Membrane binding properties of blood coagulation Factor V and derived peptides

The interactions of factor V and factor Va light chain with phospholipid vesicles were compared. The results showed that the factor Va light chain bound with the same parameters as factor V when the proteins were present at similar densities on the membrane. The protein-vesicle collisional efficiency was 30-50% for both factor V and factor Va light chain. The factor Va light chain bound at a higher density, and the additional binding interactions had lower affinity. The dissociation process showed negative cooperativity, possibly due to competition for acidic phospholipids in the membrane. The higher molar packing density produced more rapid protein-membrane dissociation rate constants. However, when factor V and Va light chains were present at similar molar densities on the vesicle, the dissociation rates, estimated by two methods, were similar. Analysis of dissociation rates also showed that factor Va interacted with factor Xa on the membrane surface while factor Va light chain did not. Factor Va generated by thrombin digestion of factor V did not result in a major loss of membrane-bound protein mass unless ethylenenediaminetetraacetic acid was present; in the latter case the mass changes indicated that all peptides were removed from the membrane except factor Va light chain. Equilibrium and dynamic measurements showed that ionic strength had a major effect on the dissociation rate but not on the association process. The salt effect indicated interaction between oppositely charged species with the product of the number of charges equal to at least -5.5. Factor Va light chain appeared to interact with phospholipids via a general charge interaction rather than via a specific charge stoichiometry.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

The interaction of bovine factor V and factor V-derived peptides with phospholipid vesicles

The binding of bovine Factor V, isolated Factor Va, and isolated activation intermediates to single bilayer phospholipid vesicles was studied by light scattering. The vesicles composed of 25% phosphatidylserine and 75% phosphatidylcholine had a mean radius of approximately 163 A as determined by quasi-elastic light scattering. When these vesicles were saturated with Factor V, the radii increased by approximately 120 A in both 0.15 and 1 M NaCl. At saturation, about 35 molecules of Factor V and 141 molecules of Factor Va were bound to each vesicle. Studies of the binding of Factor V and Factor Va at various ionic strengths showed little change in either Kd or n, suggesting that the binding is not electrostatic. The dissociation constants (Kd) and the lipid to protein ratios at saturation, moles/mol (n), obtained by relative light scattering intensities were: Factor V (Kd = 4.3 X 10(-8) M, n = 214); isolated Factor Va (Kd = 1.7 X 10(-7) M, n = 57); component B, Mr = 205,000 (Kd = 1.8 X 10(-7) M, n = 140); component C, Mr = 150,000 (Kd = 7.0 X 10(-7) M, n = 136); component D, Mr = 94,000 (no binding could be demonstrated); component E, Mr = 74,000 (Kd = 3.8 X 10(-7) M, n = 42). The results presented here indicate that the lower Kd exhibited by Factor V compared to Factor Va (components D and E) is primarily due to the interaction present within the component C portion of the molecule which is destroyed when component C is further cleaved to give component D. The interactions responsible for the binding of Factor Va are expressed in component E as well as in its precursor peptide component B. Dissociation of components D and E by the addition of EDTA indicate that component E alone is responsible for the interaction of bovine Factor Va with phospholipid.     

Membrane penetration of bovine factor V and Va detected by labeling with 5-iodonaphthalene-1-azide

The membrane-binding properties of Factor V and Factor Va were investigated using the lipophyllic, photoactivable probe 5-[125I]iodonaphthalene-1-azide. In the presence of vesicles composed of 75% phosphatidylcholine and 25% phosphatidylserine, both Factor V and Va were found to be labeled by the probe. The label was almost exclusively localized to the carboxyl-terminal-derived component E of Factor Va. The results are consistent with the interpretation that component E is the membrane binding subunit of Factor Va and that the interaction between Factor V or Factor Va and the membrane involves the penetration of the protein into the lipid bilayer.     

The interaction of bovine factor IX, its activation intermediate, factor IX alpha, and its activation products, factor IXa alpha and factor IXa beta, with acidic phospholipid vesicles of various compositions.

The interactions of bovine factor IX, its activation intermediate, Factor IX alpha, and its activation products, Factor IXa alpha and Factor IXa beta, with phospholipid vesicles, of mean radius of approx. 30 nm, containing various amounts of phosphatidylserine (PS) and phosphatidylcholine (PC), have been examined. For Factor IX, Factor IX alpha, Factor IXa alpha and Factor IXa beta, the dissociation constants, at saturating levels of Ca2+, are independent of the PS concentration in the vesicle after levels of 20-30% (w/w) have been reached, and attain minimum values of approx. 1.7, 1.7, 0.7 and 1.0 microM, respectively, with vesicles containing 50% PS. The amount of protein bound per vesicle particle is independent of the PS content, above 20% PS, for Factor IX and Factor IXa beta, with values of approx. 995-1197 and 1128-1566 molecules/vesicle, respectively. With Factor IX alpha, a dependence on the amount of protein bound with the content of PS is seen, which ranges from 338 to 619 molecules/vesicle with membranes containing 30-50% PS. For Factor IXa alpha, no regularity is noted and a range of 583-1083 molecules of protein/vesicle is observed with the systems employed. Examination of the radii of the proteins on the vesicle demonstrates that Factors IX alpha and IXa alpha occupy considerably more of the surface than do Factors IX and IXa beta, suggesting that a reason for the decreased number of binding sites for the former two proteins on the vesicle may be related to their greater surface spatial requirements.     

Structural model of factors V and Va based on scanning transmission electron microscope images and mass analysis

Coagulation factor V (fV) is a single-chain glycoprotein (Mr 330,000; domain structure A1-A2-B-A3-C1-C2) that is activated to factor Va (fVa; Mr 174,000) by thrombin, which cleaves away the B domain leaving a heterodimeric structure composed of a heavy chain (A1-A2; Mr 94,000) and a light chain (A3-C1-C2; Mr 74,000). We analyzed the ultrastructure of scanning transmission electron microscope images of bovine and human fV, bovine fVa, and its constituent light chains and heavy chains. Factor V molecules had irregularly globular (10-12 nm) to oblong (8-14 nm) core structures which commonly displayed a peripheral satellite appendage of variable morphology attached to the core by a narrow stalk. Scanning transmission electron microscope mass analyses indicated that monomolecular bovine fV molecules had a mass of 322 +/- 45 kDa and human fV, 315 +/- 31 kDa. Factor Va molecules were irregular, globular (8-12 nm) structures that resembled the fV core structure, lacked the satellite appendage representing B domainal structures, and had a mass of 180 +/- 22 kDa. Our findings permit us to propose a structural model of fV suggesting the relative orientation of its closely associated light chain and heavy chain core components and indicating that these constituents remain associated in the transition from fV to fVa.     

Interaction of bovine blood clotting factor Va and its subunits with phospholipid vesicles

Thrombin-activated factor Va and factor Va subunit binding to large-volume vesicles was investigated by a technique based on the separation by centrifugation of phospholipid-bound protein from the bulk solution. This technique allows the direct measurement of free-protein concentration. It is concluded that the phospholipid binding site on factor Va is located on a basic factor Va subunit with Mr 80 000 (factor Va-LC). The effects of phospholipid vesicle composition, calcium concentration, pH, and ionic strength on the equilibrium constants of factor Va- and factor Va-LC-phospholipid interaction were studied. Factor Va and factor Va-LC binding to phospholipid requires the presence of negatively charged phospholipids. It is further demonstrated that the following occur: (a) Calcium ions compete with factor Va and factor Va-LC for phospholipid-binding sites. (b) The dissociation constant of protein-phospholipid interaction increases with the ionic strength, whereas the maximum protein-binding capacity of the phospholipid vesicle was not affected by ionic strength. (c) The dissociation constant for factor Va-phospholipid interaction depends on pH when the vesicle consists of phosphatidic acid. It is concluded that factor Va-phospholipid interaction is primarily electrostatic in nature, where positively charged groups on the protein directly interact with the phosphate group of net negatively charged phospholipids. The results suggest that factor Va, like factor Xa and prothrombin, has the characteristics of an extrinsic membrane protein.     

Role of regulatory exosite I in binding of thrombin to human factor V, factor Va, factor Va subunits, and activation fragments.

The blood coagulation proteinase, thrombin, converts factor V into factor Va through a multistep activation pathway that is regulated by interactions with thrombin exosites. Thrombin exosite interactions with human factor V and its activation products were quantitatively characterized in equilibrium binding studies based on fluorescence changes of thrombin covalently labeled with 2-anilinonaphthalene-6-sulfonic acid (ANS) linked to the catalytic site histidine residue by Nalpha-[(acetylthio)acetyl]-D-Phe-Pro-Arg-CH2Cl ([ANS]FPR-thrombin). Exosite I was shown to play a predominant role in the binding of factor V and factor Va from the effect of the exosite I-specific ligand, hirudin54-65, on the interactions. Factor V and factor Va bound to exosite I of [ANS]FPR-thrombin with similar dissociation constants of 3.4 +/- 1.3 and 1.1 +/- 0.4 microM and fluorescence enhancements of 182 +/- 41 and 127 +/- 17%, respectively. Native thrombin and labeled thrombin bound with similar affinity to factor Va. Among factor V activation products, the factor Va heavy chain was shown to contain the site of exosite I binding, whereas exosite I-independent, lower affinity interactions were observed for activation fragments E and C1, and no detectable binding was observed for the factor Va light chain. The results support the conclusion that the factor V activation pathway is initiated by exosite I-mediated binding of thrombin to a site in the heavy chain region of factor V that facilitates the initial cleavage at Arg709 to generate the heavy chain of factor Va. The results further suggest that binding of thrombin through exosite I to factor V activation intermediates may regulate their conversion to factor Va and that similar binding of thrombin to the factor Va produced may reflect a mode of interaction involved in the regulation of prothrombin activation.     

Coagulation Factor V contains copper ion

Preparations of bovine and human coagulation Factor V were analyzed for copper using both atomic absorption and atomic emission spectroscopy. All preparations of the bovine and human protein were found to contain copper ion at a ratio of 1 copper ion bound per mol (Mr = 330,000) of Factor V. As a result of copper binding and sequence homology between ceruloplasmin and Factor V, bovine Factor V and thrombin-activated Factor V (Va) were assessed with respect to their visible and near ultraviolet absorption spectra and to their ability to oxidize N,N-dimethyl-p-phenylenediamine (a substrate for ceruloplasmin). Factor V and Factor Va exhibited absorption spectra with no maxima at either 310 or 610 nm, indicating that the copper is not bound in a site analogous to Type I or Type III copper sites in ceruloplasmin. Further, Factor V and Factor Va are not capable of serving as catalysts for the oxidation of N,N-dimethyl-p-phenylenediamine under solution conditions that are optimum for ceruloplasmin oxidase activity. These data suggest that the copper ion bound to Factor V may be functionally and structurally distinct from the Type I and Type III copper ion bound to ceruloplasmin.     

The role of the membrane in the inactivation of factor va by plasmin. Amino acid region 307-348 of factor V plays a critical role in factor Va cofactor function

The mechanism of inactivation of bovine factor Va by plasmin was studied in the presence and absence of phospholipid vesicles (PCPS vesicles). Following 60-min incubation with plasmin (4 nm) membrane-bound factor Va (400 nm) is completely inactive, whereas in the absence of phospholipid vesicles following a 1-h incubation period, the cofactor retains 90% of its initial cofactor activity. Amino acid sequencing of the fragments deriving from cleavage of factor Va by plasmin demonstrated that while both chains of factor Va are cleaved by plasmin, only cleavage of the heavy chain correlates with inactivation of the cofactor. In the presence of a membrane surface the heavy chain of the bovine cofactor is first cleaved at Arg(348) to generate a fragment of M(r) 47,000 containing the NH(2)-terminal part of the cofactor (amino acid residues 1-348) and a M(r) 42,000 fragment (amino acid residues 349-713). This cleavage is associated with minimal loss in cofactor activity. Complete loss of activity of the membrane-bound cofactor coincides with three cleavages at the COOH-terminal portion of the M(r) 47,000 fragment: Lys(309), Lys(310), and Arg(313). These cleavages result in the release of the COOH terminus of the molecule and the production of a M(r) 40,000 fragment containing the NH(2)-terminal portion of the factor Va molecule. Factor Va was treated with plasmin in the absence of phospholipid vesicles followed by the addition of PCPS vesicles and activated protein C (APC). A rapid inactivation of the cofactor was observed as a result of cleavage of the M(r) 47,000 fragment at Arg(306) by APC and appearance of a M(r) 39,000 fragment. These data suggest a critical role of the amino acid sequence 307-348 of factor Va. A 42-amino acid peptide encompassing the region 307-348 of human factor Va (N42R) was found to be a good inhibitor of factor Va clotting activity with an IC(50) of approximately 1.3 microm. These data suggest that plasmin is a potent inactivator of factor Va and that region 307-348 of the cofactor plays a critical role in cofactor function and may be responsible for the interaction of the cofactor with factor Xa and/or prothrombin.     

Platelet-derived microparticles express high affinity receptors for factor VIII.

Factor VIII is a cofactor in the tenase enzyme complex which assembles on the membrane of activated platelets. A critical step in tenase assembly is membrane binding of factor VIII. Platelet membrane factor VIII-binding sites were characterized by flow cytometry using either fluorescein maleimide-labeled recombinant factor VIII or a fluorescein-labeled monoclonal antibody against factor VIII. Following activation by thrombin, most platelets bound factor VIII within 90 s. In addition, over the course of several minutes, membranous vesicles (microparticles) were shed from the platelet plasma membrane and each microparticle bound as much factor VIII as a stimulated platelet. Over 30 min, stimulated platelets (but not microparticles) lost the capacity to bind factor VIII. Factor VIII bound saturably to microparticles from platelets stimulated with thrombin, thrombin plus collagen, or the complement proteins C5b-9. The binding of factor VIII was compared to factor V, a structurally homologous coagulation cofactor. Analysis of microparticle binding kinetics yielded similar on and off rates for factor VIII and factor Va and KD values of 2-10 nM. In the presence of 20 nM factor Va, the binding of factor VIII to microparticles was increased, and there was a comparable increase in platelet tenase activity. At higher factor Va concentrations, factor VIII binding and tenase activity were inhibited. Conversely, factor VIII had a similar dose-dependent effect on factor Va binding and platelet prothrombinase activity. Synthetic phospholipid vesicles containing phosphatidylserine competed with microparticles for binding of factor VIII and factor Va. These studies indicate that activated platelets express a transient increase in high affinity receptors for factor VIII, whereas platelet-derived microparticles express a sustained increase in receptors. The binding characteristics of platelet membrane receptors for factor VIII are similar to those for factor Va.     

The contribution of bovine Factor V and Factor Va to the activity of prothrombinase.

The rates of prothrombin activation under initial conditions of invariant concentrations of prothrombin and Factor Xa were studied in the presence of various combinations of Ca2+, homogeneous bovine Factor V, Factor Va, phosphatidylcholine-phosphatidylserine vesicles, and activated bovine platelets. Reactions were monitored continuously through the enhanced fluorescence accompanying the interaction of newly formed thrombin with dansylarginine-N-(3-ethyl-1,5-pentanediyl) amide. The complete prothrombinase (Factor Xa, Ca2+, phospholipid, and Factor Va) behaved as a "typical" enzyme and catalyzed the activation of prothrombin with an apparent Vmax of 2100 mol of thrombin/min/mol of Factor Va or Factor Xa, whichever was the rate-limiting component. Regardless of whether the enzymatic complex was composed of Factor Xa, Ca2+, and plasma Factor Va plus phospholipid vesicles, or activated platelets in the place of the latter components, similar specific activity values were observed. The combination of Factor Va, Ca2+, and phospholipid enhanced the rate of the Factor Xa-catalyzed activation of prothrombin by a factor of 278,000. Factor Va itself when added to Factor Xa, Ca2+, and phospholipid, enhanced the rate of prothrombin activation by a factor of 13,000. Unactivated Factor V appears to possess 0.27% of the procoagulant activity of thrombin-activated Factor Va. From the kinetics of prothrombinase activity, an interaction between Factor Xa and both Factor V and Factor Va was observed, with apparent 1:1 stoichiometries and dissociation constants of 7.3 x 10(-10) M for Factor Va and 2.7 x 10(-9) M for Factor V. The present data, combined with data on the equilibrium binding of prothrombinase components to phospholipid, indicate that the model prothrombinase described in this paper consists of a phospholipid-bound, stoichiometric complex of Factor Va and Factor Xa, with bound Factor Va serving as the "binding site" for Factor Xa, in concert with its proposed role in platelets.     

Identification of a minimal myosin Va binding site within an intrinsically unstructured domain of melanophilin

Myosin V is a molecular motor that transports a variety of cellular cargo, including organelles, vesicles, and messenger RNA. The proper peripheral distribution of melanosomes, a dense pigment-containing organelle, is dependent on actin and the activity of myosin Va. The recruitment of myosin Va to the melanosome and proper transport of the melanosome requires melanophilin, which directly binds to myosin Va and is tethered to the melanosome membrane via Rab27a. Here we use highly purified proteins to demonstrate that the globular tail domain of myosin Va binds directly to an intrinsically unstructured domain of melanophilin. The myosin Va binding domain of melanophilin lacks stable secondary structure, and (1)H NMR measurements indicate that the protein is unfolded. This domain is extremely sensitive to mild proteolysis and has a hydrodynamic radius that is consistent with a random coil-like polypeptide. We show that myosin Va binding does not induce the global folding of melanophilin. Truncations of melanophilin were utilized to define a short peptide sequence (26 residues) within melanophilin that is critical for myosin Va binding. We demonstrate that a peptide corresponding to these residues binds directly to the globular tail domain with the same affinity as melanophilin. We discuss the possible implications of protein intrinsic disorder in recruitment and maintenance of myosin Va on melanosome membranes.     

Annexin V and vesicle membrane electroporation

The method of membrane electroporation (ME) has been used as an analytical tool to quantify the effect of membrane curvature on transient electric pore for-mation, and on the adsorption of the protein annexin V (M_r = 35,800) to the outer surface of unilamellar lipid vesicles (of radii 25 ≤ a/nm ≤ 200). Relaxation kinetic studies using optical membrane probes of the diphenylhexatriene type suggest that electric pore formation is induced by ionic interfacial polarization causing entrance of the (more polarizable) water into the lipid bilayer membrane yielding (hydrophobic and hydrophilic) pore states with a mean stationary pore radius r_p=0.35 (±0.05) nm. Extent and rate of ME, compared at the same induced transmembrane voltage, were found to decrease both with increasing vesicle radius and with increasing protein concentration. This `inhibitory' effect of annexin V is apparently allosteric and saturates at about [AN_T]_sat = 4 μm annexin V for vesicles of a = 100 nm at 1 mm total lipid concentration, 0.13 mm total Ca²⁺ concentration and at T = 293 K. Data analysis in terms of Gibbs area-difference-elasticity energy suggests that the bound annexin V reduces the gradient of the lateral pressure across the membrane. At [AN_T]_sat, about 20% of the vesicle surface is covered by the bound protein, but it is only 0.01% of the surface of the outer lipid leaflet in which a part of the protein, perhaps the aromatic residue of the tryptophan (W 187), is inserted. Insertion leads to a denser packing of the lipid molecules in the outer membrane leaflet. As a consequence, the radius of the electropores in the remaining membrane part, not covered by annexin V decreases (r_p/nm = 0.37, 0.36 and 0.27) with increasing adsorption of the protein ([AN_T] = 0, 2 and 4 μm, respectively). Received: 9 January 1997 / Accepted: 21 April 1997     

Interaction of prothrombin with factor Va-phospholipid complexes

The effects of factor Va and the phospholipid-binding fragment of factor Va [factor Va light chain (LC), Mr 80000] on the binding of prothrombin, factor X, and factor Xa to phospholipid vesicles are reported. Equilibrium binding experiments were performed that utilized large-volume vesicles, which can be removed from the bulk solution by centrifugation. Factor Va decreased the dissociation constant of the prothrombin-phospholipid complex 50-fold, from 2.0 X 10(-7) M to 4.0 X 10(-9) M. For the factor X-phospholipid complex the decrease was 60-fold (1.8 X 10(-7) M to 3.0 X 10(-9) M) and for factor Xa, 160-fold (1.6 X 10(-7) M to 1.0 X 10(-9) M). The ratios of moles of protein bound to moles of total added factor Va at saturation of phospholipid-bound factor Va indicate an 1:1 stoichiometric complex of either factor Xa, factor X, or prothrombin and phospholipid-bound factor Va. In the presence of factor Va LC, the dissociation constants of factor Xa- and prothrombin-phospholipid complexes were increased, while the maximal protein-binding capacities of the vesicles were not affected by factor Va LC. The data suggest a competitive interaction between factor Xa and factor Va LC binding as well as between prothrombin and factor Va LC binding at the phospholipid surface. From this, it is concluded that the phospholipid-binding fragment of factor Va alone does not serve as the binding site for interactions of factor Xa and prothrombin with factor Va.     

Proteolytic alterations of factor Va bound to platelets

The coagulation protein Factor Va forms the receptor for the serine protease Factor Xa at the platelet surface. This membrane-bound complex of Factor Va and Factor Xa plus calcium constitutes the enzymatic complex prothrombinase, which effects the conversion of prothrombin to the clotting enzyme, thrombin. Studies were undertaken to investigate the proteolytic events accompanying the inactivation of platelet-bound Factor Va by activated protein C as well as the ability of Factor Xa to protect Factor Va from activated protein C inactivation. During the course of these studies, observations were made which indicated that Factor Va was also cleaved by both a platelet-associated protease, as well as Factor Xa. When Factor Va was incubated with washed platelets, electrophoresis and autoradiography of solubilized platelet pellets indicated that three Factor Va peptides were associated with the platelet: component D (Mr = 94,000), component E (Mr = 74,000), and a 90,000-dalton peptide (component D') which appeared with time as the result of a platelet-associated protease cleavage of component D. The Factor Va peptides bound to platelets were proteolytically inactivated by activated protein C, resulting in five peptide products, all of which remained associated with the platelet-membrane surface. Factor Va was protected from activated protein C proteolysis by complex formation with Factor Xa or active site-blocked Factor Xa. However, active Factor Xa cleaved platelet-bound Factor Va to peptide products which also remained associated with the platelet. Whereas activated protein C rapidly cleaved components D and D' with secondary cleavages occurring in component E, Factor Xa rapidly cleaved component E with secondary cleavages occurring in components D and D'. The Factor Xa-cleaved Factor Va is catalytically functional. To determine whether cleavage was necessary for function, prothrombin conversion reaction mixtures were monitored for thrombin formation and Factor Va cleavage with time in a defined phospholipid vesicle model system. The results indicated that Factor Xa cleavage of Factor Va is not essential for Factor Va activity but may promote its ability to function in the prothrombinase complex.     

The factor V activation paradox

The prothrombinase complex consists of the protease factor Xa, Ca2+, and factor Va assembled on an anionic membrane. Factor Va functions both as a receptor for factor Xa and a positive effector of factor Xa catalytic efficiency and thus is key to efficient conversion of prothrombin to thrombin. The activation of the procofactor, factor V, to factor Va is an essential reaction that occurs early in the process of tissue factor-initiated blood coagulation; however, the catalytic sequence leading to formation of factor Va is a subject of disagreement. We have used biophysical and biochemical approaches to establish the second order rate constants and reaction pathways for the activation of phospholipid-bound human factor V by native and recombinant thrombin and meizothrombin, by mixtures of prothrombin activation products, and by factor Xa. We have also reassessed the activation of phospholipid-bound human prothrombin by factor Xa. Numerical simulations were performed incorporating the various pathways of factor V activation including the presence or absence of the pathway of factor V-independent prothrombin activation by factor Xa. Reaction pathways for factor V activation are similar for all thrombin forms. Empirical rate constants and the simulations are consistent with the following mechanism for factor Va formation. alpha-Thrombin, derived from factor Xa cleavage of phospholipid-bound prothrombin via the prethrombin 2 pathway, catalyzes the initial activation of factor V; generation of factor Va in a milieu already containing factor Xa enables prothrombinase formation with consequent meizothrombin formation; and meizothrombin functions as an amplifier of the process of factor V activation and thus has an important procoagulant role. Direct activation of factor V by factor Xa at physiologically relevant concentrations does not appear to be a significant contributor to factor Va formation.     

Bovine coagulation factor V visualized with electron microscopy. Ultrastructure of the isolated activated forms and of the activation fragments

Single chain bovine factor V (Mr = 330,000) was isolated and visualized by means of high resolution transmission electron microscopy of negatively stained samples. Both factor Va, activated by thrombin or by the factor V activator from Russell's viper venom, and the isolated fragments, D (Mr = 105,000), C1 (Mr = 150,000), and F1F2 (Mr = 72,000), were studied. Single chain factor V appeared as a multidomain structure with three globular domains of similar size (diameter approximately 80 A), and oriented around a somewhat larger central domain (diameter approximately 140 A). The distance between the center of the molecule and the center of each of the peripheral domains was 120 A and the maximum length of factor V was 300 A. The structure was essentially identical with that recently shown for human single chain factor V (Dahlb?ck, B. (1985) J. Biol. Chem. 260, 1347-1349). Isolated thrombin-activated factor Va (containing fragments D and F1F2) was composed of two domains of similar size, each of which was approximately 80 A in diameter and corresponded in size and shape to the peripheral domains seen in intact factor V. The isolated activation fragment C1 appeared as an irregular structure with an approximate diameter of 140 A and corresponded in size and shape to the larger central domain in intact factor V. The activator from Russell's viper venom only cleaves the bond(s) between C1 and F1F2, which results in two fragments, a larger fragment (Mr = 220,000) bearing the D, E, and C1 region and a smaller one corresponding to the F1F2 fragment. The venom-activated factor Va in the electron microscope demonstrated a multidomain structure similar in size and shape to that obtained with intact factor V. A model for factor V and the molecular events involved in activation is proposed.     

The binding of factor Va to phospholipid vesicles

The analysis of free sulfhydryl groups in factor Va using dithiobis-(nitrobenzoic acid) (DTNB) indicated the presence of one accessible thiol in each of the two subunits of the cofactor. Intact factor Va contained one readily accessible sulfhydryl group under native conditions and approximately two such groups after denaturation. A comparison of the rate of modification of the accessible thiol in factor Va under native conditions to those observed with the isolated subunits indicated that the thiol present in component D of the cofactor was readily accessible to reaction with DTNB. Factor Va was reacted with the sulfhydryl-directed fluorophore N-(1-pyrene)maleimide, resulting in the concomitant loss of the accessible thiol with no detectable alteration in the activity of the cofactor. This fluorescent derivative of factor Va (Pyr-Va) was used to examine the binding of factor Va to phospholipid vesicles by fluorescence polarization. Fluorescence polarization of the pyrene moiety increased saturably when Pyr-Va was titrated with increasing concentrations of vesicles composed of phosphatidylcholine and phosphatidylserine (PS). Systematic analysis of the binding of Pyr-Va to PCPS (75% phosphatidylcholine, 25% PS) indicated that the binding interaction was characterized by a dissociation constant of 2.7 x 10(-9) M with 42 mol of PCPS bound per mol of Va at saturation. The data obtained by varying the PS content of the vesicles are consistent with the interpretation that the Va-combining site on the vesicle surface is composed of a discrete number of PS molecules. The binding of Pyr-Va to PCPS was independent of added calcium ion and could be reversed by the addition of unlabeled Va or isolated component E but not by component D. Analysis of the displacement curves indicated that native factor Va or isolated component E and Pyr-Va mutually excluded each other on the vesicle surface with identical affinities. Competition experiments conducted using component E digested by factor Xa or the isolated derivative peptides indicated that the cleavage of component E by factor Xa had no effect on the PCPS binding properties of this subunit. Further, the data obtained with the isolated peptides suggest that the lipid-binding domain of component E is present in the amino-terminal region of this subunit.