Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.6 (thromboplastin)
 13,278 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

The Ca2+ dependence of factor Xa binding to phospholipid vesicles was measured in the presence and absence of factor Va. The increase in polarization of a fluorescently labeled derivative of factor Xa, [5-(dimethylamino)-1-naphthalenesulfonyl] glutamylglycylarginyl factor Xa (Dns-EGR-Xa), was used as a probe to measure the interaction of factor Xa with phospholipid. The Ca2+ concentration required for half-maximal binding of Dns-EGR-Xa to phospholipid vesicles was 3.5 X 10(-4) M in the presence of factor Va and 9.5 X 10(-4) M in the absence of factor Va. At a Ca2+ concentration of 5 X 10(-4) M, the binding of Dns-EGR-Xa to phospholipid-bound factor Va was near maximal, whereas there was no detectable interaction of Dns-EGR-Xa with phospholipid alone at this Ca2+ concentration as detected by fluorescence polarization. These results were qualitatively confirmed by high-performance liquid chromatography. The rate of hydrolysis of the factor Xa synthetic substrate, benzoylisoleucylglutamylglycylarginine p-nitroanilide, by factor Xa in the presence of factor Va and phospholipid decreased in a Ca2+-dependent manner. These data were analyzed as fraction of factor Xa bound to the phospholipid. A Ca2+ concentration of 2.7 X 10(-4) M resulted in half-maximal binding by this technique. The relationship observed between rates of prothrombin activation and Ca2+ concentration could be predicted quantitatively from calculations of local enzyme and substrate concentrations.
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PMID:Differentiation of enzyme and substrate binding in the prothrombinase complex. 662 14

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

We have recently shown that amino acid region 307-348 of factor Va heavy chain (42 amino acids, N42R) is critical for cofactor activity and may contain a binding site for factor Xa and/or prothrombin [(2001) J. Biol. Chem. 276, 18614-18623]. To ascertain the importance of this region for factor Va cofactor activity, we have synthesized eight overlapping peptides (10 amino acid each) spanning amino acid region 307-351 of the heavy chain of factor Va and tested them for inhibition of prothrombinase activity. The peptides were also tested for the inhibition of the binding of factor Va to membrane-bound active site fluorescent labeled Glu-Gly-Arg human factor Xa ([OG488]-EGR-hXa). Factor Va binds specifically to membrane-bound [OG488]-EGR-hXa (10nM) with half-maximum saturation reached at approximately 6 nM. N42R was also found to interact with [OG488]-EGR-hXa with half-maximal saturation observed at approximately 230 nM peptide. N42R was found to inhibit prothrombinase activity with an IC50 of approximately 250 nM. A nonapeptide containing amino acid region 323-331 of factor Va (AP4') was found to be a potent inhibitor of prothrombinase. Kinetic analyses revealed that AP4' is a noncompetitive inhibitor of prothrombinase with respect to prothrombin, with a K(i) of 5.7 microM. Thus, the peptide interferes with the factor Va-factor Xa interaction. Displacement experiments revealed that the nonapeptide inhibits the direct interaction of factor Va with [OG488]-EGR-hXa (IC50 approximately 7.5 microM). The nonapeptide was also found to bind directly to [OG488]-EGR-hXa and to increase the catalytic efficiency of factor Xa toward prothrombin in the absence of factor Va. In contrast, a peptadecapeptide from N42R encompassing amino acid region 337-351 of factor Va (P15H) had no effect on either prothrombinase activity or the ability of the cofactor to interact with [OG488]-EGR-hXa. Our data demonstrate that amino acid sequence 323-331 of factor Va heavy chain contains a binding site for factor Xa.
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PMID:Identification of a binding site for blood coagulation factor Xa on the heavy chain of factor Va. Amino acid residues 323-331 of factor V represent an interactive site for activated factor X. 1237 14

We have recently demonstrated that amino acid region 323-331 of factor Va heavy chain (9 amino acids, AP4') contains a binding site for factor Xa (Kalafatis, M., and Beck, D. O. (2002) Biochemistry 41, 12715-12728). To ascertain which amino acids within this region are important for the effector and receptor properties of the cofactor with respect to factor Xa, we have synthesized three overlapping peptides (5 amino acids each) spanning the amino acid region 323-331 and tested them for their effect on prothrombinase complex assembly and function. Peptide containing amino acids 323EYFIA327 alone was found to increase the catalytic efficiency of factor Xa but had no effect on the fluorescent anisotropy of active site-labeled factor Xa (human factor Xa labeled in the active site with Oregon Green 488; [OG488]-EGR-hXa). In contrast, peptide containing the sequence 327AAEEV331 was found to interact with [OG488]-EGR-hXa with half-maximal saturation reached at approximately 150 microm, but it was unable to produce a cofactor effect on factor Xa. Peptide 325FIAAE329 inhibited prothrombinase activity and was able to partially decrease the fluorescent anisotropy of [OG488]-EGR-hXa but could not increase the catalytic efficiency of factor Xa with respect to prothrombin. A control peptide with the sequence FFFIA did not increase the catalytic efficiency of factor Xa, whereas a peptide with the sequence AAEMI was impaired in its capability to interact with [OG488]-EGR-hXa. Two mutant recombinant factor Va molecules (Glu323 --> Phe/Tyr324 --> Phe, factor VaFF; Glu330 --> Met/Val331 --> Ile, factor VaMI) showed impaired cofactor activity when used at limiting cofactor concentration, whereas the quadruple mutant (Glu323 --> Phe/Tyr324 --> Phe and Glu330 --> Met/Val331 --> Ile, factor VaFF/MI) had no cofactor activity under similar experimental conditions. Our data demonstrate that amino acid residues Glu323, Tyr324, Glu330, and Val331 of factor Va heavy chain are critical for expression of factor Va cofactor activity.
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PMID:Amino acids Glu323, Tyr324, Glu330, and Val331 of factor Va heavy chain are essential for expression of cofactor activity. 1273 85

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

Because all three protein components of prothrombinase, factors (f) Xa and Va and prothrombin, bind to negatively charged membrane phospholipids, the exact role of the membrane in the prothrombinase reaction has not been fully understood. In this study, we prepared deletion derivatives of fXa and prothrombin in which both the Gla and first EGF-like domains of the protease (E2-fXa) as well as the Gla and both kringle domains of the substrate (prethrombin-2) had been deleted. The fVa-mediated catalytic activity of E2-fXa toward prethrombin-2 was analyzed in both the absence and presence of phospholipids composed of 80% phosphatidylcholine (PC) and 20% phosphatidylserine (PS). PCPS markedly accelerated the initial rate of prethrombin-2 activation by E2-fXa, with the cofactor exhibiting saturation only in the presence of phospholipids (apparent K(d) of approximately 60 nM). Competitive kinetic studies in the presence of the two exosite-1-specific ligands Tyr(63)-sulfated hirudin(54-65) and TM456 suggested that while both peptides are highly effective inhibitors of the fVa-mediated activation of prethrombin-2 by E2-fXa in the absence of PCPS, they are ineffective competitors in the presence of phospholipids. Since neither E2-fXa nor prethrombin-2 can interact with membranes, these results suggest that interaction of fVa with PCPS improves the affinity of the activation complex for proexosite-1 of the substrate. Direct binding studies employing OG(488)-EGR-labeled fXa and E2-fXa revealed that the interaction of the Gla domain of fXa with PCPS also induces conformational changes in the protease to facilitate its high-affinity interaction with fVa.
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PMID:Membrane-dependent interaction of factor Xa and prothrombin with factor Va in the prothrombinase complex. 1937 73