A further insight into the binding of blood clotting factors to membranes

The active site of factor Xa, labelled with dansylglutamylglycylarginine (DnsEGR) is sensitive to association with Ca2+, factor Va and phospholipids. When bound to factor Va, DnsEGR-factor-Xa does not change the composition of the binding site of factor Va, as shown by fluorescence energy-transfer experiments between the Trp residues of factor Va and pyrene-labelled phospholipids. Prothrombin was cleaved by alpha-chymotrypsin into two parts: N-terminal residues 1-41 (peptide 1-41) containing the gamma-carboxyglutamic acid residues (Gla), and des-(1-41)-prothrombin; their membrane association was investigated. Peptide 1-41 contains the aromatic residues Tyr and Trp in positions 24 and 41, respectively, and is suitable for fluorescence spectroscopy. The absence of fluorescence energy transfer between these residues suggests that they are more than 2.8 nm apart. Binding of Ca2+ and of phospholipids involves essentially the Tyr residue, while the C-terminal characteristics of the Trp residue remain unchanged. The conformational change which takes place on binding does not shorten the distance between Tyr and Trp beyond 2.8 nm. Our conclusion is that peptide 1-41 has an extended conformation. This result is compatible with the disordered character of the Gla region found in the crystalline structure of fragment 1 of prothrombin. Ca2+ induces a greater fluorescence energy transfer between prothrombin and membranes labelled with pyrene but has no influence on the binding of des-(1-41)-prothrombin. Moreover, the binding curves of des(1-41)-prothrombin are similar to those of prothrombin in the absence of Ca2+. It is concluded that the Ca2+-independent association of prothrombin with membranes involves essentially that part of the prothrombin molecule deleted in the Gla region.     

Interaction of an amphitropic protein (factor Xa) with membrane models in a complex system

Phosphatidylserine (PS) plays a crucial role, in the conversion of prothrombin into thrombin by the protease, factor Xa. Physiologically, the conversion occurs in the prothrombinase complex. The question of how water-soluble proteins that normally circulate in plasma bind remains to be unambiguously determined. We previously found that the amphitropic proteins (prothrombin, factors V and Va) penetrate into phospholipid layers. AC polarography has allowed the detection for the first time of insertion of factor Xa into condensed monolayers containing phosphatidylserine (PS) and phosphatidylcholine (PC) either 100% PS or 25% PS in the presence of Ca2+. This observation demonstrates that part of factor Xa can cross the phospholipid polar headgroup/hydrocarbon chain interface. In parallel experiments, radioactive surface measurements permitted measuring binding of tritium-labeled factor Xa onto a PS monolayer and calculate an association constant, 6x10(6) M(-1). Penetration of factor Xa into PS-containing vesicles was investigated also using photoactivable 5-[125I]iodonaphthalene-1-azide, which binds selectively to the lipid embedded domains of the protein. These experiments suggest that Factor Xa penetrates preferentially by its heavy chain, an alternative mode of binding to the commonly accepted binding via its Gla domain. Interaction of factor Xa with PS vesicles also changes its apparent K(m) for S 2222.     

Metal and phospholipid binding properties of partially carboxylated human prothrombin variants

To study the specific role of gamma-carboxyglutamic acid (Gla) residues in prothrombin, we have isolated a series of partially carboxylated prothrombin variants from a patient with a hereditary defect in vitamin K-dependent carboxylation (Goldsmith, G. H., Pence, R. E., Ratnoff, O. D., Adelstein, D. A., and Furie, B. (1982) J. Clin. Invest. 69, 1253-1260). The three variant prothrombins, purified by DEAE-Sephacel, immunoaffinity chromatography, and preparative gel electrophoresis, were indistinguishable from prothrombin in molecular weight, amino acid composition, and NH2-terminal amino acid sequence, with the exception of Gla residues. Variant prothrombin 1, with 8 Gla residues, had 66% of the coagulant activity of prothrombin, one high affinity metal-binding site (Kd = 15 nM), and three lower affinity sites (Kd = 2.7 microM); prothrombin contained two high affinity (36 nM) and four lower affinity sites (Kd = 1 microM). Ca(II) induced a 23% decrease in the intrinsic fluorescence of variant prothrombin 1 fragment 1, compared to a 35% decrease in that of prothrombin fragment 1. The phospholipid binding activity of variant prothrombin 1 was 44% that of prothrombin. Variant prothrombin 2 and variant prothrombin 3, with 4 and 6 Gla residues, respectively, had about 5% of prothrombin coagulant activity and a single high affinity and two lower affinity metal-binding sites and exhibited no phospholipid binding activity. Variant prothrombin 3 fragment 1 and variant prothrombin 2 fragment 1 demonstrated 18 and 13% of Ca(II)-induced fluorescence quenching, respectively. Abnormal prothrombin, with 1 Gla residue, had 8% of prothrombin coagulant activity, a single lower affinity (1 microM) metal-binding site, and 13% Ca(II)-induced fluorescence quenching of the fragment 1 species and did not bind to phospholipid. These results indicate that Gla residues define the metal binding properties of prothrombin. Most, if not all, of the Gla residues are required for complete prothrombin function, and the prothrombin coagulant activity correlates to the phospholipid binding activity of the prothrombin species.     

The Gla domain of human prothrombin has a binding site for factor Va

The role of the Gla domain of human prothrombin in interaction with the prothrombinase complex was studied using a peptide with the sequence of the first 46 residues of human prothrombin, PT-(1-46). Intrinsic fluorescence measurements showed that PT-(1-46) undergoes a conformational alteration upon binding calcium; this conclusion is supported by one-dimensional (1)H NMR spectroscopy, which identifies a change in the chemical environment of tryptophan 41. PT-(1-46) binds phospholipid membranes in a calcium-dependent manner with a K(d) of 0.5 microm and inhibits thrombin generation by the prothrombinase complex with a K(i) of 0.8 microm. In the absence of phospholipid membranes, PT-(1-46) inhibits thrombin generation by factor Xa in the presence but not absence of factor Va, suggesting that PT-(1-46) inhibits prothrombin-factor Va binding. The addition of factor Va to PT-(1-46) labeled with the fluorophore sulfosuccinimidyl-7-amino-4-methylcoumarin-3-acetic acid (PT-(1-46)AMCA) caused a concentration-dependent quenching of AMCA fluorescence, providing direct evidence of a PT-(1-46)-factor Va interaction. The K(d) for this interaction was 1.3 microm. These results indicate that the N-terminal Gla domain of human prothrombin is a functional unit that has a binding site for factor Va. The prothrombin Gla domain is important for interaction of the substrate with the prothrombinase complex.     

Isolation and characterization of the vitamin K dependent domain of human prothrombin

Chymotryptic cleavage of human prothrombin produces two fragments, prothrombin (des 1–44) previously characterized and peptide 1–41. This peptide has been purified by barium citrate adsorption and Sephadex G100 chromatography. It contains the 10 γ-carboxyglutamic residues of prothrombin. Added to a prothrombin activation mixture containing FXa, phospholipid and Ca⁺⁺, peptide 1–41 inhibits thrombin generation with the same potency as prothrombin fragment 1. Ca⁺⁺ produces a 20 % quenching of the intrinsic fluorescence of the peptide. So do Mn⁺⁺ and Mg⁺⁺ although to a lesser extent. Phospholipid enhances the Ca⁺⁺ induced quenching by a factor of 1.7 and shifts the midpoint of the transition from 0.34 to 0.46 mM Ca⁺⁺. The major difference with titration curves obtained with prothrombin F1 is the absence of cooperativity. Hence peptide 1–44 has retained some of the prothrombin properties to interact with Ca⁺⁺ and phospholipid and represents the vitamin K dependent domain of the molecule. The presence of the remaining part of F1 (residues 44–155) however is necessary for the expression of cooperativity.     

Phospholipid binding properties of bovine prothrombin peptide residues 1-45

The present study investigates the unique contribution of the NH2-terminal 33 residues of prothrombin, the gamma-carboxyglutamic acid (Gla) domain, to the Ca(II) and phospholipid-binding properties of prothrombin. Two Gla domain peptides, 1-42 and 1-45, produced by chymotryptic cleavage of prothrombin fragment 1 (residues 1-156 of the amino terminus of bovine prothrombin) and isolated by anion-exchange chromatography were utilized to characterize the Gla domain of prothrombin. This investigation utilized several experimental approaches to examine the properties of the Gla domain peptides. These studies were somewhat hampered by the metal ion-induced insolubility of the peptides. However, the 1-45 peptide was specifically radioiodinated, which facilitated the study of this peptide at low concentrations. In contrast to prothrombin fragment 1, the intrinsic fluorescence of both 1-42 and 1-45 was not quenched upon the addition of 1 mM Ca(II) or any concentration of Mg(II). Equilibrium dialysis studies revealed that the 1-42 peptide bound three Ca(II) ions noncooperatively, whereas fragment 1 binds seven Ca(II) ions in a positive cooperative manner. Ca(II)-promoted conformational changes are observed by comparison of electrophoretic mobility changes in the presence of increasing Ca(II) concentrations. Prothrombin, fragment 1, and the Gla domain peptides 1-42 and 1-45 exhibited similar electrophoretic mobility behavior in the presence of Ca(II) ions. The radiolabeled 1-45 peptide was found to comigrate with phospholipid vesicles on gel permeation chromatography in the presence of Ca(II). Fragment 1 was shown to inhibit this Ca(II)-dependent phospholipid binding of 1-45, demonstrating that the 1-45 peptide does possess the necessary phospholipid-binding structure. Furthermore, a metal ion-dependent conformational monoclonal antibody, F9.29, was inhibited from binding fragment 1 by the 1-42 peptide.     

Structure of Ca2+ prothrombin fragment 1 including the conformation of the Gla domain

The structure of Ca2+ prothrombin fragment 1 has been solved at 2.8-A resolution by X-ray crystallographic methods. Most of the Gla domain of fragment 1 (residues 1-48), which is high homologous with the N-terminal regions of six other blood proteins, cannot be identified in the electron density map of the apo structure. This is not the case when crystals are grown in the presence of Ca2+ ions where the Gla domain exhibits a well-defined folded structure. The folding of the Gla domain is dominated by secondary structure: (a) 3.0 turns of alpha-helix (25%) and (b) five short beta-strands arranged into two beta-structural units (40%). The Cys18-Cys23 disulfide of the small conserved loop of Gla domains is close to a cluster of conserved aromatic residues. The resulting interaction is probably responsible for the fluorescence quenching event accompanying Ca2+ ion binding. Since the Gla domain approximates a discoid, all the Gla residues are easily accessible to solvent. The arrangement of the paired Gla residues (7-8, 20-21, 26-27) is highly suggestive in that they essentially line one edge of the Gla domain creating a potentially intense electronegative environment. This region might well be that associated with phospholipid binding. The kringle structure of Ca2+ fragment 1 is essentially indistinguishable from that of the apoprotein at this stage.     

The omega-loop region of the human prothrombin gamma-carboxyglutamic acid domain penetrates anionic phospholipid membranes

The hydrophobic omega-loop within the prothrombin gamma-carboxyglutamic acid-rich (Gla) domain is important in membrane binding. The role of this region in membrane binding was investigated using a synthetic peptide, PT-(1-46)F4W, which includes the N-terminal 46 residues of human prothrombin with Phe-4 replaced by Trp providing a fluorescent probe. PT-(1-46)F4W and PT-(1-46) bind calcium ions and phospholipid membranes, and inhibit the prothrombinase complex. PT-(1-46)F4W, but not PT-(1-46), exhibits a blue shift (5 nm) and red-edge excitation shift (28 nm) in the presence of phosphatidylserine (PS)-containing vesicles, suggesting Trp-4 is located within the motionally restricted membrane interfacial region. PS-containing vesicles protect PT-(1-46)F4W, but not PT-(1-46), fluorescence from potassium iodide-induced quenching. Stern-Volmer analysis of the quenching of PT-(1-46)F4W in the presence and absence of 80% phosphatidylcholine/20% PS vesicles suggested that Trp-4 is positioned within the membrane and protected from aqueous quenching agents whereas Trp-41 remains solvent-accessible in the presence of PS-containing vesicles. Fluorescence quenching of membrane-bound PT-(1-46)F4W is optimal with 7- and 10-doxyl-labeled lipids, indicating that Trp-4 is inserted 5 to 7 A into the bilayer. This report demonstrates that the omega-loop region of prothrombin specifically interacts with PS-containing membranes within the interfacial membrane region.     

Chemical modification of bovine prothrombin fragment 1 in the presence of Tb3+ ions

The formaldehyde-morpholine method for the conversion of gamma-carboxyglutamyl (Gla) residues to gamma-methyleneglutamyl (gamma-MGlu) residues has been applied to the modification of bovine prothrombin fragment 1. In the absence of Tb3+ ions or at Tb3+ ion concentrations of 2 Km app and 25 Km app the action of 10,000-fold molar excess of formaldehyde and morpholine, pH 5.0, converts the 10 Gla residues of the protein into 10 gamma-MGlu residues. Modification of the protein using the same conditions but increasing the Tb3+ concentration to 100 Km app provided a homogeneous protein containing 3 gamma-MGlu and 7 Gla residues, bovine 3 gamma-MGlu-fragment 1. The modified protein binds the same number of Ca2+ ions (6-7) as bovine fragment 1. However, the positive cooperatively associated with Ca2+ binding is abolished and the overall affinity for Ca2+ ions is reduced. Fluorescence titrations of 3 gamma-MGlu-fragment 1 using either Ca2+ or Mg2+ ions indicate that the modified protein retains a fluorescence quenching behavior similar to that of the native protein. The modified protein does not bind to phosphatidylserine/phosphatidylcholine vesicles in the presence of Ca2+ ions. Thus the metal ion-induced fluorescence transition exhibited by the bovine protein appears to be a necessary but not sufficient condition for phospholipid binding.     

Dicoumarol-induced 9-gamma-carboxyglutamic acid prothrombin: isolation and comparison with the 6-, 7-, 8-, and 10-gamma-carboxyglutamic acid isomers.

The role of gamma-carboxyglutamic acid (Gla) in prothrombin function can be effectively evaluated by characterizing dicoumarol-induced, Gla-deficient prothrombin structural isomers. In addition to the isolation of 8-, 7-, 6-, 5-, 3-, 2-, 1-, and 0-Gla isomers, we have now purified a variant prothrombin containing 9(8.80) Gla residues by barium citrate adsorption, elution, and finally by DEAE-cellulose and immunoaffinity chromatographies. Agar gel electrophoretic mobilities of the 9-Gla isomer and its fragment 1 were slower than those of the respective 10-Gla (normal) prothrombin and fragment 1, both in the absence and presence of Ca(II). In the presence of Ca(II), both 9- and 10-Gla fragments 1 moved slower than 8- and 7-Gla fragments 1. However, in the absence of metal ions, 9- and 7-Gla fragments 1 migrated at the same rate, but slower than 10- and 8-Gla fragments. Similarly, the 9-Gla fragment 1 electrofocused cathodically to 10- and 8-Gla, but comparably with 7-Gla fragment 1. The 9-Gla fragment 1 exhibited a Ca(II)-induced 44% decrease in the intrinsic fluorescence, compared with a 40% decrease in that of 10-Gla; 8-Gla fragment 1 revealed only 23% quenching. Ca(II)-dependent anti-normal prothrombin antibodies are not specific for 10-Gla prothrombin, since only a twofold molar excess of the 9-Gla isomer was required to displace equal amounts of labeled normal prothrombin. The most critical Gla residue for influencing the functional, thrombin-generating properties of prothrombin appears to be the one present in the 9-Gla isomer but absent in the 8-Gla variant, since 9-Gla prothrombin possesses four times the normal coagulant activity (78 versus 20%) of the 8-Gla isomer.     

Distribution of gamma-carboxyglutamic acid residues in partially carboxylated human prothrombins

The role of gamma-carboxyglutamic acid in prothrombin has been examined using partially carboxylated variant prothrombins isolated from a person with a hereditary defect in vitamin K-dependent carboxylation. These species differ in gamma-carboxyglutamic acid content, distribution, and function, as monitored by metal binding properties, conformational transitions, phospholipid binding, and calcium-dependent coagulant activity (Borowski, M., Furie, B. C., Goldsmith, G. H., and Furie, B. (1985) J. Biol. Chem. 260, 9258-9264). The distribution of gamma-carboxyglutamic acids in the variant prothrombin species was determined by specific tritium incorporation into gamma-carboxyglutamic acid residues, thermal decarboxylation, and automated Edman degradation. gamma-Carboxyglutamic acid residues in the partially carboxylated prothrombins were identified by the assay of tritium in the resultant glutamic acid residues in the acarboxyprothrombins. The results indicate that variant prothrombins 1-3 are nearly homogeneous populations of partially carboxylated prothrombins. The ability of prothrombin to undergo a metal-induced conformational change and to bind to phospholipid vesicles correlated closely to the presence of a gamma-carboxyglutamic acid at residue 16. This residue is likely involved in the formation of a critical high affinity metal-binding site, possibly formed by Gla 16 and Gla 25 and/or Gla 26. A second high affinity metal-binding site, present in all of the variant prothrombin species, is defined, as an upper limit, by Gla 6, Gla 14, Gla 19, and Gla 20. This region is likely responsible for the interaction of certain of the conformation-specific antibodies to the metal-stabilized conformer of prothrombin.     

Interaction of α-thrombin and prethrombin 2, with phosphatidylserine-containing monolayers

Prothrombin activation complex is located at a phospholipid surface on activated platelets. To see whether the thrombin domain of the molecule plays a role in the interaction with lipids, we investigated the direct interaction of human α-thrombin and its precursor prethrombin 2 with phospholipid monolayers of varous compositions (PS/PC). Adsorption of the labeled proteins was determined by surface radioactive measurements. Penetrations of the proteins in the lipid layer was inferred from capacitance variation of the monolayer measured by a palarography. Disulfide bridges reduced at the electrode were determined by cycle voltametry.In all the cases studied, although in different manners thrombin was found both to adsorb and penetrate the lipid layer, whereas prethrombin 2 did not penetrate pure phosphatidylcholine (PC). In the case of thrombin but not of prethrombin 2, penetration is accompanied by S-S reduction which is maximum at 10 per cent of phosphatidylserine (PS). This indicate a different orientation for prethrombin 2 and thrombin in the lipid layer. This observation might be of importance for the comprehension of the architecture of the prothrombin might be of for the regulation of thrombin formation within the complex.     

Chemical modification of bovine prothrombin fragment 1 in the presence of Tb3+ ions. Sequence studies on 3-gamma-MGlu-fragment

Chemical modification of the gamma-carboxyglutamyl (Gla) residues of bovine prothrombin fragment 1 using the formaldehyde-morpholine method in the presence of 100 Kappm Tb3+ ions at pH 5.0 provided a modified protein containing 3 gamma-methyleneglutamyl residues (gamma-MGlu) and 7 Gla residues (bovine 3-gamma-MGlu-fragment 1). The modified protein bound the same number of Ca2+ ions as the native protein (six to seven), exhibited 28Mg2+-binding properties identical to native fragment 1 (five Mg2+ ions bound), exhibited the metal ion-promoted quenching of the intrinsic fluorescence in a manner similar to the native protein, but did not bind to phosphatidylserine (PS)/phosphatidylcholine (PC) vesicles in the presence of Ca2+ ions. Modification of the bovine protein using [14C]formaldehyde-morpholine provided a 14C-labeled 3-gamma-MGlu-fragment 1 suitable for sequence analysis. Edman sequencing of the peptides released by a tryptic digest of the reduced and carboxymethylated bovine [14C]3-gamma-MGlu-fragment 1 indicated that Gla residues at positions 7, 8, and 33 had been converted to [14C]gamma-methyleneglutamyl residues. In addition Lys97 was found to contain a 14C label. Similar analysis of the human [14C]3-gamma-MGlu-fragment 1 indicated that Gla residues at positions 7 and 32 were major modification sites and that Gla residues at positions 6 and 14 were partially modified. Lysine 96 was also modified in the human protein. The incorporation of a 14C label at Lys97 in bovine 3-gamma-MGlu-fragment 1 protein is not responsible for the loss of Ca2+-promoted binding to PS/PC vesicles. We suggest that Gla residues 7, 8, and 33 are elements of the first Ca2+-binding site; occupancy of this site establishes the Ca2+-specific conformation which is essential for the Ca2+-promoted interaction of the bovine protein with PS/PC vesicles. These studies also suggest that the loss of Gla residues at positions 7 and 32 prevents the formation of the initial Ca2+-binding site in the human protein.     

The kinetics of activation of normal and gamma-carboxyglutamic acid-deficient prothrombins

The kinetics of activation of normal and gamma-carboxyglutamic acid (Gla)-deficient prothrombins isolated from cattle maintained for extended periods on the vitamin K antagonist dicoumarol were studied. The catalyst was prothrombinase, comprising isolated Factor Xa, Factor Va, phospholipid vesicles, and calcium ion. The Km and kcat values for prothrombins with 0, 1, 2, 5, 7, and 10 Gla residues were determined both by initial rate analysis and by integrated Michaelis-Menten-Henri analysis. Each of the Gla-deficient prothrombins exhibited kcat values similar to that of normal 10-Gla prothrombin but Km values that were 8- to 20-fold greater than that of the normal molecule. The increased Km coincided with a loss of Ca2+- and phospholipid-binding properties of the Gla-deficient prothrombins. The magnitude of the defect in both the kinetics of activation and Ca2+ and phospholipid binding is not progressive with the loss of Gla residues but rather appears abruptly with the loss of as few as 3 of the 10 Gla residues present in the normal substrate. The theoretical relationship between Km(app) and the dissociation constant (Kd) of the prothrombin-phospholipid interactions was derived. According to the result, the increase in apparent Km observed with the Gla-deficient prothrombins corresponds to at least a 100- to 1000-fold decrease in affinity for phospholipid compared to the affinity of normal prothrombin. In addition, the products of the activation of 10-Gla prothrombin were found to inhibit the activation of the Gla-deficient prothrombins.     

Adsorption of bovine prothrombin to spread phospholipid monolayers

The interaction of bovine prothrombin with phospholipids was measured, using as the lipid source monolayers spread at the air-buffer interface. Fluorescence spectroscopy was implemented to determine the equilibrium concentration of free prothrombin in the aqueous subphase of the protein-monolayer suspensions, in a continuous assay system. The increase in surface pressure (pi) from the protein-monolayer adsorption was also measured and, with values of the adsorbed protein concentration (c[s]), was used to calculate dc(s)/d(pi). At a particular phosphatidylserine (PS) content of liquid-expanded (LE) phosphatidylcholine (PC)/PS monolayers, dc(s)/d(pi) was independent of the initial surface pressure (pi[i]), when this latter value exceeded 30 mN/m. However, dc(s)/d(pi) varied significantly with the relative PS content of the monolayer. Values of the equilibrium dissociation constants calculated from the concentration dependence of delta(pi) indicated that the affinity of prothrombin for LE monolayers was higher at higher PS contents and lower packing densities. The affinity of prothrombin for liquid-condensed (LC) PC/PS monolayers was found to be much weaker relative to LE monolayers of similar phospholipid composition. This approach, employing spread monolayers to study prothrombin-phospholipid binding, coupled with a simple and accurate method to determine the free protein concentration in protein-monolayer suspensions, offers significant advantages for the investigation of protein-membrane interaction. The equilibrium characteristics that describe the interaction of prothrombin with the different phospholipid monolayers under various conditions also provide support for previous results which indicated that hydrophobic interactions are involved in the adsorption of vitamin K-dependent coagulation and anticoagulation proteins to model membrane systems.     

Association of protein kinase C with phospholipid monolayers: two-stage irreversible binding

The association of protein kinase C (PKC) with phospholipid (PL) monolayers spread at the air-water interface was examined. PKC-PL binding induced surface pressure changes that were dependent on the amount of PKC, the phospholipid composition of the monolayers, the presence of Ca2+, and the initial surface pressure of the monolayer (pi 0). Examination of surface pressure increases induced by PKC as a function of phospholipid surface pressure, pi 0, revealed that PKC-phosphatidylserine (PS) association had a critical pressure of 43 dyn/cm. Above this surface pressure, PKC cannot cause further surface pressure changes. This high critical pressure indicated that PKC should be able to penetrate many biological membranes which appear to have surface pressures of about 30 dyn/cm. PKC-induced surface pressure changes were Ca2+ dependent only for PL monolayers spread at a pi 0 greater than 26 dyn/cm. PKC alone (in the absence of PL) formed a film at the air-water interface with a surface pressure of about 26 dyn/cm. Calcium-dependent binding was studied at the higher surface pressures which effectively excluded PKC from the air-water interface. Subphase depletion measurements suggested that association of PKC with PS monolayers consisted of two stages: a rapid Ca2+-dependent interaction followed by a slower process that resulted in irreversible binding of PKC to the monolayer. The second stage appeared to involve penetration of PKC into the hydrocarbon region of the phospholipid. The commonly used in vitro substrates for PKC, histone and protamine sulfate, also associated with and penetrated PS monolayers with critical pressures of 50 and 60 dyn/cm, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Preparation and properties of derivatives of bovine factor X and factor Xa from which the gamma-carboxyglutamic acid containing domain has been removed

Limited proteolysis of bovine blood coagulation Factor X by chymotrypsin produces a derivative in which the light chain is cleaved between Tyr 44 and Lys 45. Two peptide products, residues 1-44 of the Factor X light chain and a modified zymogen, Factor X(-GD) have been isolated and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, elution behavior on anion-exchange chromatography, amino acid composition, and by partial amino acid sequence determination. Factor X(-GD) no longer contains the 12 gamma-carboxyglutamic acid residues of the native zymogen and thus serves as a model for investigation of the properties conferred on Factor X by the presence of gamma-carboxyglutamic acid. Cleavage of Factor X at Tyr 44 by chymotrypsin is inhibited by Ca2+ and Mg2+ ions. Factor X(-GD) is activated by the coagulation factor activator of Vipera russellii venom, but at less than 1% of the rate of activation of native Factor X. The susceptibility of Tyr 44 to chymotryptic cleavage implies that this residue is on the surface of the light chain of Factor X. Factor Xa(-GD) is indistinguishable from native Factor Xa in its activity on Benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide, on prothrombin alone, and on prothrombin plus Factor Va. In the presence of phospholipid the rate of prothrombin activation catalyzed by Factor Xa(-GD) is the same as in the absence of phospholipid.     

Comparison of coagulation factor Xa and des-(1-44)factor Xa in the assembly of prothrombinase

The gamma-carboxyglutamic acid (Gla)-domain region of factor X (residues 1-44 of the light chain) was selectively removed by limited proteolysis with alpha-chymotrypsin. The Gla-domainless factor X was then activated by the factor X coagulant protein of Russell's viper venom. Apparent dissociation constants Kd' values for the interaction of factor Va with either factor Xa or Gla-domainless factor Xa were determined kinetically using prothrombin as the substrate. In the absence of phospholipid, factor Va interacted with Gla-domainless factor Xa with lower affinity (Kd' 4 X 10(-6) M) than with factor Xa (Kd' = 5 X 10(-8) M). At saturating concentrations of factor Va, maximal rates of thrombin formation were similar for either enzyme. The addition of phospholipid increased the affinity of factor Va for factor Xa approximately 75-fold (Kd' = 3.3 X 10(-10) M). In contrast, phospholipid had no effect on the affinity of Gla-domainless factor Xa for factor Va (Kd' = 4 X 10(-6) M). The maximal rate of thrombin formation increased approximately 300-fold with the addition of phospholipid to the factor Xa-factor Va system. Under the same conditions, phospholipid had no effect on the rate of thrombin formation when Gla-domainless factor Xa was the enzymatic moiety. These results demonstrate phospholipid has little or no effect on factor Va function when factor Xa has lost its Gla-mediated Ca2+-binding sites.     

The cytoplasmic region of mouse Fc gamma RIIb1, but not Fc gamma RIIb2, binds phospholipid membranes

The cytoplasmic regions of the mouse low-affinity Fc gamma RII isoforms, Fc gamma RIIb1 and Fc gamma RIIb2, play key roles in signal transduction by mediating different cellular functions. The Fc gamma RIIb1 (94 residues) and Fc gamma RIIb2 (47 residues) cytoplasmic regions are generated by differential mRNA splicing in which a single aspartic acid residue in Fc gamma RIIb2 is replaced by a 48-residue insert in Fc gamma RIIb1. In previous work, quantities of the mFc gamma RIIb1 and mFc gamma RIIb2 cytoplasmic regions were generated, and their secondary structures were examined in different solutions with circular dichroism [Chen, L., Thompson, N. L., and Pielak, G. J. (1997) Protein Sci. 6, 1038-1046]. In the work described here, steady-state light scattering was used to investigate possible interactions of the two isolated cytoplasmic regions with phospholipid vesicles. Three phospholipid compositions were examined: phosphatidylserine/phosphatidylcholine (PS/PC) (25/75, mol/mol); phosphatidylinositol bisphosphate/phosphatidylcholine (PIP2/PC) (25/75, mol/mol); and pure phosphatidylcholine (PC). Binding was examined in the presence and absence of Ca2+. The mFc gamma RIIb1 cytoplasmic peptide binds PS/PC vesicles weakly in the absence of Ca2+ and more strongly in the presence of Ca2+. For PIP2/PC vesicles, the behavior is reversed; binding is weak in the presence of Ca2+ and stronger in its absence. The mFc gamma RIIb1 peptide also weakly binds pure PC vesicles, in a Ca2+-independent manner. The mFc gamma RIIb2 cytoplasmic peptide does not bind, in the presence or absence of Ca2+, to PS/PC, PIP2/PC, or PC vesicles. The implications of these results for the mechanisms of signal transduction mediated by the two mFc gamma RII cytoplasmic regions are discussed.     

Phospholipid interactions of synthetic peptides representing the N-terminus of HIV gp41

Peptides representing the N-terminal 23 residues of the surface protein gp41 of LAV1a and LAVmal strains of the human immunodeficiency virus were synthesized and their interactions with phospholipid vesicles studied. The peptides are surface-active and penetrate lipid monolayers composed of negatively charged but not neutral lipids. Similarly, the peptides induce lipid mixing and solute (6-carboxyfluorescein) leakage of negatively charged, but not neutral, vesicles. Circular dichroism and infrared spectroscopy show that at low peptide:lipid ratios (approximately 1:200), the peptides bind to negatively charged vesicles as alpha-helices. At higher peptide:lipid ratios (1:30), a beta conformation is observed for the LAV1a peptide, accompanied by a large increase in light scattering. The LAVmal peptide showed less beta-structure and induced less light scattering. With neutral vesicles, only the beta conformation and a peptide:lipid ratio-dependent increase in vesicle suspension light scattering were observed for both peptides. We hypothesize that the inserted alpha-helical form causes vesicle membrane disruption whereas the surface-bound beta form induces aggregation.