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 relationship between activation of resting chick embryo fibroblasts by proteases and proteolytic alteration of the cell surface has been investigated. Five different proteases were examined: trypsin, collagenase, plasmin, alpha-chymotrypsin, and thrombin. All of these proteases, when added to the culture medium at concentrations of 0.08-2.2 mug/ml, stimulated deoxyglucose uptake and induced cell division. The absolute levels of stimulation depended on the specific protease. Activation ranged from a doubling in cell number in 24 hr for trypsin and thrombin down to a 47% increase in cell number for alpha-chymotrypsin. Except in the case of thrombin, the stimulatory effects of these proteases correlated with breakdown of Z, a protein which is the major chick surface protein as revealed by lactoperoxidase-catalyzed iodination and which disappears upon transformation. In the case of thrombin, stimulatory concentrations brought about no detectable loss of surface components. Thus loss of Z is not a necessary condition for activation of chick fibroblasts; it may be a sufficient condition for activation of part of the cell population.
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PMID:Effect of proteases on activation of resting chick embryo fibroblasts and on cell surface proteins. 17 Oct 80

Binding of human [125I]thrombin to washed human platelets was studied in order to analyze the nonenzymic aspects of the thrombin stimulation of platelets. Highly purified alpha-thrombin that was iodinated with lactoperoxidase retained full clotting and esterase activities and full activity toward platelets, was not distinguished from native thrombin by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or gel chromatography, and bound to platelets the same as unlabeled thrombin. Bound and free [125I]thrombin were measured after rapid separation of platelets from the suspending medium by centrifugation through oil. Maximum binding was within 15 s, the shortest time measured. At concentrations of thrombin sufficient to cause less than maximal platelet stimulation, 90% of the total thrombin was free in the suspending solutions. Equilibrium binding was established, with both free thrombin and free platelets retaining activity, and with rapid reequilibration after dilution or addition of unlabeled thrombin. The equilibrium was complex, with the apparent number of binding sites and dissociation constants dependent on thrombin concentration. Analysis of bound thrombin as a function of thrombin concentration by double-reciprocal and Scatchard plots indicated 300-400 high affinity sites (Kdiss = 1.8-2 nM); these correlate with thrombin stimulation of Ca2+ secretion, which shows half maximal effect at 1.5 nM thrombin and maximal effect with 500-600 thrombins bound per platelet.
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PMID:Equilibrium binding of thrombin to platelets. 99 Feb 49

A young Italian man (A.P.) has a lifelong history of bleeding from gums and mucocutaneous tissue. Electron microscopy showed a wide diversity of platelet size including giant forms. In citrated platelet-rich plasma (PRP), platelet aggregation induced by adenosine diphosphate (ADP) and other agonists was much reduced. Both secretion and clot retraction were normal. The aggregation of washed platelets with ADP was improved but remained subnormal, as was aggregation with collagen and thrombin. Fibrinogen-binding was analyzed by flow cytometry using platelets in whole blood or PRP and was markedly decreased. Crossed immunoelectrophoresis of Triton X-100 extracts of (A.P.) platelets showed that GP IIb-IIIa levels were 40% to 50% of normal. Glycoprotein (GP) IIb and GP IIIa were of usual migration in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but their labeling was much reduced during lactoperoxidase-catalyzed iodination. Binding to (A.P.) platelets of four different 125I-labeled monoclonal antibodies to GP IIb-IIIa complexes was reduced to 12% to 20% of normal levels. However, when the patient's platelets were stimulated with alpha-thrombin, monoclonal antibody binding showed the same increase (approximately 20,000 sites) as normal platelets. Both flow cytometry and immunocytochemical studies showed that the distribution of residual surface GP IIb-IIIa within the total (A.P.) platelet population was heterogeneous and not related to platelet size. Staining of ultrathin sections confirmed the presence of an internal pool of GP IIb-IIIa. Monoclonal antibodies to other membrane glycoproteins bound normally to (A.P.) platelets. The patient has a selective deficiency of the surface pool of GP IIb-IIIa complexes that is manifested clinically by a mild Glanzmann's thrombasthenia-like syndrome.
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PMID:A defect of platelet aggregation associated with an abnormal distribution of glycoprotein IIb-IIIa complexes within the platelet: the cause of a lifelong bleeding disorder. 163 23

Recent evidence suggests the presence in resting platelets of centrally located compartments of glycoprotein (GP) IIb-IIIa. We have employed an experimental procedure which dissociates and antigenically denatures the surface compartment of GP IIb-IIIa and allows internal compartments of GP IIb-IIIa to be studied immunochemically and functionally in intact platelets. When gel-filtered platelets are incubated with 0.25 mM EGTA at 37 degrees C for 30 min, and then supplemented for 30 min with 5 mM calcium, they lose their ability to bind GP IIb-IIIa complex-specific monoclonal antibody Fab fragments. However, when such platelets are subsequently stimulated with thrombin, GP IIb-IIIa-specific Fabs are again able to bind in large amounts to the platelet surface, in concert with the appearance of substantial amounts of receptors for fibrinogen and fibronectin. In immunoprecipitation experiments, we have found that this thrombin-displayed pool of GP IIb-IIIa originates from a pool that is not labeled by lactoperoxidase-catalyzed radioiodination of intact resting platelets. In immunofluorescence experiments, we have found that EGTA-incubated platelets contain a large sequestered internal pool of GP IIb-IIIa which upon thrombin stimulation is translocated to the platelet surface. Additional experiments suggest that this centrally located compartment may be surface connected in resting platelets and that it is accessible to some extracellular proteins, but not others.
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PMID:Resting platelets contain a substantial centrally located pool of glycoprotein IIb-IIIa complex which may be accessible to some but not other extracellular proteins. 242 67

To investigate the association of lipid with the cytoskeleton of platelets during aggregation, rabbit and human platelets were isolated and labeled with [3H]palmitic acid; lipid extraction showed approximately 80% in phospholipid. Limited aggregation was induced with ADP or thrombin, and the cytoskeleton was isolated after lysis with 1% Triton X-100, 5 mM EGTA. Cytoskeleton from unactivated platelets had approximately 0.03% of the total label in the platelets, but after aggregation with ADP (2 microM) or thrombin (0.1 U/ml) for 20-30 s, 1.5-8% of the label was with the cytoskeleton. Fibrinogen enhanced aggregation and the association of label with the cytoskeleton; incorporation of label increased exponentially as aggregation proceeded, decreased exponentially during deaggregation, and appeared to be related to the number of sites of contact. Inhibitors that increase cyclic AMP inhibited aggregation and cytoskeletal labeling, but aspirin had no effect. Some experiments were done with DNase I and Ca2+ in the Triton X-100 lysis medium to cause actin depolymerization, under conditions in which the Ca2+-dependent protease activity was inhibited. This greatly reduced the association of label with the cytoskeleton at early time points, but when aggregation had proceeded further, a large proportion of the label was not dissociated by this treatment. These findings, electron microscopy, and the enrichment of the cytoskeleton of aggregated platelets with only some of the membrane proteins that were labeled by the 125I-lactoperoxidase method, indicated that with limited aggregation, the 3H-labeled lipid was mainly associated with the cytoskeleton and not with trapped membrane fragments resulting from incomplete lysis. Since the pattern of cytoskeleton labeling ([3H]palmitate) and the selective association of some membrane proteins with the cytoskeleton/lipid complex was the same with ADP and thrombin, the reactions must be dependent on aggregation and not on events associated with the release of granule contents.
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PMID:Aggregation-related association of lipid with the cytoskeleton of rabbit and human platelets prelabeled with [3H]palmitic acid. Similar effects of adenosine diphosphate- and thrombin-induced aggregation. 282 26

Glycoprotein Ia (GP Ia) is a relatively minor component of human blood platelets thought to be a receptor involved in collagen-induced platelet activation. However, some difficulties exist with the definition of this glycoprotein. The expression of GP Ia on resting (prostacyclin analogue-treated) and thrombin-activated platelets was compared by surface labeling with 125I-lactoperoxidase. Intact platelets or platelets solubilized in sodium dodecyl sulfate were labeled with periodate/[3H]NaBH4. Analysis on two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels showed that GP Ia is very poorly labeled in resting platelets. After activation a new spot (GP Ia*) appears with the same relative molecular mass as GP Ia under reducing conditions. GP Ia and Ia* can be clearly separated by two-dimensional nonreduced/reduced gel electrophoresis. Therefore, two glycoproteins which have been termed GP Ia exist in platelets with similar molecular weight and pI under reducing conditions. One of these (GP Ia*) is only surface-labeled when platelets are activated, indicating that it is only exposed on the surface of activated platelets. Supernatant from activated platelets contains this glycoprotein as well as other granule components. This glycoprotein is missing in platelets from two patients with collagen-response defects.
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PMID:Human platelet glycoprotein Ia. One component is only expressed on the surface of activated platelets and may be a granule constituent. 290 36

Human factor VIII was purified from commercial factor VIII concentrate with a 12% yield. The specific coagulant activity of purified factor VIII was 8,000 units/mg. In the presence of SDS the purified factor VIII consisted of a variety of polypeptides on polyacrylamide gels, ranging between Mr 80,000 and Mr 208,000. In the absence of SDS the purified factor VIII showed an apparent molecular weight of 270,000 upon Sephadex G200 gel-filtration. The purified factor VIII could be activated by thrombin, which resulted in the disappearance of Mr 108,000-208,000 polypeptides in favor of an Mr 92,000 polypeptide. Treatment with factor Xa also activated factor VIII, whereas treatment with activated protein C resulted in the inactivation of coagulant activity. Coagulant-active 125I-factor VIII was prepared using a lactoperoxidase radioiodination procedure. This 125I-factor had the same characteristics as unlabeled factor VIII. All polypeptides could be precipitated with monoclonal antibodies directed against factor VIII. With 125I-factor VIII a pIapp of 5.7 was found in the presence of urea.
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PMID:Human factor VIII: purification from commercial factor VIII concentrate, characterization, identification and radiolabeling. 309 64

Radioiodination of the two tyrosine residues (Tyr-99 and Tyr-138) of ox testis calmodulin was performed using several methods, and studied through the specific activity, and the [125I]iodoamino acid analysis of the radiolabeled calmodulins. Hydrolysis by thrombin of 125I-calmodulin labeled by the lactoperoxidase method and subsequent isolation of peptides TM1 and TM2 by gel electrophoresis showed preferential labeling by 125I of Tyr-99 (TM1) over Tyr-138 (TM2). Analysis of [125I]iodoamino acids of radiolabeled TM1, TM2 and calmodulin demonstrated that [125I]monoiodotyrosine was predominant, the remainder being [125I]diiodotyrosine. Radioiodination of wheat germ calmodulin, which contains a single tyrosine residue (Tyr-139), showed that only TM2 was labeled by 125I on the Tyr-139 residue and also on the His-108 residue (radiolabeled monoiodotyrosine, diiodotyrosine and monoiodohistidine being present).
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PMID:Radioiodination of tyrosine residue(s) of ox testis and of wheat germ calmodulins. 370 67

Heparin cofactor II (HC II) is a heparin-dependent inhibitor of thrombin, distinct from antithrombin III (AT III). This study was designed to evaluate its metabolism in healthy subjects. Purified HC II was labelled with 125I by the lactoperoxidase-glucose oxidase technique. The biological activity of the HC II was unchanged after labelling as was its migratory pattern by crossed immunoelectrophoresis in the presence of heparin or dermatan sulfate. Three healthy volunteers were injected with 10 microCi and the plasma radioactivity was measured daily. The data were approximated by a sum of two exponential terms and the metabolism of HC II was described by a two compartment mamillary system. The mean values of fractional catabolic rate, intravascular fraction and half-life of the elimination phase were respectively: 0.44 d-1, 0.60 and 2.53 d. These parameters are of the same order of magnitude as those reported in the literature for AT III. The plasma HC II concentration in the 3 subjects ranged from 61 to 82 micrograms/ml as estimated using our purified preparation. Accordingly, the absolute catabolic rate ranged from 1.17 to 1.36 mg X kg-1 X d-1.
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PMID:Turnover study of heparin cofactor II in healthy man. 384 20

Platelet glycoprotein (GP) Ib from 131 healthy Japanese was analyzed using SDS-polyacrylamide gel electrophoresis and specific staining with peroxidase-coupled wheat germ agglutinin after it was transferred to nitrocellulose membranes. Four slightly different species of GPIb were observed and designated as A, B, C, and D for glycoproteins with molecular weights of 168,000, 162,000, 159,000, and 153,000 daltons, respectively. The respective gene frequencies were calculated to be .073, .011, .561, and .355 for A-, B-, C-, D-type GPIb. Portions from each type of GPIb molecule (alpha-chain and glycocalicin) showed heterogeneity with the same molecular weight difference, indicating that the variance would be derived from the polypeptide portion that is exposed to the outer medium. The different types of GPIb were the same with respect to their accessibility to lactoperoxidase, reactivity to lectins, and affinity to TLCK-thrombin. Although Bolin et al reported patients with a bleeding tendency whose platelets have double GPIb bands, here we found that platelets with different GPIb phenotypes showed no significant differences in aggregating activity and platelet retention. Analysis of GPIb phenotype should be important for structural and physiologic studies on GPIb and glycocalicin.
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PMID:Genetic polymorphism of platelet glycoprotein Ib. 623 67


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