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 1985 we isolated a new vascular anticoagulant protein VAC alpha, now called annexin V, with a high binding affinity (Kd less than 10(-10) M) for phospholipids. Its anticoagulant effect was attributed to displacement of coagulation factors from the phospholipid membrane. The present study demonstrates that the inhibition of prothrombinase activity by annexin V strongly depends on the curvature of the membrane surface and on the calcium concentration. Half-maximal inhibition of prothrombinase on and binding of annexin V to small vesicles, composed of 20% phosphatidylserine and 80% phosphatidylcholine, requires 2-3 mM calcium. With large vesicles and planar bilayers considerably less calcium is required for inhibition of prothrombinase and for lipid binding. Half-maximal binding of annexin V to large vesicles and to planar bilayers occurs at 0.7 and 0.2 mM calcium, respectively. This seemingly confirms the displacement model. The displacement of coagulation factors, however, proved to be incomplete, with residual surface concentrations of factors Xa, Va, and prothrombin sufficient for effective production of thrombin. Cryoelectron microscopy revealed that annexin V binding to large vesicles caused planar facets, indicating the formation of large sheets of clustered annexin V. Apparently, the formation of these two-dimensional arrays is promoted by calcium and hampered by high surface curvature. It is speculated that the complete inhibition (greater than 99%) of prothrombinase activity by annexin V is caused by the reduced lateral mobility of prothrombin and factor Xa in rigid sheets of annexin V covering the membrane.
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PMID:Clustering of lipid-bound annexin V may explain its anticoagulant effect. 138 43

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

Proteins of the annexin/lipocortin family act as in vitro anticoagulants by binding to anionic phospholipid vesicles. In this study, we investigated whether annexin V (placental anticoagulant protein I) would bind to human platelets. Annexin V bound to unstimulated platelets in a reversible, calcium-dependent reaction with an apparent Kd of 7 nM and 5000-8000 sites/platelet. Additional binding sites could be induced by several platelet agonists in the following order of effectiveness: A23187 greater than collagen + thrombin greater than collagen greater than thrombin. However, neither ADP nor epinephrine induced additional binding sites. Three other proteins of the annexin family (annexins II, III, and IV) competed for annexin V platelets binding sites with the same relative potencies previously observed for binding to phospholipid vesicles. Phospholipid vesicles containing phosphatidylserine completely inhibited binding of annexin V to platelets. Annexin V completely blocked binding of 125I-factor Xa to thrombin-stimulated platelets. These results support the hypothesis that phosphatidylserine exposure occurs during platelet activation and may be necessary for assembly of the prothrombinase complex on platelet membranes.
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PMID:Binding of annexin V/placental anticoagulant protein I to platelets. Evidence for phosphatidylserine exposure in the procoagulant response of activated platelets. 214 74

An anticoagulant protein was purified from the soluble fraction of human placenta by ammonium sulfate precipitation and column chromatography on DEAE-Sepharose, Sephadex G-75, and Mono S (Pharmacia). The yield of the purified protein was approximately 20 mg from one placenta. The purified protein gave a single band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular weight of 36,500. This protein prolonged the clotting time of normal plasma when clotting was induced either by brain thromboplastin or by kaolin in the presence of cephalin and Ca2+. It also prolonged the factor Xa induced clotting time of platelet-rich plasma but did not affect thrombin-induced conversion of fibrinogen to fibrin. The purified placental protein completely inhibited the prothrombin activation by reconstituted prothrombinase, a complex of factor Xa-factor Va-phospholipid-Ca2+. The placenta inhibitor had no effect on prothrombin activation when phospholipid was omitted from the above reaction. Also, it neither inhibited the amidolytic activity of factor Xa, nor did it bind to factor Xa. The placenta inhibitor, however, did bind specifically to phospholipid vesicles (20% phosphatidylserine and 80% phosphatidylcholine) in the presence of calcium ions. These results indicate that the placental anticoagulant protein (PAP) inhibits coagulation by binding to phospholipid vesicles. The amino acid sequences of three cyanogen bromide fragments of PAP aligned with those of two distinct regions of lipocortin I and II with a high degree of homology, showing that PAP is a member of the lipocortin family.
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PMID:Human placental anticoagulant protein: isolation and characterization. 296 Mar 76

Endotoxin-stimulated monocytes can elicit a dual procoagulant response. They express tissue factor and expose phosphatidylserine in the outer leaflet of the plasma membrane. Tissue factor, a membrane glycoprotein, is the cellular trigger of blood coagulation reactions. Phosphatidylserine is an essential anionic phospholipid for surface amplification of thrombin generation. In this study the distribution of these two procoagulant entities between activated monocytes and derived microparticles was assessed after stimulation by LPS. The presence of CD14, CD11a, and CD18, and possible associated adhesion potential were examined, particularly on microparticles. Tissue factor was evidenced by using a specific functional assay and flow cytometry. Phosphatidylserine exposure was monitored through its catalytic activity in a thrombin generation assay and by flow cytometry with the use of FITC-conjugated annexin V, a protein probe of anionic phospholipids. CD14, CD11a, and CD18 were detected by flow cytometry. The interaction of microparticle CD11a/CD18 with intracellular adhesion molecule-1 was demonstrated by using immobilized recombinant intracellular adhesion molecule-1 fusion protein. The major part of tissue factor and phosphatidylserine-dependent procoagulant activity was associated with microparticles after LPS stimulation. This was confirmed by flow cytometry. The presence of functional CD11a/CD18, and CD14 on microparticles testifies to an associated adhesion potential. These results show that membrane vesiculation could be responsible for dissemination of inducible monocyte procoagulant activities and suggest that derived microparticles could also participate in endothelium stimulation. This emphasizes the role of monocyte as a central element in the coupling between inflammation/infection and thrombosis.
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PMID:Monocyte vesiculation is a possible mechanism for dissemination of membrane-associated procoagulant activities and adhesion molecules after stimulation by lipopolysaccharide. 752 56

One of the essential roles for platelets in haemostasis is in the potentiation of blood clotting due to the contribution of anionic phospholipid from the surface of the cells, as an essential cofactor to the proteolytic reactions of coagulation (platelet procoagulant activity). Only a limited number of agonists are known to initiate platelet procoagulant activity. In this study the rate of thrombin formation on the platelet surface was observed to increase in a dose-dependent manner upon treatment of washed platelets with heavy-metal compounds. Unlike the immediate increase observed upon treatment of platelets with calcium ionophore, A23187, the change due to these agents was progressive, approaching a maximum after 10 min. The maximum-fold acceleration of the rate of thrombin formation compared with control platelets was calculated for HgCl2 (56-fold), AgNO3 (42-fold) phenylmercuriacetate (24-fold) and thimerosal (14-fold), compared with 70-fold observed for calcium ionophore. The increase in procoagulant activity due to HgCl2 coincided with a large increase in intracellular calcium and phosphorylation of 22 and 45 kDa proteins. It is considered that the mechanism responsible for the increase in procoagulant activity is exposure of anionic phospholipids. This was detected by a 2-fold increase in the binding of 125I-annexin V upon addition of HgCl2, compared with resting platelets (3-fold on treatment of platelets with calcium ionophore). In contrast to the generation of activity by A23187 and other known agonists of this reaction, heavy-metal compounds appeared to cause little or no release of microparticles from the platelet surface. Since HgCl2 did not cause aggregation of platelets or significant release of serotinin, these findings may give further support to the need for exposure and ligation of glycoprotein IIb:IIIa for vesiculization to occur. Treatment of platelets with heavy metals may constitute a new approach to investigating the early changes in the cell membrane which lead to increased expression of anionic phospholipid.
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PMID:Increased expression of procoagulant activity on the surface of human platelets exposed to heavy-metal compounds. 775 58

Annexins are a family of calcium-binding proteins that have been implicated in a wide range of intracellular processes. We have previously reported that stimulation of platelets with thrombin can induce the association of intracellular annexin V with membranes in two distinct ways. First, in such a way that it can be eluted from the membrane with EGTA and secondly in a manner such that it is tightly bound to the membrane and requires the non-ionic detergent Triton X-100 for its solubilization. We report that exposure of platelets to the calcium ionophore A23187 mimics the relocation induced by stimulation with thrombin. In separate experiments we demonstrate that a calcium ion concentration [Ca2+] of 0.8 microM is sufficient for maximum binding of the EGTA-resistant form to membranes. In contrast a higher [Ca2+] was required to induce maximal binding of the annexin V which could be extracted with EGTA. We demonstrate that following temperature-induced phase separation in Triton X-114, the membrane-associated annexin V partitions predominantly into the aqueous phase. We also show that the isoelectric point of annexin V does not change following membrane association. These observations suggest that a covalent modification, of annexin V itself, is not responsible for its association with the membrane. Millimolar [Ca2+] is required for maximal binding of purified annexin V to phospholipid vesicles. We show that binding to phospholipids can be reversed entirely by subsequent treatment with EGTA. This suggests that the EGTA-resistant form of annexin V is binding to a membrane component other than phosphatidylserine. Annexin V has previously been shown to bind to protein kinase C. Relocation of annexin V to membranes paralleled that of protein kinase C in thrombin-stimulated cells but not in cells treated with A23187, suggesting that these proteins are not functionally linked in platelet activation. Using bifunctional cross-linking reagents we have identified an 85 kDa complex containing annexin V. This may represent an association between annexin V and an annexin V-binding protein with a molecular mass of approximately 50 kDa.
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PMID:Ca2+ concentration during binding determines the manner in which annexin V binds to membranes. 777 46

Annexins are a family of proteins that have been implicated in a range of intracellular processes. In this paper we confirm the existence of annexin V in human platelets (0.02 +/- 0.005% of cell protein). We also demonstrate that 13.7 +/- 6.8% of intracellular annexin V becomes tightly associated with membranes in response to platelet activation by the physiological agonist thrombin and requires non-ionic detergent for solubilization. Thrombin stimulation also induces the association of annexin V (11.0 +/- 4.6% of the total) with the membrane in a manner which requires prolonged treatment with EGTA for its release from the membrane.
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PMID:Thrombin stimulates the intracellular relocation of annexin V in human platelets. 803 48

Annexin V binds with high affinity to procoagulant phospholipid vesicles and thereby inhibits the procoagulant reactions catalysed by these surfaces in vitro. In vivo, vascular endothelial cells are known to catalyse the formation of thrombin by the expression of binding sites at which procoagulant complexes can assemble. Here, we have studied the binding capacity of recombinant annexin V (rANV) to quiescent, phorbol 12-myristate 13-acetate (PMA)- and tumour necrosis factor alpha (TNF-alpha)-stimulated cultured human umbilical-vein endothelial cells (HUVEC). The dissociation constant (Kd) was 15.5 +/- 3.3 nM and the number of binding sites was 8.8 (+/- 3.9) x 10(6)/cell. These binding parameters did not change significantly during a 30 h incubation period with PMA or TNF-alpha. rANV inhibited HUVEC-mediated factor Xa formation via the extrinsic as well as the intrinsic route. Activation of factor X by the tissue factor-factor VII-factor X complex and tenase complex was inhibited with IC50 values of 43 +/- 30 nM and 33 +/- 24 nM respectively. Endothelial-cell-mediated generation of thrombin by the prothrombinase complex was inhibited by rANV with an IC50 of 16 +/- 12 nM. Preincubation of rANV with the endothelial cells did not significantly influence the IC50 values. These results show that rANV binds to the same extent to quiescent, PMA- and TNF-stimulated HUVEC, and, as a result of this binding, rANV efficiently inhibits endothelial-cell-mediated thrombin formation.
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PMID:Binding of recombinant annexin V to endothelial cells: effect of annexin V binding on endothelial-cell-mediated thrombin formation. 806 19

The effect of hyperlipidaemia on endothelial cell haemostatic properties was examined using ex vivo studies on aortic segments obtained from fat-fed Chinchilla rabbits, mounted in a template device which exposed the luminal surface. Exposure of arterial endothelium to lipids resulted in marked enhancement of externally exposed anionic phospholipids, detected using either fluorescence microscopy with the probe merocyanine 540 or by binding of 125I-polymyxin B and 125I-Annexin V. Consistent with the known procoagulant properties of anionic phospholipid, following the lipid and cholesterol-rich diet intake, intact endothelial cells demonstrated enhanced binding of radioiodinated factors IX/IXa and Xa, and enhanced factor IXa/VIII-dependent factor X activation and factor Xa-factor Va-mediated prothrombin activation. Both factor Xa and thrombin formation were blocked, in large part, by polymyxin B, suggesting dependence of the reaction on anionic phospholipids. Consistent with these results, evidence of increased activation of the coagulation mechanism in vivo was observed in hyperlipidaemic animals, as assessed by a three-fold increase in levels of circulating antithrombin-protease complexes, compared with normolipidaemic controls.
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PMID:Intrinsic procoagulant surface induced by hypercholesterolaemia on rabbit aortic endothelium. 829 24


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