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)

Platelet pseudopodia were compared to platelet cell bodies with respect to their lipid composition, fatty acid distribution and protein composition. The methodology for producing pseudopodial preparations of platelets stimulated with thrombin, ADP or calcium ionophore was established. The separation of pseudopodia and cell bodies was verified by electron microscopic examination of the respective platelet components. Lipid analyses demonstrated a preponderance of lysophospholipids and sphingomyelin in pseudopodial preparations and a large increase in mono-, di- and tri-ene fatty acids as compared to cell bodies. Changes were also evident in the protein composition evaluated by one- and two-dimensional SDS-polyacrylamide gel electrophoresis and by [32P]ATP labeling of exofacial membrane proteins. A protein of approximately 68 kDa which reacted strongly with antibody to PlA1, was prominantly displayed in platelet pseudopodia. Thus, our studies demonstrate a heterogeneous distribution of lipids and proteins in a mammalian membrane system which may have important implications for the functional behavior of the cell.
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PMID:Unequal distribution of membrane components between pseudopodia and cell bodies of platelets. 222 2

Human rheumatoid synovial cells in culture secrete at least three related metalloproteinases that digest extracellular matrix macromolecules. One of them, termed matrix metalloproteinase 2 (MMP-2), has been purified as an inactive zymogen (proMMP-2). The final product is homogeneous on SDS/PAGE with Mr = 72,000 under reducing conditions. The NH2-terminal sequence of proMMP-2 is Ala-Pro-Ser-Pro-Ile-Ile-Lys-Phe-Pro-Gly-Asp-Val-Ala-Pro-Lys-Thr, which is identical to that of the so-called '72-kDa type IV collagenase/gelatinase'. The zymogen can be rapidly activated by 4-aminophenylmercuric acetate to an active form of MMP-2 with Mr = 67,000, and the new NH2-terminal generated is Tyr-Asn-Phe-Phe-Pro-Arg-Lys-Pro-Lys-Trp-Asp-Lys-Asn-Gln-Ile. However, following 4-aminophenylmercuric acetate activation, MMP-2 is gradually inactivated by autolysis. Nine endopeptidases (trypsin, chymotrypsin, plasmin, plasma kallikrein, thrombin, neutrophil elastase, cathepsin G, matrix metalloproteinase 3, and thermolysin) were tested for their abilities to activate proMMP-2, but none had this ability. This contrasts with the proteolytic activation of proMMP-1 (procollagenase) and proMMP-3 (prostromelysin). The optimal activity of MMP-2 against azocoll is around pH 8.5, but about 50% of activity is retained at pH 6.5. Enzymic activity is inhibited by EDTA, 1,10-phenanthroline or tissue inhibitor of metalloproteinases, but not by inhibitors of serine, cysteine or aspartic proteinases. MMP-2 digests gelatin, fibronectin, laminin, and collagen type V, and to a lesser extent type IV collagen, cartilage proteoglycan and elastin. Comparative studies on digestion of collagen types IV and V by MMP-2 and MMP-3 (stromelysin) indicate that MMP-3 degrades type IV collagen more readily than MMP-2, while MMP-2 digests type V collagen effectively. Biosynthetic studies of MMPs using cultured human rheumatoid synovial fibroblasts indicated that the production of both proMMP-1 and proMMP-3 is negligible but it is greatly enhanced by the treatment with rabbit-macrophage-conditioned medium, whereas the synthesis of proMMP-2 is constitutively expressed by these cells and is not significantly affected by the treatment. This suggests that the physiological and/or pathological role of MMP-2 and its site of action may be different from those of MMP-1 and MMP-3.
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PMID:Matrix metalloproteinase 2 from human rheumatoid synovial fibroblasts. Purification and activation of the precursor and enzymic properties. 226 96

We have discovered and characterized a novel coagulation factor Xa inhibitor from the salivary gland of the black fly, Simulium vittatum. Salivary glands were surgically dissected from the flies and a crude salivary gland extract was tested for inhibition of a number of coagulation assays. The gland extract inhibited both thrombin and factor Xa. To purify further the factor Xa inhibitor, a factor Xa affinity column was utilized. Final purification of the black fly factor Xa inhibitor was achieved by reverse-phase C8 microbore high pressure liquid chromatography. Inhibition of factor Xa was nearly stoichiometric by the purified inhibitor with no inhibitor of thrombin detected. SDS-polyacrylamide gel electrophoresis indicated the inhibitor had a molecular weight of 18,000 and sequence analysis of the inhibitor revealed a blocked amino terminus. These data indicate that the blood-sucking black fly has evolved a highly potent inhibitor of mammalian coagulation factor Xa to disrupt its host normal hemostatic clotting mechanisms.
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PMID:Isolation and characterization of a coagulation factor Xa inhibitor from black fly salivary glands. 227 May 32

Recently, we demonstrated that tissue plasminogen activator directly releases fibrinopeptides A and B (FPA and FPB) from fibrinogen. The purpose of this study was to determine whether urokinase has similar activity. Incubation of urokinase with fibrinogen or heparinized plasma results in concentration-dependent FPB release unaccompanied by FPA cleavage. For equivalent amidolytic activity, high molecular weight urokinase releases twofold more FPB than the low molecular weight species. In contrast, prourokinase does not release FPB until activated to urokinase. Contaminating thrombin or plasma is not responsible for urokinase-mediated FPB release because this activity is unaccompanied by FPA or B beta 1-42 cleavage, and is unaffected by heparin, hirudin, a monospecific antibody against thrombin, aprotinin, or alpha 2-antiplasmin. FPB release reflects a direct action of urokinase on fibrinogen because release is completely inhibited by a monospecific antibody against the enzyme. Further, urokinase releases FPB from the FPB-containing substrate B beta 1-42, thus confirming its specificity for the B beta 14 (Arg)-B beta 15 (Gly) bond. In addition to FPB release, SDS-PAGE analysis of the time course of urokinase-mediated fibrinogenolysis indicates progressive proteolysis of both the A alpha- and B beta-chains of fibrinogen that occurs after FPB release is completed. As a consequence of urokinase-mediated fibrinogenolysis, there is progressive prolongation of the thrombin clotting time. These studies indicate that urokinase has direct catalytic activity against fibrinogen. By releasing FPB, a potent chemoattractant, and by rendering fibrinogen less clottable by thrombin, urokinase may participate in processes extending beyond fibrinolysis.
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PMID:Urokinase has direct catalytic activity against fibrinogen and renders it less clottable by thrombin. 236 16

The thienopyridines, ticlopidine and PCR 4099, inhibit ex vivo aggregation in response to ADP and other agonists. It has been shown that ticlopidine induces a functional defect in the binding of fibrinogen to its platelet membrane receptor. We have studied the effects on platelet functions of PCR 4099 in rat and in man. The aim of the study was to check the possibility of a direct modification of the fibrinogen binding site on the GP IIb-IIIa complex. Washed platelet suspensions were used for aggregation and fibrinogen binding studies. Platelet lysates were submitted to SDS-polyacrylamide gel electrophoresis, crossed immunoelectrophoresis and immunoprecipitation. We found that administration of PCR 4099 inhibited selectively and irreversibly ADP-induced aggregation. Although the effect of ADP on aggregation was blocked, PCR 4099 did not modify ADP-induced shape change. Only the effects of low concentrations of thrombin on platelet aggregation were inhibited. Fibrinogen binding was dramatically inhibited in rat and in man when platelets were stimulated with ADP and low concentrations of thrombin. At high concentration of thrombin there still remained a part of fibrinogen binding inhibition although aggregation was not impaired. Electrophoretic and immunoelectrophoretic studies showed no difference before and after treatment by PCR 4099. In particular, the GP IIb-IIIa-complex was not dissociated, its electrophoretic mobility was not changed and three monoclonal anticomplex antibodies recognized it in the same manner before and after treatment. We conclude that PCR 4099 selectively inhibits the ADP aggregation pathway and that the inhibition of fibrinogen binding is probably not due to a direct modification of the GP IIb-IIIa complex.
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PMID:The thienopyridine PCR 4099 selectively inhibits ADP-induced platelet aggregation and fibrinogen binding without modifying the membrane glycoprotein IIb-IIIa complex in rat and in man. 237 65

Congenital heterozygous dysfibrinogenemia was diagnosed in a young woman with bleeding tendency. 3 other asymptomatic members of her family (mother and the 2 sisters) had abnormal fibrinogen. The proposita's plasma exhibited prolonged thrombin and reptilase times. Plasma fibrinogen concentration determined by functional assay was 0.3 g/l, whereas immunologic assay revealed normal fibrinogen levels. Turbidity curves, representing the rate of thrombin-induced fibrin formation, were markedly delayed both in the presence and absence of Ca2+. Isoelectric focusing and SDS electrophoresis of reduced fibrinogen showed normal charge and size of the subunit chains. Release of fibrinopeptide B by thrombin was normal, whereas HPLC elution diagrams of fibrinopeptide A showed an abnormal peak A* with a slightly shorter retention time than the normal fibrinopeptide A. The amino acid analysis showed that the arginine in peak A* is replaced by histidine (A alpha 16 Arg----His).
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PMID:Fibrinogen Milano. VI: A heterozygous dysfibrinogenemia (A alpha 16 Arg----His) with bleeding tendency. 237 62

Purification of vitronectin by identical procedures from serum instead of plasma results in the coisolation of an additional protein component with mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of 82 kDa. We show that this component is the thrombin-antithrombin III complex based on the following evidence. Similar to a complex constructed using purified thrombin and antithrombin III, the 82-kDa component has a reduced molecular size of 69 kDa if it is not boiled prior to SDS-PAGE. Upon prolonged boiling in SDS it dissociates into 56- and 32-kDa components which co-migrate in SDS-PAGE with purified antithrombin III and thrombin, respectively. The 82- and 56-kDa components react with an antiserum against antithrombin III, and an antiserum prepared against the 82-kDa complex reacts with purified antithrombin III. Thrombin-antithrombin III complex, from either serum or recalcified clotted plasma, bound to vitronectin immobilized on Sepharose or plastic. However, purified antithrombin III which had not reacted with thrombin lacked affinity for vitronectin as did antithrombin III from citrated plasma. Purified antithrombin III acquired affinity for immobilized vitronectin if it was complexed with thrombin or was modified by radioiodination. Binding of vitronectin to antithrombin III coated on plastic was demonstrated using enzyme-linked immunosorbent assay. These results demonstrate that vitronectin binds thrombin-antithrombin III complexes through a cryptic site in antithrombin III which can be exposed when antithrombin III is radioiodinated, bound to plastic, or complexed with thrombin. Since vitronectin can interact with cells, the binding of vitronectin to the thrombin-antithrombin III complex may serve to facilitate the interaction of this complex with cell surfaces.
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PMID:Association of thrombin-antithrombin III complex with vitronectin in serum. 241 21

A monoclonal antibody named TM60, which inhibited both thrombin- and ristocetin-induced platelet aggregations, was obtained by hybridoma technique. TM60 inhibited binding of von Willebrand factor to platelets under the presence of ristocetin. The subclass of TM60 was IgG2a. TM60 did not inhibit ADP-, collagen-A-23187-, arachidonic acid- and PAF-induced platelet aggregations, but inhibited polylysine-, polybrene- and cationized ferritin-induced platelet aggregations. ATP-release from platelets induced by thrombin was also inhibited by TM60. Immunoprecipitation and SDS-PAGE experiments demonstrated that TM60 recognized an epitope on GPIb whose molecular weight was 165,000 under non-reduced and 145,000 under reduced conditions.
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PMID:Monoclonal antibody to glycoprotein Ib inhibits both thrombin- and ristocetin-induced platelet aggregations. 241 61

An assay for the quantification of pentosan polysulphate (PPS) in plasma is described. As PPS has been shown to potentiate thrombin inhibition by the second heparin cofactor (HC II), the principle of this assay was to measure the formation of covalent complexes between HC II and the thrombin generated in plasma after contact activation and recalcification. The complexes were quantified by using purified 125I-HC II added to the plasma as a tracer and SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The assay was sensitive to low PPS concentrations (limit of detection: 0.1 microgram/ml) and therefore suitable for the measurement of PPS in plasma after its administration to man. The clearance of PPS was studied in 3 subjects receiving respectively 10, 50 and 100 mg intravenously (IV) and in 3 subjects receiving 35 mg subcutaneously (SC). PPS was still detectable 8 h after 50 and 100 mg IV and 6 h after 35 mg SC. The activated partial thromboplastin time (APTT) was, in comparison, relatively insensitive but for concentrations above 1 microgram/ml the values derived from the APTT and from the SDS-PAGE method fitted. The results were also in general agreement with those reported by McGregor et al. (5) who used a sensitive competitive binding assay. This indicates that low concentrations of PPS previously measured chemically are also pharmacologically active in plasma.
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PMID:Effect of pentosan polysulphate administration in man on the formation of covalent complexes between heparin cofactor II and thrombin generated in plasma by contact activation. 243 30

Antithrombin III (ATIII) is the main inhibitor of thrombin in adult plasma; alpha 2-macroglobulin (alpha 2M) and heparin cofactor II (HCII) are of lesser importance. The relative contributions of these inhibitors to the inactivation of thrombin may differ during the neonatal period and infancy, when plasma concentrations of alpha 2M are about twice as high as those of ATIII. We therefore compared the relative importance of these anti-proteases for the inhibition of 125I-thrombin in defibrinated pooled adult and neonatal plasma. Observations were also made in pooled plasma of 6 months old infants. 125I-thrombin-inhibitor complexes were quantitated after SDS-PAGE and autoradiography by scanning densitometry. Thrombin (2.5 NIH U/ml) was inhibited more slowly in neonatal than in adult plasma. However, both plasmas inhibited 88% of the added thrombin by 5 minutes. Alpha 2M inhibited consistently a larger fraction of thrombin in neonatal than in adult plasma. Consequently, the ratio of thrombin bound to ATIII over thrombin bound to alpha 2M was significantly lower in neonatal (less than 2.5) than in adult plasma (greater than 4.5; p less than 0.0001). In infant plasma, this ratio was less than 2.0. Upon addition of therapeutic amounts of heparin (0.4 U/ml), differences between the contributions of ATIII and alpha 2M to the inhibition of thrombin were no longer apparent, as over 90% of complexed thrombin was bound to ATIII in heparinized plasmas of all age groups. We conclude that alpha 2M is an important progressive inhibitor of thrombin in young infants. This finding may explain why healthy newborns rarely suffer from thrombosis, despite their low plasma ATIII levels.
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PMID:Alpha-2-macroglobulin is an important progressive inhibitor of thrombin in neonatal and infant plasma. 248 48


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