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 addition of excess sodium citrate to plasma was found to inhibit fibrin polymerisation (clot opacity) from patients with cirrhosis, hepatitis and hepatoma but not from normal controls. Abnormal clot opacity in plasma from patients with liver disease could be partly or completely abolished by removal of citrate ions by dialysis against citrate-free buffer, but not by dialysis against buffer containing citrate. Similar results were observed in plasma freed of calcium ions by treatment with EGTA. Treatment of plasma with neuraminidase largely abolished the inhibitory effect of excess citrate, and the thrombin times and clot opacity of asialofibrinogen were less affected by citrate than native fibrinogen. In addition, the effects of citrate on the clotting of purified, calcium-free fibrinogen from cirrhotic patients correlated with the sialic acid content. It is concluded that binding of citrate ions to fibrinogen renders the molecule acutely more sensitive to elevations in the sialic acid content, and that a simple plasma clot opacity test in the presence of excess citrate may be a useful aid in the differential diagnosis of liver disease. These findings may also explain why defects in fibrin polymerisation observed in plasma are not always reproduced in purified fibrinogen or fibrin monomer preparations.
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PMID:The role of sodium citrate in the dysfibrinogenaemia of liver disease. 672 77

Culture-produced subendothelium (SE) has been prepared from cultured porcine aortic endothelial cells (ECs) by a rapid freeze-thaw, ice-shearing method. En face preparations of this in situ SE material are essentially free of intact or damaged cells and cell debris and consisted of an extensive meshwork of microfibrillar and amorphous material. Washed porcine platelets reacted extensively with this SE material and were associated with the SE as single adherent platelets, single spread platelets, and varying-sized platelet aggregates or 'microthrombi'. Platelet aggregates were associated only with the damaged or frayed edges of the SE, and the platelets had undergone extensive SE-induced contraction and degranulation, as indicated by transmission electron microscopy. Platelet-SE interaction was affected by pH, calcium, platelet concentration, rapid shaking and exposure time. Platelet-SE interaction was significantly enhanced by the addition of 0.1-1% citrated plasma or purified porcine F.VIIIR:WF. Pretreatment of the SE with thrombin, elastase, neuraminidase or hyaluronidase had no effect on platelet-SE interaction, whereas pretreatment with pepsin, plasmin, trypsin, alpha-chymotrypsin or collagenase decreased or completely abolished all platelet-SE interaction. Extraction of the SE with various solutions (high salt, detergents, etc.) had no effect on platelet-SE interaction, only solutions containing sodium dodecyl sulfate completely abolished all platelet-SE interaction.
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PMID:Culture-produced subendothelium. I. Platelet interaction and properties. 680 25

The specificity of five monoclonal antibodies (P1-P6) against platelet surface components was determined by immunoprecipitation of surface-labelled platelets from normal donors and patients with known platelet glycoprotein defects, followed by analysis by gel electrophoresis. Three (P2, P4 and P6) precipitated glycoproteins IIb and IIIa and, in addition, P2 precipitated glycoprotein Ia. P1 precipitated normally only glycoprotein Ib also Ia when the platelets were pretreated with neuraminidase. P3 precipitated principally glycoprotein Ia but glycoprotein Ib was also weakly precipitated. The effects of the monoclonals on platelet function were tested. P1 and P2 completely inhibited and P3 slightly inhibited thrombin-induced platelet aggregation. P2 also inhibited collagen-induced aggregation and partially inhibited ADP-induced platelet aggregation. P3, P4 and P6 partially inhibited ADP-induced platelet aggregation. None had any effect on ristocetin-induced aggregation despite P1 and P3 binding to glycoprotein Ib. These results confirm the role of glycoproteins IIb and IIIa in aggregation induced by various agents and suggest that the function of glycoprotein Ib in thrombin-induced aggregation is more important than previously suspected and that glycoprotein Ia may also be involved in platelet functions.
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PMID:Monoclonal antibodies against platelet membrane glycoproteins. Characterization and effect on platelet function. 683 59

To further evaluate the role of sialic acid in the dysfibrinogenemia associated with liver disease, we studied the effect of removal of excess sialic acid residues from the fibrinogen of five patients with liver disease on the thrombin time and fibrin monomer aggregation. Patient fibrinogens containing 1.4-3.4 residues of sialic acid per molecule in excess of normal controls, with thrombin times 12-22 sec longer than normal and with abnormal fibrin monomer aggregation, were stripped of their excess sialic acid by incubation with Vibrio cholerae neuraminidase, followed by rapid removal of the enzyme by antineuraminidase antibody affinity chromatography. These partially desialylated patient fibrinogens, with a normal number of sialic acid residues remaining, exhibited normal thrombin times and normal fibrin monomer aggregation. Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of reduced normal, patient, and partially desialylated patient (sialyl-3H)-fibrinogen exhibited 60% of the radioactivity in the B beta chain and 40% in the gamma chain. There was no radioactivity detectable in the A alpha chain. These studies provide additional evidence that the increased sialic acid content of the acquired dysfibrinogenemia of liver disease is responsible for its functional defect and that the excess sialic acid is distributed on the B beta chain and gamma chains of the fibrinogen.
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PMID:The role of sialic acid in the dysfibrinogenemia associated with liver disease: distribution of sialic acid on the constituent chains. 683 20

The objective of the present study was to characterize the interaction between human platelets and vaccinia virus and to examine possible impairment of platelet functions. The vaccinia virus was selected for our model system because it lacks detectable neuraminidase activity. Platelets were incubated with purified viral particles labeled with 3H-thymidine and binding parameters were analyzed. Binding reached saturation with an average of 5 particles/platelet. It was not affected by the plasma but was sensitive to temperature and to metabolic inhibitors. 3H-thymidine-labeled vaccinia virus and formaldehyde-fixed platelets were used to measure viral adsorption. The adsorption was temperature-independent but was affected by ionic strength, indicating electrostatic interactions. Treatment of the fixed platelets with neuraminidase or with alkaline phosphatase reduced viral adsorption, indicating that sialate and phosphate residues on the platelet surface may be involved in the adsorption. Platelet activities were markedly affected by vaccinia virus. The virus caused a dramatic 14C-serotonin release with no added inducer. The release was inhibited by aspirin, a known inhibitor of serotonin release related to prostaglandin synthesis. Furthermore, the virus inhibited platelet aggregation, induced by either ADP, collagen, or thrombin. This study demonstrates that although vaccinia virus lacks neuraminidase activity, it does bind to platelets and affects their function.
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PMID:Interaction between vaccinia virus and human blood platelets. 705 66

The removal, with time, of sialic acid residues from human fibrinogen by neuraminidase, can be explained, as a result of a mathematical analysis, by the summation of two first order reactions with clearly different rate constants. Quantitative studies on the bound sialic acid to isolated fibrinogen chains, after reduction and alkylation of the partially desialylated fibrinogen, show that the fast reaction takes place with the sialic acid bound to the B beta chains. This indicates that the carbohydrate located in the gamma chains is somehow protected or less accessible to the enzyme. Coagulation with thrombin of desialylated fibrinogen shows that the aggregation rate increases linearly as the amount of sialic acid residues decreases, regardless of their location in the fibrinogen molecule.
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PMID:Desialylation of fibrinogen with neuraminidase. Kinetic and clotting studies. 714 11

51Cr-prelabelled endothelial cells (EC) in confluent monolayers were incubated in RPMI 1640 + foetal calf serum 20% (v/v) to which purified thrombin was added. Thrombin (greater than or equal to 0.1 NIH U/ml) significantly accelerated 51Cr-release and caused extensive but reversible cell "contraction". Thrombin-exposed EC reacted to a new dose of thrombin with no appreciable shape change, but 51Cr-efflux was again accelerated. EC exposed to thrombin pretreated with N-bromosuccinimide (modifying the macromolecular site) or phenylmethylsulfonyl fluoride (blocking the serine site) retained normal morphology and did not leak excess amounts of 51Cr. Antithrombin III also inhibited the effect of thrombin. Pretreatment of EC with either indomethacin, aspirin, sulfinpyrazone, pronase or neuraminidase did not influence the effect of subsequent thrombin exposure.
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PMID:Effects of thrombin on the integrity of monolayers of cultured human endothelial cells. 717 6

Platelets are required for certain experimental metastases. Several lines of animal tumor cells aggregate platelets in vitro and in vivo. Previous studies with one of these lines, an SV40-transformed 3T3 mouse fibroblast (SV3T3) have revealed that the platelet-aggregating material is an extractable membrane-associated sialolipoprotein which requires divalent cation, complement, and a heat-stable plasma component for activity. Little information is available on the interaction of human tumors with platelets. We now report on the ability of two human adenocarcinomas of the colon (LoVo and HCT-8) and an anaplastic mouse tumor (Hut-20) to aggregate platelets by a different mechanism, the generation of thrombin. These spontaneous cell lines aggregate human or rabbit platelet-rich plasma after a 1- to 2-min lag period. This is often followed by a visible clot. Unlike SV3T3 cells, aggregation by LoVo, HCT-8, and Hut-20 cells is not inhibited by neuraminidase, trypsin, or cobra venom factor. These three cell lines markedly shorten the recalcification time of citrated plasma, whereas SV3T3 cells do not. Phospholipase A2 treatment inhibits the shortening of the recalcification time for the three tumors; this parallels its inhibitory effect on platelet aggregation. LoVo, HCT-8, and Hut-20 cells generate thrombin via the "tissue factor" coagulation pathway (using coagulation factor-deficient substrates). Dansylarginine-N-(3-ethyl-1,5-pentanediyl)amide, a highly specific, potent antithrombin antagonist, inhibits LoVo-, HCT-8-, and Hut-20-induced platelet aggregation at 4 to 15 microM, whereas its effect on SV3T3 cells is negligible. If platelets are required for certain human tumor metastases, dansylarginine-N-(3-ethyl-1, 5-pentanediyl)amide, or other antithrombin agents, may prove to be valuable therapeutic agents.
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PMID:Inhibition of the platelet-aggregating activity of two human adenocarcinomas of the colon and an anaplastic murine tumor with a specific thrombin inhibitor, dansylarginine N-(3-ethyl-1,5-pentanediyl)amide. 730 74

The final stage in a series of blood coagulating reactions is fibrinogen-fibrin conversion by thrombin. This reaction consists of fibrinopeptide A and fibrinopeptide B release, polymerization of fibrin monomer, and stabilized fibrin formation by factor XIII. The latter two reactions require calcium. In the present study there was no difference in the rate of thrombin-induced fibrinopeptide release between fibrinogen and asialofibrinogen where sialic acid in the terminal end of carbohydrate moiety of fibrinogen was removed by neuraminidase, but turbidity associated with asialofibrin clot formation was increased more rapidly. In asialo-derivatives, the dissolution time of the clots in high concentrated urea solution tended to be shortened and rigidity as a gel tended to be decreased. In measurement by thromboelastography there was no difference in the reaction time (r) between fibrinogen and asialofibrinogen, but the maximum amplitude (ma) was obviously decreased in asialofibrinogen. Furthermore, when the rate of cross-link formation between gamma chains by F-XIII was compared, the production of gamma-dimer in the same reaction time was found to be lower and formation of stabilized fibrin tended to be retarded in asialofibrinogen. Sialic acid in fibrinogen thus may clearly influence the polymerization of fibrin-monomer and the formation of cross-linked fibrin in a series of reactions for fibrinogen-fibrin conversion. This may be consistent with the theory that fibrinogen sialic acid residues are low affinity calcium-binding sites and influence fibrin assembly.
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PMID:Sialic acid in fibrinogen: effects of sialic acid on fibrinogen-fibrin conversion by thrombin and properties of asialofibrin clot. 836 89

The serine proteinase catalyzed hydrolysis of N-ethoxycarbonyl-D-phenylalanyl-L-prolyl-alpha-azalysine p- nitrophenyl ester (Eoc-D-Phe-Pro-azaLys-ONp) was investigated at pH 6.2 and 21.0 degrees C. The results are consistent with the minimum three-step catalytic mechanism. The acylation step is rate limiting for human (Lys 77 species) and porcine plasmin, and for bovine beta-trypsin, the deacylation rate being limiting, on the other hand, for human and bovine alpha-, beta- and gamma-thrombin. Moreover the M(r) 33,000 species of human urokinase and the neuraminidase-treated porcine pancreatic beta-kallikrein-B do not catalyze the hydrolysis of the tripeptide. According to the specificity properties of the serine proteinases considered. Eoc-D-Phe- Pro-azaLys-ONp shows the characteristics of a novel, high selective and optimal chromogenic active site titrant for human and bovine alpha-, beta- and gamma-thrombin.
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PMID:N-ethoxycarbonyl-D-phenylalanyl-L-prolyl-alpha-azalysine p-nitrophenyl ester: a novel, high selective and optimal chromogenic active site titrant for human and bovine alpha-, beta- and gamma-thrombin. 875


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