Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Factor Va”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 19 recordsLinked to original sources
PubMed1983

A monoclonal antibody which inhibits the factor Va:factor Xa interaction.

An immunoprecipitation technique has been used to determine the subunit specificity of two of the monoclonal antibodies to bovine Factor V(Va) developed by this laboratory. One of the antibodies is specific for the 74,000-dalton subunit (the E chain) of Factor Va, and the other antibody is specific for the 94,000-dalton subunit (the D chain). The binding of Factor Va to phospholipid was studied by light scattering, and the interaction of Factor Xa with phospholipid-bound Factor Va was examined using 5-dimethylaminonaphthalene-1-sulfonyl-glutamyl-glycyl-arginyl-Xa (Dns-EGR-Xa). Neither the antibody specific for the E chain nor the antibody specific for the D chain inhibit the binding of Factor Va to phospholipid vesicles. The antibody specific for the E chain blocks the increase in fluorescence polarization seen when Factor Va is added to a solution of Dns-EGR-Xa, phospholipid vesicles and calcium. This antibody also inhibits the association of Dns-EGR-Xa with phospholipid-bound Factor Va as determined by gel-exclusion high pressure liquid chromatography. The antibody specific for the D chain of Factor Va does not block the increase in polarization seen when Factor Va is added to a solution of Dns-EGR-Xa, phospholipid, and calcium. It was concluded that the antibody specific for the E chain of Factor Va binds at or near the Factor Xa-binding site on the E chain and that the Factor Va E chain plays a significant role in binding Factor Xa.

Animals↗
PubMed1979

Phospholipid-binding properties of bovine factor V and factor Va.

Factor V and factor Va binding to single bilayer phospholipid vesicles was investigated by light-scattering intensity measurements. This technique allows the measurement of free and phospholipid-bound protein concentrations from which equilibrium constants can be obtained. As controls, the Ca2+-dependent phospholipid binding of prothrombin and factor X were also studied. The average values obtained for the dissociation constants (Kd) and lipid to protein ratio at saturation, moles/mole (n), for prothrombin (Kd = 2.3 X 10(-6) M, n = 104) and factor X (Kd = 2.5 X 10(-6) M, n = 46) binding to vesicles containing 25% Folch fraction III and 75% phosphatidylcholine in the presence of 2 mM Ca2+ were in agreement with those reported in the literature. The average factor V and factor Va values for the dissociation constants and lipid to protein ratio at saturation (moles/mole) were Kd = 7.2 X 10(-8) M and n = 270 for factor V and Kd = 4.4 X 10(-7) M and n = 76 for factor Va. In contrast to prothrombin and factor X, factor V and factor Va demonstrated Ca2+-independent lipid binding. In addition, the number of factor V and factor Va molecules bound per vesicle was found to be dependent both on the phosphatidylserine content of the vesicle and the ionic strength of the buffer.

Animals↗
PubMed1995

Peptide bond cleavages and loss of functional activity during inactivation of factor Va and factor VaR506Q by activated protein C.

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)

Arginine↗
PubMed1995

Effects of protein S and factor Xa on peptide bond cleavages during inactivation of factor Va and factor VaR506Q by activated protein C.

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.

Amino Acid Sequence↗
PubMed1981

Coordinate binding of factor Va and factor Xa to the unstimulated platelet.

The interaction of Factor Xa and Factor Va at the platelet surface was investigated by direct, simultaneous binding measurements of both proteins to platelets and by immunochemical and kinetic techniques. Binding measurements of 125I-Factor Xa and 131I-Factor Va to unstimulated platelets indicate that the amount of Factor Xa bound is proportional to the amount of Factor Va bound. At saturating concentrations of Factor Xa, the ratio of platelet-bound Factor Xa to platelet-bound Factor Va was unity. At saturating levels of Factor Va (1.2 X 10(-8) M), 2300 molecules of Factor Xa are bound to the platelet, whereas at an equivalent concentration of unactivated Factor V, only 800 molecules of Factor Xa are bound. In the absence of exogenous Factor V or Va, thrombin-treated washed platelets bound only 200 Factor Xa molecules per platelet, suggesting that under these conditions, platelet Factor Va is the limiting component. Monovalent Fab fragments of burro antibovine Factor V inhibit, to the same degree, the rate of thrombin generation and the binding of Factor Va and Factor Xa to the platelet surface. Anti-Factor V Fab decreased the extent of Factor Va and Factor Xa binding equivalently. When the interaction of Factor Xa with platelets is modeled as Factor Xa binding to platelet-bound Factor Va, double reciprocal plots are linear, yielding a stoichiometry of 1.04 and a dissociation constant of 6 X 10(-10) M. Kinetic experiments indicate the presence of approximately 900 functional Factor Va platelet sites (Kd = 1.5 to 2.2 X 10(-10) M). This number of functional sites is equivalent to the number (837 +/- 48) of Factor Va high affinity binding sites (Kd = 4.0 X 10(-10) M). These sites most likely represent the Factor Xa binding sites involved in the function of the prothrombinase complex at the platelet surface.

Animals↗
PubMed1993

Phosphorylation of factor Va and factor VIIIa by activated platelets.

Platelet activation leads to the incorporation of 32[PO4(2-)] into bovine coagulation factor Va and recombinant human factor VIII. In the presence of the soluble fraction from thrombin-activated platelets and (gamma-32P) adenosine triphosphate, radioactivity is incorporated exclusively into the M(r) = 94,000 heavy chain (H94) of factor Va and into the M(r) = 210,000 to 90,000 heavy chains as well into the M(r) = 80,000 light chain of factor VIII. Proteolysis of the purified phosphorylated M(r) = 94,000 factor Va heavy chain by activated protein C (APC) gave products of M(r) = 70,000, 24,000, and 20,000. Only the intermediate M(r) = 24,000 fragment contained radioactivity. Because the difference between the M(r) = 24,000 and M(r) = 20,000 fragments is located on the COOH-terminal end of the bovine heavy chain, phosphorylation of H94 must occur within the M(r) = 4,000 peptide derived from the carboxyl-terminal end of H94 (residues 663 through 713). Exposure of the radioactive factor VIII molecule to thrombin ultimately resulted in a nonradioactive light chain and an M(r) = 24,000 radioactive fragment that corresponds to the carboxyl-terminal segment of the A1 domain of factor VIII. Based on the known sequence of human factor VIII, phosphorylation of factor VIII by the platelet kinase probably occurs within the acidic regions 337 through 372 and 1649 through 1689 of the procofactor. These acidic regions are highly homologous to sequences known to be phosphorylated by casein kinase II. Results obtained using purified casein kinase II gave a maximum observed stoichiometry of 0.6 mol of 32[PO4(2-)]/mol of factor Va heavy chain and 0.35 mol of 32[PO4(2-)]/mol of factor VIII. Phosphoamino acid analysis of phosphorylated factor Va by casein kinase II or by the platelet kinase showed only the presence of phosphoserine while phosphoamino acid analysis of phosphorylated factor VIII by casein kinase II showed the presence of phosphothreonine as well as small amounts of phosphoserine. The platelet kinase responsible for the phosphorylation of the two cofactors was found to be inhibited by several synthetic protein kinase inhibitors. Finally, partially phosphorylated factor Va was found to be more sensitive to APC inactivation than its native counterpart. Our findings suggest that phosphorylation of factors Va and VIIIa by a platelet casein kinase II-like kinase may downregulate the activity of the two cofactors.

Amino Acid Sequence↗
PubMed1995

Prothrombin contributes to the assembly of the factor Va-factor Xa complex at phosphatidylserine-containing phospholipid membranes.

The activation of prothrombin is catalyzed by prothrombinase, a complex of factor Xa and factor Va assembled on a negatively charged phospholipid membrane. We used a tubular flow reactor to identify the relative contributions of factor Va, prothrombin, and the negatively charged phosphatidylserine to the assembly of prothrombinase. Perfusion of phospholipid-coated capillaries with a mixture of factor Xa, factor Va, and prothrombin resulted in a steady-state rate of thrombin production that increased with (i) the phosphatidylserine content of the phospholipid bilayer, (ii) the factor Va concentration, and, most interestingly, (iii) the prothrombin concentration of the perfusion solution. Incorporation of 20 mol % phosphoatidylethanolamine, a phospholipid with poor ability to promote prothrombinase activity, into a 5 mol % phosphatidylserine membrane also increased the steady-state rate of thrombin production. Direct measurements of the amount of prothrombinase in the flow reactor demonstrated that increased catalytic activities were the result of an increased steady-state amount of membrane-associated prothrombinase. Thus, similar turnover numbers of prothrombin activation (3100 min-1) were calculated, irrespective of the phosphatidylserine content of the membrane. We established for membranes with low phosphatidylserine content (< 10 mol%) a linear relationship between the prothrombinase activity and the arithematical product of the factor Va concentration in the perfusion solution and the prothrombin concentration near the catalytic surface. Our results indicate that, in addition to factor Va, prothrombin also is essential to the assembly of prothrombinase at macroscopic surfaces with low phosphatidylserine content. The data further suggest that the prothrombin concentration near the surface, controlled by the prothrombinase activity and mass transfer, is an important regulator of the prothrombinase surface density.

Factor Va↗
PubMed1982

Factor Va-factor Xa interaction. Effects of phospholipid vesicles of varying composition.

The interaction between factor Xa and factor Va was investigated both in solution and in the presence of phospholipid vesicles with varying contents of phosphatidylserine. The binding parameters were inferred from the kinetics of prothrombin activation. Factor Xa and factor Va form in solution an equimolar complex with a dissociation constant of 3.3 X 10(-9) M. Phospholipid vesicles promote the formation of the factor Xa-Va complex. The Kd of complex formation is dependent on both the phospholipid concentration and the composition of the phospholipid vesicle. For the interaction between factor Xa and factor Va in the presence of phospholipid vesicles containing 40 mol % dioleoylphosphatidylserine (DOPS) and 60 mol % dioleoylphosphatidylcholine (DOPC), the Kd increases linearly with increasing phospholipid concentration. In the presence of 10 microM phospholipid (DOPS/DOPC, 40/60 mol/mol) Kd = 3 X 10(-11) M. When the mole percentage of DOPS in the phospholipid vesicles is lowered from 20 to 5 mol %, there is a gradual increase of the Kd. In the presence of 10 microM phospholipid vesicles containing 5 mol % DOPS and 95 mol % DOPC Kd = 2.8 X 10(-10) M. The Kd measured in the presence of phospholipid vesicles containing 5 mol % DOPS and 95 mol % DOPC is independent of the phospholipid concentration. Two models are discussed that can quantitatively explain the effect of phospholipid vesicles on the complex formation between factor Xa and factor Va. Studies on the effect of the polypeptides with Mr 80 000 and Mr 94000 of which factor Va is composed on the Kd of the factor Xa-Va complex suggest that factor Xa binding to factor Va requires a Ca2+-mediated interaction between the two polypeptides.

Animals↗
PubMed1987

Proteolysis of factor Va by factor Xa and activated protein C.

Bovine Factor Va, produced by selective proteolytic cleavage of Factor V by thrombin, consists of a heavy chain (D chain) of Mr = 94,000 and a light chain (E chain) of Mr = 74,000. These peptides are noncovalently associated in the presence of divalent metal ion(s). Each chain is susceptible to proteolysis by activated protein C and by Factor Xa. Sodium dodecyl sulfate electrophoretic analysis indicates that cleavage of the E chain by either activated protein C or Factor Xa yields two major fragments: Mr = 30,000 and Mr = 48,000. Amino acid sequence analysis indicates that the Mr = 30,000 fragments have identical NH2-terminal sequences and that this sequence corresponds to that of intact E chain. The Mr = 48,000 fragments also have identical NH2-terminal sequences, indicating that activated protein C and Factor Xa cleave the E chain at the same position. Sodium dodecyl sulfate electrophoretic analysis indicates that activated protein C cleavage of the D chain yields two products: Mr = 70,000 and Mr = 24,000. Amino acid sequence analysis indicates that the Mr = 70,000 fragment has the same NH2-terminal sequence as intact D chain, whereas the Mr = 24,000 fragment does not. Factor Xa cleavage of the D chain also yields two products: Mr = 56,000 and Mr = 45,000. The Mr = 56,000 fragment corresponds to the NH2-terminal end of the D chain and Factor V. Functional studies have shown that both chains of Factor Va may be entirely cleaved to products by Factor Xa without loss of activity, whereas activated protein C cleavage results in loss of activity. Since activated protein C and Factor Xa cleave the E chain at the same position, the cleavage of the D chain by activated protein C is responsible for the inactivation of Factor Va.

Amino Acid Sequence↗
PubMed1994

The activities of recombinant gamma-carboxyglutamic-acid-deficient mutants of activated human protein C toward human coagulation factor Va and factor VIII in purified systems and in plasma.

The dependence of the activity of recombinant activated human protein C (r-APC) on each of its nine gamma-carboxyglutamic (Gla) residues (sequence positions 6, 7, 14, 16, 19, 20, 25, 26, and 29) has been assessed in purified systems and in plasma using r-mutants in which each Gla residue of r-APC was individually altered to an Asp (D) residue. The assays employed included a factor Va inactivation assay in the prothrombinase system with purified components and in plasma. In addition, a factor VIII inactivation assay in the tenase system, also with purified components, was utilized. Compared to wild-type protein (wtr-APC), the r-mutants that possessed nearly full activity in all assays were the Gla6-->D variant ([Gla6D]r-APC]) as well as [Gla14D]r-APC and [Gla19D]r-APC. In addition, another mutant (Q32-->Gla) in which a Gla was substituted for Gln (Q) at position 32, a situation that exists with other vitamin-K-dependent clotting proteins (e.g., factor IX and prothrombin), displayed full activity in all assays. Those mutants that possessed very-low-to-no activity in all assays included [Gla16D]r-APC and [Gla26D]r-APC. The other mutants showed partial and, in some cases, differential activity in these assay systems, with [Gla25D]r-APC being the most remarkable example. In this case, the factor V/Va plasma assay and the plasma-based activated partial thromboplastin time assay yielded < 25% activity, whereas nearly full activity was observed for this variant in the prothrombinase and tenase assays with purified components.(ABSTRACT TRUNCATED AT 250 WORDS)

1-Carboxyglutamic Acid↗
PubMed1984

Loss of prothrombin and of factor Xa-factor Va interactions upon inactivation of factor Va by activated protein C.

Activated factor V (factor Va) is composed of two nonidentical subunits which can be dissociated on chelation of the bound Ca2+ with EDTA. The isolated subunits can be recombined in the presence of Ca2+ to form factor Va. The factor Va heavy chain (Mr = 94,000) binds to prothrombin in a specific and Ca2+-independent fashion. Following inactivation of either factor Va or the factor Va heavy chain by limited proteolysis with activated protein C, factor Va no longer binds to the immobilized prothrombin. Factor Va also binds specifically to (p-amidinophenyl)-methanesulfonyl-factor Xa-Affi-Gel 15. However, neither isolated subunit binds to this column. Factor Va inactivated by activated protein C is no longer retained by the factor Xa column. This data suggests that both subunits are required for optimal factor Va-factor Xa interaction and that inactivation of factor Va with activated protein C reduces the affinity of factor Va for both prothrombin and factor Xa.

Animals↗
PubMed1990

Studies of the role of factor Va in the factor Xa-catalyzed activation of prothrombin, fragment 1.2-prethrombin-2, and dansyl-L-glutamyl-glycyl-L-arginine-meizothrombin in the absence of phospholipid.

In order to specifically evaluate the role of Factor Va in the prothrombinase complex, studies of the activation of prothrombin, Fragment 1.2-prethrombin-2, and active-site-blocked meizothrombin were carried out, both in the absence of phospholipid and at concentrations of substrates and Factor Va sufficient to approach saturation in all components. Km values were independent of Factor Va concentrations, whereas kcat (apparent) values approached saturation with respect to Factor Va concentrations. The three respective substrates exhibited the following parameters of kinetics (Km, microM; kcat, s-1 at saturating [Factor Va]): prothrombin (9.0 +/- 0.4; 31 +/- 1); Fragment 1.2-prethrombin-2 (5.4 +/- 0.4; 13 +/- 2); and meizothrombin (3.6 +/- 0.3; 51 +/- 5). Models of kinetics were constructed to interpret the results, and two of these were formally consistent with experimental results. Both models indicated that the variation of kcat(app) with concentrations of Factor Va reflects the formation of a Factor Va-Factor Xa binary complex. Analysis of kinetics indicated Kd values for this interaction of 1.3 +/- 0.1, 3.0 +/- 0.5, and 1.0 +/- 0.1 microM for the three respective substrates. The models differed in the interpretation of Km. One indicated that Km reflects a binary interaction between Factor Xa and prothrombin, whereas the other indicated a binary interaction between Factor Va and prothrombin. Both indicated that two of the three possible binary interactions between the three components would be reflected in Km and kcat values but not the third. To distinguish these models, the binary interactions were studied by extrinsic fluorescence (Va.Xa), light-scattering (Factor Va.prothrombin), and competition kinetics (Xa.II). The first two interactions were detected and were characterized by Kd values of 2.7 +/- 0.1 microM (Va.Xa) and 8.8 +/- 0.8 microM (Factor Va.prothrombin). No active-site-dependent interaction between prothrombin and Factor Xa could be detected in the absence of Factor Va. The results of these studies suggest that Factor Va interacts with both Factor Xa and prothrombin and effectively presents one to the other in the formation of a ternary enzyme-substrate-cofactor complex. In addition, a comparison of the parameters of kinetics of conversion of prothrombin and its intermediates indicates that meizothrombin is the major intermediate of prothrombin activation in the absence, as well as in the presence of phospholipid.

Amino Acid Sequence↗
PubMed1983

The light chain of factor Va contains the activity of factor Va that accelerates protein C activation by thrombin.

Protein C, a vitamin K-dependent protein, circulates in plasma as an inactive precursor. Once activated, it possesses potent anticoagulant activity through the inactivation of factors Va and VIIIa. Thrombin, the only known physiologic activator of this protein, is catalytically inefficient. Thrombomodulin, a protein purified from rabbit lungs, has been reported to enhance protein C activation by thrombin. We have previously demonstrated that factor Va, a substrate for activated protein C, is also a thrombin cofactor in the activation of protein C (Salem, H.H., Broze, G.J., Miletich, J. P., and Majerus, P.W. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 1584-1588). When factor Va is fractionated to its individual components, only the light chain (Mr 78,000) has thrombin cofactor activity. Although factor Va and thrombomodulin can both stimulate thrombin-catalyzed protein C activation, the physiological relationship between these two proteins remains to be determined.

Blood Coagulation Factors↗
PubMed1990

Identification and characterization of a phospholipid-binding site of bovine factor Va.

Coagulation factor Va is a cofactor which combines with the serine protease factor Xa on a phospholipid surface to form the prothrombinase complex. The phospholipid-binding domain of bovine factor Va has been reported to be located on the light chain of the molecule and more precisely on a fragment of Mr = 30,000 which is obtained after digestion of factor Va light chain by factor Xa. This proteolytic fragment is located in the NH2-terminal part of factor Va light chain (residues 1564-1765). In order to further characterize the lipid-binding domain of bovine factor Va, isolated bovine light chain was preincubated with synthetic phospholipid vesicles (75% phosphatidylcholine, 25% phosphatidylserine) and digested with trypsin, chymotrypsin, and elastase. Two peptide regions protected from proteolytic cleavage were identified and characterized from each proteolytic digestion. A comparison of the NH2-terminal sequence and amino acid composition of the two tryptic peptides with the deduced sequence of human factor V indicates a match with residues 1657-1791 of the light chain of human factor V for one peptide and residues 1546-1656 for the other peptide. When chymotrypsin or elastase were used for digestion, the NH2-terminal sequence of one peptide showed a match with residues 1667-1797 of the light chain, while the other peptide presented an NH2-terminal sequence identical with the previously described for the bovine factor Va light chain. When these peptides were assayed for direct binding to phospholipid vesicles, only the tryptic and the chymotryptic peptides covering the middle region of the A3 domain of the bovine factor Va light chain demonstrated an ability to interact with phospholipid vesicles. Thus, knowing that the factor Xa cleavage site on the factor Va light chain is located between residues 1765 and 1766 of the light chain this lipid-binding region of the bovine factor Va is further localized to amino acid residues 1667-1765.

Amino Acid Sequence↗
PubMed1994

Structure of membrane-bound human factor Va.

Coagulation factor Va is an essential cofactor which combines with the serine protease factor Xa on a phospholipid surface to form the prothrombinase complex. In the present study, the structure of factor Va interacting with lipid surfaces containing phosphatidylserine was studied by electron microscopy. Two-dimensional crystals of factor Va were obtained on planar lipid films under quasi-physiological conditions. The two-dimensional projected structure of factor Va was calculated at a resolution of 2 nm, revealing dimers of factor Va arranged on the surface lattice with the symmetry of the plane group p2. Average unit cell dimensions are a = 14.4 nm, b = 8.8 nm, gamma = 107 degrees. Each factor Va molecule presents two distinct domains of protein density consisting of one small domain, of 3 nm in diameter, connected to a larger domain of about 6 nm x 4.5 nm. The projected structure of factor Va covers an area equivalent to about fifty phospholipid molecules. In addition, edge-on views of factor Va molecules bound to liposomes reveal a globular structure connected through a thin stem to the liposome surface. A three-dimensional model of membrane-bound factor Va is proposed.

Cell Membrane↗
PubMed1992

Characterization of the interaction between the heavy and light chains of bovine factor Va.

Bovine factor Va has been previously been shown to consist of heavy (M(r) = 94,000) and light chains (M(r) = 81,000), that interact in a manner dependent upon the presence of either calcium or manganese ions. In an attempt to understand the mechanism of subunit interaction we have studied the effects of temperature and ions on factor Va stability. The rates of formation of factor Va from isolated chains and dissociation were temperature-dependent with an energy of activation of 6.2 and 1.3 kcal mol-1, respectively. The yield of factor Va from isolated chains was inversely related to the amount of time the chains were incubated at 4 degrees C. Incubation of individual chains revealed that the heavy chain is cold-labile, an effect that is reversible. Manganese ion was observed to prevent the conversion to the inactive form. High salt tends to stabilize the two-chain structure of factor Va, but is inhibitory to its formation from isolated chains. High concentrations of either manganese or calcium ions also inhibited reconstitution of activity. The light chain, in particular, was sensitive to the presence of manganese or calcium ion. Heavy chain that had been cleaved by activated protein C had a weakened interaction with the light chain, and the resulting complex had no procoagulant activity. Cooling of the heavy chain to 4 degrees C enhanced its intrinsic fluorescence. Manganese ion prevented some of this enhancement. The heavy chain fluorescence returned to the room temperature value with a half-life of approximately 10 min. In the presence of manganese ion relaxation was accelerated. The intrinsic fluorescence of activated protein C-cleaved heavy chain was not increased when the temperature was decreased. These data suggest that the heavy chain can exist in two forms. Elevated temperature converts it to a form that can bind ions and have a productive interaction with the light chain. However, conditions that prevent the heavy chain from combining with the light chain also stabilize the two subunit structure, suggesting that the high affinity of the complex is due to conformational changes that occur after chain interaction.

Animals↗
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↗
PubMed1993

The influence of factor Va on the active site of factor Xa.

The interaction of factor Xa with factor Va on the membrane surface results in a 3,000-fold increase in the kcat for the activation of prothrombin catalyzed by factor Xa. The reaction between the transition state irreversible inhibitor dansylglutamyl-glycyl-arginyl chloromethyl ketone (DEGRck) and factor Xa was characterized and employed to evaluate changes in the active site of factor Xa resulting from its interaction with factor Va, which may account for the increased catalytic efficiency of the enzyme complex. Inhibition studies indicated a two-step inhibition reaction involving a reversible binding step (Ki = 1.13 microM) and an irreversible alkylation step (ki = 0.65 s-1). The interaction between factors Va and Xa in solution or on membranes resulted in a small decrease in the overall second-order rate constant (ki/Ki) for the inhibition reaction. The incorporation of DEGRck into the active site of factor Xa results in a large change in the fluorescence intensity of the dansyl moiety. The fluorescence change was employed to study the reaction between enzyme and inhibitor directly by stopped-flow fluorescence measurements. The fluorescence traces were biphasic, indicating that the association of DEGRck with factor Xa and the subsequent covalent modification of the active site could be resolved because of differences in fluorescence intensities of the intermediate and product. This interpretation was verified by rapid chemical quench experiments. The reaction between DEGRck and factor Xa was characterized by a second-order association rate constant of 8.38 +/- 0.28 x 10(5) M-1.s-1 and an intrinsic rate constant for the alkylation step of 1.67 +/- 0.25 s-1. The rate constant for the alkylation step was unchanged in the presence of factor Va and membranes, whereas the association rate constant was modestly decreased by approximately 50%. The decrease in the association rate constant did not result from the partitioning of factor Xa to the membrane surface and could therefore be ascribed to an effect of factor Va on the protease. The data suggest that the interaction between factors Va and Xa on the membrane surface does not detectably alter the catalytic residues but may result in changes in the binding or accessibility of substrate to extended macromolecular recognition sites on the protease.

Amino Acid Chloromethyl Ketones↗