Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.5 (thrombin)
 33,306 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Phospholipids bearing a proportion of anionic species such as phosphatidylserine are necessary to promote the anticoagulant potential of the protein C pathway. Factor Xa (200 or 350 pM) was found to activate protein C in a thrombomodulin-independent reaction requiring only phospholipids in Al(OH)3,-adsorbed plasma resupplemented with physiological concentrations of protein C (70 nM) and protein S (130 nM). All experiments were performed in the presence of an excess of hirudin. The activity of activated protein C was assessed by the survival of factor Va. The optimal phospholipid concentration range was 5 to 25 microM with a proportion of phosphatidylserine of 50% (mol/mol) resulting in a half-life of factor Va of 7.5 min in the absence of protein S and 4.2 min in its presence. Dns-EGR-Xa, an inactive derivative of factor Xa, behaved as an apparent protector of factor Va. When replacing factor Xa, thrombin at 10 nM was not an efficient protein C activator in the absence of purified human placenta thrombomodulin. In the presence of 100 pM activated protein C, factor Va half-life was 2 min in the absence of protein S and 1.1 min in its presence in the above optimal phospholipid concentration range. The presence of protein S allowed reduction of phospholipid requirements. Annexin-V (placental anticoagulant protein-I), a potent phospholipid antagonist, fully protected factor Va from degradation by phospholipid-dependent mechanisms. Factor Va was partially protected in the plasma of a patient having experienced thrombosis associated with lupus-like anticoagulant and anti-phospholipid auto-antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:The catalytic role of anionic phospholipids in the activation of protein C by factor Xa and expression of its anticoagulant function in human plasma. 179 56

A quantitative and non-occlusive deep vein thrombosis model was developed in rabbits. We used this model to test the antithrombotic activity of the prothrombinase complex inhibitors factor rXai and its chemical analog glutamyl-glycyl-arginyl chloromethyl ketone inactivated human factor Xa (EGR-Xai), along with the thrombin inhibitors D-phenylalanyl-prolyl-arginyl chloromethyl ketone (PPACK) and heparin. Dose dependent effects of the inhibitors during constant infusion were monitored. Measurements included thrombus weights, hemostatic parameters and both cuticle and ear bleeding times. In this model, factor rXai and EGR-Xai had comparable in-vivo efficacy, and showed 80%-93% inhibition at plasma levels of 6.5 nM (rXai) and 8 nM (EGR-Xai). Effects on ex-vivo clotting times varied among the inhibitors. At 80-100% thrombus inhibition, factor rXai and EGR-Xai had no statistically significant effect, while PPACK extended thrombin clotting time (TCT) times 2.3-fold, and heparin prolonged both activated partial thromboplastin time (APTT), prothrombin time (PT) and TCT ex-vivo clotting times 6.9-, 1.2-, and 7-fold respectively. At these dosages, cuticle and ear bleeding times were prolonged for all inhibitors and showed increases of 177%-389% (cuticle) and 45%-129% (ear). Our results demonstrate that direct inhibition of prothrombinase complex assembly is effective in arresting venous thrombosis.
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PMID:A comparative study of prothrombinase and thrombin inhibitors in a novel rabbit model of non-occlusive deep vein thrombosis. 802 1

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

We evaluated the in vivo anti-metastatic activity of recombinant Ancylostoma caninum Anticoagulant Peptide (rAcAP), a potent (Ki = 265 pM) and specific active site inhibitor of human coagulation factor Xa originally isolated from bloodfeeding hookworms. Subcutaneous injection of SCID mice with rAcAP (0.01-0.2 mg/mouse) prior to tail vein injection of LOX human melanoma cells resulted in a dose dependent reduction in pulmonary metastases. In order to elucidate potential mechanisms of rAcAP's anti-metastatic activity, experiments were carried out to identify specific interactions between factor Xa and LOX. Binding of biotinylated factor Xa to LOX monolayers was both specific and saturable (Kd = 15 nM). Competition experiments using antibodies to previously identified factor Xa binding proteins, including factor V/Va, effector cell protease receptor-1, and tissue factor pathway inhibitor failed to implicate any of these molecules as significant binding sites for Factor Xa. Functional prothrombinase activity was also supported by LOX, with a half maximal rate of thrombin generation detected at a factor Xa concentration of 2.4 nM. Additional competition experiments using an excess of either rAcAP or active site blocked factor Xa (EGR-Xa) revealed that most of the total factor Xa binding to LOX is mediated via interaction with the enzyme's active site, predicting that the vast majority of cell-associated factor Xa does not participate directly in thrombin generation. In addition to establishing two distinct mechanisms of factor Xa binding to melanoma, these data raise the possibility that rAcAP's antimetastatic effect in vivo might involve novel non-coagulant pathways, perhaps via inhibition of active-site mediated interactions between factor Xa and tumor cells.
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PMID:Ancylostoma caninum anticoagulant peptide blocks metastasis in vivo and inhibits factor Xa binding to melanoma cells in vitro. 960 44

Optimal rates of factor X (FX) activation require occupancy of receptors for factor IXa (FIXa), factor VIII (FVIII), and FX on the activated platelet surface. The presence of FVIII and FX increases 5-fold the affinity of FIXa for the surface of activated platelets, and the presence of FVIII or FVIIIa generates a high affinity, low capacity specific FX-binding site on activated platelets. We have now examined the effects of FX and active site-inhibited FIXa (EGR-FIXa) on the binding of both FVIII and FVIIIa to activated platelets and show the following: (a) von Willebrand factor inhibits FVIII binding (K(i) = 0.54 nM) but not FVIIIa binding; (b) thrombin and the thrombin receptor activation peptide (SFLLRN amide) are the most potent agonists required for FVIII-binding site expression, whereas ADP is inert; (c) FVa does not compete with FVIIIa or FVIII for functional platelet-binding sites; and (d) Annexin V is a potent inhibitor of FVIIIa binding (IC(50) = 10 nM) to activated platelets. The A2 domain of FVIII significantly increases the affinity and stoichiometry of FVIIIa binding to platelets and contributes to the stability of the FX-activating complex. Both FVIII and FVIIIa binding were specific, saturable, and reversible. FVIII binds to specific, high affinity receptors on activated platelets (n = 484 +/- 59; K(d) = 3.7 +/- 0.31 nM) and FVIIIa interacts with an additional 300-500 sites per platelet with enhanced affinity (K(d) = 1.5 +/- 0.11 nM). FVIIIa binding to activated platelets in the presence of FIXa and FX is closely coupled with rates of F-X activation. The presence of EGR-FIXa and FX increases both the number and the affinity of binding sites on activated platelets for both FVIII and FVIIIa, emphasizing the validity of a three-receptor model in the assembly of the F-X-activating complex on the platelet surface.
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PMID:Structural and functional characterization of platelet receptor-mediated factor VIII binding. 1077 12

To test the hypothesis that factor Xa (fXa) interacts with protein S, fXa was labeled active-site specifically with a dansyl (D) dye via a Glu-Gly-Arg (EGR) tether to yield DEGR-fXa(i). When protein S was added to phosphatidylcholine/phosphatidylserine (PC/PS, 4:1) vesicle-bound DEGR-fXa(i), the anisotropy of the dansyl moiety was altered from 0.219 +/- 0.002 to 0.245 +/- 0.003. This change in dansyl anisotropy was not observed when DEGR-Xa(i) was titrated with protein S in the absence of PC/PS vesicles, or in the presence of 100% PC vesicles, or when PC/PS vesicle-bound DEGR-fXa(i) was titrated with thrombin-cleaved protein S. The protein S-dependent dansyl fluorescence change was specific for fXa because it was not observed for two homologous and similarly labeled DEGR-fIXa(i) and DEGR-fVIIa(i). Furthermore, protein S specifically and saturably altered the fluorescence anisotropy of PC/PS-bound active site-labeled LWB-FPR-fXa(i) (Kd = 33 nm) and was photocross-linked to PC/PS-bound LWB-FPR-fXa(i) analog, independently confirming the above results. Chemically synthesized microprotein S, comprising residues 1-116 of protein S and including the gamma-carboxyglutamic-rich domain, the thrombin-sensitive region (TSR), and the first epidermal growth factor-like domain (EGF1) of protein S, altered the anisotropy of PC/PS-bound DEGR-fXa(i) from 0.219 to 0.242, similar to the effect of the protein S titration (Kd = 303 nm), suggesting that microprotein S binds to DEGR-fXa(i). To identify individual protein S domain(s) that binds DEGR-fXa(i), the EGF1 and TSR domains were chemically synthesized and studied. The TSR altered the anisotropy of DEGR-fXa(i) by approximately 16% (Kd = 3.9 microm), but the EGF1 domain had no effect on the signal. In controls, the TSR domain did not alter the anisotropy of DEGR-fIXa(i) and DEGR-fVIIa(i), respectively. These data demonstrate that membrane-bound fXa binding to protein S involves the TSR of protein S.
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PMID:The thrombin-sensitive region of protein S mediates phospholipid-dependent interaction with factor Xa. 1878 85

A number of alanine and more conservative mutants of residues in the fourth domain of thrombomodulin (TM) were prepared and assayed for protein C activation and for thrombin binding. Several of the alanine mutations appeared to cause misfolding or structural defects as assessed by poor expression and/or NMR HSQC experiments, while more conservative mutations at the same site appeared to allow correct folding and preserved activity. Several of the conservative mutants bound more weakly to thrombin despite the fact that the fourth domain does not directly contact thrombin in the crystal structure of the thrombin-TM complex. A few of the mutant TM fragments bound thrombin with an affinity similar to that of the wild type but exhibited decreases in k cat for protein C activation. These mutants were also less able to cause a change in the steady state fluorescence of fluorescein-EGR-chloromethylketone bound to the active site of thrombin. These results suggest that some residues within the fourth domain of TM may primarily interact with protein C but others are functionally important for altering the way TM interacts with thrombin. Residues in the fourth domain that primarily affect k cat for protein C activation may do this by changing the active site of thrombin.
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PMID:Mutations in the fourth EGF-like domain affect thrombomodulin-induced changes in the active site of thrombin. 1880 1