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

---

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

Factor Va, the essential cofactor for prothrombinase, is phosphorylated on the acidic COOH terminus of the heavy chain of the cofactor, at Ser692, by a platelet membrane-associated casein kinase II (CKII). Consistent with this observation, phosphorylation of the factor Va heavy chain by the platelet kinase was inhibited by heparin. The membrane-associated platelet CKII kinase was partially purified using heparin-agarose, phosphocellulose, and ion exchange chromatography. CKII antigen was monitored using a polyclonal antibody to the alpha-subunit, and kinase activity in the various fractions was confirmed using human factor Va as a substrate. Immunoblotting experiments using polyclonal antibodies raised against synthetic peptides mimicking a portion of the deduced amino acid sequence of the alpha-, alpha'-, and beta-subunits of human CKII demonstrated the coexistence of both alpha- and alpha'-subunits in platelets and suggested that the platelet CKII kinase may exist in part as an alpha alpha'beta2 complex. It is also possible that there are two distinct populations of CKII in platelets, one that is alphaalpha/betabeta and one that is alpha'alpha'/betabeta. In the presence of the purified platelet-derived CKII, human factor Va incorporates between 0.8 and 1.3 mol of phosphate/mol of factor Va depending on the concentration of the beta-subunit in the kinase preparation. A peptide mimicking the sequence 687-705 of the human factor V molecule incorporates radioactivity in the presence of purified CKII and inhibits factor Va heavy chain phosphorylation by the platelet CKII. In contrast, a peptide with an alanine instead of a serine at position 692 neither incorporates phosphate nor inhibits factor Va phosphorylation by the platelet CKII. Human factor Va is inactivated by activated protein C following three cleavages of the heavy chain at Arg506, Arg306, and Arg679. Cleavage at Arg506 is necessary for efficient exposure of the inactivating cleavage site at Arg306. The phosphorylated cofactor has increased susceptibility to inactivation by activated protein C, since phosphorylated factor Va was found to be inactivated approximately 3-fold faster than its native counterpart. Acceleration of the inactivation process of the phosphorylated cofactor occurs because of acceleration of the rate of cleavage at Arg506. These data suggest a critical role for factor Va phosphorylation on the surface of platelets in regulating cofactor activity.
...
PMID:Identification and partial characterization of factor Va heavy chain kinase from human platelets. 952 59

Factor Va (FVa), derived from plasma or released from stimulated platelets, is the essential protein cofactor of the prothrombinase complex. Plasma-derived factor V (FV) is synthesized by the liver, whereas the source of the platelet-derived cofactor has not been unambiguously identified. Megakaryocytes, platelet precursors, are known to synthesize platelet proteins and to endocytose proteins from plasma (ie, fibrinogen) and then package these proteins into alpha-granules. To determine which mechanism accounts for FV presence in platelets, two patients heterozygous for FVLeiden who underwent allogeneic transplantation from homozygous FV wild-type donors (bone marrow [BM] or liver) were studied. Patient JMW, whose skin biopsy specimen showed heterozygous FVLeiden, received a BM transplant from a wild-type homozygous FV donor as analyzed from posttransplant peripheral blood cells. Patient FW, whose native liver is heterozygous for FVLeiden, received a homozygous wild-type FV liver. Because each individual has two distinct genetic pools of factor V in liver and megakaryocytes, it was possible to determine whether secretable platelet-derived FV was normal or contained the FVLeiden mutation. Platelet-derived FVa released from thrombin-activated platelets from a normal individual, an individual heterozygous for the FVLeiden mutation, and the two patients was incubated with phospholipid vesicles and activated protein C (APC). Western blotting analyses using a monoclonal antibody that allows distinction between platelet-derived FVa and FVaLeiden subsequent to APC-catalyzed cleavage were then performed. Based on the accumulation of proteolytic fragments derived from APC-induced cleavage, analyses of platelet-derived FVa from JMW demonstrated both normal FVa and FVaLeiden consistent with a plasma-derived origin of the secretable platelet-derived FVa. Western blotting analyses of the APC-cleaved platelet-derived FVa from FW showed a wild-type phenotype, despite the presence of a FVLeiden allele in her megakaryocyte genome, also consistent with a plasma origin of her secretable platelet-derived FVa. Platelets do not appear to endocytose the plasma cofactor, because a 35-hour incubation of platelet-rich plasma with 125I-factor V showed no specific association/uptake of the radiolabeled ligand with the platelet pellet. Collectively, these results show for the first time that the majority of secretable platelet-derived factor V is endocytosed by megakaryocytes from plasma and is not exclusively synthesized by these cells, as previously believed.
...
PMID:Secretable human platelet-derived factor V originates from the plasma pool. 1033 71

Platelet activation is an inevitable consequence of blood-material interactions. The ability of those activated platelets and platelet-derived microparticles to enhance coagulation reactions leading to thrombin/fibrin formation has not been well studied despite its potential significance. Activated platelets and platelet-derived microparticles are known to dramatically enhance the catalytic efficiencies of the tenase and prothrombinase complexes. In this paper, a modified Russell viper venom coagulation time test is used to quantitate material-induced procoagulant activity due to the generation of activated phospholipid surfaces. In our test system, polyethylene and Silastic tubes were filled with heparinized whole blood and left to gently flow back and forth at 37 degrees C. After 1 h, the blood within the tubes was gravity drained and the plasma fraction assayed for procoagulant activity. The clotting times were determined by a Coag-A-Mate X2 instrument after the automated addition of Russell viper venom (to activate factors V and X) and calcium ions. Appreciable procoagulant activity was generated during whole blood contact within polyethylene and Silastic tubes although significantly greater activity was generated by the latter surface. As previously reported, platelet-derived microparticles also were detected by flow cytometry. Filtration of the plasma after material contact through a 0.1 microm filter led to substantial gains in clotting times and to near complete removal of microparticles, indicating that the material-induced microparticles likely were responsible for the procoagulant activity.
...
PMID:Assessment of material-induced procoagulant activity by a modified Russell viper venom coagulation time test. 982 86

The aim of this study was to investigate the effect of factor Xa inhibitors on the prothrombinase activity of platelet-derived microparticles in vitro and in vivo. The factor Xa inhibitors studied were DX9065A (a direct factor Xa inhibitor) and Sanorg34006 (an antithrombin (AT)-mediated factor Xa inhibitor). Microparticles formed from the platelet surface following activation were isolated by size exclusion gel chromatography. After purification, their presence was detected by their procoagulant activity and by flow cytometry. Our results show that factor Xa and/or factor Va were present at the surface of the platelet-derived microparticles. Prothrombinase formed on the microparticles was inhibited by factor Xa inhibitors at IC50 values of 0.45+/-0.05 and 0.045+/-0.005 microM for DX9065A and AT-Sanorg34006 respectively. In an experiment aimed at determining the kinetics of microparticles formation we demonstrated that thrombin traces were sufficient to induce the formation of a significant quantity of microparticles. Both factor Xa inhibitors delayed the formation of microparticles by delaying thrombin generation. The thrombogenic effect of the microparticles were studied in vivo in a modified arterio-venous shunt model in the rat. In this model, the increase in the thrombus weigh due to microparticles or phospholipids did not differ significantly (33% and 23% respectively). In these conditions, prothrombinase activity seemed to play a lesser role in the thrombogenic effect than phospholipids. Nevertheless, factor Xa inhibitors were efficient and inhibited thrombus formation in a dose-dependent manner. These results demonstrate that platelet-derived microparticles display a potent prothrombotic effect in vivo and show that factor Xa inhibitors are potent antithrombotic compounds when thrombosis was induced by microparticles.
...
PMID:Effect of factor Xa inhibitors on the platelet-derived microparticles procoagulant activity in vitro and in vivo in rats. 1105 68

Recent studies have indicated that factor Va bound to activated platelets is partially protected from inactivation by activated protein C (APC). To explore whether this sustained factor Va activity could maintain ongoing thrombin generation, the kinetics of platelet factor Va-dependent prothrombinase activity and its inhibition by APC were studied. In an attempt to mimic physiologically relevant conditions, platelets were adhered to collagen type I-coated discs. These discs were then spun in solutions containing prothrombin and factor Xa either in the absence or presence of APC. The experiments were performed in the absence of platelet-derived microparticles, with thrombin generation and inhibition confined to the surface of the adherent platelets. APC completely inactivated platelet-associated prothrombinase activity with an overall second order rate constant of 3.3 x 10(6) m(-)1 s(-)1, which was independent of the prothrombin concentration over a wide range around the apparent K(m) for prothrombin. Kinetic studies on prothrombinase assembled at a planar phospholipid membrane composed of 25 mol % phosphatidylserine and 75 mol % phosphatidylcholine revealed a similar second order rate constant of inhibition (2.5 x 10(6) m(-1) s(-1)). Collectively, these data demonstrate that ongoing platelet factor Va-dependent thrombin generation at the surface of collagen-adherent platelets is effectively inhibited by APC. No differences were observed between the kinetics of APC inactivation of plasma-derived factor Va or platelet factor Va as part of the prothrombinase associated with, respectively, a planar membrane of synthetic phospholipids or collagen-adherent platelets.
...
PMID:Regulation of platelet factor Va-dependent thrombin generation by activated protein C at the surface of collagen-adherent platelets. 1111 37

Factor X(a) (FX(a)) binding to factor V(a) (FV(a)) on platelet-derived membranes containing surface-exposed phosphatidylserine (PS) forms the "prothrombinase complex" that is essential for efficient thrombin generation during blood coagulation. There are two naturally occurring isoforms of FV(a), FV(a1) and FV(a2). These two isoforms differ by a 3-kDa polysaccharide chain (at Asn(2181) in human FV(a1) (Kim, S. W., Ortel, T. L., Quinn-Allen, M. A., Yoo, L., Worfolk, L., Zhai, X., Lentz, B. R., and Kane, W. H. (1999) Biochemistry 38, 11448-11454)) and have different coagulant activities. We examined the interaction of the two bovine isoforms with active site-labeled FX(a), finding no significant difference. A soluble form of PS (C6PS) bound to FV(a1) and FV(a2) with comparable affinities (K(d) = 11-12 microm) and changes in FV(a) intrinsic fluorescence. At concentrations well below its critical micelle concentration, C6PS binding to bovine FV(a2) enhanced its affinity for FX(a) in solution by nearly 3 orders of magnitude (K(d)(eff) = 40-2 nm over a C6PS range of 30-400 microm) but had no effect on the affinity of FV(a1) for FX(a) (K(d) = 1 microm). This results in a soluble complex between FX(a) and FV(a2), whose expected molecular weight was confirmed by calibrated native gel electrophoresis. This complex behaved as a normal Michaelis-Menten enzyme in its ability to produce thrombin from meizothrombin (apparent k(cat)/K(m) congruent with 10(9) m(-1) s(-1)). The ability of soluble PS to trigger formation of a soluble prothrombinase complex suggests that exposure of PS molecules during platelet activation is likely the key event responsible for the assembly of an active membrane-bound complex.
...
PMID:Soluble phosphatidylserine triggers assembly in solution of a prothrombin-activating complex in the absence of a membrane surface. 1204 94

This article addresses the role of platelet membrane phosphatidylserine (PS) in regulating the production of thrombin, the central regulatory molecule of blood coagulation. PS is normally located on the cytoplasmic face of the resting platelet membrane but appears on the plasma-oriented surface of discrete membrane vesicles that derive from activated platelets. Thrombin, the central molecule of coagulation, is produced from prothrombin by a complex ("prothrombinase") between factor Xa and its protein cofactor (factor V(a)) that forms on platelet-derived membranes. This complex enhances the rate of activation of prothrombin to thrombin by roughly 150,000 fold relative to factor X(a) in solution. It is widely accepted that the negatively charged surface of PS-containing platelet-derived membranes is at least partly responsible for this rate enhancement, although there is not universal agreement on mechanism by which this occurs. Our efforts have led to an alternative view, namely that PS molecules bind to discrete regulatory sites on both factors X(a) and V(a) and allosterically alter their proteolytic and cofactor activities. In this view, exposure of PS on the surface of activated platelet vesicles is a key regulatory event in blood coagulation, and PS serves as a second messenger in this regulatory process. This article reviews our knowledge of the prothrombinase reaction and summarizes recent evidence leading to this alternative viewpoint. This viewpoint suggests a key role for PS both in normal hemostasis and in thrombotic disease.
...
PMID:Exposure of platelet membrane phosphatidylserine regulates blood coagulation. 1281 44

During myocardial infarction (MI), high levels of circulating procoagulant microparticles (MP) shed from endothelial cells and platelets diffuse prothrombotic and proinflammatory potentials crucial for the coronary prognosis. In addition to conventional treatments, we evaluated whether vitamin C treatment could modify circulating levels of procoagulant MP. Upon admission, 61 patients with MI were prospectively randomized for immediate additional vitamin C treatment. Circulating MP were quantified by functional prothrombinase assay before and after 5 days of vitamin C administration (1 g day-1). The cellular origin of MP was also assessed. In vitamin C-treated patients, the reduction in platelet-derived MP was 10% higher (P = 0.01). In patients with diabetes mellitus, dyslipidemia or more than two cardiovascular risk factors, vitamin C decreased endothelial and platelet-derived MP levels by approximately 70% and 13%, respectively. This early effect on circulating platelet and endothelial-derived MP, testifies to the importance of oxidative stress during MI. Vitamin C could prove beneficial for the outcome of patients at higher thrombotic risk.
...
PMID:Protective effects of vitamin C on endothelium damage and platelet activation during myocardial infarction in patients with sustained generation of circulating microparticles. 1287 55

Platelet- and plasma-derived factor Va (FVa) serve essential cofactor roles in prothrombinase-catalyzed thrombin generation. Platelet-derived FV/Va, purified from Triton X-100 platelet lysates was composed of a mixture of polypeptides ranging from approximately 40 to 330 kDa, mimicking those visualized by Western blotting of platelet lysates and releasates with anti-FV antibodies. The purified, platelet-derived protein expressed significant cofactor activity such that thrombin activation led to only a 2-3-fold increase in cofactor activity yet expression of a specific activity identical to that of purified, plasma-derived FVa. Physical and functional differences between the two cofactors were identified. Purified, platelet-derived FVa was 2-3-fold more resistant to activated protein C-catalyzed inactivation than purified plasma-derived FVa on the thrombin-activated platelet surface. The heavy chain subunit of purified, platelet-derived FVa contained only a fraction ( approximately 10-15%) of the intrinsic phosphoserine present in the plasma-derived FVa heavy chain and was resistant to phosphorylation at Ser(692) catalyzed by either casein kinase II or thrombin-activated platelets. MALDI-TOF mass spectrometric analyses of tryptic digests of platelet-derived FV peptides detected an intact heavy chain uniquely modified on Thr(402) with an N-acetylglucosamine or N-acetylgalactosamine, whereas Ser(692) remained unmodified. N-terminal sequencing and MALDI-TOF analyses of platelet-derived FV/Va peptides identified the presence of a full-length heavy chain subunit, as well as a light chain subunit formed by cleavage at Tyr(1543) rather than Arg(1545) accounting for the intrinsic levels of cofactor activity exhibited by native platelet-derived FVa. These collective data are the first to demonstrate physical differences between the two FV cofactor pools and support the hypothesis that, subsequent to its endocytosis by megakaryocytes, FV is modified to yield a platelet-derived cofactor distinct from its plasma counterpart.
...
PMID:Unique in vivo modifications of coagulation factor V produce a physically and functionally distinct platelet-derived cofactor: characterization of purified platelet-derived factor V/Va. 1459 14

During myocardial infarction (MI), platelet activation and endothelial apoptosis are responsible for the release of procoagulant membrane-derived microparticles (MP) in the blood flow. MP prothrombotic and proinflammatory properties may be crucial for coronary prognosis. Elevated amounts of circulating procoagulant MP were described in diabetes mellitus (DM), and could be of particular significance in a MI context. We evaluated the prothrombotic status of DM and non-DM (NDM) patients at days 1 and 6 after MI, by measurement of circulating procoagulant MP and soluble GPV (sGPV), the platelet glycoprotein V major fragment released upon thrombin cleavage. Variations were compared to values measured in healthy volunteers (HV). Procoagulant MP were captured onto insolubilized annexin V and quantified by prothrombinase assay. Their cellular origin was assessed. With respect to HV, the levels of procoagulant MP detected at D1 and D6 were elevated in DM and NDM, MP being significantly higher in DM vs. NDM. The high amounts of platelet-derived MP and the correlation between procoagulant MP and sGPV, testify to the central role of thrombin-activated platelets during MI in both DM and NDM subsets. The release of platelet and endothelial cell-derived MP persisted at D6 and was more important in DM, the associated prothrombotic risk being also reflected by higher levels of sGPV. The endothelial damage revealed by endothelial-derived MP was twice that observed in NDM patients. In DM patients presenting cardio-vascular events at 6 month follow-up, MP levels were significantly higher at D1 after MI than in those without complication (24.9 +/- 4.8 vs. 12.3 +/- 2.7 nM PhtdSer, p = 0.02), suggesting a prognostic potential for MP.
...
PMID:Sustained elevated amounts of circulating procoagulant membrane microparticles and soluble GPV after acute myocardial infarction in diabetes mellitus. 1496 Nov 63


<< Previous 1 2 3 4 Next >>

---