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 authors used an immunogold labeling procedure to investigate the redistribution of platelet receptors and their ligands on the surface of contact-activated adherent platelets before and after thrombin stimulation. During the initial stage of platelet adhesion, a typical segregation of receptors occurred. Gold particles identifying glycoprotein (GP) Ib (CD42b) and GPIIb-IIIa (CD41a) remained distributed over the entire platelet surface, whereas gold particles identifying GPIa-IIa (CDw 49b) and GPIV (CD36) were found essentially overlying the granulomere; p24 (CD9) was present at the peripheral platelet rim and over the cell body. An increased labeling of GPIIb-IIIa, GPIV and p24 was also observed on pseudopods, with GPIIb-IIIa and GPIV concentrated at the enlarged extremities and at sites of contact between two platelets, whereas GPIb was absent from pseudopods. After thrombin stimulation of adherent platelets, GPIb underwent a relocation to the cell center, in contrast to GPIIb-IIIa which still remained randomly distributed over the cell body. To investigate whether ligand distribution paralleled this receptor segregation, platelet released von Willebrand factor (vWF), fibrinogen (Fg) and thrombospondin (TSP) were visualized. During the early stages of platelet activation, surface labeling for all three adhesive proteins was minimal and almost undetectable. Occasionally, intragranular Fg and vWF was accessible to gold-coupled antibodies, with vWF exhibiting the typical eccentric alpha-granular localization. At later stages of activation and especially after thrombin stimulation, no surface labeling for vWF was observed, whereas immunogold particles identifying vWF were still present inside enlarged clear vacuoles. In contrast, labeling of Fg and TSP was increased over the granulomere and extended to the cell periphery and the pseudopods, but was absent from the hyalomere, despite the presence of GPIIb-IIIa molecules. Double labeling experiments showed colocalization of Fg and TSP, GPIV and TSP, as well as Fg and GPIIb-IIIa, although no typical coclustering of GPIIb-IIIa and GPIV or GPIIb-IIIa and p24 was apparent. Our results further suggest that 1) on surface activated adherent platelets, not all GPIIb-IIIa molecules become competent to bind Fg, 2) GPIa-IIa is not anchored to the platelet membrane skeleton, and 3) during the early stage of platelet activation, a communication exists between the alpha granules and the platelet surface.
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PMID:Dynamic redistribution of major platelet surface receptors after contact-induced platelet activation and spreading. An immunoelectron microscopy study. 130 61

Recently, we described a platelet antibody against a putative collagen receptor (P62), which was found in a patient with idiopathic thrombocytopenic purpura (ITP) (Blood 69:1712). We now report a deficiency of the P62 receptor in a young man whose platelets showed defective collagen-induced platelet aggregation. He had a mild bleeding tendency and slight thrombocytopenia. The results of coagulation and fibrinolysis studies were normal. The patient's platelets were partially unresponsive to collagen, although aggregation in response to ADP, thrombin, ristocetin, and calcium ionophore (A23187) was almost normal. Adhesion of his platelets to bovine collagen was markedly reduced. Addition of collagen caused no synthesis of thromboxane (TX)B2 in platelet rich plasma (PRP) from this patient. Furthermore, collagen produced no rise of cytosolic free calcium ([Ca2+]i) in fura2-loaded platelets. In contrast, thrombin caused TXB2 formation and an increase of [Ca2+]i in his platelets. These results suggest defective interaction between the platelets and collagen. The IgG from the ITP-patient induced irreversible aggregation in normal PRP, but caused no aggregation of the young man's platelets. Immunoblot studies showed that normal platelets had antigens with a molecular weight of 62 KDa under reducing conditions and of 57 KDa under nonreducing conditions. In contrast, the young man's platelets had no P62 band, although GPIa/IIa and thrombospondin were normally present. These results indicate that impaired collagen-induced aggregation in the patient's platelets was due to a deficiency of P62 and confirm that P62 may play a crucial role as a collagen receptor in platelet activation.
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PMID:Deficiency of P62, a putative collagen receptor, in platelets from a patient with defective collagen-induced platelet aggregation. 131 Nov 44

This review highlights the increasing knowledge of the biochemistry, pathology, and cell and molecular biology of platelet receptors. A receptor for ADP has been identified using the affinity label FSBA as aggregin, a 100-kDa membrane protein responsible for shape change, aggregation, and exposure of fibrinogen binding sites. A variety of putative receptors for collagen have been described, with GPIa/IIa and GPIV receiving the most attention recently. A thromboxane A2 receptor has been identified using receptor antagonists and photoaffinity labels. The alpha 2-adrenergic receptor has been cloned and expressed. The platelet thrombin receptor has been tentatively identified as GPIb. Following binding of thrombin to this receptor, activation of calpain occurs, with cleavage of aggregin leading to exposure of GPIIb/III alpha and platelet aggregation. Isolation, expression, or both of the ADP, collagen, and thrombin receptors as single gene products of the human platelet responsible for activation, and more complete understanding of stimulus-response coupling, should allow for greater specificity of drugs with selective therapeutic actions.
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PMID:Receptors that activate platelets. 164 41

Very late activation antigens (VLAs) are glycoproteins (GPs) that play a major role in platelet adhesion to extracellular matrix. These GPs, members of the integrin family, are heterodimer complexes with different alpha subunits noncovalently associated with a common beta 1 subunit known as GPIIa. GPIa-IIa (also known as VLA2), GPIc-IIa (VLA5), and GPIc*-IIa (VLA6) are involved, respectively, in platelet adhesion to collagen, fibronectin, and laminin. At this stage, very little is known about the role of GPIIa in platelet adhesive functions. In this study, we have generated a monoclonal antibody (MoAb) (LYP22) directed against GPIIa. Immunoaffinity chromatography using LYP22 combined with two-dimensional nonreduced-reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that the antibody brings down all VLA subunits. Western blots indicate that the binding site of LYP22 on GPIIa is disulfide bridge-dependent. The number of LYP22 binding sites is not increased on stimulation with thrombin and is in the range of what is observed with another anti-GPIIa MoAb (A-1A5). LYP22 is the first anti-GPIIa MoAb to inhibit aggregation and secretion of washed platelets stimulated with collagen, thrombin, or arachidonic acid. Moreover, the lag-phase usually observed on collagen stimulation is significantly prolonged (by 60 seconds) in the presence of LYP22. This lag-phase, mediated by LYP22, is also observed in the presence of plasma proteins and is coupled with a reduced effect on collagen-induced platelet aggregation. In addition, LYP22 affects the adhesion of resting platelets to type III collagen, but not to fibronectin, laminin, or type I collagen. These results strongly indicate that the site on GPIIa, bearing the LYP22 epitope, is an active participant in signal transduction controlling platelet functions.
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PMID:Role of glycoprotein IIa (beta 1 subunit of very late activation antigens) in platelet functions. 171 78

We have investigated the impaired secretion response of neonatal platelets. We compared the response of washed neonatal and adult platelets to thrombin and collagen, and to specific activators of calcium flux (inositol trisphosphate) and protein kinase C activation (oleoyl-acetyl glycerol). Neonatal platelets show no impairment of aggregation, secretion of [14C]serotonin or phosphorylation of specific intracellular proteins in response to thrombin, inositol trisphosphate, or oleoyl-acetyl glycerol. However, neonatal platelets have a markedly decreased response to collagen. To further evaluate this deficient response, we examined specific aspects of the collagen activation pathway. Collagen-platelet interaction as measured by adhesion of platelets to collagen-coated dishes showed no difference in adhesion of neonatal platelets compared to adult controls (20.1 +/- 11.6 versus 18.6 +/- 9.3%). The presence of GPIa/IIa, a Mg2(+)-dependent collagen receptor, was evaluated by flow cytometric analysis of binding of fluorescein-tagged monoclonal antibody, 6F1 (directed against GPIa/IIa). There was no difference either in the percent of platelets that bound antibody (80 versus 81%) or in the mean fluorescence intensity of the adult and neonatal samples. Phosphoinositide hydrolysis was decreased in neonatal platelets in response to collagen but normal in response to thrombin. Neonatal platelets released more arachidonic acid than adult platelets in response to thrombin (29.5 +/- 3.2 versus 19.6 +/- 1.8%) but less than adult platelets in response to 10 micrograms/mL collagen (3.2 +/- 1.1 versus 9.3 +/- 3.0%).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Deficient collagen-induced activation in the newborn platelet. 211 40

Platelets are heterogeneous in the content of membrane glycoprotein (GP)IIb/IIIa complex. To determine whether this heterogeneity is related to changes associated with platelet aging in the circulation, newly released platelets, obtained during recovery from nonimmune-mediated acute experimental thrombocytopenia in baboons, were studied. Monoclonal antibody (MoAb) binding to epitopes expressed on GPIIb/IIIa complex (LJ-CP8), GMP-140 (S12), and GPIa/IIa (12F1) was measured on control platelets (comprising platelets with a normal age distribution; mean age 60 to 72 hours) and newly formed platelets (mean age 12 hours), both in the resting state and after thrombin stimulation. Whereas LJ-CP8 binding to resting control platelets increased by 34% upon stimulation by gamma-thrombin from 30,885 +/- 1,171 to 41,458 +/- 1,311 molecules/platelet at saturating concentrations of antibody, LJ-CP8 binding to resting young platelets did not increase significantly upon thrombin stimulation (31,878 +/- 3,330 and 33,791 +/- 3,486 molecules/platelet, respectively). Similarly, binding of antibody S12 in response to maximal thrombin stimulation was reduced by 42% from 10,246 +/- 834 molecules/platelet at saturating concentrations of S12 for control platelets to 5,971 +/- 665 molecules/platelet for young platelets (P = .001). S12 binding to unstimulated platelets was less than 10% of the binding observed after thrombin stimulation at all concentrations of S12 for both control and young platelets. However, maximal binding of antibody 12F1 to resting control platelets did not differ significantly from that observed with resting young platelets (2,926 +/- 167 and 2,857 +/- 208 molecules/platelet, respectively), and 12F1 binding was unchanged after thrombin stimulation for both control and young platelets. We conclude that the thrombin-induced increase in the expression of epitopes on platelet membrane GPIIb/IIIa complex and GMP-140 is a function of platelet age.
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PMID:Thrombin-induced increase in surface expression of epitopes on platelet membrane glycoprotein IIb/IIIa complex and GMP-140 is a function of platelet age. 247 8

The binding of collagen to platelet glycoprotein VI (GPVI) leads to the subsequent activation of phospholipase Cgamma2 through a pathway that is dependent on the Fc receptor gamma (FcR gamma) chain and the tyrosine kinase p72syk. We have investigated the role of platelet Src-family kinases in this signalling pathway. The selective Src-family kinase inhibitor PP1 prevented collagen-stimulated increases in whole-cell tyrosine phosphorylation and tyrosine phosphorylation of the FcR gamma chain and p72syk. A similar set of observations was made for a collagen-related peptide (CRP), which binds to GPVI but not to the integrin alpha2beta1 (GPIa/IIa). These effects were seen at a concentration of PP1 that inhibited platelet aggregation, dense granule release and Ca2+ mobilization induced by CRP, but not aggregation and Ca2+ mobilization mediated by the G-protein-coupled receptor agonist thrombin. After stimulation by CRP or collagen, the Src-family kinases p59fyn and p53/56lyn became associated with several tyrosine-phosphorylated proteins including the FcR gamma chain. This was not true of the other platelet Src-family kinases. The association between the FcR gamma chain and p59fyn was also seen under basal conditions, and was stable only in the weak detergent Brij96 but not in Nonidet P40, suggesting a non-SH2-dependent interaction. These results provide strong evidence for the involvement of p59fyn and p53/56lyn in signalling via GPVI, with p59fyn possibly acting upstream of FcR gamma chain phosphorylation.
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PMID:Evidence for the involvement of p59fyn and p53/56lyn in collagen receptor signalling in human platelets. 993 17

In the present study we have investigated whether the collagen receptor alpha2beta1 (GPIa-IIa; GP, glycoprotein) regulates protein tyrosine phosphorylation in platelets directly through activation of tyrosine kinases or indirectly through modification of the response to GPVI. The interaction of collagen with alpha2beta1 was inhibited in two distinct ways, using the metalloprotease jararhagin, which cleaves the beta1 subunit, or the antibody P1E6 which competes with binding of collagen to the integrin. The two inhibitors caused a shift to the right in the collagen concentration response curves for protein tyrosine phosphorylation and platelet activation consistent with a causal relationship between the two events. There was no change in the overall pattern of tyrosine phosphorylation in response to high concentrations of collagen in the presence of alpha2beta1 blockade demonstrating that the integrin is not required for this event. In contrast, jararhagin and P1E6 had a small, almost negligible inhibitory effect against responses to the GPVI-selective agonist collagen-related peptide (CRP) and the G protein-coupled receptor agonist thrombin. Crosslinking of alpha2beta1 in solution or by adhesion to a monolayer using a variety of antibodies to either subunit of the integrin did not induce detectable protein tyrosine phosphorylation in whole cell lysates. The snake venom toxin trimucytin-stimulated a similar pattern of tyrosine phosphorylation to that induced by crosslinking of GPVI which was maintained in the presence of jararhagin. Trimucytin may therefore induce activation via GPVI rather than alpha2beta1 as previously thought. These observations show that the integrin alpha2beta1 is not required for regulation of tyrosine phosphorylation by collagen.
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PMID:Evidence against a direct role of the integrin alpha2beta1 in collagen-induced tyrosine phosphorylation in human platelets. 1072 49

Wolfram syndrome (WS) is a recessively inherited disorder associated with recognised clinical features. Bleeding tendency was noticed in some of our patients, although this has not been reported before. We therefore studied this problem in all our WS patients and tried to postulate a possible pathogenesis. At the same time, a genetic linkage study provided evidence of locus heterogeneity of this syndrome and showed that the majority of our patients belong to the second WS locus identified in that study. Our study group consisted of 13 WS patients, belonging to WSF2 locus (group I). Controls consisted of 4 healthy siblings of WS patients (group II) and 7 diabetics who do not have WS (group III). Relevant clinical data were obtained, and a coagulation screen was carried out for all groups. All individuals in the three study groups have normal platelet count, thrombin time (TT), prothrombin time (PT), activated partial thromboplastin time (aPTT), clot retraction, Factor VIII activity (FVIIIc) and von Willebrand factor antigen (vWAg). Eleven of the WS patients have prolonged template bleeding time (BT) compared with both control groups. Patients with WS have a longer BT (mean 9.6 min, 95% CL 8.61-10.53 min) than the siblings group (mean 6.75 min, 95% CL 5.52-7.98 min) and the diabetic group (mean 5.49 min, 95% CL 4.56-6.42 min). The differences between the study group and controls are statistically significant, p = 0.02 and 0.0002, respectively. In the three groups, platelet aggregation studies were normal using adenosine diphosphate (ADP), ristocetin and epinephrine. Aggregation with collagen was either absent or impaired, with failure of secondary wave being noticed in 11 of the WS patients (85%) and normal in the control groups. The pathogenesis of this problem is not known, but could be due to an inhibitory effect of vWAgII, deficiency of thrombospondin or a defect in the platelet membrane GPIa/IIa. Bleeding diathesis is a new additional feature to the clinical spectrum of WS, which is probably a feature of the disorder WFS2 and not WFS1, as bleeding has never been reported in the latter. This provides further evidence for the phenotypic and genotypic heterogeneity of this complex disorder and may provide clues to the search for the second gene responsible for this phenotype.
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PMID:Bleeding tendency in Wolfram syndrome: a newly identified feature with phenotype genotype correlation. 1131 48

Thrombocytopenia (TP) often accompanies chronic liver diseases. The causes are numerous and include impaired production of blood platelets, spleen sequestration, and the immune factors, e.g. antiplatelet autoantibodies. ELISA (GTI-PAKPLUS) examinations were conducted in order to estimate the rate of autoimmune thrombocytopenia occurrence in patients with thrombocytopenia in the course of chronic hepatitis (10 patients) and liver cirrhosis (20 patients). Blood platelet activity was also evaluated as well as the expression of platelet glycoproteins (GPIIb, GPIIIa, and GPIX) in platelets of the patients and the controls. It was observed that autoimmune TP occurred in 30% of patients with liver cirrhosis and in 10% of patients with chronic hepatitis, in which anti-GPIIb/IIIa, GPIa/IIa, and HLA class I antibodies were detected. In all patients there occurred significant/marked platelet activation with CD61P expression. Thrombocytopenia in patients showed a similar activity after thrombin stimulation to that in healthy individuals. Expression on GPIIb platelet receptors was markedly increased and GPIX decreased in patients in comparison to the controls. There was no correlation between the occurrence of certain types of anti-platelet autoantibodies and the expression of GP on thrombocytes in these patients.
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PMID:[Autoimmune thrombocytopenia in chronic liver disease]. 1189 44


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