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M Kalafatis

Publications and source records attributed to M Kalafatis.

At least 37 records · Page 2Linked to original sources
PubMed1996

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.

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.

Amino Acid Sequence↗
PubMed1996

Comparison of activated protein C/protein S-mediated inactivation of human factor VIII and factor V.

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.

Factor VIIIa↗
PubMed1996

Regulation and regulatory role of gamma-carboxyglutamic acid containing clotting factors.

Blood coagulation is initiated following damage of the vessel wall. The large number of proteins that participate in the reactions that regulate blood coagulation must behave in a concerted manner to generate alpha-thrombin. This most abundant enzyme product of the coagulation process acts on the soluble blood protein fibrinogen to create the insoluble fibrin clot, trigger platelet activation, and initiate a variety of vascular processes ranging from coagulation inhibition and activation of fibrinolysis to cell growth. The majority of enzymes that participate in the blood-clotting process circulate in blood as inactive zymogens and procofactors. Following mechanical injury to the vessel wall, a cell-associated protein, tissue factor, is exposed and initiates a series of enzymatic reactions localized on a membrane surface generally provided by cells and accumulating platelets. The binding of the enzymes and zymogen substrates to the exposed membrane surface and their activation are in part governed by the gamma-carboxyglutamic acid residues (gla) of each protein. Recently, it has been demonstrated that the gla residues are necessary for Ca2+ binding and subsequent exposure of the hydrophobic membrane binding regions to the cell bilayer. Thus, the gla residues play a preeminent role during the blood-clotting process. Absence of gamma-carboxylation of one or more of the critical glutamic acid residues results in an impaired coagulation/anticoagulation process which may lead to a bleeding diathesis or thrombosis.

1-Carboxyglutamic Acid↗
PubMed1995

The mechanism of inactivation of human platelet factor Va from normal and activated protein C-resistant individuals.

The inactivation of human platelet factor Va by activated protein C (APC) was analyzed by functional assessment of cofactor activity and Western blotting analysis to visualize the factor Va fragments accompanying proteolysis. Platelets were treated with thrombin to facilitate both their activation as well as the release and further activation of platelet factor Va, followed by APC addition. The rates of inactivation were donor-dependent such that 15-60% of the initial cofactor activity was lost within 5 min of APC addition with as much as 10-20% of the activity still remaining after 2 h of incubation. Western blot analysis using a monoclonal antibody that recognizes an epitope between amino acid residues 307 and 506 of the factor V molecule suggested that the factor Va activity resistant to APC inactivation was due to residual heavy chain. Furthermore, in contrast to studies with normal plasma-derived factor Va, two possible cleavage mechanisms could explain the platelet factor Va fragments observed. APC can cleave platelet factor Va initially at Arg506, with subsequent cleavages occurring at Arg306 and Arg679. Alternatively, APC can cleave at Arg306 initially, with further cleavage at Arg679 then at Arg506 or at Arg506 followed by cleavage at Arg679. Similar results were obtained if platelets were removed from the inactivation mixtures and phospholipid vesicles were used to supply the membrane surface required for inactivation, suggesting that the order of platelet factor Va peptide bond cleavage or the amount of cofactor activity remaining was not altered by either of these surfaces. Thus, APC is unable to effect the complete inactivation of platelet factor Va, even though it would appear that the same cleavages which render the plasma cofactor inactive are occurring in the platelet cofactor. Analogous protocols were used to study an individual heterozygous for the Arg506-->Gln506 mutation (Factor V Leiden, Factor VR506Q). With respect to the mutant platelet factor Va in the presence of APC, > 70% of the initial cofactor activity remained after 1 min, with 30% activity still remaining after 2 h. As seen in studies of the APC-catalyzed inactivation of plasma factor VaR506Q, proteolysis of the mutant platelet factor Va confirms that even though cleavage at Arg306 will occur in the absence of cleavage at Arg506, the rate of inactivation is slower. Collectively these data suggest that when compared to normal plasma factor Va, differences in normal platelet factor Va which define: 1) whether the heavy chain is susceptible to cleavage at Arg306 or Arg506 and 2) the extent to which it is cleaved initially at Arg306, in contrast to cleavage of Arg506, will define both the extent and rate of inactivation.

Amino Acid Sequence↗
PubMed1995

Characterization of the molecular defect in factor VR506Q.

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.

Arginine↗
PubMed1995

Biochemical prototype for familial thrombosis. A study combining a functional protein C mutation and factor V Leiden.

Resistance to activated protein C (APC) is associated with a single amino acid substitution in factor V (Arg506-->Gln, factor V Leiden) that results in delayed inactivation of the molecule by APC. The mutation is present in 20% of patients with a first episode of deep venous thrombosis. Arterial and venous thromboses are also associated with the type II protein C deficiency (protein CVermont). In protein CVermont, the substitution Glu20-->Ala alone (rPC gamma 20A) is responsible for the defective anticoagulant properties of PCVermont. It was recently established that a thrombotic episode occurred in 73% of family members who are heterozygous for both a functional protein C gene mutation and the factor V Leiden mutation. We evaluated the molecular defect that would accrue in the combined deficiency state of factor VR506Q/VaR506Q and rAPC gamma 20A using recombinant APC and natural purified factor VR506Q from patients homozygous for the Arg506-->Gln substitution. While wild-type recombinant APC (rAPC) slowly cleaves and inactivates factor VR506Q and factor VaR506Q, minimal cleavage of membrane-bound factor VR506Q and VaR506Q by rAPC gamma 20A at Arg306 and Arg679 occurs, and no loss in cofactor activity is observed. Our data demonstrate that rAPC gamma 20A cannot inactivate either factor VR506Q or factor VaR506Q at biologically relevant rates because of impaired cleavage at Arg306 and Arg679.(ABSTRACT TRUNCATED AT 250 WORDS)

Factor V↗
PubMed1994

The mechanism of inactivation of human factor V and human factor Va by activated protein C.

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.

Amino Acid Sequence↗
PubMed1994

Determination of the disulfide bridges in factor Va heavy chain.

The M(r) = 94,000 heavy chain of bovine factor Va contains 10 cysteine residues which are distributed in the 2 A domains which make up this portion of the factor V molecule. The A1 domain contains four cysteines while the A2 domain contains six cysteines. The locations of disulfide bridges and free cysteines in bovine factor Va heavy chain were analyzed using iodo[14C]acetamide-labeled factor Va heavy chain digested with trypsin, plasmin, V-8 protease, and cyanogen bromide. Following HPLC separation of the resulting peptides, free cysteines were identified by the incorporation of radioactivity while disulfide-containing peptides were detected using an SBD-F fluorometric assay after reduction. All cysteine-containing peptides were analyzed by amino acid sequence analysis. The four cysteines in the A1 domain are associated with two disulfide bonds, Cys139-Cys165 and Cys220-Cys301. One disulfide bond was explicitly identified in the A2 domain; Cys471-Cys497, and a free cysteine was found in the A2 domain at Cys538. Significant difficulties were encountered in preparing identifiable or soluble peptides which would permit the explicit identification of the three remaining cysteines in the A2 domain. On the basis of homology, it is likely that Cys589 is a free SH while a disulfide bridge exists between Cys579 and Cys660. Thus, three major disulfide bonding patterns, characterized as "alpha", "beta", and "gamma" loops, are found in factor V. Each A domain contains a 26 residue "alpha loop at positions 139-165, 471-497, and 1684-1710. The A1 and A2 domains each contain 81 amino acid residue "beta" loops at 220-301 and 579-660.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

A model for the tissue factor pathway to thrombin. I. An empirical study.

The activation of prothrombin, factor V, factor VIII, factor IX, and factor X by the tissue factor-factor VIIa complex, in vitro, in a system in which each precursor protein was present at plasma concentration, was evaluated using a combination of activity assays, immunoblots, active-site blots, and autoradiography. The thrombin generation curves observed were distinctly nonlinear and typically displayed a time lag in which little or no thrombin was observed. This was followed by an almost linear propagation phase of thrombin formation. The lag was a function of tissue factor/factor VIIa concentration and represented primarily the interval of factor V and factor VIII activation. The postlag propagation phase of thrombin generation was nearly independent of the initial activator (factor VIIa or tissue factor) concentration over a 10(3)-fold range in factor VIIa-tissue factor concentration. Maximum thrombin generation rates were observed when less than 1% of the factor IX and X present was activated but when nearly 100% activation of the cofactors, factor V and factor VIII, was achieved. Analyses of the activation pattern of factor V indicated that the cofactor is activated by both factor Xa and thrombin which are formed at low levels during the lag phase of the reaction. When the initial reaction mixture contained factor Va instead of factor V, the lag was substantially reduced. When factor V was deleted from the reaction mixture, no thrombin formation was observed. When either factor VII or factor IX was deleted from the reaction system, the propagation phase of thrombin formation (at 5 pM tissue factor-factor VIIa complex) was only one-third that observed for reactions which contained factor VIII and factor IX. The addition of factor XI to the experimental system increased the rate of thrombin formation by 15% during the propagation phase but had no effect upon the lag phase of the reaction. Our data suggest that normal hemostasis may be initiated by the factor VIIa-tissue factor complex and support the concept of multiple feedback reactions which amplify and propagate the hemostatic response.

Blood Coagulation Factors↗
PubMed1994

Loss of membrane-dependent factor Va cleavage: a mechanistic interpretation of the pathology of protein CVermont.

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.

1-Carboxyglutamic Acid↗
PubMed1994

Contribution of the heavy and light chains of factor Va to the interaction with factor Xa.

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)

Amino Acid Sequence↗
PubMed1994

Platelet coagulation factor Va: the major secretory platelet phosphoprotein.

Platelet-derived coagulation factor Va is the primary secreted substrate for a thrombin-stimulation-dependent platelet kinase. Human platelet factor Va, consisting of a molecular weight (M(r)) 105,000 heavy chain and an M(r) 74,000 light chain, incorporates phosphate in at least two sites on the light chain. Phosphorylated factor Va represents 50% of the secreted protein-associated phosphate. This modification occurs exclusively at serine residues and is inhibited by H-7 and staurosporine, which suggests a protein kinase C (PKC)-mediated event. Purified plasma factor V and Va are phosphorylated in the light chain region by rat brain PKC. The activity of platelet factor Va in prothrombinase on platelets is not altered when phosphorylation is inhibited by staurosporine. Plasma-derived factor Va in the presence of thrombin stimulated platelets is phosphorylated on both the heavy chain and the light chain. Plasma factor V and factor Va heavy chain phosphorylation occurs without light chain phosphorylation in the presence of added 32P gamma-ATP and non-stimulated or collagen-stimulated platelets or casein kinase II. This differential phosphorylation of factor Va heavy and light chain shows two independent platelet kinase activities that act on factor Va. The heavy chain factor V/Va kinase activity is similar to casein kinase II, which we have demonstrated previously to act on factor Va and accelerate activated protein C inactivation of the cofactor. Our data show platelet-dependent phosphorylation of platelet and plasma factor V and Va resulting in significant covalent modifications of the cofactor. These modifications may play a role in directing the extracellular distribution of factor V and factor Va.

Blood Platelets↗
PubMed1994

Cooperative interaction of divalent metal ions, substrate, and tissue factor with factor VIIa.

Factor VIIa-tissue factor (TF) complex formation in the presence of EDTA or divalent cations (Me2+) was investigated. The influence of Me2+ on the amidolytic activity of factor VIIa and factor VIIa-TF complex was evaluated using low molecular weight synthetic substrates possessing substituted aminonaphthalenesulfonamides as detecting groups. Factor VIIa expressed low amidolytic activity in the presence of EDTA. In the presence of EDTA and saturating concentrations of TF, the amidolytic activity of factor VIIa was increased approximately 90-fold. Gel electrophoresis and sedimentation velocity studies demonstrated complex formation between factor VIIa and TF in the presence of EDTA. Substrate titration curves obtained at fixed factor VIIa and TF concentrations gave sigmoidal shapes, indicating that substrates influenced factor VIIa amidolytic activity in the presence of TF. In the absence of Me2+, the KD,app of the factor VIIa-TF complex was influenced by substrate structure and varied from 3.9 to 34 nM. All Me2+ used increased the amidolytic activity of factor VIIa approximately 8-fold compared with experiments in the presence of EDTA. The KD,app values for factor VIIa-Ca2+ complex and factor VIIa-Mn2+ complex were independent of substrate and were 270 and 40 microM, respectively. The KD,app for factor VIIa-Mg2+ complex varied from 3 to 12 mM and was substrate structure dependent. The presence of TF had no influence upon the KD,app for the factor VIIa-Ca2+ complex. The amidolytic activity of factor VIIa was enhanced by TF significantly in the presence of Ca2+, and similar results were obtained with Mg2+ and Mn2+.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium↗
PubMed1994

Factor Va-membrane interaction is mediated by two regions located on the light chain of the cofactor.

Factor Va was incubated with 1-azidopyrene, a fluorescent lipophilic probe, in the presence of phospholipid vesicles composed of various proportions of phosphatidylcholine (PC) and phosphatidylserine (PS). The majority of the label was associated with the light chain of factor Va. The light chain was found to be labeled in the presence of phospholipid vesicles containing either 100% PC or 100% PS. After cleavage by factor Xa and incubation with PC/PS vesicles composed of 75% PC and 25% PS, label was found both on the M(r) = 30,000 fragment, derived from the NH2-terminal portion of the bovine factor Va light chain (residues 1537-1752), and on the M(r) = 46,000/48,000 carboxyl-terminal fragment of the factor Va light chain (residues 1753-2183). The M(r) = 46,000/48,000 fragment incorporated 1-azidopyrene independent of the phospholipid composition, while label incorporation into the M(r) = 30,000 fragment required phospholipid vesicles containing PC. No labeling of the M(r) = 30,000 fragment was observed with phospholipid vesicles composed of 100% PS. The label incorporation into the two portions of the molecule was found to be independent of the ionic strength in the presence of phospholipid vesicles containing 75% PC and 25% PS. In contrast, the labeling of the M(r) = 46,000/48,000 fragment with phospholipid vesicles composed of 100% PS was ionic strength dependent.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Role of the membrane in the inactivation of factor Va by activated protein C.

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.

Amino Acid Sequence↗
PubMed1993

Determination of the disulfide bridges in factor Va light chain.

The 74-kDa light chain of bovine factor Va is composed of three domains: the NH2-terminal A3 domain and the COOH-terminal C1 and C2 domains. In total, the light chain has eight cysteines: two in the A3 domain and three in each C domain. To determine the locations of the disulfide bridges, peptides were obtained from factor Va and iodo[1-14C]acetamide-labeled factor Va light chains by digestion with trypsin, activated protein C, lysylendopeptidase, and V8 protease. After HPLC purification, amino acid sequence and composition analyses showed that each domain of bovine Va light chain possesses a disulfide bond. The sites are Cys1684-Cys1710 (A3), Cys1866-Cys2020 (C1), and Cys2025-Cys2180 (C2). One free cysteine is located in each C domain, i.e., Cys1953 and Cys2100. The locations of the disulfide bonds in human Va and VIIIa light chains are anticipated to be similar to those of bovine Va light chain, because the cysteines involved are conserved.

Amino Acid Sequence↗