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)

A very highly purified von Willebrand factor (vWF) concentrate was analyzed in order to evaluate its suitability for the treatment of von Willebrand's disease (vWD). The functional activity of vWF assessed by its ristocetin cofactor activity (vWF:RCo) correlated with the level of vWF antigen (vWF:Ag), with the vWF:RCo/vWF:Ag ratios ranging from 0.56 to 1.02, and the specific activity being always greater than 50 IU vWF:RCo/mg protein. Electrophoretic analysis showed a normal pattern of high, intermediate and low-molecular-weight multimers of vWF. The biological activity of vWF was also evaluated by studying its ability to bind to collagen and to platelet receptors in the presence of either ristocetin or thrombin. Furthermore, these functional activities of vWF were confirmed by the capacity of this concentrate to induce platelet adhesion to collagen in a perfusion system. Moreover, the vWF present in this preparation was able to bind factor VIII. All these in vitro data suggest that this preparation is likely to be effective in the treatment of vWD patients.
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PMID:In vitro evaluation of a very-high-purity, solvent/detergent-treated, von Willebrand factor concentrate. 194 3

Essential mixed cryoglobulinemia (EMC) is a rheumatic disorder characterized by widespread vasculitis. To better define the nature of the vasculitic process and to possibly outline assessment methods reliable for using in a clinical context, we studied plasma levels of three endothelial related peptides: fibronectin (FN), von Willebrand factor (vWF) and tissue plasminogen activator (t-PA), and those of thrombin-antithrombin III complexes (TAT) as markers of activation of the coagulation in 21 patients and in 16 controls. In EMC we found a picture consisting of reduced FN and increased vWF, t-PA, and TAT levels, suggesting a condition of endothelial cell damage with thrombin formation in vivo. Since we previously demonstrated the presence of chronic disseminated intravascular coagulation in these patients, we may assume that endothelial cells stressed by cryoprecipitation or stimulated by soluble mediators may be actively involved in the vasculitic process and possibly express procoagulant properties. This is a good example of the complex interplay existing between autoimmunity and coagulation mechanisms. We also suggest that FN, vWF, t-PA and TAT should be considered as additional clinical parameters when evaluating patients with EMC.
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PMID:Clinical significance of endothelial damage markers in essential mixed cryoglobulinemia. 195 Mar 76

We have used the polymerase chain reaction (PCR) and differential oligonucleotide melting to screen for mutations in selected CpG dinucleotides in the factor VIII genes of haemophilia A patients. By this means we have identified and confirmed by sequencing a novel point mutation in codon 372 (CGC) of the factor VIII gene of a moderately severe CRM+ haemophiliac. The first C of this codon has been substituted by T resulting in the non-conservative substitution of cysteine for arginine at an essential thrombin cleavage site in factor VIII. Analysis of three intragenic restriction fragment length polymorphisms was uninformative in the patient's family. However, DNA analysis for the specific mutation shows one sister and the patient's mother to be carriers, and the other sister to be normal. This DNA analysis confirmed the results of phenotype analysis by factor VIII coagulant to von Willebrand factor antigen ratios for the females at risk. The two carrier females had low factor VIII coagulant activity and excess VIII antigen as predicted but the non-carrier sister also had anomalously high VIII antigen in her plasma. When feasible, mutation specific DNA analysis is able to resolve the difficulties posed by variable phenotype data and unknown level of mutation in sporadic haemophilia A.
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PMID:CRM+ haemophilia A due to a missense mutation (372----Cys) at the internal heavy chain thrombin cleavage site. 197 1

Vascular permeability factor (VPF), a tumor-secreted heparin-binding protein (Mr approximately 38,000), is responsible for increased vessel permeability and fluid accumulation associated with tumor growth. Vascular permeability factor also promotes the growth of human umbilical vein endothelial cells (EC) and bovine pulmonary ECs in vitro. It is shown for the first time that guinea pig VPF (half-maximal and maximal dose approximately 0.4 and 22 pmol/l (picomolar), respectively), as well as human VPF, are potent stimuli for human ECs resulting in [Ca2+]i increases (maximal three- to fourfold) and inositol triphosphate (IP3) formation. Unlike the maximal responses to thrombin and histamine, the [Ca2+]i response to a maximal VPF dose was preceded by a characteristic 10- to 15-second delay. Guinea pig VPF also selectively increased [Ca2+]i in cultured aortic and pulmonary artery ECs, but not aortic smooth muscle cells, human fibroblasts, or neutrophils. Affinity-purified rabbit antibody (raised to a synthetic peptide representing VPF N-terminal amino acids 1 to 24) adsorbed all vessel permeability-increasing activity, EC growth-promoting activity, and specifically all activity responsible for increasing EC [Ca2+]i. Similar to other mediators that increase [Ca2+]i in cultured ECs, VPF also induced a 200% increase in von Willebrand factor release. Together these data indicate that VPF acts directly on ECs and that rapid cellular events in its in vivo/in vitro actions are likely to involve phospholipase C activation, [Ca2+]i increase, and von Willebrand factor release.
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PMID:Tumor-secreted vascular permeability factor increases cytosolic Ca2+ and von Willebrand factor release in human endothelial cells. 198 67

Exhaustive extraction of human platelets with 6 M guanidine-HCl, and 5% beta-mercaptoethanol, followed by 5% SDS resulted in a sedimentable material which showed fibrous structure by transmission electron microscopy. When platelets treated with 8 M urea, 50 mM DTT and 2% SDS were applied on a 3% solubilizable acrylamide gel a high molecular weight material could be also isolated which was highly crosslinked by epsilon(gamma-glutamyl)lysine bonds. Its amino acid composition was Asp 110, Glu 119, Ser 55, Gly 70, Arg 33, Thr 41, Ala 112, Pro 93, Tyr 35, Val 18, Met 55, Cys 46, IIe 47, Leu 71, Phe 27, Lys 76 expressed as residue per 1000. The quantity of platelet polymer material as well as the amount of epsilon(gamma-glutamyl)lysine bond was slightly higher in thrombin activated platelets. The insoluble matrix of resting platelets reacts with antibodies against spectrin, alpha-actinin, actin, myosin, tropomyosin. The matrix from activated platelets has shown reaction with additional antibodies including ones against blood coagulation factor XIIIa, fibrinogen, von Willebrand factor, thrombospondin, tubulin and filamin. The presence of an epsilon(gamma-glutamyl)lysine cross-linked cell matrix in platelets is consistent with the observation of a similar structure in other cells.
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PMID:The presence of a covalently cross-linked matrix in human platelets. 200 80

In this study, the question of whether glycoprotein Ib (GPIb) mediates both high and moderate affinity pathways of alpha-thrombin-induced platelet activation was examined. Flow cytometric studies, using a panel of monoclonal antibodies (MoAbs), showed that Serratia marcescens protease treatment removed greater than 97% of the glycocalicin portion of GPIb but did not affect the changes in the expression of GPIX or GMP-140 that were induced by high concentrations of alpha-thrombin (10 nmol/L). However, Serratia treatment almost completely abolished the increase in platelet surface GMP-140 induced by low concentrations of alpha-thrombin (0.5 nmol/L) and diminished the downregulation of platelet surface GPIX by 60.9% +/- 5.6% (mean +/- SEM, n = 3). When present in 20-fold molar excess, an MoAb directed against the alpha-thrombin/von Willebrand factor (vWf) binding domains of GPIb completely blocked the ristocetin-dependent binding of vWf to platelets but inhibited only to about 50% the binding of alpha-thrombin and the activation-dependent binding of vWf. In platelets treated with Serratia marcescens protease to remove GPIb, a concentration of this MoAb 16,000-fold in excess of the maximum possible remaining copies of GPIb failed to inhibit platelet activation by alpha-thrombin. These studies demonstrate that activation of intact platelets by alpha-thrombin proceeds by both GPIb-dependent and GPIb-independent mechanisms.
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PMID:Glycoprotein Ib (GPIb)-dependent and GPIb-independent pathways of thrombin-induced platelet activation. 201

We have previously reported the purification of a 37 kDa platelet agglutinating protein (PAP p37) from the plasma of a patient with thrombotic thrombocytopenic purpura (TTP) that was shown to be present in a subset of TTP patients. To gain further insight into the interaction between PAP p37 and platelets, we have studied the properties of PAP p37 binding to platelets. Washed human platelets from two normal donors and two TTP patients after recovery were used for the experiments. The PAP p37 binding to platelets was specific, concentration dependent and saturable. Scatchard analysis demonstrated about 20,564-27,090 PAP p37 binding sites per platelet. Stimulation of platelets with thrombin or ADP did not have any significant effect on its binding. Thiol- and serine-specific protease inhibitors did not inhibit PAP p37 binding to the platelets. Sugars such as glucose, fructose, mannose, and sialic acid, at 40 mM, inhibited its binding to platelets by 44%, 73%, 79%, and 91% respectively, but galactose and amino sugars did not have any significant effect. At 250 micrograms/ml, Concanavalin-A inhibited 42% of binding, but other lectins, such as phytohemagglutinin-P, potato lectin and helix pomatia lectin (snail), did not. Pre-incubation of 125I-PAP p37 with the adult human IgG, decreased its binding to the platelets. The monoclonal antibodies to GP Ib (6D1) and GP IIb-IIIa complex (10E5) did not inhibit the binding of 125I-PAP p37 to platelets. Fibrinogen and von Willebrand factor did not affect the binding either. These results suggest that PAP p37 binds to platelets on the sites other than GP Ib or GP IIb-IIIa complex.
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PMID:Binding of platelet agglutinating protein p37 from the plasma of a patient with thrombotic thrombocytopenic purpura to human platelets. 202 44

The role of glycoprotein (GP) IIb-IIIa complexes and of adhesive proteins in mediating platelet aggregation is now well defined. However, less is known of the changes that occur once aggregation has begun. We report immunogold staining of thin sections of platelets or platelet aggregates, embedded in Lowicryl K4M, after the use of polyclonal antibodies to GP IIb or GP IIIa, fibrinogen (Fg), von Willebrand factor (vWF), and thrombospondin (TSP). Bound immunoglobulin G (IgG) was located by species-specific anti-IgG coupled to 5-nm gold particles and by electron microscopy. Initial experiments with platelet-rich plasma confirmed the feasibility of visualizing adhesive proteins between platelets in aggregates. Experiments then continued, using stirred suspensions of washed platelets incubated with alpha-thrombin. After 20 seconds, platelets were in contact without detectable release, although giant secretory vesicles containing adhesive proteins were seen. Internal pools of GP IIb-IIIa were progressively externalized within the aggregate. Secreted Fg was readily detected between platelets at 40 seconds. After 3 minutes, when most of the secretion had occurred, Fg had a patchwork-like distribution within the aggregate. After 6 minutes, zones with closely interspaced surface membranes, usually representing pseudopods, were dominant and Fg free. Results for vWF and TSP were similar to those for Fg. Nonetheless, GP IIb-IIIa complexes continued to be located between adjacent surface membranes throughout the aggregate. Thrombin-induced platelet aggregates were isolated, and sodium dodecyl sulfate-soluble extracts were obtained. Western blot experiments showed that, although fibrinopeptide A had been cleaved, degradation of adhesive proteins by platelet proteases had not occurred. These results emphasize that a platelet aggregate is a dynamic structure and suggest that not all surface-contact interactions are mediated by Fg or the other adhesive proteins tested in this study.
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PMID:Thrombin-induced platelet aggregates have a dynamic structure. Time-dependent redistribution of glycoprotein IIb-IIIa complexes and secreted adhesive proteins. 202 7

Endothelial cells are the principal source of plasma and basement membrane von Willebrand factor (vWF). To arrive at its biologically active multimeric form, vWF undergoes a series of intracellular processing steps. The protein is synthesized as a large precursor pro-vWF, which dimerizes in the endoplasmic reticulum through disulfide bonds located in the carboxyl-terminal portion of the subunit. Only dimers are transported to the Golgi apparatus. Expression of truncated pro-vWF subunits, which lack the last 20 kd, abolishes the requirement for dimerization and thereby allows the monomeric protein to be secreted. Another requirement for intracellular transport from the endoplasmic reticulum is N-linked glycosylation. Inhibition of N-linked glycosylation prevents exit of both the wild-type and the truncated vWF from the endoplasmic reticulum. In the acidic environment of the trans-Golgi and post-Golgi compartments, pro-vWF dimers multimerize by a second set of interchain disulfide bonds. The presence of the vWF propolypeptide and acidic pH conditions are necessary for the multimerization process. The largest vWF multimers are stored in endothelial cell-specific organelles called Weibel-Palade bodies. At the site of vascular injury and inflammation, physiologic secretagogues such as thrombin, fibrin, and histamine may cause release of these large, biologically potent vWF multimers from the Weibel-Palade bodies into the surrounding blood and subendothelium.
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PMID:von Willebrand factor and the endothelium. 204 1

The glycoprotein Ib/IX complex on platelets is responsible for the first stage of haemostasis as an essential component in the primary adhesion of platelets to damaged vessel walls. Glycocalicin is the extracellular part of platelet glycoprotein Ib alpha and contains the von Willebrand factor and thrombin binding sites. Disulphide bonds are implicated in the von Willebrand binding site and studies with peptides point towards a region of glycocalicin with four cysteines as containing the binding sites for both von Willebrand factor and thrombin. The position and linkage of these two disulphide bonds are now determined to be 209-248 and 211-264 and the relevance of this double-loop structure for glycoprotein Ib/IX function is discussed.
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PMID:Identification of the disulphide bonds in human platelet glycocalicin. 207 Jul 94


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