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)

In order to elucidate the role of protein C (PC) in the rat, we expressed, purified, and characterized recombinant rat PC. The purified recombinant rat PC was 70-90% two-chain (41 kDa heavy chain; 22 and 23 kDa light chain) and 10-30% single-chain (61 kDa). Amino acid analysis confirmed the presence of 10 moles of gamma-carboxyglutamic acid residues per mol of protein. For comparison, plasma rat PC was purified from a barium citrate precipitate using similar method. Plasma rat PC was a two-chain form (41 kDa heavy chain; 22 kDa light chain) with no detectable single-chain nor 23 kDa light chain. For determination of the in vitro secreted species, primary cultured rat hepatocytes were incubated for 6 h with methionine-free MEM containing vitamin K1, aprotinin, and [35S]methionine. The supernatant was immunoprecipitated and analyzed by SDS-PAGE followed by autoradiography. Approximately 90% of the PC radioactivity migrated as a two-chain molecule. These results indicate that rat PC is secreted mainly as a two-chain molecule from the liver. PROTAC-activated forms of recombinant rat PC, plasma rat PC, and plasma human PC hydrolyzed the S-2366 chromogenic substrate at the same rate. Recombinant rat PC was also activated by the thrombin-thrombomodulin complex at a rate similar to plasma rat PC. The anticoagulant activities of the three activated PCs were examined in rat plasma. Both recombinant and plasma rat PC prolonged the activated partial thromboplastin time in a dose-dependent manner, but plasma human PC was less effective.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Recombinant rat protein C: comparative studies of structure, function and synthesis with plasma protein C. 816 47

The interactions of the isolated heavy and light chains of factor Va with factor Xa were evaluated using active-site-modified factor Xa [(carboxytetramethyl)rhodamine-Glu-Gly- Arg-factor Xa (ctr-EGR-Xa)]. The Kd for the factor Va heavy-chain interaction with ctr-EGR-Xa was 60 microM. A series of monoclonal antibodies directed against bovine factor Va were tested for their ability to inhibit thrombin formation in an assay using the fluorescent thrombin inhibitor dansylarginine N,N-(3-ethyl-1,5-pentanediyl)amide (DAPA). Monoclonal antibody alpha BFV-4, which recognizes the light chain of the cofactor, was found to inhibit the formation of thrombin. Similarly, monoclonal antibody alpha BFV-5, which is directed against the heavy chain of the cofactor, was found to inhibit thrombin formation. In contrast, monoclonal antibody alpha BFV-1, also directed against the heavy chain of the cofactor, did not inhibit thrombin generation by the prothrombinase complex. Monoclonal antibodies alpha BFV-4 and alpha BFV-5 inhibited the interaction of active-site-modified radiolabeled factor Xa (125I-Xa-EGR) with factor Va bound to PC/PS-coated microtiter wells, whereas nonimmune mouse IgG did not have any effect on the 125I-Xa-EGR.membrane-bound factor Va interaction. The antibodies effect upon the phospholipid-independent interaction between the cofactor and ctr-EGR-Xa was evaluated by analytical ultracentrifugation. Both alpha BFV-4 and alpha BFV-5 inhibited the phospholipid-independent interaction between factor Va and ctr-EGR-Xa.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Contribution of the heavy and light chains of factor Va to the interaction with factor Xa. 820 89

Factor VIII and factor V function as cofactors in the blood coagulation cascade to accelerate the rate of activation of factor X and prothrombin, respectively. Both cofactors require proteolytic activation by either activated factor X or thrombin for functional activity. Human factor VIII and factor V expressed in mammalian cells are both modified by posttranslational sulfation of tyrosine residues. In the present study, the posttranslational addition of sulfate in factor V expressed in transfected Chinese hamster ovary (CHO) cells was demonstrated by [35S]sulfate incorporation into the thrombin-cleaved 94-kDa heavy chain and the 150-kDa activation peptide. The presence of tyrosine sulfate in recombinant factor V was confirmed by barium hydroxide hydrolysis and two-dimensional thin-layer electrophoresis. The importance of sulfation for factor V secretion and activity was evaluated by characterizing factor V expressed in Chinese hamster ovary cells grown in the presence of sodium chlorate, a potent inhibitor of posttranslational sulfation in intact cells. Increasing concentrations of sodium chlorate inhibited the incorporation of [35S]sulfate into factor V but did not inhibit the synthesis or secretion of factor V. However, the specific activity of factor V secreted in the presence of sodium chlorate was reduced 5-fold. The reduced activity was attributed to (1) slower cleavage and activation by thrombin and (2) a reduced intrinsic activity of factor Va. In contrast, sulfation of factor V did not affect the rate of activation mediated by factor Xa. These results show that sulfation of factor V is required for efficient thrombin activation but not for activation by factor Xa.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Posttranslational sulfation of factor V is required for efficient thrombin cleavage and activation and for full procoagulant activity. 820 29

A recombinant human prothrombin was prepared in which Arg155 was replaced by Ala. The recombinant prothrombin was converted into a meizothrombin derivative (R155A meizothrombin) that was resistant to autocatalytic removal of the fragment 1 domain. R155A meizothrombin appeared to be a potent factor V activator in reaction mixtures that contained negatively charged phospholipid vesicles. Factor V activation by R155A meizothrombin was characterized by second-order rate constants of 0.06 x 10(6) M-1 S-1 in the absence of phospholipid and 18 x 10(6) M-1 S-1 in the presence of 60 microM phospholipid vesicles composed of a 10:90 mol/mol mixture of phosphatidylserine (PS) and phosphatidylcholine (PC). The rate constant for thrombin-catalyzed activation of factor V was hardly affected by the presence of phospholipid vesicles and was 4.0 x 10(6) M-1 S-1. The initial rate of activation of 3 nM factor V by R155A meizothrombin was a function of the concentration of PS/PC vesicles present in the reaction mixture, and the calculated rate constant reached a plateau value at > or = 50 microM PS/PC. Gel electrophoretic analysis of factor V activation showed that R155A meizothrombin and thrombin cleaved the susceptible peptide bonds in factor V at different rates. However, both activators finally generated a factor Va molecule composed of a heavy chain with an M(r) of 104,000 and a light chain doublet with M(r) values of 74,000 and 71,000. Since meizothrombin is one of the major reaction products formed during the initial phase of prothrombin activation, these findings are indicative of a significant contribution of meizothrombin to in vivo factor V activation.
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PMID:Activation of human factor V by meizothrombin. 820 91

The activation of factor VIII (fVIII) by thrombin is associated with heavy chain cleavages at Arg372 and Arg740 and light chain cleavage at Arg1689. In a defined, plasma-free assay of fVIII activation and at physiological ionic strength and pH, heparin inhibited the rate of activation of either human or porcine fVIII by thrombin in either the presence or absence of von Willebrand factor (vWf). The inhibitory effect of heparin was associated with inhibition of all three thrombin-catalyzed bond cleavages. At plasma concentrations of fVIII (approximately 1 nM) and vWf (approximately 35 nM), the rate of fVIII activation was inhibited by 50% at approximately 0.1 unit/ml heparin, which is below the normal range of heparin concentrations in plasma during therapeutic anticoagulation (0.2-0.7 unit/ml). We propose that, in addition to catalyzing the inhibition of thrombin and other intrinsic pathway coagulation proteases by antithrombin, heparin functions as an anticoagulant by direct inhibition of the activation of the fVIII-vWf complex by thrombin.
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PMID:Inhibition by heparin of thrombin-catalyzed activation of the factor VIII-von Willebrand factor complex. 827 57

Electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry (MS/MS) have been used for the first time to study the interaction of human alpha-thrombin with methyl 3-(2-methyl-1-oxopropoxy)[1]benzothieno[3,2-b]furan-2-carbox ylate (LY806303; 1), a potent and selective inhibitor whose mechanism of action was never fully defined. Using ESI-MS, it is shown that inhibitor 1 covalently modifies human alpha-thrombin as evidenced by a shift in the molecular weight of the native protein by 72 Da, which is consistent with isobutyrylation (C4H7O; 71 Da) of the enzyme at a single site. Tryptic digestion of the modified protein and tandem mass spectral analysis of isolated peptide fragments indicate that compound 1 acylates Ser-205 of the heavy chain of alpha-thrombin. Ser-205, along with His-43 and Asp-99 make up the catalytic triad within the active site of thrombin.
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PMID:Characterization of the interaction between human alpha-thrombin and methyl 3-(2-methyl-1-oxopropoxy)[1]benzothieno[3,2-b]furan-2-carboxylate (LY806303) using electrospray mass spectrometry and tandem mass spectrometry. 836 Aug 80

Thrombin-induced platelet aggregation has been suggested to play an important role in reocclusion following thrombolytic therapy of angioplasty for treatment of myocardial infarction. We previously demonstrated that aggregation of washed platelets by thrombin is accompanied by cleavage of aggregin, a putative ADP receptor, and that these events are indirectly mediated by calpain, expressed on the surface of the external membrane. High-molecular-mass kininogen (HK) contains, in its heavy chain, domain 2, which is responsible for its action as a potent inhibitor of platelet calpain. Domain 3 of the heavy chain of HK directly inhibits binding of thrombin to platelets, confounding mechanistic studies using the entire molecule. Moreover, HK, a protease of 120 kDa, is unsuitable as a potential pharmacological agent. The highly conserved sequence Gln-Val-Val-Ala-Gly, present in HK and its evolutionary precursors, the cystatins, is thought to be involved in the binding of cysteine proteases but is, itself, not inhibitory. An affinity analog, Phe-Gln-Val-Val-Cys(Npys)-Gly-NH2(Npys, 3-nitro-2-sulfenylpyridine), P1, corresponding to the thiol-protease-binding sequence in HK and containing a ligand, Npys, that can react with the free sulfhydryl group in the active site of calpain, was synthesized. P1 was an irreversible inhibitor of platelet calpain. P1 selectively inhibited thrombin-induced aggregation of washed platelets and platelets in plasma, but did not inhibit the aggregatory effects of other platelet agonists. P1 did not inhibit the amidolytic activity and coagulant activity of thrombin. Unlike HK, P1 did not inhibit binding of thrombin to washed platelets. P1 did not inhibit thrombin-induced platelet-shape change. P1 neither raised intracellular levels of cAMP nor did it interfere with the ability of thrombin to antagonize the rise in intracellular levels of cAMP induced by iloprost, an analog of prostaglandin I2. The design and synthesis of P1 could leave to the development of a new class of inhibitors that selectively block thrombin-induced platelet aggregation while sparing other functions of this pathophysiological protease and without inhibiting the action of other platelet agonists.
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PMID:Modulation of thrombin-induced platelet aggregation by inhibition of calpain by a synthetic peptide derived from the thiol-protease inhibitory sequence of kininogens and S-(3-nitro-2-pyridinesulfenyl)-cysteine. 838 1

Activated protein C (APC) exerts its physiologic anticoagulant role by proteolytic inactivation of the blood coagulation cofactors Va and VIIIa. The synthetic peptide-(311-325) (KRNRTFVLNFIKIPV), derived from the heavy chain sequence of APC, potently inhibited APC anticoagulant activity in activated partial thromboplastin time (APTT) and Xa-1-stage coagulation assays in normal and in protein S-depleted plasma with 50% inhibition at 13 microM peptide. In a system using purified clotting factors, peptide-(311-325) inhibited APC-catalyzed inactivation of factor Va in the presence or absence of phospholipids with 50% inhibition at 6 microM peptide. However, peptide-(311-325) had no effect on APC amidolytic activity or on the reaction of APC with the serpin, recombinant [Arg358]alpha 1-antitrypsin. Peptide-(311-325) surprisingly inhibited factor Xa clotting activity in normal plasma, and in a purified system it inhibited prothrombinase activity in the presence but not in the absence of factor Va with 50% inhibition at 8 microM peptide. The peptide had no significant effect on factor Xa or thrombin amidolytic activity and no effect on the clotting of purified fibrinogen by thrombin, suggesting it does not directly inhibit these enzymes. Factor Va bound in a dose-dependent manner to immobilized peptide-(311-325). Peptide-(311-315) inhibited the binding of factor Va to immobilized APC or factor Xa.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Interactions and inhibition of blood coagulation factor Va involving residues 311-325 of activated protein C. 840 Dec 32

Factor Va is an essential cofactor in factor Xa-catalyzed prothrombin activation. Purified human factor Va appears to consist of a heavy chain (M(r) approximately 105,000) and a light chain doublet with M(r) approximately 74,000 and approximately 71,000. We separated factor Va by chromatography on a Mono-S column into two fractions, designated factors Va1 and Va2. Factor Va1 contains the light chain with M(r) approximately 74,000, and factor Va2 exclusively contains the light chain with M(r) approximately 71,000. The two forms of factor Va express different cofactor activities when prothrombin is activated at low phospholipid concentrations or on membranes containing low amounts of phosphatidylserine in phosphatidylcholine. Compared with factor Va2, much higher amounts of factor Va1 are required for factor Xa. Va complex formation at the membrane surface. Once incorporated into the prothrombinase complex, factors Va1 and Va2 are equally active in prothrombin activation. This indicates that the two forms of factor Va do not differ in their ability to promote the catalytic activity of factor Xa or to interact with prothrombin. Direct binding experiments show that the different cofactor activities are explained by a greatly impaired ability of factor Va1 to bind to negatively charged membranes. Factor V is also separated into two protein peaks after chromatography on a Mono-S column. Upon incubation with thrombin, the first peak yields factor Va1 and the second peak factor Va2. The same two forms of factor Va were generated when freshly prepared plasma samples or platelet suspensions were treated with thrombin. This shows that the heterogeneity of the light chain domain is an intrinsic property of both plasma and platelet factor V. It is hypothesized that the heterogeneity is caused by small differences in the carboxylterminal C2 domain of factor V that are introduced as the result of post-ribosomal processing.
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PMID:Characterization of two forms of human factor Va with different cofactor activities. 840 49

Platelet activation leads to the incorporation of 32[PO4(2-)] into bovine coagulation factor Va and recombinant human factor VIII. In the presence of the soluble fraction from thrombin-activated platelets and (gamma-32P) adenosine triphosphate, radioactivity is incorporated exclusively into the M(r) = 94,000 heavy chain (H94) of factor Va and into the M(r) = 210,000 to 90,000 heavy chains as well into the M(r) = 80,000 light chain of factor VIII. Proteolysis of the purified phosphorylated M(r) = 94,000 factor Va heavy chain by activated protein C (APC) gave products of M(r) = 70,000, 24,000, and 20,000. Only the intermediate M(r) = 24,000 fragment contained radioactivity. Because the difference between the M(r) = 24,000 and M(r) = 20,000 fragments is located on the COOH-terminal end of the bovine heavy chain, phosphorylation of H94 must occur within the M(r) = 4,000 peptide derived from the carboxyl-terminal end of H94 (residues 663 through 713). Exposure of the radioactive factor VIII molecule to thrombin ultimately resulted in a nonradioactive light chain and an M(r) = 24,000 radioactive fragment that corresponds to the carboxyl-terminal segment of the A1 domain of factor VIII. Based on the known sequence of human factor VIII, phosphorylation of factor VIII by the platelet kinase probably occurs within the acidic regions 337 through 372 and 1649 through 1689 of the procofactor. These acidic regions are highly homologous to sequences known to be phosphorylated by casein kinase II. Results obtained using purified casein kinase II gave a maximum observed stoichiometry of 0.6 mol of 32[PO4(2-)]/mol of factor Va heavy chain and 0.35 mol of 32[PO4(2-)]/mol of factor VIII. Phosphoamino acid analysis of phosphorylated factor Va by casein kinase II or by the platelet kinase showed only the presence of phosphoserine while phosphoamino acid analysis of phosphorylated factor VIII by casein kinase II showed the presence of phosphothreonine as well as small amounts of phosphoserine. The platelet kinase responsible for the phosphorylation of the two cofactors was found to be inhibited by several synthetic protein kinase inhibitors. Finally, partially phosphorylated factor Va was found to be more sensitive to APC inactivation than its native counterpart. Our findings suggest that phosphorylation of factors Va and VIIIa by a platelet casein kinase II-like kinase may downregulate the activity of the two cofactors.
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PMID:Phosphorylation of factor Va and factor VIIIa by activated platelets. 842 63


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