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)

Polytransfused patients often develop platelet-reactive antibodies (PRAb). These give positive reactions in the platelet immunofluorescence test (PIFT) and may be either lymphocytotoxic (LCTAb) or platelet-specific antibodies (PSAb). The latter may be detected in the PIFT using chloroquine-treated platelets (Chl-PIFT) or by immunoblotting. Serial samples from 106 multiply transfused patients with bone marrow failure were screened by PIFT using a microplate method and flow-cytometric analysis. PSAb activity was confirmed by Chl-PIFT. In 45 (42%) of the patients studied PSAb were detected; 37 (35%) formed LCTAb and 19 (51%) had co-existent PSAb. Sera from 25 of 27 patients with a positive Chl-PIFT, retested by immunoblotting, recognised determinants of Mr 82-160 kD on whole platelets. A large group became sensitised to a component of Mr 105-115 kD reduced (99 kD non-reduced) with similar electrophoretic mobility to GPIIIa using a monoclonal anti-GPIIIa and two human polyclonal anti-HPA-1a sera; some also produced anti-GPIIb. The largest group recognised a determinant of Mr 80-83 kD, probably glycoprotein V (GPV). Three sera were immunoblotted against thrombin-treated platelets and the results confirmed GPV specificity.
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PMID:Antibodies to platelet glycoprotein V in polytransfused patients with haematological disease. 848 49

Six monoclonal IgG1-k antibodies (LK2, LK3r, LK4-55, LK5, LK6-55, LK7r) were raised against platelet membrane GPIIIa in order to study the structure-function relationship of this molecule. Antibodies were selected on their ability to react with GPIIIa by ELISA on adherent platelets, by immunoblot on platelet lysates and by fluorescence flow cytometry on intact platelets. Fluorescence reactivity varied from 3- to 202-fold greater than isotype control fluorescence. Two MoAbs reacted on immunoblot under reduced conditions (LK7r and LK3r). Two reacted with a 55 kD chymotrypsin/subtilisin digest of GPIIIa which is likely to exclude amino acids 121-348 (LK4-55 and LK6-55). Four of the MoAbs (LK5, LK3r, LK2 and LK4-55) inhibited tyrosine phosphorylation of one to four distinct bands on immunoblot. LK4-55 reacted with an N-terminal 66 amino acid fusion protein of GPIIIa near the PLA epitope (Leu 33). LK7r reacted with a 212-222 peptide reported to be an RGD fibrinogen binding site. LK2 reacted near a disintegrin-RGD binding site. Except for LK5, all inhibited ADP, collagen and thrombin-induced platelet aggregation in a heterogeneous fashion. Percentage inhibition of 125I-fibrinogen binding to platelets varied from 18% to 98%. No correlation was noted between inhibition of fibrinogen binding, location of MoAb binding on GPIIIa, reactivity of MoAb binding with GPIIIa, inhibition of thrombin-induced tyrosine phosphorylation or inhibition of platelet aggregation induced by ADP, collagen or thrombin. Thus MoAbs, binding to platelet GPIIIa at different sites, inhibit platelet aggregation in a heterogeneous manner.
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PMID:Heterogenous inhibition of platelet aggregation by monoclonal antibodies binding to multiple sites on GPIIIa. 854 51

During platelet activation, the glycoprotein (GP) complex IIb-IIIa (alpha 11b beta 3-integrin) changes its conformation, resulting in binding of adhesive proteins of RGD containing an amino acid sequence as well as in expression of new ligand-induced binding sites (LIBS) on the GPIIb-IIIa molecule. Like its F(ab)-fragments, the monoclonal antibody CRC54, whose epitope is located in the N-terminal part of the GPIIIa molecule, binds to no more than 10% of GPIIb-IIIa on the resting platelet surface. However, the binding of CRC54 increases considerably during activation of platelets by thrombin, platelet adhesion on plastic, GPIIb-IIIa interaction with RGDS-peptide as well as during dissociation of the complex in the presence of EDTA. These finding suggest that CRC54 is specifically directed against the LIBS epitope on the GPIIIa molecule. This epitope differs from those of other known conformation-dependent antibodies against GPIIb-IIIa (LIBS1, LIBS6, PMI-1, pl55 and p180), since those antibodies did not block the CRC54 binding to GPIIb-IIIa on the surface of adhering platelets. Unlike whole platelets, the binding of GPIIb-IIIa from lysates of platelets treated with Triton X-100 with immobilized CRC54 did not depend on the presence of the RGDS peptide. Under these conditions another anti-LIBS-antibody, p180 specifically directed against GPIIb, preserved its ability to discriminate the RGDS-occupied and resting conformations of GPIIb-IIIA. CRC54 and its F(ab) fragments induced platelet aggregation in both platelet-enriched plasma and in suspensions of washed platelets. CRC54 also stimulated the binding to platelets of GPIIb-IIIa ligand fibrinogen, labelled with 125I as well as adhesion of 51Cr-labelled platelets to immobilized ligands-fibrinogen and fibronectin. The CRC54-dependent aggregation was fully blocked by RGDS-peptide and antibody CRC64 inhibiting the GPIIb-IIIa binding to the ligands. However, the platelet activation inhibitor, prostaglandin EI, and the mixture of metabolic inhibitors, deoxyglucose-sodium azide, only party inhibited the CRC54-dependent aggregation. Incubation of platelets with CRC54 induced the binding to platelets of the anti-GPIIb LIBS antibody p180 and of the anti-GPIIb-IIIa activation-dependent antibody p155. The binding of GPIIb-IIIa from lysates of CRC54-treated platelets with immobilized p180 and p155 was also several times as high as that of GPIIb-IIIa from control platelet lysates. The data obtained indicate that the GPIIb-IIIa transition to the active state and its interaction with ligands induces conformational changes in the N-terminal part of GPIIIa and that the CRC54 binding to the N-terminal part of GPIIIa stimulates conformational changes in GPIIb-IIIa, complex interaction with ligands and platelet aggregation.
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PMID:[Conformational changes of the platelet membrane glycoprotein IIb-IIIa complex stimulated by a monoclonal antibody to the N-terminal segment of glycoprotein IIIa]. 872 99

In an in vitro study the effect of various thrombin inhibitors (argatroban, efegatran, DuP 714, recombinant hirudin and PEG-hirudin) on platelet activation in whole blood was investigated. Blood was drawn from normal human volunteers using the double syringe technique without use of a tourniquet to avoid autoaggregation of platelets. Blood was anticoagulated with either argatroban, efegatran, DuP 714, hirudin or PEG-hirudin at final concentrations of 10 micrograms/ml. Blood samples were then incubated at 37 degrees C either with saline, r-tissue factor, arachidonic acid, adenosine diphosphate or collagen. At definite times (1, 2.5, 5, 10 min) aliquots were taken and after various steps of fixative procedure the percentage of platelet activation was measured using fluorescent monoclonal antibodies to platelet surface receptors GPIIIa (CD-61) and P-selectin (CD-62). Flow cytometric analysis showed a platelet activation after all agonists used. All thrombin inhibitors studied caused a nearly complete inhibition of r-tissue factor-mediated platelet activation. In contrast, after activation with the other agonists an increased percent CD-62 expression was found with a maximum after 2.5 to 5 min. The results show that in whole blood thrombin inhibitors are effective in preventing platelet activation induced by r-tissue factor. The formation of active serine proteases including thrombin may be effectively inhibited by these agents. The observations further suggest that while thrombin inhibitors may control serine proteases, these agents do not inhibit the activation of platelets mediated by other agonists.
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PMID:Flow cytometric evaluation of the effect of various thrombin inhibitors on platelet activation in whole blood. 873 29

The present review has summarized the expression, production and effects of the human interleukins (IL) 1-11 and myelopoietic colony stimulating factors (CSF) in the established myeloid leukemia cell lines and in cells from patients with acute myeloid leukemia as well as the oncogene expression reported in these myeloid leukemia cell lines. The genetic dissection of leukemic myelopoiesis may provide new perspectives for the control of myeloid leukemias. Based on their expression of phenotypic markers (e.g., surface antigens, cytochemical staining, etc.), myeloid cell lines can be further subdivided into myelogenous, monocytic, erythroid and megakaryoblastic leukemia cell lines. Due to the close relationship of erythroid and megakaryoblastic progenitor cells and to the existence of a probably common precursor cell giving rise to these two different cell lineages, many megakaryoblastic cell lines express erythroid markers (e.g., expression of hemoglobin or glycophorin A) and conversely cell lines with a predominant erythroid profile might display megakaryoblastic features (e.g., platelets peroxidase or glycoproteins CD41, CD42b or CD61). The recent cloning of the specific cytokine: thrombopoietin (TPO) and its receptor generated a strong interest in these particular myeloid cell lines that are discussed in more detail in the present review. Both normal and leukemic megakaryocytopoiesis are stimulated by granulocyte-macrophage colony stimulating factor (GM-CSF), IL-3, GM-CSF/IL-3 fusion protein, IL-6, IL-11 and TPO but inhibited by IL-4, interferon-alpha (IFN-alpha) and IFN-gamma. Human megakaryoblastic leukemia cell lines have common biological features: high expression of the megakaryocytic specific antigen (CD41); high expression of early myeloid antigens (CD34, CD33 and CD13); constitutive expression of IL-6 and platelet-derived growth factor; a complex karyotype picture; expression of c-kit (the stem cell factor receptor); growth-dependency or -stimulation by IL-3 and/or GM-CSF; and in vivo tumorigenicity in mice associated with marked fibrosis. Whereas numerous chemical and biologic agents induce granulocytic and/or monocytic differentiation of myeloid leukemia cell lines, only a few agents including phorbol myristate acetate, vitamin D3, IFN-alpha, IL-6 and thrombin have been reported to induce megakaryocytic differentiation in the megakaryoblastic leukemia cells.
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PMID:Interleukins and colony stimulating factors in human myeloid leukemia cell lines. 875 Jun 18

The purpose of this study was to monitor platelet activation by following alpha IIb beta 3 integrin (GpIIb/IIIa complex or CD41/CD61) on the platelet surface by flow cytometry (FCM) analysis using workshop mAbs. The results obtained with the mAbs showed increased expression of the GpIIb/IIIa complex (about 40%) on the platelet membrane surface under thrombin stimulation.
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PMID:Platelet activation studies with anti-CD41/61 monoclonal antibodies. 889 25

Ligand-induced binding sites (LIBS) are neoantigenic regions of glycoprotein (GP)IIb-IIIa that are exposed upon interaction of the receptor with the ligand fibrinogen or the ligand recognition sequence (RGDS). LIBS have been suggested to contribute to postreceptor occupancy events such as full-scale platelet aggregation, adhesion to collagen, and clot retraction. This study examined the induction requirements of a GPIIIa LIBS with regard to ligand specificity. Through the use of the anti-LIBS D3, we report that this complex-activating antibody induces fibrinogen- and von Willebrand factor-binding to GPIIb-IIIa on intact platelets. Bound ligand was detected by flow cytometric analysis and platelet aggregation assays. These bound ligands increased the number of D3-binding sites and altered the affinity of D3 for GPIIb-IIIa on platelets. In contrast, activation of platelet GPIIb-IIIa by D3 did not increase the binding of another RGD-containing ligand, vitronectin. Furthermore, bound vitronectin on thrombin-stimulated platelets did not cause the expression of the D3 LIBS epitope. We conclude direct activation of GPIIb-IIIa in the absence of platelet activation results in selective ligand interaction and that D3 LIBS induction requires the binding of the multivalent ligands, fibrinogen or von Willebrand factor. Thus, the region of GPIIIa recognized by D3 may be an important regulatory domain in ligand-receptor interactions that directly mediate platelet aggregation.
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PMID:Selective induction of a glycoprotein IIIa ligand-induced binding site by fibrinogen and von Willebrand factor. 891 46

The effect of activated protein C (APC) on agonist-induced platelet activation and on thrombin generation after intrinsic (IA) and extrinsic (EA) activation of the coagulation system was studied by flow cytometry and by measuring levels of prothrombin fragment F1+2. In platelet activation studies blood drawn from healthy volunteers was anticoagulated with 10 micrograms/ml APC and incubated at 37 degrees C either with saline, recombinant tissue factor (r-TF), arachidonic acid (AA), ADP or collagen. At definite times aliquots were taken and processed for flow studies. Platelet activation was measured using fluorescent monoclonal antibodies to platelet surface receptors GPIIIa (CD-61) and P-selectin (CD-62). Flow cytometric analysis showed platelet activation after all agonists used. APC did not influence AA-, ADP- and collagen-induced platelet activation but completely inhibited activation of platelets induced by r-TF. The effect of APC on r-TF-mediated platelet activation was concentration-dependent in the range of 0.5 to 20 micrograms/ml showing an increase in CD-62 expression at lower concentrations. In citrated and APC-anticoagulated blood the generation of thrombin was studied after IA and EA. At 10 and 20 micrograms/ml APC effectively prevented blood clotting which rapidly occurred especially after EA. The amount of thrombin generated via the extrinsic pathway was reduced by APC whereas after IA F1+2 levels measured in the presence of APC were still strongly increased. These results indicate that small amounts of thrombin generated by r-TF are sufficient to activate platelets as well as blood coagulation. APC exerts strong concentration-dependent anticoagulant actions and effectively prevents activation of platelets.
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PMID:In vitro studies on the effect of activated protein C on platelet activation and thrombin generation. 925 10

Genetic defects of the blood platelet membrane glycoproteins, GPIIb-IIIa (alpha IIb/beta 3; CD41/CD61) and GPIb-V-IX (CD42) are the origin of several rare bleeding disorders, the best known of which are Glanzmann's thrombasthenia, Bernard-Soulier syndrome, and platelet-type von Willebrand's disease. In Glanzmann's thrombasthenia, GPIIb-IIIa are missing or defective and platelet aggregation is lacking or reduced. Either gene can be affected and mutations leading to lack of expression or to expression of poorly functional forms have been described. In Bernard-Soulier syndrome, GPIb-V-IX are missing or defective, leading to poor platelet adhesion at high-shear stress to damaged vessel wall and reduced platelet response to thrombin. Mutations in both GPIb alpha (CD42b) and GPIX (CD42a) have been described. Mutations in GPIb alpha can also lead to platelet-type von Willebrand's disease in which GPIb-V-IX are expressed normally but bind von Willebrand's factor spontaneously, which leads to platelet aggregation and thrombocytopenia.
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PMID:Molecular abnormalities in Glanzmann's thrombasthenia, Bernard-Soulier syndrome, and platelet-type von Willebrand's disease. 937 10

The HHLGGAKQAGDV (H12) sequence at the carboxyl termini of the y chains and the RGD sequences in the Aalpha chains of human fibrinogen are potential recognition sites for the binding of soluble fibrinogen to glycoprotein IIb-IIIa (GPIIb-IIIa) on activated human platelets. Thus, addition of either H12 or RGD-containing peptides inhibits aggregation of and fibrinogen binding to human platelets. In contrast, we reported previously that RGDS had relatively little inhibitory effect on these functions of rabbit platelets. In the present study, we found that H12 inhibited ADP- and thrombin-induced aggregation of rabbit platelets in a dose-dependent manner. Specificity was demonstrated by the failure of the variant HHLGGAKQAGEV peptide to inhibit ADP-induced aggregation. Furthermore, flow cytometric analyses demonstrated that H12 inhibited the binding of FITC-fibrinogen to ADP-activated rabbit platelets in a dose-dependent manner. To examine the direct interaction of H12 with rabbit GPIIb-IIIa, we performed affinity chromatography by applying an octylglucoside extract of rabbit platelet proteins onto an affinity matrix containing the fibrinogen gamma chain sequence. Proteins of approximately 135 kDa and approximately 95 kDa were specifically eluted by soluble H12, and the 95 kDa protein band was immunoblotted by anti-LIBS1, a monoclonal antibody against human GPIIIa. In control samples, no detectable protein from rabbit platelet lysates was eluted from an RGD affinity matrix by GRGDSP. Collectively, our results demonstrated that H12 inhibits aggregation of and fibrinogen binding to rabbit platelets by directly interacting with rabbit GPIIb-IIIa. These findings suggest that rabbit platelets would serve as a suitable thrombosis model for testing the efficacy of peptide mimetics derived from H12.
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PMID:The fibrinogen gamma chain dodecapeptide inhibits agonist-induced aggregation of rabbit platelets and fibrinogen binding to rabbit glycoprotein IIb-IIIa. 1061 55


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