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PubMed2000

Homocysteine inhibits inactivation of factor Va by activated protein C.

We report the effect of homocysteine on the inactivation of factor Va by activated protein C (APC) using clotting assays, immunoblotting, and radiolabeling experiments. Homocysteine, cysteine, or homocysteine thiolactone have no effect on factor V activation by alpha-thrombin. Factor Va derived from homocysteine-treated factor V was inactivated by APC at a reduced rate. The inactivation impairment increased with increasing homocysteine concentration (pseudo first order rate k = 1.2, 0.9, 0.7, 0.4 min(-1) at 0, 0.03, 0.1, 1 mm homocysteine, respectively). Neither cysteine nor homocysteine thiolactone treatment of factor V affected APC inactivation of derived factor Va. Western blot analyses of APC inactivation of homocysteine-modified factor Va are consistent with the results of clotting assays. Factor Va, derived from factor V treated with 1 mm beta-mercaptoethanol was inactivated more rapidly than the untreated protein sample. Factor V incubated with [(35)S]homocysteine (10-450 micrometer) incorporated label within 5 min, which was found only in those fragments that contained free sulfhydryl groups: the light chain (Cys-1960, Cys-2113), the B region (Cys-1085), and the 26/28-kDa (residues 507-709) APC cleavage products of the heavy chain (Cys-539, Cys-585). Treatment with beta-mercaptoethanol removed all radiolabel. Plasma of patients assessed to be hyperhomocysteinemic showed APC resistance in a clot-based assay. Our results indicate that homocysteine rapidly incorporates into factor V and that the prothrombotic tendency in hyperhomocysteinemia may be related to impaired inactivation of factor Va by APC due to homocysteinylation of the cofactor by modification of free cysteine(s).

Blotting, Western↗
PubMed1989

Ca2+-dependent structural changes in bovine blood coagulation factor Va and its subunits.

The calcium dependence of the structures of bovine blood coagulation factor Va and its subunits (Vh and Vl) has been examined spectroscopically in order to characterize the conformational changes which accompany the binding of Ca2+ to Vh and Vl to form factor Va. The far-UV CD spectra of the isolated subunits indicate that the secondary structures of both Vh and Vl are predominantly beta-sheet (greater than 45%), with little alpha-helix content (less than 15%). No change in the far-UV CD spectrum was observed when factor Va was formed by the addition of Ca2+ to an equimolar mixture of Vl and Vh. Hence, no detectable change in secondary structure occurs during the formation of factor Va. In contrast, the addition of Ca2+ to an equimolar mixture of Vh and Vl caused a small (2%) increase in the total intrinsic fluorescence intensity and a blue shift in the emission spectrum that resulted from a tertiary structural change and/or the association of nonpolar surfaces at the subunit interface. This fluorescence change correlated closely with the appearance of functional factor Va, since the rate of the spectral change was the same as the rate of recovery of cofactor activity, and since both were half-maximal near 50 microM Ca2+. This fluorescence change required both subunits, was reversed by the addition of EDTA, and was observed only with metal ions that can substitute for Ca2+ in reconstituting factor Va activity from Vh and Vl (Mn2+ and Tb3+; not Mg2+). When a sample containing ANS (8-anilino-1-naphthalenesulfonate) and an equimolar mixture of calcium-free Vh and Vl was titrated with Ca2+, the ANS emission intensity decreased by about 30%, most likely because the association of Vl and Vh caused nonpolar regions at the subunit-subunit interface to become inaccessible for ANS binding. The calcium dependence of this spectral change yielded a Kd of 51 +/- 2 microM, and the rate of the decrease in ANS fluorescence occurred at nearly the same rate as the recovery of factor Va activity. Thus, both intrinsic and extrinsic fluorescence data, as well as other data, indicate that the calcium binding site in factor Va has an apparent Kd of 50 microM under our conditions and that the calcium-mediated binding between Vl and Vh involves hydrophobic interactions between the subunits.(ABSTRACT TRUNCATED AT 400 WORDS)

Anilino Naphthalenesulfonates↗
PubMed1984

Functional properties of factor Va subunits after proteolytic alterations by activated protein C.

The two-subunit structure of the factor Va molecule is essential to its function in the prothrombinase complex. In the presence of phospholipids, the cleavage of the light chain of bovine factor Va by activated protein C proceeded at the same rate as the cleavage of the heavy chain. The limited proteolysis of factor Va is accompanied by a parallel loss of factor Va activity. Evidence that loss of activity was solely the result of the cleavage of the heavy chain, was obtained from reconstitution experiments utilizing cleaved and intact chains. The pseudo first-order rate constant of factor Va inactivation by activated protein C was found to be dependent on the amount of phospholipid-bound activated protein C and not on the amount of phospholipid-bound factor Va. However, phospholipids enhance the rate of proteolysis of the phospholipid-binding subunit, i.e. the light chain, and not the cleavage of the heavy chain. Cleavage of the heavy chain and as a consequence the inactivation of factor Va by activated protein C is mediated by phospholipid-bound light chain. After cleavage of the light chain, the 'two-subunit' structure, as well as the phospholipid-binding properties of factor Va were found to be conserved.

Animals↗
PubMed1997

Prothrombin kringle 1 domain interacts with factor Va during the assembly of prothrombinase complex.

The kringle 2 domain of prothrombin has been shown to interact with factor Va during the activation of prothrombin by the prothrombinase complex composed of factor Xa, factor Va, negatively charged phospholipids and Ca2+ ions. However, contradictory results have been reported about the role of the kringle 1 domain of prothrombin during the assembly of the prothrombinase complex. In an attempt to clarify the role of the kringle 1 domain of prothrombin, its effect on the activation of prothrombin by the prothrombinase complex and its direct binding to human factor Va were assessed. Comparative evaluation with the effects caused by other prothrombin structural components [a fragment 1 (gamma-carboxyglutamic acid and kringle 1 domains), a kringle 2 domain and a catalytic protease domain] was also performed. In the presence of factor Va, each kringle 1 and kringle 2 fragment significantly inhibited the factor Xa-catalysed prothrombin activation in the absence of phospholipids. However, in the absence of both factor Va and phospholipids, kringle 2 fragment, but not kringle 1 fragment, inhibited prothrombin activation. Evaluation of the molecular interaction of the kringle domains with factor Va in assays with solid-phase phospholipid vesicles showed that each kringle 1 and kringle 2 fragment inhibited the prothrombinase complex activity. Assessment of the direct binding of prothrombin and each kringle domain of prothrombin with factor Va by fluorescence polarization showed that prothrombin, kringle 1 and kringle 2 fragments bind directly to factor Va with dissociation constants of 1.9+/-0.1, 2.3+/-0.1 and 2.0+/-0.4 microM (means+/-S.D.) respectively. These findings suggest that both kringle 1 and 2 domains of prothrombin interact with factor Va during the assembly of the prothrombinase complex.

Blood Coagulation↗
PubMed1984

Coagulation factor Va binds to human umbilical vein endothelial cells and accelerates protein C activation.

In vitro the rate of protein C activation by thrombin is significantly accelerated by two distinct cofactors (a) the endothelial cell surface protein, thrombomodulin, and (b) human coagulation Factor Va. We have recently reported that the activity of Factor Va is contained in the 78,000-D light chain. In this study we have investigated the effects of Factor Va and its light chain on the activation of protein C in the presence of cultured endothelial cells. Thrombin-catalyzed protein C activation on human umbilical vein endothelial cells was enhanced by Factor Va. The ability of Factor Va to stimulate protein C activation on these cells was saturated at 50 nM Factor Va and was observed at several protein C concentrations. Isolated Factor Va light chain in concentrations up to 50 nM also accelerated protein C activation on endothelial cells, but higher concentrations inhibited the reaction. The effects of Factor Va or its light chain on protein C activation were also shown on a mouse hemangioma cell line but not on human fibroblasts nor on a human amelanotic melanoma cell line. Protein C activation on endothelial cells was partially inhibited by a goat anti-thrombomodulin antibody and further addition of a polyclonal rabbit anti-Factor V(Va) antibody resulted in additional inhibition. Endothelial cells grown in medium supplemented with human serum, devoid of Factor V coagulant activity, contained cell surface Factor V(Va) (approximately 15,000 molecules/cell) as measured by the binding of a monoclonal IgG directed against Factor V(Va). These cells also bound an additional 6,000-10,000 molecules Factor Va per cell as determined by direct binding studies using 125I-Factor Va. We suggest that thrombomodulin and Factor Va act in concert to regulate protein C activation on the surface of endothelial cells.

Cells, Cultured↗
PubMed2004

Trp2063 and Trp2064 in the factor Va C2 domain are required for high-affinity binding to phospholipid membranes but not for assembly of the prothrombinase complex.

Interactions between factor Va and membrane phosphatidylserine (PS) regulate activity of the prothrombinase complex. Two solvent-exposed hydrophobic residues located in the C2 domain, Trp(2063) and Trp(2064), have been proposed to contribute to factor Va membrane interactions by insertion into the hydrophobic membrane bilayer. However, the prothrombinase activity of rHFVa W(2063, 2064)A was found to be significantly impaired only at low concentrations of PS (5 mol %). In this study, we find that 10-fold higher concentrations of mutant factor Va are required for half-maximal prothrombinase activity on membranes containing 25% PS. The ability of the mutant factor Va to interact with factor Xa on a membrane was also impaired since 4-fold higher concentrations of factor Xa were required for half-maximal prothrombinase activity. The interaction of factor Va with 25% PS membranes was also characterized using fluorescence energy transfer and surface plasmon resonance. We found that the affinity of mutant factor Va for membranes containing 25% PS was reduced at least 400-fold with a K(d) > 10(-7) M. The binding of mutant factor Va to 25% PS membranes was markedly enhanced in the presence of factor Xa, indicating stabilization of the factor Va-factor Xa-membrane complex. Our findings indicate that Trp(2063) and Trp(2064) play a critical role in the high-affinity binding of factor Va to PS membranes. It remains to be determined whether occupancy of this PS binding site in factor Va is also required for high-affinity binding to factor Xa.

Alanine↗
PubMed1987

Interaction of blood coagulation factor Va with phospholipid vesicles examined by using lipophilic photoreagents.

Two different lipophilic photoreagents, [3H]adamantane diazirine and 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine (TID), have been utilized to examine the interactions of blood coagulation factor Va with calcium, prothrombin, factor Xa, and, in particular, phospholipid vesicles. With each of these structurally dissimilar reagents, the extent of photolabeling of factor Va was greater when the protein was bound to a membrane surface than when it was free in solution. Specifically, the covalent photoreaction with Vl, the smaller subunit of factor Va, was 2-fold higher in the presence of phosphatidylcholine/phosphatidylserine (PC/PS, 3:1) vesicles, to which factor Va binds, than in the presence of 100% PC vesicles, to which the protein does not bind. However, the magnitude of the PC/PS-dependent photolabeling was much less than has been observed previously with integral membrane proteins. It therefore appears that the binding of factor Va to the membrane surface exposes Vl to the lipid core of the bilayer, but that only a small portion of the Vl polypeptide is exposed to, or embedded in, the bilayer core. Addition of either prothrombin or active-site-blocked factor Xa to PC/PS-bound factor Va had little effect on the photolabeling of Vl with TID, but reduced substantially the covalent labeling of Vh, the larger subunit of factor Va. This indicates that prothrombin and factor Xa each cover nonpolar surfaces on Vh when the macromolecules associate on the PC/PS surface. It therefore seems likely that the formation of the prothrombinase complex involves a direct interaction between Vh and factor Xa and between Vh and prothrombin.(ABSTRACT TRUNCATED AT 250 WORDS)

Adamantane↗
PubMed1983

Human coagulation factor Va is a cofactor for the activation of protein C.

During blood clotting in vitro, protein C is converted in part to protein Ca, Protein Ca, in turn, inactivates factor Va. This is evidenced by the rapid inactivation of factor Va coagulant activity after clot formation which is associated with the cleavage of the Mr 110,000 peptide of factor Va. When exogenous factor Va is added to serum, it is inactivated only after a lag of 10-20 min. Using purified coagulation factors in the presence of EDTA, we demonstrated that factor Va enhances the rate of protein C activation by thrombin by 50-fold. The Km for factor Va in the reaction is 14 nM, 100 times higher than its Km for accelerating platelet surface prothrombin activation by factor Xa. By this mechanism, factor Va can act as a procoagulant as well as limit dissemination of the coagulation process through the activation of protein C and the subsequent inactivation of both factor Va and factor VIIIa.

Blood Coagulation↗
PubMed1999

Phospholipid-bound tissue factor modulates both thrombin generation and APC-mediated factor Va inactivation.

Hemostasis is initiated by tissue factor (TF) exposed on cellular phospholipid (PL) membranes, leading to thrombin generation. The binding of thrombin to thrombomodulin (TM), activates the protein C pathway, resulting in the inactivation of factors Va and VIIIa by activated protein C (APC) and a negative feedback effect on thrombin generation. A new assay system was developed for simultaneous measurement of thrombin and APC generation in defibrinated plasma induced by large unilamellar PL vesicles complexed with full-length recombinant TF (TF:PL). TF:PL preparations with a low TF concentration induced an initial rate of thrombin generation below 100 nM/min, and resulted in less thrombin formation in the presence of TM than in its absence. In contrast, TF:PL preparations with a high concentration of TF induced a higher rate of thrombin generation, and APC-mediated feedback inhibition did not occur, despite maximal APC generation. We used the same TF:PL surfaces to study factor Va inactivation by APC in a non-plasma reaction system, and found an inverse correlation between TF surface density and the rate of factor Va inactivation. This observation suggests a previously unrecognized hemostatic effect of TF, namely a non-enzymatic surface density-based inhibition of the anticoagulant effect of APC. In this model, high concentrations and surface density of TF exert complementary effects by promoting the regular procoagulant cascade and by inhibiting the protein C pathway, thereby maximizing hemostasis after vascular injury.

Animals↗
PubMed2003

The contribution of amino acid region ASP695-TYR698 of factor V to procofactor activation and factor Va function.

There is strong evidence that a functionally important cluster of amino acids is located on the COOH-terminal portion of the heavy chain of factor Va, between amino acid residues 680 and 709. To ascertain the importance of this region for cofactor activity, we have synthesized five overlapping peptides representing this amino acid stretch (10 amino acids each, HC1-HC5) and tested them for inhibition of prothrombinase assembly and function. Two peptides, HC3 (spanning amino acid region 690-699) and HC4 (containing amino acid residues 695-704), were found to be potent inhibitors of prothrombinase activity with IC(50) values of approximately 12 and approximately 10 microm, respectively. The two peptides were unable to interfere with the binding of factor Va to active site fluorescently labeled Glu-Gly-Arg human factor Xa, and kinetic analyses showed that HC3 and HC4 are competitive inhibitors of prothrombinase with respect to prothrombin with K(i) values of approximately 6.3 and approximately 5.3 microm, respectively. These data suggest that the peptides inhibit prothrombinase because they interfere with the incorporation of prothrombin into prothrombinase. The shared amino acid motif between HC3 and HC4 is composed of Asp(695)-Tyr-Asp-Tyr-Gln(699) (DYDYQ). A pentapeptide with this sequence inhibited both prothrombinase function with an IC(50) of 1.6 microm (with a K(D) for prothrombin of 850 nm), and activation of factor V by thrombin. Peptides HC3, HC4, and DYDYQ were also found to interact with immobilized thrombin. A recombinant factor V molecule with the mutations Asp(695) --> Lys, Tyr(696) --> Phe, Asp(697) --> Lys, and Tyr(698) --> Phe (factor V(2K2F)) was partially resistant to activation by thrombin but could be readily activated by RVV-V activator (factor Va(RVV)(2K2F)) and factor Xa (factor Va(Xa)(2K2F)). Factor Va(RVV)(2K2F) and factor Va(Xa)(2K2F) had impaired cofactor activity within prothrombinase in a system using purified reagents. Our data demonstrate for the first time that amino acid sequence 695-698 of factor Va heavy chain is important for procofactor activation and is required for optimum prothrombinase function. These data provide functional evidence for an essential and productive contribution of factor Va to the activity of prothrombinase.

Amino Acids↗
PubMed1993

Binding of protein S to factor Va associated with inhibition of prothrombinase that is independent of activated protein C.

Since plasma protein S serves an anticoagulant function by mechanisms which are not completely understood, its possible interaction with Factor Va was investigated. Human protein S bound to immobilized human Factor Va in a calcium-dependent, saturable, and reversible manner and Factor Va bound similarly to immobilized protein S. Binding of protein S to immobilized Factor V was greatly enhanced by pretreatment of the surface-bound Factor V with increasing doses of thrombin up to 1 unit/ml. Binding of protein S to Factor Va was also demonstrated in fluid phase with a Kd of 33 +/- 9 nM. Biotin-labeled heavy chain of Factor Va bound to immobilized protein S, and this binding was reversed by a 17-fold molar excess of intact unlabeled Factor Va. Protein S competed efficiently with prothrombin for binding to immobilized Factor Va. The prothrombinase activity in a reaction mixture of purified clotting factors was inhibited by protein S and exhibited a pattern of mixed inhibition. The concentration of protein S needed for 50% inhibition of the prothrombinase activity of a mixture containing 1 nM Factor Xa, 20 pM Factor Va, and 50 microM phospholipids was about 16 nM. Since not all protein S preparations exhibited this degree of prothrombinase inhibitory activity, extensive control experiments were performed to verify that the inhibitory activity was associated with protein S during immunoaffinity chromatography and was not caused by traces of activated protein C in the protein S preparations. These data show that protein S has an anticoagulant function which is independent of activated protein C and, at least in part, that this is because of its competition with prothrombin for direct binding to Factor Va.

Binding, Competitive↗
PubMed1987

Thrombin-induced platelet factor Va formation in patients with a gray platelet syndrome.

The present study was initiated to establish the functional factor V concentration in platelets of patients with a mild bleeding disorder ascribed to a gray platelet syndrome. This inherited platelet disorder has been characterized by a specific deficiency of alpha-granules and subsequent deficiencies in the alpha-granule proteins. We found that the concentration of plasma factor V was slightly decreased (70% of normal values). In contrast, platelet factor Va formation was severely impaired. Besides a much lower factor V content than in control platelets (10-20% of normal), the dependency of platelet factor Va formation on thrombin concentration was altered. Increasing the thrombin concentration 4-fold compared to the concentration that results in maximal factor Va generation from normal platelets did not result in a maximal factor Va formation from gray platelets. When a suspension of washed gray platelets was incubated with a prostacyclin analogue prior to the stimulation with thrombin, a 10-fold lower factor Va activity was measured. Thus, thrombin-induced factor Va formation in a suspension of gray platelets is the result of a release reaction, followed by the thrombin-catalyzed activation of released factor V. Whereas the kinetics of the former reaction are apparently impaired, the kinetics of the latter one were found to be identical to those observed for normal platelet and plasma factor V activation.

Blood Platelet Disorders↗
PubMed1993

Effects of Ca2+ binding on the protease module of factor Xa and its interaction with factor Va. Evidence for two Gla-independent Ca(2+)-binding sites in factor Xa.

The assembly of macromolecular complexes containing factors Xa and Va on suitable phospholipid surfaces is crucial for rapid activation of prothrombin. We have used quantitative affinity chromatography to characterize the interaction between factor Va and intact factor Xa on the one hand and between factor Va and factor Xa lacking the gamma-carboxyglutamic acid (Gla)-containing module on the other. The dissociation constants were found to be 1.0 +/- 0.1 and 9.5 +/- 1.8 microM, respectively. There was good agreement between these dissociation constants and the concentrations of active site-inhibited factor Xa and Gla-domainless factor Xa that caused half-maximal inhibition of prothrombin activation. To investigate whether the noncatalytic modules of factor Xa interacted directly with factor Va, intact modules were isolated from proteolytic digests of factor X and used as inhibitors of prothrombin activation. The inhibitory effect observed with the isolated Gla module in the absence of phospholipid was due to inhibition of the amidolytic activity of factor Xa rather than to an interaction with factor Va. The epidermal growth factor-like modules did not inhibit prothrombin activation. Using antibodies specific for calcium-dependent epitopes in the serine protease module of factor Xa we demonstrated that Ca2+ binding to the Gla module alters the conformation of the catalytic module. Half-maximal binding was observed at approximately 0.8 mM Ca2+. Evidence was also obtained for the presence of two Gla-independent Ca(2+)-binding sites in factor Xa. One of these sites, located in the NH2-terminal epidermal growth factor-like module, was half-saturated at approximately 60 microM Ca2+ in intact factor Xa and at approximately 1.2 mM Ca2+ in Gla-domainless factor Xa. This site appeared not to influence the conformation of the protease module. The second site, which was half-saturated at approximately 0.16 mM Ca2+, appeared to reside in the serine protease module and to alter its conformation as judged by binding of antibodies specific for calcium-dependent epitopes.

1-Carboxyglutamic Acid↗
PubMed2006

A control switch for prothrombinase: characterization of a hirudin-like pentapeptide from the COOH terminus of factor Va heavy chain that regulates the rate and pathway for prothrombin activation.

Membrane-bound factor Xa alone catalyzes prothrombin activation following initial cleavage at Arg(271) and prethrombin 2 formation (pre2 pathway). Factor Va directs prothrombin activation by factor Xa through the meizothrombin pathway, characterized by initial cleavage at Arg(320) (meizo pathway). We have shown previously that a pentapeptide encompassing amino acid sequence 695-699 from the COOH terminus of the heavy chain of factor Va (Asp-Tyr-Asp-Tyr-Gln, DYDYQ) inhibits prothrombin activation by prothrombinase in a competitive manner with respect to substrate. To understand the mechanism of inhibition of thrombin formation by DYDYQ, we have studied prothrombin activation by gel electrophoresis. Titration of plasma-derived prothrombin activation by prothrombinase, with increasing concentrations of peptide, resulted in complete inhibition of the meizo pathway. However, thrombin formation still occurred through the pre2 pathway. These data demonstrate that the peptide preferentially inhibits initial cleavage of prothrombin by prothrombinase at Arg(320). These findings were corroborated by studying the activation of recombinant mutant prothrombin molecules rMZ-II (R155A/R284A/R271A) and rP2-II (R155A/R284A/R320A) which can be only cleaved at Arg(320) and Arg(271), respectively. Cleavage of rMZ-II by prothrombinase was completely inhibited by low concentrations of DYDYQ, whereas high concentrations of pentapeptide were required to inhibit cleavage of rP2-II. The pentapeptide also interfered with prothrombin cleavage by membrane-bound factor Xa alone in the absence of factor Va increasing the rate for cleavage at Arg(271) of plasma-derived prothrombin or rP2-II. Our data demonstrate that pentapeptide DYDYQ has opposing effects on membrane-bound factor Xa for prothrombin cleavage, depending on the incorporation of factor Va in prothrombinase.

Blood Coagulation↗
PubMed2004

The critical role of the 185-189-loop in the factor Xa interaction with Na+ and factor Va in the prothrombinase complex.

The S1 site (Asp(189)) of factor Xa (fXa) is located on a loop (residues 185-189) that contains three solvent-exposed charged residues (Asp(185), Lys(186), and Glu(188)) below the active-site pocket of the protease. To investigate the role of these residues in the catalytic function of fXa, we expressed three mutants of the protease in which the charges of these residues were neutralized by their substitutions with Ala (D185A, K186A, and E188A). Kinetic studies revealed that E188A has a normal catalytic activity toward small synthetic and natural substrates and inhibitors of fXa; however, the same activities were slightly ( approximately 2-fold) and dramatically ( approximately 20-50-fold) impaired for the D185A and K186A mutants, respectively. Further studies revealed that the affinity of D185A and K186A for interaction with Na(+) has also been altered, with a modest impairment ( approximately 2-fold) for the former and a dramatic impairment for the latter mutant. Both prothrombinase and direct binding studies indicated that K186A also has an approximately 6-fold impaired affinity for factor Va. Interestingly, a saturating concentration of factor Va restored the catalytic defect of K186A in reactions with prothrombin and the recombinant tick anticoagulant peptide that is known to interact with the Na(+) loop of fXa, but not with other substrates. These results suggest that factor Va interacts with 185-189-loop for fXa, which is energetically linked to the Na(+)-binding site of the protease.

Alanine↗
PubMed1986

A domain of membrane-bound blood coagulation factor Va is located far from the phospholipid surface. A fluorescence energy transfer measurement.

The larger subunit of blood coagulation factor Va was covalently labeled with iodoacetamido derivatives of fluorescein and rhodamine without loss of functional activity, as measured by either the one-stage clotting assay or the ability to accelerate prothrombin activation in a purified system. The spectral properties of the dyes were not altered by the presence or absence of the smaller subunit of factor Va, Ca2+, prothrombin, factor Xa, or phosphatidylcholine/phosphatidylserine (PC/PS, 4:1) vesicles. When fluorescein-labeled protein (factor VaF) was titrated with PC/PS vesicles containing either octadecylrhodamine or 5-(N-hexadecanoylamino)eosin, fluorescence energy transfer was observed between the protein-bound donor dyes and the acceptor dyes at the outer surface of the phospholipid bilayer. The extent of energy transfer correlated directly with the extent of protein binding to the vesicles monitored by light scattering. The distance of closest approach between the fluorescein on factor Va and the bilayer surface averaged 90 A for the two different acceptors. Association of factor VaF with factor Xa on the phospholipid surface reduced this separation by 7 A, but association with prothrombin did not alter the distance between the labeled domain on factor VaF and the surface. The efficiency of diffusion-enhanced energy transfer between rhodamine-labeled factor Va and terbium dipicolinate entrapped inside PC/PS vesicles was less than 0.01, consistent with the location of the dye far above the inner surface of the vesicle. Thus, a domain of membrane-bound factor Va is located a minimum of 90 A above the phospholipid surface.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

Kinetic studies of prothrombin activation: effect of factor Va and phospholipids on the formation of the enzyme-substrate complex.

The kinetic parameters of bovine prothrombin activation by factor Xa were determined in the absence and presence of factor Va as a function of the phospholipid concentration and composition. In the absence of factor Va, the Km for prothrombin increases proportionally with the phospholipid concentration and correlates well with the affinity of prothrombin for the different membranes. Phospholipid vesicles with a high affinity for prothrombin yield low Km values compared to membranes with less favorable binding parameters. At limited phospholipid concentrations, the Vmax of prothrombin activation correlates with the binding affinity of factor Xa for the various phospholipid vesicles. Membranes with a high affinity for factor Xa have high Vmax values, while for membranes with a low affinity a low Vmax is observed. Extrapolation of double-reciprocal plots of 1/Vmax vs. 1/[phospholipid] to infinite phospholipid concentrations, a condition at which all factor Xa would participate in prothrombin activation, yields a kcat of 2-4 min-1 independent of the type and amount of acidic phospholipid present in the vesicles. Also, in the presence of factor Va the Km for prothrombin varies proportionally with the phospholipid concentration. There is, however, no correlation between the binding parameters and the Km. Factor Va drastically lowers the Km for prothrombin for vesicles that have a low affinity for prothrombin. Vesicles composed of 20 mol % phosphatidylglycerol and 80 mol % phosphatidylcholine have a Km of 0.04 microM when factor Va is present, compared to 2.2 microM determined in the absence of factor Va.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1996

Insights into the complex association of bovine factor Va with acidic-lipid-containing synthetic membranes.

The mechanism of binding of blood coagulation cofactor factor Va to acidic-lipid-containing membranes has been addressed. Binding isotherms were generated at room temperature using the change in fluorescence anisotropy of pyrene-labeled bovine factor Va to detect binding to sonicated membrane vesicles containing either bovine brain phosphatidylserine (PS) or 1,2-dioleoyl-3-sn-phosphatidylglycerol (DOPG) in combination with 1-palmitoyl-2-oleoyl-3-sn-phosphatidylcholine (POPC). The composition of the membranes was varied from 0 to 40 mol% for PS/POPC and from 0 to 65 mol % for DOPG/POPC membranes. Fitting the data to a classical Langmuir adsorption model yielded estimates of the dissociation constant (Kd) and the stoichiometry of binding. The values of Kd defined in this way displayed a maximum at low acidic lipid content but were nearly constant at intermediate to high fractions of acidic lipid. Fitting the binding isotherms to a two-process binding model (nonspecific adsorption in addition to binding of acidic lipids to sites on the protein) suggested a significant acidic-lipid-independent binding affinity in addition to occupancy of three protein sites that bind PS in preference to DOPG. Both analyses indicated that interaction of factor Va with an acidic-lipid-containing membrane is much more complex than those of factor Xa or prothrombin. Furthermore, a change in the conformation of bound pyrene-labeled factor Va with surface concentration of acidic lipid was implied by variation of both the saturating fluorescence anisotropy and the binding parameters with the acidic lipid content of the membrane. Finally, the results cannot support the contention that binding occurs through nonspecific adsorption to a patch or domain of acidic lipids in the membrane. Factor Va is suggested to associate with membranes by a complex process that includes both acidic-lipid-specific and acidic-lipid-independent sites and a protein structure change induced by occupancy of acidic-lipid-specific sites on the factor Va molecule.

Adsorption↗