Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.5 (thrombin)
 33,306 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A high molecular weight glycoprotein (450,000) was obtained from thrombin-treated duck thrombocytes by barium citrate adsorption technique followed by heparin-agarose affinity chromatography. Amino acid composition (high number of acidic amino acids and cystine) as well as carbohydrate contents (1.3 per cent hexosamine, 0.9 per cent sialic acid and 1.5 per cent hexose) showed similarity to mammalian platelet thrombospondin.
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PMID:Avian thrombocyte thrombospondin. 334 67

Platelets from a patient with a severe lifelong bleeding tendency, which later spontaneously disappeared, lacked intact thrombospondin and glycoprotein (GP) Ia. Before disappearance of the bleeding disorder, results of coagulation studies and platelet aggregation in response to adenosine diphosphate (ADP), arachidonic acid, thrombin, A23187, epinephrine, and ristocetin were normal. In contrast, aggregation only occurred in the presence of collagen or wheat germ agglutinin at unusually high doses of these agonists. The platelets adhered normally to purified bovine and human type I collagen, and they did not spread in the presence of methylated type I collagen. No collagen-induced clot retraction was observed. Two-dimensional gel electrophoretic analyses of platelet proteins and immunologic studies showed that intact thrombospondin and GP Ia were absent. Aggregation in response to collagen could be restored by adding thrombospondin. Disappearance of the bleeding tendency occurred at the onset of menopause; subsequent analyses revealed that thrombospondin and GP Ia were present in platelets and that collagen-induced platelet aggregation was normal. These results suggest that both thrombospondin and GP Ia are essential in collagen-induced platelet aggregation. The spontaneous disappearance of the bleeding tendency may have been related to hormonal influences.
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PMID:Deficiency of intact thrombospondin and membrane glycoprotein Ia in platelets with defective collagen-induced aggregation and spontaneous loss of disorder. 335 88

Human thrombospondin, a 450-kDa glycoprotein isolated from platelets and endothelial cells, specifically interacts with osteonectin, a protein of 30 kDa isolated from bovine bones and human platelets. Using ELISA, purified osteonectin binds to solid-phase-adsorbed thrombospondin with a dissociation constant (Kd) of 0.7 nM. Binding of thrombospondin to solid-phase-adsorbed osteonectin was also observed (Kd = 0.86 nM). The interaction of thrombospondin with solid-phase-adsorbed osteonectin was significantly decreased (81% inhibition) when using an excess of fluid-phase osteonectin. Thrombospondin-osteonectin complex formation was calcium-dependent as shown by a 50-80% inhibition in the presence of EDTA. None of the proteins known to interact with thrombospondin (fibrinogen, fibronectin, collagen, plasminogen) had a significant inhibitory effect on thrombospondin-osteonectin complex formation. This selective interaction was confirmed by affinity chromatography. Iodinated osteonectin, previously incubated with purified thrombospondin, specifically bound to an anti-thrombospondin monoclonal antibody (P10) linked to protein-A--Sepharose 4B. Elution of the anti-thrombospondin antibody from protein A allowed the recovery of the thrombospondin-osteonectin complex in the eluate, as judged by SDS/polyacrylamide gel electrophoresis and autoradiography. Blotting of purified thrombospondin to osteonectin adsorbed onto nitrocellulose further confirmed complex formation. In addition, when released from thrombin-stimulated platelets, thrombospondin and osteonectin bound to anti-thrombospondin IgG-coated plates indicating that osteonectin was complexed to thrombospondin once the platelet-release reaction has occurred.
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PMID:Complex formation of human thrombospondin with osteonectin. 340 55

Essential thrombocythemia is a myeloproliferative disorder characterized by frequent bleeding and thrombotic complications. On a molecular level, two abnormalities of platelet thrombospondin have been identified: abnormal glycosylation of the intact 185,000-dalton chain has been detected and a shortened form of the thrombospondin chain is present. We have used two monoclonal antibodies and Lens culinaris lectin to probe the structure of thrombospondin in the platelets from three patients with essential thrombocythemia; one patient with polycythemia vera and two patients with secondary thrombocytosis. The presence of abnormal thrombospondin fragments with molecular weights of 160,000 and 30,000 was detected in the intact platelets and in the supernatant from thrombin-treated platelets, in all of the individuals except one of the secondary thrombocytosis patients. Monoclonal antibody binding studies indicate that both fragments are produced by proteolysis at a single site, which results in the removal of a 30,000-dalton fragment from the NH2-terminal. Lens culinaris lectin-binding studies revealed that some of the carbohydrate moieties of thrombospondin are near this cleavage site. The results are consistent with the hypothesis that the abnormal thrombospondin fragments observed under conditions of increased platelet production are due to increased susceptibility to proteolysis which, in turn, may be due to defective glycosylation.
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PMID:Thrombospondin in essential thrombocythemia. 351 Jun 84

The distribution and transport in platelets of human coagulation Factor V was investigated by immunofluorescent and immunoelectron microscopy. In resting intact platelets, little surface staining was observed by immunofluorescence. In permeable resting cells, punctate staining similar to that reported for fibrinogen (Fbg), thrombospondin (TSP), fibronectin (Fn), von Willebrand factor (VWF), B-thromboglobulin (BTG), and platelet Factor 4 (PF4) was observed. Double label immunofluorescent staining for Fbg and Factor V demonstrated colocalization, suggesting their presence in the same intracellular structure. Thrombin stimulation induced the appearance of larger (approximately 0.5 mu) immunofluorescent masses of these proteins which exactly colocalized. Thus, at the light level, Factor V and Fbg are localized in the same structure in resting and thrombin-stimulated cells. On the ultrastructural level, an alpha granule localization for Fbg has previously been established. We have extended our immunofluorescent observations regarding the localization of Factor V in human platelets by use of transmission electron microscopy of antibody-stained ultrathin frozen sections. In resting cells, staining of virtually all alpha granules was observed for Factor V. In contrast, consistent staining was absent from other organelles including plasma membranes, mitochondria, and vacuolar structures which may represent the open canalicular system. These data thus establish at the ultrastructural level an alpha granule localization of human coagulation Factor V.
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PMID:Ultrastructural localization of coagulation factor V in human platelets. 352 62

Thrombospondin with fibrinogen, fibronectin, and von Willebrand factor binds to platelets stimulated with agonists and support platelet adhesive functions. The receptors for the latter three proteins are associated with membrane glycoprotein GPIIb-IIIa. Thrombasthenic platelets deficient in GPIIb-IIIa have been utilized to examine the role of this membrane protein in the interactions of thrombospondin with platelets. Radioiodinated thrombospondin bound to thrombin-stimulated platelets from normal and thrombasthenic donors with a similar affinity and capacity. As monitored with a monoclonal antibody to thrombospondin, the divalent ion-dependent and -independent pathways for the expression of the endogenous pool of thrombospondin on the surface of thrombin-stimulated platelets from normal and thrombasthenic donors were also qualitatively and quantitatively similar. GPIIb-IIIa or ligands associated with GPIIb-IIIa thus are not essential for the binding of thrombospondin to platelets. Therefore, thrombospondin interacts with unique receptors on platelets.
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PMID:Identification of a new class of inducible receptors on platelets. Thrombospondin interacts with platelets via a GPIIb-IIIa-independent mechanism. 378 77

Sixty-eight patients with malignant disease were divided into two groups based on the results of the platelet antithrombin test (PAT). The normal group had a PAT clotting time ranging from 21.4 to 29.8 seconds, which was equivalent to 25% to 65% inactivation of the 2 U of thrombin added to the test system. The other group showed abnormal PAT clotting time, less than 21.4 seconds or less than 25% thrombin inactivation. The polypeptide composition of platelets from the two patient groups was analyzed by sodium dodecyl sulfate (SDS)-electrophoresis on 7.5% polyacrylamide gels. A polypeptide of 180,000 apparent mol wt was decreased or absent in both Coomassie blue- and Alcian blue-stained gels of the platelets from patients whose PAT was abnormal; this polypeptide comigrated with purified platelet thrombospondin. Tritium labeling of platelet surface glycoproteins by the periodate-borohydride method followed by two-dimensional electrophoresis was performed on platelets of seven patients with abnormal PAT. When they were compared with ten patients with normal PAT, a glycoprotein of 140,000 apparent mol wt with a pl of 4.5 to 5.2 was decreased in platelets of all seven patients with abnormal PAT. Nitrocellulose replicas of one-dimensional gels of platelets from 13 of 14 patients with abnormal PAT showed decreased reaction with an anti-human platelet glycocalicin antiserum. Platelets of these same patients also showed a decreased or absent platelet agglutination induced by ristocetin. Patients with normal PAT had a mean agglutination slope of 1.25 +/- 0.6 (n = 26) as compared with 0.37 +/- 0.34 (n = 26) for the abnormal PAT group (P less than .001). Results indicate that platelets from a subpopulation of tumor patients characterized by decreased platelet antithrombin activity have alterations in two platelet glycoproteins, identified as GPIb and thrombospondin.
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PMID:Platelet antithrombin defect in malignancy: platelet protein alterations. 380 64

Fibrinogen, fibronectin, von Willebrand factor, and thrombospondin are four large glycoproteins that bind to thrombin-stimulated platelets and influence cellular adhesive functions. The effects of five monoclonal antibodies that react with platelet membrane glycoproteins (GP) IIb and/or IIIa on the binding of these four molecules to stimulated platelets were assessed. Tab and PMI-1, antibodies recognizing GPIIb, had no effect, whereas 10E5 and 2G12, antibodies that immunoprecipitate both GPIIb and IIIa in the presence of calcium, inhibited binding of all four ligands by greater than 85%. T10, an antibody specific for the GPIIb-IIIa complex, produced partial inhibition (60% to 80%) of the binding of each ligand. Inhibitory antibodies were effective in the same dose range for all four proteins and also inhibited binding of fibrinogen, fibronectin, and von Willebrand factor to receptors fixed in an induced state (thrombin-stimulated platelets fixed with paraformaldehyde). Thrombospondin did not bind to these fixed cell preparations. The results suggest that these four adhesive proteins have a related mechanism of binding to thrombin-stimulated platelets. This related mechanism may entail the sharing of some, but not necessarily all, binding sites for the four ligands or a proximal relationship between these binding sites.
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PMID:Related binding mechanisms for fibrinogen, fibronectin, von Willebrand factor, and thrombospondin on thrombin-stimulated human platelets. 387 76

Thrombospondin is a principal glycoprotein secreted by thrombin-stimulated platelets and has known affinities for fibrinogen and fibrin. We studied the distribution of thrombospondin in clots formed in situ on Formvar-coated coverslips at 37 degrees C for intervals up to 17 hours. The distributions of three other major platelet granular proteins--fibrinogen, fibronectin, and von Willebrand factor (vWF)--were also determined. The portions of the clots adhering to the coverslips after stripping, washing, and fixation with formaldehyde were stained for the four proteins by the peroxidase-antiperoxidase technique. Monoclonal antibodies were used to localize thrombospondin, fibronectin, and vWF; affinity-purified polyclonal antibodies were used to localize fibrinogen. Platelets stained positively for all four proteins. Thrombospondin was maximally present in the fibrin meshwork from 1 1/2 to 2 hours, after which the intensity of staining decreased until only trace amounts of thrombospondin were detectable between four and 17 hours. Antifibrinogen and, to a lesser extent, antifibronectin stained the fibrin meshwork at all time points. The vWF was not detectable in the fibrin meshwork at any time point. Staining of polymorphonuclear leukocytes (PMNLs) in a fine granular pattern was found with antithrombospondin. The fraction of PMNLs staining positively was 6% to 14% at 1/2 to 4 hours and increased at eight hours to 27%. At 17 hours, 52% of the PMNLs stained for thrombospondin. More than 48% of the PMNLs stained with antifibrinogen at all time points. PMNLs did not stain for either fibronectin or vWF. These studies indicate that thrombospondin is a transient component of the temporary fibrin meshwork and has a unique spatial and temporal distribution in the hemostatic plug.
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PMID:Localization of thrombospondin in clots formed in situ. 390 20

The thrombin-platelet interaction was investigated by analysis of the changes in the nature of platelet-associated thrombin during incubations for as long as 30 min. Washed human platelets were incubated with 125I-labeled human alpha-thrombin at either 22 or 37 degrees C. The saturably bound thrombin (total bound minus that bound in the presence of hirudin) was measured after collection of the platelets by centrifugation through a nonaqueous fluid. The rate of dissociation of bound thrombin was measured by addition of hirudin at intervals before centrifugation of the thrombin-platelet mixtures. Four states of thrombin-platelet complexes were identified: an initial rapidly equilibrating state; a more slowly dissociating state that formed within 5 min; and a nondissociable state and a large sodium dodecyl sulfate-stable complex that formed within 30 min. Transition from the rapidly equilibrating to the slowly dissociable state required a period of occupancy of activated platelets, but it did not require catalytically active thrombin. The nondissociable state represented 50-80% of the total saturably bound thrombin after a 30-min incubation at 37 degrees C. It formed only slightly at 22 degrees C or with inhibited thrombin. Formation of the sodium dodecyl sulfate-stable complex represented about 50% of platelet-associated thrombin after 30 min at 37 degrees C; only slight amounts were detected after incubation at 22 degrees C or with inhibited thrombin. The sodium dodecyl sulfate-stable complex was also found in the supernatant solution, with only about 20% of the total complex bound to platelets. Immunoprecipitation revealed that the complex included the platelet alpha-granule protein glycoprotein G (thrombin-sensitive protein, thrombospondin). It was concluded that only the initial rapidly equilibrating thrombin-platelet association could include binding necessary for platelet activation; transition to the other states requires platelet activation. By better describing the changes that occur in the nature of the binding of thrombin to platelets, this study explains most of the large discrepancies among published descriptions of the binding of thrombin to platelets.
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PMID:Analysis of the fate of platelet-bound thrombin. 391 49


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