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:3.4.21.6 (thromboplastin)
13,278 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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

Since plasma protein S serves an anticoagulant function by mechanisms which are not completely understood, its possible interaction with Factor Va was investigated. Human protein S bound to immobilized human Factor Va in a calcium-dependent, saturable, and reversible manner and Factor Va bound similarly to immobilized protein S. Binding of protein S to immobilized Factor V was greatly enhanced by pretreatment of the surface-bound Factor V with increasing doses of thrombin up to 1 unit/ml. Binding of protein S to Factor Va was also demonstrated in fluid phase with a Kd of 33 +/- 9 nM. Biotin-labeled heavy chain of Factor Va bound to immobilized protein S, and this binding was reversed by a 17-fold molar excess of intact unlabeled Factor Va. Protein S competed efficiently with prothrombin for binding to immobilized Factor Va. The prothrombinase activity in a reaction mixture of purified clotting factors was inhibited by protein S and exhibited a pattern of mixed inhibition. The concentration of protein S needed for 50% inhibition of the prothrombinase activity of a mixture containing 1 nM Factor Xa, 20 pM Factor Va, and 50 microM phospholipids was about 16 nM. Since not all protein S preparations exhibited this degree of prothrombinase inhibitory activity, extensive control experiments were performed to verify that the inhibitory activity was associated with protein S during immunoaffinity chromatography and was not caused by traces of activated protein C in the protein S preparations. These data show that protein S has an anticoagulant function which is independent of activated protein C and, at least in part, that this is because of its competition with prothrombin for direct binding to Factor Va.
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PMID:Binding of protein S to factor Va associated with inhibition of prothrombinase that is independent of activated protein C. 842 62

Resistance to activated protein C (APC), which is the most prevalent pathogenetic risk factor of thrombosis, is linked to a single point-mutation in the factor V (FV) gene, which predicts replacement of Arg (R) at position 506 with a Gln (Q). This mutation modifies one of three APC-cleavage sites in the heavy chain of activated FV (FVa), suggesting that mutated FVa (FVa:Q506) is at least partially resistant to APC-mediated degradation. To elucidate the molecular mechanisms of APC-resistance and to investigate the functional properties of FV in APC resistance, FV:Q506 was purified from an individual with homozygosity for the Arg to Gln mutation. Intact and activated FV:Q506 were demonstrated to convey APC resistance to FV-deficient plasma. Thrombin- or factor Xa-activated FV:Q506 were found to be approx. 10-fold less sensitive to APC-mediated degradation than normal FVa, at both high and low phospholipid concentrations. The degradation pattern observed on Western blotting suggested that FVa:Q506 was not cleaved at position 506. However, it was slowly cleaved at Arg306, which explains the partial APC sensitivity of FVa:Q506. FV is initially activated during clotting and then rapidly inactivated in a process which depends on the integrity of the protein C anticoagulant system. During clotting of APC-resistant plasma, FV:Q506 was activated in a normal fashion, but then only partially inactivated. In conclusion, the reduced sensitivity of FVa:Q506 to APC-mediated degradation is the molecular basis for the life-long hypercoagulable state which constitutes a risk factor for thrombosis in APC-resistant individuals.
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PMID:Molecular mechanisms of activated protein C resistance. Properties of factor V isolated from an individual with homozygosity for the Arg506 to Gln mutation in the factor V gene. 857 79

A specific activator of blood coagulation factor X was purified from the venom of Bungarus fasciatus by gel filtration and by ion-exchange chromatography on a Mono-Q column (FPLC). It consisted of a single polypeptide chain, with a mol. wt of 70,000 in reducing and non-reducing conditions. The enzyme had an amidolytic activity towards the chromogenic substrates S-2266 and S-2302 but it did not hydrolyse S-2238, S2251 or S-2222, which are specific substrates for thrombin, plasmin and factor Xa, respectively. The enzyme activated factor X in vitro and the effect was Ca2+ dependent with a Hill coefficient of 7.9. As with physiological activators, the venom activator cleaves the heavy chain of factor X, producing the activated factor Xa alpha. The purified factor X activator from B. fasciatus venom did not activate prothrombin, nor did it cleave or clot purified fibrinogen. The amidolytic activity and the factor X activation activity of the factor X activator from B. fasciatus venom were readily inhibited by serine protease inhibitors such as diisopropyl fluorophosphate (DFP), phenylmethanesulfonyl fluoride (PMSF), benzamidine and by soybean trypsin inhibitor but not by EDTA. These observations suggest that the factor X activator from B. fasciatus venom is a serine protease. It therefore differs from those of activators obtained from Vipera russelli and Bothrops atrox venoms, which are metalloproteinases.
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PMID:An activator of blood coagulation factor X from the venom of Bungarus fasciatus. 859 79

We have previously showed that factor X activator of Russell's viper venom (RVV-X) contains six N-linked oligosaccharide chains: four in the heavy chain and one in each of the two light chains [Gowda, D.C., Jackson, C.M., Hensley, P., & Davidson, E.A. (1994) J. Biol. Chem. 269, 10644-10650]. In the present study, we have investigated the role of the carbohydrate moieties in the structure and functional activity of RVV-X. Sequential removal of sugar residues from the terminal ends by exoglycosidases, up to 50% of total carbohydrates, did not significantly alter the activity of RVV-X, demonstrating that the peripheral carbohydrate moieties are not involved in interactions with factor X. However, removal of whole oligosaccharide chains by N-glycanase caused an almost total loss of the ability of RVV-X to activate factor X to factor Xa. In parallel with these observations, circular dichroism spectroscopy showed that complete deglycosylation, but not the removal of peripheral sugars, caused a significant change in the secondary structure. Together, these data demonstrate that the oligosaccharide chains are necessary for the functional activity, and that the trimannosylchitobiose core residues are sufficient for the maintenance of the native polypeptide structure.
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PMID:Core sugar residues of the N-linked oligosaccharides of Russell's viper venom factor X-activator maintain functionally active polypeptide structure. 863 44

Prekallikrein, the precursor to the serine proteinase kallikrein, circulates in plasma in an equimolar complex with H-kininogen. The binding to H-kininogen is mediated by the kallikrein heavy chain consisting of four "apple" domains, A1-A4, which attaches to H-kininogen with high specificity and affinity (KD = 83 nM). At least two distinct portions of the kallikrein heavy chain form this H-kininogen binding site: a proximal segment located in the NH2-terminal fragment of the heavy chain encompassing A1, and distal segment(s) located in COOH-terminal fragment spanning domains A2-A4. The proximal binding segment has been located to amino acid positions 56-86 of A1. To precisely map the distal binding segment, we have identified monoclonal antibodies directed to the COOH-terminal fragment which interfere with the H-kininogen-prekallikrein complex formation. Monoclonal antibody 13G11 binds to recombinant apple domain A4 but not to domain A3 of the prekallikrein heavy chain. Deletion mutagenesis of domain A4 narrowed down the target epitope of 13G11 to the center portion of domain A4, positions 284-331. Direct binding studies of H-kininogen to various domain A4 constructs revealed that the distal H-kininogen binding portion is located on a segment of 48 residues, which overlaps the 13G11 epitope. Hence the tight interaction of H-kininogen and prekallikrein is mediated by at least two separate sequence segments located in domains A1 and A4, respectively, of the prekallikrein heavy chain. The isolated distal binding segment significantly prolongs the partial thromboplastin time of reconstituted Williams plasma thus stressing the critical role of the prekallikrein-H-kininogen complex formation in the initiation of the endogenous blood coagulation cascade.
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PMID:Mapping of the discontinuous kininogen binding site of prekallikrein. A distal binding segment is located in the heavy chain domain A4. 866 5

The activation of factor X by the extrinsic coagulation system results from the action of an enzyme complex composed of factor VIIa bound to tissue factor on phospholipid membranes in the presence of calcium ions (extrinsic Xase complex). Proteolysis at the Arg52-Ile53 peptide bond in the heavy chain of factor X leads to the formation of the serine protease, factor Xa, and the generation of a heavily glycosylated activation peptide comprising residues 1-52 of the heavy chain. The role of the activation peptide region in mediating substrate recognition and cleavage by the extrinsic Xase complex is unclear. The protease Agkistrodon rhodostoma hydrolase gamma (ARHgamma), from the venom of the Malayan pit viper, was used to selectively cleave human factor X in the activation peptide region. Three cleavage sites were found within this region and gave products designated Xdes1-34, Xdes1-43, and Xdes1-49. The products were purified to yield Xdes 1-49 and a mixture of Xdes 1-34 and Xdes 1-43. Reversed phase high pressure liquid chromatography analysis indicated that the cleaved portion of the activation peptide was likely removed during purification. All cleaved species were inactive and could be completely activated to factor Xa by the extrinsic Xase complex or by a purified activator from Russell's viper venom. Steady state kinetic studies using tissue factor reconstituted into membranes yielded essentially equivalent kinetic constants for the activation of intact factor X and the cleaved derivatives under a wide range of conditions. Since Xdes 1-49 lacks all but three residues of the activation peptide and is devoid of the carbohydrate present in this region, the data suggest that the specific recognition of human factor X by the extrinsic Xase complex is not achieved through specific interactions with residues 1-49 of the activation peptide or with carbohydrate structures attached to these residues.
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PMID:Role of the activation peptide domain in human factor X activation by the extrinsic Xase complex. 866 1

Blood coagulation factor Xa (FXa) has recently been shown to function as a plasminogen receptor in the presence of procoagulant phospholipid (phosphatidylserine; PS) and Ca2+. In the current work, the possible effect of autoproteolytic and plasmin-mediated cleavage of FXa on complex formation with plasminogen was investigated. 125I-plasminogen binding to derivatives of FXa electrotransferred to polyvinylidene difluoride revealed that the autoproteolytic conversion of FXaalpha to FXabeta was required for the expression of a plasminogen binding site. In the presence of PS and Ca2+, plasmin was shown to convert FXaalpha to a FXabeta-like species at least 3 orders of magnitude faster than the autoproteolytic mechanism. This also resulted in the exposure of a plasminogen binding site. Further processing by plasmin generated a fragment (33 kDa) due to cleavage at Gly331 in the FXa heavy chain. Production of this species enhanced apparent plasminogen binding compared with FXabeta and resulted in the loss of FXa amidolytic and clotting activity. In the absence of either PS or Ca2+, the plasmin-mediated fragmentation of FXaalpha was altered to include a FXabeta-like molecule and a species (40 kDa) with intact beta-heavy chain disulfide linked to a COOH-terminal fragment of the light chain starting at Tyr44. Neither of these products was observed to interact with plasminogen. The 40-kDa species had amidolytic activity comparable with FXaalpha but inhibited clotting activity. Cumulatively the data provide the first evidence for a functional difference between the FXa subforms and suggest a mechanism where autoproteolysis and plasmin-mediated cleavage modulate the function of FXaalpha from a procoagulant enzyme to a profibrinolytic plasminogen receptor.
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PMID:Autoproteolysis or plasmin-mediated cleavage of factor Xaalpha exposes a plasminogen binding site and inhibits coagulation. 866 21

A soluble form of the endothelial cell protein C receptor (EPCR) was analyzed for the ability to modulate the functional properties of protein C and activated protein C (APC). In a plasma clotting system initiated with factor Xa, EPCR blocked the anticoagulant activity of APC in a dose-dependent fashion. EPCR had no influence on clotting in the absence of APC. Consistent with the plasma results, EPCR slowed the proteolytic inactivation of factor Va by slowing both of the key proteolytic cleavages in the heavy chain of factor Va. EPCR did not prevent protein C activation by the soluble thrombin-thrombomodulin complex, did not alter the inactivation of APC by alpha1-antitrypsin or protein C inhibitor, and did not influence the kinetics of peptide paranitroanilide substrate cleavage significantly. We conclude that EPCR binds to an exosite on APC that selectively modulates the enzyme specificity in a manner reminiscent of the influence of thrombomodulin on thrombin.
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PMID:The endothelial cell protein C receptor. Inhibition of activated protein C anticoagulant function without modulation of reaction with proteinase inhibitors. 866 74


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