in rats was reduced from 40% (vehicle) to 22% and 18% by 10 and 30 mg/kg of G619 i.v., respectively. G619 (30 mg/kg) had no effect on the U46619-induced vasopressor response or sudden death in rats, and had no effect on TxB2 formation. Our results indicate that G619 is a broad-spectrum platelet aggregation inhibitor and may have its effect on a common mechanism for platelet aggregation besides an effect on the thromboxane A2 receptor.
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PMID:Pharmacological profile of G619, a new platelet aggregation inhibitor. 141

Porcine von Willebrand factor (PvWF) induces platelet aggregation which is thought to be responsible for the thrombocytopenia that occurs in haemophilic patients treated with commercial preparations of porcine factor VIII. This study demonstrates that such aggregation can be completely inhibited by a monoclonal antibody against human platelet glycoprotein GPIb and partially inhibited by an antibody directed against platelet GPIIb/IIIa. The interaction of PvWF with GPIb is also demonstrated by the inhibitory effect of purified glycocalycin on aggregation. The binding site of PvWF to GPIb is very close to that of human vWF, since a recombinant peptide blocks the binding of both molecules to GPIb. When platelets are incubated with PvWF, the GPIIb/IIIa receptor is activated and binds fibrinogen. PvWF also binds to GPIIb/IIIa when platelets are stimulated with thrombin, suggesting that the molecule has the same RGD sequence as other adhesive proteins (human vWF, fibrinogen, fibronectin and vitronectin). These findings identify the dual mechanisms responsible for in vivo platelet aggregation induced by PvWF, i.e. binding to GPIb and activation of the GPIIb/IIIa receptor.
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PMID:Interaction of porcine von Willebrand factor with the platelet glycoproteins Ib and IIb/IIIa complex. 141 6

Glycoprotein (GP) IIb and IIIa are major constituents of the platelet membrane which are involved in forming the fibrinogen receptor on activated platelets. We used flow cytometry to study the effects of ethylene-diamine tetraacetic acid (EDTA) on the membrane GPIIb/IIIa complexes of platelets and microparticles, and to study the effects of cations on dissociated GP complexes. Microparticles were detected by both the volume signal and by fluorescence using an FITC-conjugated anti-GPIb antibody (NNKY5-5). When platelets were stimulated with ADP, calcium ionophore A23187, or thrombin, fibrinogen binding to the platelet surface increased markedly. However, fibrinogen binding to microparticles showed little increase in response to such agonists. Microparticle GPIIb/IIIa complexes were dissociated by incubation with EDTA at 37 degrees C but did not reassociate after treatment with divalent cations (Ca2+, Mg2+, and Mn2+) in contrast to platelet GPIIb/IIIa complexes. These results suggest that some interaction of GPIIb/IIIa and linked structures like the platelet cytoskeleton may be involved in the reassociation of dissociated GPIIb and GPIIIa, perhaps explaining the failure of reassociation of microparticle GPIIb/IIIa (i.e., the fibrinogen binding to microparticles).
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PMID:Differences between platelet and microparticle glycoprotein IIb/IIIa. 145 94

Fibrinolytic therapy is aimed at dissolving the fibrin clot. Its use now represents the optimal therapy for an acute evolving myocardial infarction. Although improved fibrinolytic agents are now being developed, the next frontier appears to be thrombolytic therapy which will combine fibrinolysis to platelet active drugs and specific inhibitors of blood coagulation, targetting the blood clot formation process itself. The most promising agents in this regard are inhibitors of the platelet glycoprotein IIb-IIIa receptors and specific thrombin inhibitors active on clot bound thrombin. This should result in more complete control of the thrombogenicity of the culprit coronary artery lesion. The comprehensive approach for the management of acute myocardial infarction should however ultimately be reperfusion therapy combining to thrombolytic therapy measures that will preserve the acutely ischemic myocardium and prevent the long-term deleterious effect of infarct expansion and left ventricular remodelling.
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PMID:[The future of fibrinolysis: from fibrinolytic treatment to reperfusion therapy]. 147 13

Although DDAVP has been shown to be haemostatically efficacious in patients with various congenital or acquired platelet disorders, no reasonable explanation has been found for this effect. We have previously shown DDAVP to increase platelet adhesiveness as measured with a platelet retention test. The aim of the present study was to investigate the mechanism of action responsible for the increased platelet retention in response to DDAVP. Patients with vWD type III and type Ia, severe haemophilia and severe thrombasthenia, as well as healthy controls, were included in the study. The effect of different concentrations of vWF in plasma and platelets was explored, as was the effect on platelet function of apyrase and monoclonal antibodies against GP IIb/IIIa and GP Ib. We found the effect of DDAVP on platelet retention to be unaffected by changes in the plasma concentration of vWF. The enhanced platelet retention after DDAVP is apparently dependent on the presence of platelet-vWF and on a normal function of the GP IIb/IIIa. The effect is not mediated via ADP or thrombin. The platelet-stimulating effect of DDAVP may be one explanation for the positive haemostatic effect in patients with certain platelet disorders.
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PMID:DDAVP-induced enhancement of platelet retention: its dependence on platelet-von Willebrand factor and the platelet receptor GP IIb/IIIa. 149 98

The localization of glycoprotein (GP) IIb/IIIa (integrin alpha IIb beta 3) in both resting and thrombin-activated platelets was studied immunocytochemically. By the preembedding method where only the GP IIb/IIIa molecules on the surface of platelets were immunostained, the distribution of protein A-colloidal gold label was randomly distributed along the surface membrane of resting platelets at a density of 18.0 +/- 2.7 gold particles/microns of membrane. At 15 s after stimulation by 0.1 U/ml of thrombin in an unstirred platelet suspension, the spheroid-shaped platelets with pseudopodia still had normal numbers of alpha-granules, and the density of gold particles was 19.7 +/- 3.6 particles/microns. At 5 min, the alpha-granules were no longer present because of the release reaction, and the density of gold particles significantly increased (27.0 +/- 3.7 particles/microns; p less than 0.01). In immuno-stained ultra-thin frozen sections, the gold particles were detected not only on the surface membrane, including the open canalicular system (OCS), but also on the alpha-granule membranes of resting platelets. At 30 s after thrombin stimulation the alpha-granules fused with the OCS, resulting in the formation of a swollen OCS, which still had gold particles on its membrane. At 5 min, the gold particles were detected on the membrane of the swollen OCS located near the surface membrane, while very few gold particles were present on the membrane of the OCS in the central part of the platelets. These results demonstrate that alpha-granule membrane GPIIb/IIIa translocates to the surface membrane through the membrane of the OCS.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Immunocytochemical evidence for the translocation of alpha-granule membrane glycoprotein IIb/IIIa (integrin alpha IIb beta 3) of human platelets to the surface membrane during the release reaction. 150 Feb 93

Alboaggregins (AL-A, AL-B, AL-C) isolated from Trimeresurus albolabris snake venom represent a new family of proteins which bind to platelet glycoprotein Ib (GPIb). These alboaggregins were purified to homogeneity with ion exchange HPLC (Mono-Q column) and hydrophobic HPLC (TSK Phenyl-5PW column). On SDS-polyacrylamide gel electrophoresis, apparent molecular weights of AL-A, AL-B and AL-C were 52 kDa, 26 kDa, and 121 kDa respectively, under nonreducing conditions. Upon reduction, each alboaggregin showed two types of chains with apparent molecular weights in the range of 15-20 kDa. All three alboaggregins agglutinated formalin-fixed platelets. Agglutination activities and binding of labeled alboaggregins to GPIb were specifically inhibited by the monoclonal antibody AK2 which is directed against the 45 kDa N-terminal region on GPIb, but not by monoclonal antibodies against other epitopes on GPIb. 125I-alboaggregin binding to platelets was not altered by the presence of thrombin. Alboaggregins did not bind to GPIIb/IIIa. Alboaggregins were competitive inhibitors for 125I-bovine vWF binding to platelets. Mutual competition studies between AL-A, AL-B and AL-C for the binding of labeled bovine vWF and AL-B to platelets demonstrated that AL-B and AL-C had a significantly higher affinity than AL-A.
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PMID:Characterization of three alboaggregins purified from Trimeresurus albolabris venom. 150 13

The presence of extracellular ionized calcium (Ca2+) is important to support the aggregation of human blood platelets and for the stability of the platelet fibrinogen receptor, the glycoprotein (GP) IIb-IIIa complex. This study was performed in order to determine the effects of intracellular calcium chelation on human platelet functions, on the expression of the fibrinogen receptor and on the stability of the GP IIb-IIIa complex. Intracellular Ca2+ of intact human platelets was extensively chelated by incorporation of high amounts (14-50 mmol/L) of the specific Ca2+ chelator quin2 after incubation of platelets with its lipophilic acetomethoxylester form (quin2-AM). We have investigated the effects of intracellular Ca2+ chelation with quin2 on platelet aggregation and fibrinogen binding induced by ADP and human alpha-thrombin and on the stability of the GP IIb-IIIa complex studied by crossed immunoelectrophoresis of Triton X-100 platelet lysates. The results were compared with control experiments performed with intact platelets treated with the impermeant Ca2+ chelator quin2 and with EDTA or EGTA. This study shows that high intracellular concentration of quin2 inhibits human platelet aggregation and fibrinogen binding but does not induce the dissociation of the GP IIb-IIIa complex. This study adds evidence for the role of external Ca2+ to maintain the integrity of a non-dissociated GP IIb-IIIa complex and suggests that intracellular Ca2+ is not involved in the stabilization of the GP IIb-IIIa complex.
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PMID:Differential effects of extra- and intracellular calcium chelation on human platelet function and glycoprotein IIb-IIIa complex stability. 152 93

The protein tyrosine phosphatase (PTPase) inhibitor pervanadate (vanadyl hydroperoxide) stimulated protein tyrosine phosphorylation 29-fold more than did thrombin in intact and saponin-permeabilized platelets. Increased tyrosine phosphorylation preceded, or was coincident with, a fall in PtdIns(4,5)P2 levels, production of PtdIns(3,4)P2 and phosphatidic acid, mobilization of intracellular Ca2+, stimulation of protein kinase C-dependent protein phosphorylation, secretion of dense and alpha-granules, increased actin polymerization, shape change and aggregation which required fibrinogen and was mediated by increased surface expression of GPIIb-IIIa. The tyrosine kinase inhibitor RG 50864 totally prevented induction of tyrosine phosphorylation by pervanadate, as well as all other responses measured; in contrast, the inactive structural analogue, tyrphostin #1, had no effect. Dense-granule secretion induced by pervanadate required protein kinase C activity; however, aggregation and alpha-granule secretion were independent of protein kinase C. In saponin-permeabilized platelets pervanadate and thrombin stimulated phospholipase C activity by GTP-independent and GTP-dependent mechanisms respectively. We conclude that PTPases are important regulators of signal transduction in platelets.
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PMID:Activation of signal transduction in platelets by the tyrosine phosphatase inhibitor pervanadate (vanadyl hydroperoxide). 153 May 76


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