Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:O95477 (membrane-bound)
 29,236 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Inactivation of membrane-bound factor Va by activated protein C (APC) proceeds via a biphasic reaction that consists of a rapid and a slow phase, which are associated with cleavages at Arg506 and Arg306 of the heavy chain of factor Va, respectively. We have investigated the effects of protein S and factor Xa on APC-catalyzed factor Va inactivation. Protein S accelerates factor Va inactivation by selectively promoting the slow cleavage at Arg306 (20-fold). Factor Xa protects factor Va from inactivation by APC by selectively blocking cleavage at Arg506. Inactivation of factor VaR506Q, which was isolated from the plasma of a homozygous APC-resistant patient and which lacks the Arg506 cleavage site, was also stimulated by protein S but was not affected by factor Xa. This confirms that the target sites of protein S and factor Xa involve Arg306 and Arg506, respectively. Factor Xa completely blocked APC-catalyzed cleavage at Arg506 in normal factor Va (1 nM) with a half-maximal effect (K1/2Xa) at 1.9 nM factor Xa. Expression of cofactor activity of factor Va in prothrombin activation required much lower factor Xa concentrations (K1/2Xa = 0.08 nM). When the ability of factor Xa to protect factor Va from inactivation by APC was determined at low factor Va concentrations during prothrombin activation much lower amounts of factor Xa were required (K1/2Xa = 0.03 nM). This indicates 1) that factor Va is optimally protected from inactivation by APC by incorporation into the prothrombinase complex during ongoing prothrombin activation, and 2) that the formation of a catalytically active prothrombinase complex and protection of factor Va from inactivation by APC likely involves the same interaction of factor Xa with factor Va. In accordance with the proposed mechanisms of action of protein S and factor Xa, we observed that the large differences between the rates of APC-catalyzed inactivation of normal factor Va and factor VaR506Q were almost annihilated in the presence of factor Xa and protein S. This observation may explain why, in the absence of other risk factors, APC resistance only results in a weak prothrombotic condition.
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PMID:Effects of protein S and factor Xa on peptide bond cleavages during inactivation of factor Va and factor VaR506Q by activated protein C. 749 57

Factor V was purified from the plasma of an activated protein C (APC)-resistant patient who is homozygous for the mutation Arg506-->Gln (factor VR506Q). Factor VR506Q was converted by thrombin into factor Va which was further purified yielding a factor Va preparation that had the same cofactor activity in prothrombin activation as normal factor Va. Inactivation of low concentrations of normal factor Va (< 5 nM) by 0.15 nM APC in the presence of phospholipid vesicles proceeded via a biphasic reaction that consisted of a rapid phase (k = 4.3 x 10(7) M-1s-1), yielding a reaction intermediate with reduced cofactor activity that was fully inactivated during the subsequent slow phase (k = 2.3 x 10(6) M-1s-1). Inactivation of factor VaR506Q proceeded via a monophasic reaction (k = 1.7 x 10(6) M-1s-1). Immunoblot analysis showed that APC-catalyzed inactivation of factor Va occurred via peptide bond cleavages in the heavy chain. The rapid phase of inactivation of normal factor Va was associated with cleavage at Arg506 and full inactivation of factor Va required subsequent cleavage at Arg306. The slow monophasic inactivation of factor VaR506Q correlated with cleavage at Arg306. Cleavage at Arg506 in normal factor Va resulted in accumulation of a reaction intermediate that exhibited 40% cofactor activity in prothrombin activation mixtures that contained a high factor Xa concentration (5 nM). Compared with native factor Va, the reaction intermediate retained virtually no cofactor activity at low factor Xa concentrations (0.3 nM). This demonstrates that factor Va that is cleaved at Arg506 is impaired in its ability to interact with factor Xa. Michaelis-Menten kinetic analysis showed that cleavage at Arg506 in membrane-bound factor Va was characterized by a low Km for factor Va (20 nM) and kcat = 0.96 s-1. For cleavage at Arg306 in factor VaR506Q the kinetic parameters were Km = 196 nM and kcat = 0.37 s-1. This means that differences between APC-catalyzed inactivation of factors Va and VaR506Q become much less pronounced at high factor Va concentrations. When factor VaR506Q was inactivated by APC in the absence of phospholipids, cleavage at Arg679 of the heavy chain also contributed to factor Va inactivation. Comparison of rate constants for APC-catalyzed cleavage at Arg306, Arg506, and Arg679 in the absence and presence of phospholipids indicated that phospholipids accelerated these cleavages to a different extent.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Peptide bond cleavages and loss of functional activity during inactivation of factor Va and factor VaR506Q by activated protein C. 767 48

Thrombomodulin (TM) is the anticoagulant endothelial cell membrane-bound protein cofactor in the thrombin-mediated activation of protein C (PC). It has been clearly demonstrated that the anticoagulant and profibrinolytic functions of the PC system are important for the prevention of a thromboembolic disease. Patients with PC, protein S, or PC "'cofactor"' deficiency and/or dysfunction develop thromboembolic diseases. However, the molecular abnormality in at least 20% to 30% of thrombophilic patients cannot be identified by hitherto recognized defects. A putative pathologic lesion in the TM gene could be one of several candidates for these prothrombotic mutations. A directed search strategy for deletions, insertions, or point mutations in the TM gene has not been performed. Therefore, in the present study, we have analyzed the entire TM gene, including the promoter region, by polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) in normal healthy volunteers and in patients presenting with a thromboembolic disease. We have identified a patient with a thromboembolic disease and a TM point mutation. In a 45-year-old Hispanic man with a documented pulmonary embolism, PCR-SSCP showed an aberrant band pattern and subsequent DNA sequence analysis showed a heterozygous substitution for G1456 to T. This substitution predicts an Asp468 to a Tyr change in the amino acid sequence that is located between the transmembrane domain and the sixth epidermal growth factor-like domain. The Asp468 to Tyr change would probably lead to significant structural changes not allowing the expression of the TM protein or to a conformational change that is not functional.
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PMID:The first mutation identified in the thrombomodulin gene in a 45-year-old man presenting with thromboembolic disease. 781 89

A poor anticoagulant response of plasma to activated protein C is correlated with a single mutation in the factor V molecule (Arg506-->Gln). Factor V was purified to homogeneity from plasma of two unrelated patients (patient I, factor VI, and patient II, factor VII), who are homozygous for this mutation. The factor V molecule from both patients has normal procoagulant activity when compared with factor V isolated from normal plasma in both a clotting time-based assay and in an assay measuring alpha-thrombin formation. The cleavage and subsequent inactivation by activated protein C (APC) of the alpha-thrombin-activated membrane-bound cofactor (factor Va) from both patients were analyzed and compared with the cleavage and inactivation of normal human factor Va. In normal factor Va, cleavage at Arg506 generates a M(r) = 75,000 fragment and a M(r) = 28,000/26,000 doublet and is necessary for the optimum exposure of the sites for subsequent cleavage at Arg306 and Arg679. Proteolysis at these sites leads to the appearance of M(r) - 45,000 and 30,000 fragments and a M(r) = 22,000/20,000 doublet. Cleavage at Arg306 is membrane-dependent and is required for complete inactivation. Following 5 min of incubation with APC (5.4 nM) membrane-bound normal factor Va (280 nM) has virtually no cofactor activity whereas under similar experimental conditions factor VaI and factor VaII retain approximately 50% of their initial activity. After 1 h of incubation with APC, factor VaI retains 20% of its initial cofactor activity whereas factor VaII has 10% remaining cofactor activity. The initial loss in cofactor activity (approximately 70%) of membrane-bound factor VaI and factor VaII during the first 10 min of the inactivation reaction is correlated with cleavage at Arg306 and appearance of a M(r) = 45,000 fragment and a M(r) = 62,000/60,000 doublet. Subsequently, the M(r) = 62,000/60,000 doublet is cleaved at Arg679 to generate a M(r) = 56,000/54,000 doublet resulting in complete loss of cofactor activity. Both procofactors, factor VI and factor VII, were inactivated following cleavage at Arg306 and Arg679, with APC inactivation rates equivalent to those observed for normal factor V. Our data demonstrate that: 1) cleavage at Arg506 is required for optimum exposure of the cleavage sites at Arg306 and Arg679 and rapid inactivation of membrane-bound factor Va; and 2) cleavage at Arg306 by APC on membrane-bound factor V occurs at the same rate in both normal and APC-resistant individuals. Thus cleavage at Arg306 and Arg679 and subsequent inactivation of the membrane-bound procofactor, factor V, does not require prior cleavage at Arg506 for optimum exposure.
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PMID:Characterization of the molecular defect in factor VR506Q. 787 54

The cleavage of human factor V and human factor Va by human activated protein C (APC) was analyzed in the absence and presence of phospholipid vesicles containing 75% phosphatidylcholine (PC) and 25% phosphatidylserine (PS). Membrane-bound human factor V (250 nM) is cleaved by APC (2.5 nM) to give M(r) = 200,000, 70,000, 45,000, and 30,000 fragments and an M(r) = 22/20,000 doublet. These fragments are released after four sequential cleavages of the membrane-bound procofactor at Arg306, Arg506, Arg679, and Lys994. No cofactor activity is observed following thrombin treatment of the membrane-bound APC-cleaved procofactor. In the absence of a membrane surface, no cleavage of factor V by APC is observed, and following thrombin activation factor Va retains full cofactor activity. Membrane-bound human factor Va (600 nM) loses more than 90% of its initial cofactor activity after 10 min of incubation with APC (10.9 nM), and virtually no cofactor activity is observed after 1 h of incubation. Under similar conditions but in the absence of PCPS vesicles, factor Va is cleaved but retains approximately 80% of its initial cofactor activity after 2 h of incubation with APC. In the presence of PCPS vesicles, the APC related loss of activity is correlated with cleavage of the heavy chain and appearance of fragments of M(r) = 45,000, 30,000, and of 28/26,000, and 22/20,000 doublets. These products correspond to three cleavages of the heavy chain (at Arg306, Arg506, and Arg679). Cleavage at Arg506 of factor Va precedes and appears to be required for cleavage at Arg306 and Arg679. In the absence of membrane, proteolysis at Arg506 produces an M(r) = 75,000 fragment which corresponds to the NH2-terminal portion of the human factor Va heavy chain (residues 1-506), and a carboxyl-terminal doublet of M(r) = 28/26,000 (residues 507-709) which is cleaved by APC at Arg679 to generate an M(r) = 22/20,000 doublet and an M(r) = 6,000 peptide. No cleavage of the light chain of the human cofactor is observed in the presence or absence of PCPS vesicles following 2 h of incubation with APC. Our data demonstrate that inactivation of human factor V and human factor Va only occurs in the presence of a membrane surface after cleavage at Arg306. However, while this cleavage site is exposed on membrane-bound human factor V, cleavage at Arg506 on the heavy chain of factor Va appears necessary for complete exposure of the cleavage site at Arg306.
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PMID:The mechanism of inactivation of human factor V and human factor Va by activated protein C. 798 61

Clinical manifestations of arterial and venous thrombosis in a family with protein C deficiency was associated with two mutations in the light chain of protein C: Glu20-->Ala and Val34-->Met. Further studies showed that the mutation Glu20-->Ala which eliminated a gamma-carboxylation site was exclusively responsible for the anticoagulant defect of activated protein C (APC). Membrane-bound human factor Va is inactivated by APC after two sequential cleavages of the heavy chain at Arg506 and Arg306. Human factor Va inactivation by human recombinant APC (rAPC) and a mutant molecule with an alanine instead of a glutamic acid at position 20 (rAPC(gamma 20A)) was investigated in the presence and absence of phospholipid vesicles. During a 2-hour incubation period of the cofactor with either rAPC or rAPC(gamma 20A). In the absence of a membrane surface, factor Va is cleaved quantitatively at Arg506 and retains approximately 60% of its initial cofactor activity. After a 2-hour incubation period with rAPC membrane-bound factor Va has no cofactor activity, whereas in the presence of a membrane surface and rAPC(gamma 20A) factor Va retains 60% of its initial cofactor activity. The completed loss in factor Va cofactor activity upon incubation of the membrane-bound cofactor with phospholipid vesicles and rAPC is associated with cleavages at Arg506 and Arg306, whereas membrane-bound factor Va cleavage at Arg306 by rAPC(gamma 20A) is impaired, resulting in a cofactor that is cleaved at Arg506. Slow cleavage at Arg306 occurs when membrane-bound factor Va is incubated with rAPC(gamma 20A) and only small amounts of fragments of M(r) = 45,000 and 30,000 are noticed. Our data show that the genetic defect which leads to the absence of a gamma-carboxylation site at Glu20 impairs membrane binding of human APC, which in turn is required for cleavage of factor Va at Arg306 and inactivation of the cofactor. The consequence of impaired membrane-dependent cleavage at Arg306 is manifested in vivo by venous and arterial thrombosis.
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PMID:Loss of membrane-dependent factor Va cleavage: a mechanistic interpretation of the pathology of protein CVermont. 804 58

Bovine factor Va inactivation by activated protein C (APC) was evaluated in the presence and absence of phospholipid vesicles and protein S. Following a 30-min incubation with APC (10 nM), membrane-bound factor Va (200 nM) is completely inactivated, whereas in the absence of phospholipid vesicles, after a 2-h incubation, the cofactor retains 60% of its initial cofactor activity. The complete loss of activity of membrane-bound factor Va is associated with the appearance of M(r) 40,000, 28,000, and 20,000 fragments derived from the heavy chain of the cofactor which correspond to cleavage at Arg306, Arg505, and Arg662. In the absence of a lipid bilayer, cleavage at Arg505 and Arg662 results in a cofactor with reduced activity. No difference is observed in the cleavage of the light chain of the cofactor by APC in the presence or absence of phospholipid vesicles. The rate of the cleavage of factor Va heavy chain at Arg306, Arg505, and Arg662 as well as the rate of the membrane-bound cofactor inactivation by APC were enhanced in the presence of protein S. Our data demonstrate that the anionic lipid-dependent cleavage of factor Va by APC at Arg306 is required for the complete inactivation of the cofactor.
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PMID:Role of the membrane in the inactivation of factor Va by activated protein C. 826 65

Human factor V is activated to factor Va by alpha-thrombin after cleavages at Arg709, Arg1018, and Arg1545. Factor Va is inactivated by activated protein C (APC) in the presence of a membrane surface after three sequential cleavages of the heavy chain. Cleavage at Arg506 provides for efficient exposure of the inactivating cleavages at Arg306 and Arg679. Membrane-bound factor V is also inactivated by APC after cleavage at Arg306. Resistance to APC is associated with a single nucleotide change in the factor V gene (G1691-->A) corresponding to a single amino acid substitution in the factor V molecule: Arg506-->Gln (factor V Leiden). The consequence of this mutation is a delay in factor Va inactivation. Thus, the success of the APC-resistance assay is based on the fortuitous activation of factor V during the assay. Plasmas from normal individuals (1691 GG) and individuals homozygous for the factor V mutation (1691 AA) were diluted in a buffer containing 5 mmol/L CaCl2, phospholipid vesicles (10 micromol/L), and APC. APC, at concentrations < or = 5.5 nmol/L, prevented clot formation in normal plasma, whereas under similar conditions, a clot was observed in plasma from APC-resistant individuals. Gel electrophoresis analyses of factor V fragments showed that membrane-bound factor V is primarily cleaved at Arg306 in both plasmas. However, whereas in normal plasma production of factor Va heavy chain is counterbalanced by fast degradation after cleavage at Arg506/Arg306, in the APC-resistant individuals' plasma, early generation and accumulation of the heavy chain portion of factor Va occurs as a consequence of delayed cleavage at Arg306. At elevated APC concentrations (>5.5 nmol/L), no clot formation was observed in either plasma from normal or APC-resistant individuals. Our data show that resistance to APC in patients with the Arg506-->Gln mutation is due to the inefficient degradation (inactivation) of factor Va heavy chain by APC.
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PMID:Proteolytic events that regulate factor V activity in whole plasma from normal and activated protein C (APC)-resistant individuals during clotting: an insight into the APC-resistance assay. 863 39

The proteolytic cleavage and subsequent inactivation of recombinant human factor VIII (rhFVIII) and human factor VIIIa (rhFVIIIa) by recombinant human activated protein C (rAPC) was analyzed in the presence and absence of human protein S and human factor V (FV). Membrane-bound rhFVIIIa spontaneously looses most of its initial cofactor activity after 15 minutes of incubation at pH 7.4. The remaining activity can be eliminated after incubation with rAPC. Complete inactivation of the membrane-bound rhFVIII and rhFVIIIa by APC correlates with cleavage at Arg336. The inactivation of rhFVIII and human plasma FV by rAPC were also compared. Under similar experimental conditions, complete inactivation of membrane-bound FVIII (60 nmol/L) by rAPC (10 nmol/L) requires 4 hours of incubation, in contrast to 5 minutes for FV (60 nmol/L). The presence of protein S (100 nmol/L) enhances rhFVIII inactivation by rAPC by 6.4-fold and FVa inactivation by twofold, whereas membrane-bound FV showed no protein S dependence during inactivation. The addition of human FV to the APC/protein S inactivation mixture increases by approximately twofold the rate of inactivation of rhFVIII. The effect of FV on the rhFVIII inactivation by APC is protein S-dependent, because FV alone has no effect on the inactivation rate of rhFVIII by APC. Western blotting using a monoclonal antibody that recognizes an epitope between amino acid residues 307 and 506 of human FV showed that FV was completely cleaved by APC at the beginning of the rhFVIII inactivation process. These data suggest that FV fragments derived from the B region of the procofactor after incubation of the membrane-bound procofactor with APC, but not intact single-chain FV, stimulate APC activity in the presence of protein S. rhFVIII, FV, and rhFVIIIa were not inactivated by Glu20-->Ala-substituted rAPC (rAPCgamma20A), and membrane-bound factor Va was only partially inactivated. Our data suggest that (1) FV and FVa are the physiologically significant substrates for APC inactivation and (2) membranes-bound APC-treated FV is a cofactor for the APC inactivation of rhFVIII only in the presence of the intact form of protein S.
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PMID:Comparison of activated protein C/protein S-mediated inactivation of human factor VIII and factor V. 863 40

Interactions between standard heparin and the physiological anticoagulant plasma protein, activated protein C (APC) were studied. The ability of heparin to prolong the activated partial thromboplastin time and the factor Xa- one-stage clotting time of normal plasma was markedly enhanced by addition of purified APC to the assays. Experiments using purified clotting factors showed that heparin enhanced by fourfold the phospholipid-dependent inactivation of factor V by APC. In contrast to factor V, there was no effect of heparin on inactivation of thrombin-activated factor Va by APC. Based on SDS-PAGE analysis, heparin enhanced the rate of proteolysis of factor V but not factor Va by APC. Coagulation assays using immunodepleted plasmas showed that the enhancement of heparin action by APC was independent of antithrombin III, heparin cofactor II, and protein S. Experiments using purified proteins showed that heparin did not inhibit factor V activation by thrombin. In summary, heparin and APC showed significant anticoagulant synergy in plasma due to three mechanisms that simultaneously decreased thrombin generation by the prothrombinase complex. These mechanisms include: first, heparin enhancement of antithrombin III-dependent inhibition of factor V activation by thrombin; second, the inactivation of membrane-bound FVa by APC; and third, the proteolytic inactivation of membrane-bound factor V by APC, which is enhanced by heparin.
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PMID:Anticoagulant synergism of heparin and activated protein C in vitro. Role of a novel anticoagulant mechanism of heparin, enhancement of inactivation of factor V by activated protein C. 916 95


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