The factor IX phospholipid-binding site is required for calcium-dependent activation of factor IX by factor XIa

To determine the functional role of the metal-dependent conformational changes in Factor IX, two populations of conformation-specific anti-Factor IX antibodies were prepared. Anti-Factor IX X Mg(II) antibodies bind to Factor IX in the presence of Mg(II) and other metal ions, but not in the absence of metal ions. Anti-Factor IX X Ca(II)-specific antibodies bind to Factor IX in the presence of Ca(II) and Sr(II), but not in the presence of Mn(II), Mg(II), and Ba(II). In the presence of a metal ion that induces the conformational transition recognized by the anti-Factor IX X Mg(II) antibodies, the concentrations of CaCl2 and SrCl2 needed for the half-maximal binding of the anti-Factor IX X Ca(II)-specific antibodies to Factor IX were reduced 3- and 20-fold, respectively. Factor IX binding to phospholipid vesicles was inhibited by the Fab fragments of the anti-Factor IX X Ca(II)-specific antibodies, but was not inhibited by the Fab fragments of the anti-Factor IX X Mg(II) antibodies. Factor XIa activation of Factor IX was also inhibited by the Fab fragments of the anti-Factor IX X Ca(II)-specific antibodies, but not by the anti-Factor IX X Mg(II) antibodies. These results support the hypothesis that Factor IX undergoes two metal-dependent conformational transitions: FIX----FIX'----FIX*. The first transition (FIX----FIX') is metal-dependent but cation-nonselective; the second transition (FIX'----FIX*) is metal-selective for Ca(II) or Sr(II). The second transition results in the expression of conformational determinants necessary for membrane binding and the Ca(II)-dependent activation of Factor IX by Factor XIa. These results suggest chemical similarity between a surface of a domain of Factor XIa and phospholipid vesicles, both of which interact with Factor IX in the presence of Ca(II).     

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.     

Isolation and functional characterization of the active light chain of activated human blood coagulation factor XI

Human blood coagulation Factor XIa was reduced and alkylated under mild conditions. The mixture containing alkylated heavy and light chains was subjected to affinity chromatography on high Mr kininogen-Sepharose. Alkylation experiments using [14C]iodoacetamide showed that a single disulfide bridge between the light and heavy chains was broken to release the light chain. The alkylated light chain (Mr = 35,000) did not bind to high Mr kininogen-Sepharose while the heavy chain (Mr = 48,000), like Factors XI and XIa, bound with high affinity. The isolated light chain retained the specific amidolytic activity of native Factor XIa against the oligopeptide substrate, pyroGlu-Pro-Arg-p-nitroanilide. Km and kcat values for this substrate were 0.56 mM and 350 s-1 for both Factor XIa and its light chain, and the amidolytic assay was not affected by CaCl2. However, in clotting assays using Factor XI-deficient plasma in the presence of kaolin, the light chain was only 1% as active as native Factor XIa. Human coagulation Factor IX was purified and labeled with sodium [3H]borohydride on its carbohydrate moieties. When this radiolabeled Factor IX was mixed with Factor XIa, an excellent correlation was observed between the appearance of Factor IXa clotting activity and tritiated activation peptide that was soluble in cold trichloroacetic acid. Factor XIa in the presence of 5 mM CaCl2 activated 3H-Factor IX 600 times faster than Factor XIa in the presence of EDTA. In the absence of calcium, Factor XIa and its light chain were equally active in activating 3H-Factor IX. In contrast to Factor XIa, the light chain in this reaction was inhibited by calcium ions such that, in the presence of 5 mM CaCl2, Factor XIa was 2000 times more effective than its light chain. Neither phospholipid nor high Mr kininogen and kaolin affected the activity of Factor XIa or its light chain in the activation of 3H-Factor IX. These observations show that the light chain region of Factor XIa contains the entire enzymatic active site. The heavy chain region contains the high affinity binding site for high Mr kininogen. Furthermore the heavy chain region of Factor XIa plays a major role in the calcium-dependent mechanisms that contribute to the activation of Factor IX.     

The reassociation of factor Va from its isolated subunits

Factor Va is an essential cofactor for the activation of prothrombin catalyzed by factor Xa. The cofactor is a heterodimer composed of a light chain and a heavy chain that are associated noncovalently in the presence of divalent metal ions. The kinetics of the formation of factor Va from the isolated and separated subunits was examined by the time-dependent regain in cofactor activity using direct assays of prothrombin activation catalyzed by prothrombinase. The rate of reassociation at saturating concentrations of calcium ions was slow with a strong temperature dependence. The product of the association reaction was indistinguishable from native factor Va on the basis of activity. The second order rate constant for the process at 37 degrees C in the presence of 2 mM CaCl2 was 1.58 X 10(5) M-1.min-1. Manganese ion increased the rate of regain of activity without influencing the extent of the reaction. The previous identification of a single reactive sulfhydryl in each subunit of factor Va permitted the modification of the separated subunits with sulfhydryl-directed fluorophores. Subunit reassociation was directly measured by fluorescence energy transfer using light chain modified with 6-acryloyl-2-dimethylaminonaphthalene (fluorescence donor) and heavy chain modified with fluorescein 5-maleimide (fluorescence acceptor). Fluorescence measurements indicate that the heavy and light chains associate tightly (Kd = 5.9 x 10(-9) M) and reversibly with a stoichiometry of 1:1. The dissociation of the subunits from the cofactor is first order with a rate constant of 1.03 X 10(-3) min-1. These interpretations were confirmed by physical measurements of subunit reassociation by sedimentation velocity studies.     

Monoclonal antibodies to human protein C: effects on the biological activity of activated protein C and the thrombin-catalyzed activation of protein C1

Thirteen monoclonal antibodies designated as MFC-1 to MFC-13 were obtained from hybridoma cells cloned after the fusion of mouse myeloma cells with spleen cells of mice immunized with purified human protein C. Studies were made to determine where the antibodies bound to the molecule of protein C and whether they affected the biological actions of protein C. By using the immunoblotting technique, six of these antibodies were shown to bind to the light chain of protein C, and five to the heavy chain of protein C and also activated protein C. The remaining two antibodies bound to neither the light chain nor the heavy chain, though both antibodies bound to the intact protein C. Antibodies specific for the light chain did not bind to the gamma-carboxyglutamic acid-domain. Two of the antibodies specific for the heavy chain (MFC-13 and -1) inhibited the amidolytic activity of activated protein C. The MFC-13 also inhibited the activity of bovine activated protein C, but not that of human Factor IXa, Factor Xa, or thrombin. In addition to these two antibodies, another one for the heavy chain (MFC-10) and two antibodies for the light chain (MFC-9 and -11) inhibited the inactivation of Factor Va by human activated protein C. One of the antibodies which inhibited the enzyme activity (MFC-1) blocked the inhibition of activated protein C by protein C inhibitor. Another one for the heavy chain (MFC-5) inhibited the activation of protein C by thrombin regardless of the presence or absence of thrombomodulin. Based on these results, we have established the positions of some monoclonal antibody-binding sites on the protein C molecule.     

Characterization of the interaction between factor Xa and bovine aortic endothelial cells

Cultured bovine aortic endothelial cells incubated with Factor Xa activate prothrombin. Factor V, synthesized by the endothelial cells, or plasma Factor V and calcium are required for the reaction. In the present study, it has been demonstrated that 125I-Factor Xa binds specifically to endothelial cells. In addition, the activation of prothrombin by Factor Xa and aortic endothelial cells has been further characterized. The binding of 125I-Factor Xa to endothelial cells was saturable and reversible. The equilibrium dissociation constant (Kd) for 125I-Factor Xa binding was 3.6 X 10(-9) M, with 39000 molecules bound per cell. 125I-Factor Xa, inactivated by diisopropylfluorophosphate did not bind specifically to endothelial cells, indicating that the active site of Factor Xa was required for binding. Factor Xa, but not activated protein C, competed with 125I-Factor Xa for binding. Autoradiograms of sodium dodecyl sulfate-polyacrylamide gels of cell lysates indicated that the radiolabeled material that bound to the cells had electrophoretic mobility identical to Factors Xa alpha and Xa beta. Although Factor X partially inhibited the binding of 125I-Factor Xa, Factor Xa did not inhibit the binding of 125I-Factor X, indicating that the zymogen and enzyme bound to different receptors. The relationship of the 125I-Factor Xa binding which was measured in these studies to aortic endothelial cell prothrombin activation is unclear since an anti-Factor V IgG blocked prothrombin activation but not Factor Xa binding. Additionally, 125I-Factor Xa binds to nonvascular cells; these cells do not activate prothrombin in the presence of Factor Xa. Moreover, the calcium requirements for each reaction and the saturation curves of 125I-Factor Xa binding and prothrombin activation differ. Although these data do not exclude a relationship between Factor Xa binding and prothrombin activation, the binding of 125I-Factor Xa to aortic endothelium measured in these studies may be related to a separate cellular function. To further characterize prothrombin activation by Factor Xa and endothelial cells, the rates of thrombin generation by intact bovine aorta or endothelial cells derived from this tissue were compared and were found to be equivalent. These data indicate that vascular endothelium may serve as a physiologic surface for hemostasis.     

Expression of blood clotting factor VIII:C gene in capillary endothelial cells.

The essential role of Factor VIII:C (FVIII:C, anti-hemophilia factor A) as a cofactor for Factor IXa-dependent activation of Factor X has been established. In this paper, we describe that capillary endothelial cells from bovine adrenal medulla express active FVIII:C gene. Accumulation of FVIII:C in conditioned media from an 8-day-old culture is approximately twice as high as that stored in the cell when immunoprecipitated FVIII:C was analyzed for its ability to convert Factor X to Factor Xa. Analysis of [35S]methionine-labeled and immunoprecipitated FVIII:C from cells or conditioned media on SDS-PAGE under fully denatured conditions indicated that the newly synthesized FVIII:C consists of heavy chain of M(r) 200,000 and light chain of M(r) 46,000. The secreted FVIII:C in the non-reduced condition however, has a molecular weight of 270,000 which suggests that in native protein, the heavy and light chains are held together by S-S bonds. Furthermore, susceptibility of the immunoprecipitated FVIII:C to N-glycanase digestion establishes that the endothelial cells derived FVIII:C contains asparagine-linked carbohydrate side chains.     

Purification and properties of an activating enzyme of blood clotting factor X from the venom of Cerastes cerastes

An activator of blood coagulation factor X was found in the venom of the horned viper Cerastes cerastes, and was purified by gel filtration, ion-exchange chromatography and chromatofocussing. The activator is a protein composed of a heavy and a light polypeptide chain linked by disulfide bonds. Two subforms of the activator were found. Both contained a heavy chain of Mr 58000 and are distinguished from each other by the presence of two different light chains of Mr 17700 and 15000. The activator appears to cleave the bond in the factor X molecule that is also cleaved by factor IXa. Factor X activation by the activator is strongly stimulated by Ca2+. The kinetic parameters for the activation reaction have been determined. A Km for factor X of 19.2 nM and a Vmax of 0.11 pmol of Xa/min per ng venom were found.     

Comparison of Clostridium botulinum toxins type D and C1 in molecular property, antigenicity and binding ability to rat-brain synaptosomes

Botulinum type D neurotoxin was purified 950-fold from the culture supernatant with an overall yield of 32%. The purified toxin had a specific toxicity of 5.8 X 10(7) mouse minimal lethal dose per mg of protein and a relative molecular mass of 140000. The purified toxin had a di-chain structure consisting of heavy and light chains with relative molecular masses of 85000 and 55000, respectively, linked by one disulfide bond. These subunits had different amino acid compositions and antigenicities. A similarity in molecular constructions and amino acid compositions was observed between type D and type C1 toxins as well as between their subunits. Among the seven kinds of monoclonal antibodies against type D toxin, six reacted with the heavy chain of type D toxin, while one of the six also reacted with the heavy chain of type C1 toxin and neutralized the toxicities of the two toxins. The other one of monoclonal antibodies reacted with the light chains of both toxins. This evidence indicates that both toxins have common antigenic sites on their heavy and light chains and that the antigenic site on the heavy chain may contribute to the neutralization of both toxins by antibody. The binding of type D toxin to rat brain synaptosomes was examined by use of 125I-labelled type D toxin. The binding was competitively inhibited not only by unlabelled type D and C1 toxins, but also by the heavy chains of both toxins, however, it was not inhibited by the light chain of type D toxin. These results suggest that the toxin receptors on synaptosomal membrane are common for type D and C1 toxins, and that the heavy chain contributes to the binding of toxin to synaptosomes and the structure of the binding sites on the heavy chains of both toxins is quite similar.     

Determination of the bifunctional properties of high molecular weight kininogen by studies with monoclonal antibodies directed to each of its chains

In normal human plasma two forms of kininogen exist, low molecular weight kininogen (LMWK) and high molecular weight kininogen (HMWK). When these proteins are cleaved they are found to have a common heavy chain and bradykinin, but each has a unique light chain. Monoclonal antibodies to the heavy and light chains of HMWK have been developed, and the effects of each on the function of this protein are defined. Initial studies showed that an antibody, C11C1, completely neutralized the coagulant activity of plasma HMWK whereas another antibody, 2B5, did not. On a competitive enzyme-linked immunosorbent assay (CELISA) the C11C1 antibody was consumed by kininogen antigen in normal plasma but not by kininogen antigen in HMWK-deficient plasma. On immunoblot, the C11C1 antibody recognized one kininogen protein in normal plasma and did not recognize any kininogen antigen in HMWK-deficient plasma. These combined studies indicated that the C11C1 antibody was directed to an epitope on the unique 46-kDa light chain of HMWK. In contrast, the 2B5 antibody on a CELISA was consumed by kininogen antigen in both normal plasma and HMWK-deficient plasma but not by total kininogen-deficient plasma. On immunoblot, the 2B5 antibody recognized both kininogens in normal plasma but only LMWK in HMWK-deficient plasma. These combined studies indicated that the 2B5 antibody was directed to the common 64-kDa heavy chain of the plasma kininogens. Utilizing direct binding studies or competition kinetic experiments, the 2B5 and C11C1 antibodies bound with high affinity (1.71 and 0.77 nM, respectively) to their antigenic determinants on the HMWK molecule. The 2B5 antibody did neutralize the ability of HMWK to inhibit platelet calpain. These studies with monoclonal antibodies directed to each of the HMWK chains indicate that HMWK is a bifunctional molecule that can serve as a cofactor for serine zymogen activation and an inhibitor of cysteine proteases.     

Construction, expression, and characterization of a chimera of factor IX and factor X. The role of the second epidermal growth factor domain and serine protease domain in factor Va binding.

The prothrombinase complex, which catalyzes the conversion of prothrombin to thrombin, consists of activated Factor X, Factor Va, a membrane surface and Ca2+. To examine the structures that support Factor Va binding to Factor X, we used in vitro mutagenesis to construct a chimeric molecule that includes regions of Factor IX and Factor X. This chimera (IXGla,E1XE2,SP) was prepared from cDNA encoding the second epidermal growth factor (EGF) and serine protease domains of Factor X linked downstream from the cDNA encoding the signal peptide, propeptide, Gla domain, and first EGF domain of Factor IX. The cDNAs encoding the Factor IX/X chimera and wild-type Factor X were each expressed in Chinese hamster ovary cells and the secreted proteins purified by affinity chromatography using polyclonal anti-Factor X antibodies. The chimera migrated as a single major band corresponding to a molecular weight of 68,000. By Western blotting, the chimeric protein stained with both polyclonal anti-Factor X and anti-Factor IX antibodies. gamma-Carboxyglutamic acid analysis demonstrated near complete carboxylation of both the wild-type Factor X and the Factor IX/X chimera. Compared with Factor X, the rate of zymogen activation of the Factor IX/X chimera was about 50% that of Factor X when activated by Factor IXa, Factor VIIIa, phospholipid, and Ca2+. The enzyme form of the Factor IX/X chimera, activated Factor IX/X, generated using the coagulant protein of Russell's viper venom, expressed full amidolytic activity compared with Factor Xa. The activated Factor IX/X chimera had about 14% of the activity of Factor Xa when employed in a prothrombinase assay; this activity reached 100% with increasing concentrations of Factor Va. A binding assay was employed to test the ability of the active site-inactivated Factor IX/Xa chimera to inhibit the binding of Factor Xa to the Factor Va-phospholipid complex, thus inhibiting the activation of prothrombin to thrombin. In this assay the active site-inactivated form of the chimera competed with Factor Xa completely but with decreased affinity for the Factor Va-phospholipid complex. These data indicate that the second EGF domain and the serine protease domain of Factor Xa are sufficient to interact with Factor Va. The Factor IX/X chimera is a good substrate for the tenase complex; the defective enzymatic activity of the activated Factor IX/X chimera can be accounted for by its decreased affinity for Factor Va relative to Factor Xa.     

Identification of molecular sites on factor VII which mediate its assembly and function in the extrinsic pathway activation complex

Factor VII-VIIa, in association with tissue factor, participates in the complex which initiates blood coagulation through the extrinsic pathway. To identify functional domains on factor VII which mediate the activation of factor X, 16 synthetic peptides corresponding to 55% of the primary structure were assayed for their ability to inhibit factor VII function. Factor Xa formation was inhibited by eight of the peptides in a dose-dependent manner. Kinetic analyses indicated noncompetitive inhibition of factor X activation by seven of these peptides. Peptide-(347-361) inhibited factor Xa cleavage of a chromogenic substrate by a competitive mechanism and was excluded from further analysis in this study. Among the seven inhibitory peptides which have the ability to prevent the factor VIIa-tissue factor-mediated conversion of factor X to factor Xa, peptide-(285-305) was most inhibitory, with a Ki value of 2.4 microM. The Ki values were in the range of 42-65 microM for peptides-(44-50), -(194-214), -(208-229), and -(376-390). The least inhibitory peptides were at positions 170-178 and 330-340, with a Ki value greater than 200 microM. Polyclonal antibodies were raised against four of these peptides; and when antisera were assayed by a solid-phase radioimmunoassay, they bound not only to their respective immunizing peptides, but also to factor VII. The Fab fragments of specific IgG preparations, affinity-purified on a factor VII-agarose column, inhibited the rate of factor X activation in a dose-dependent manner. Six of the seven inhibitory peptides represent amino acid sequences within the heavy chain of factor VII, and the remaining one corresponds to a sequence within the light chain. The corresponding regions in the x-ray crystal structure of chymotrypsin represented by the six heavy chain inhibitory peptides are found to be located in three distinct regions, one region located spatially distal to the active site and the other two regions located relatively closer to the active site and the substrate-binding pocket. The results suggest that at least three specific regions in the heavy chain and one region in the light chain of factor VII mediate its interaction with the factor X activation complex.     

Idiotypic determinants of monoclonal antibodies that bind to 3-fucosyllactosamine

Many monoclonal antibodies that react with the lacto-N-fucopentaose III (LNF III) antigenic determinant, Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3Gal beta 1-4Glc, have been described recently. The terminal trisaccharide of this determinant, fucosyllactosamine, is present on glycolipids and glycoproteins and on the surface of granulocytes, monocytes, and other cells. To study the structural and genetic diversity of these antibodies, syngeneic anti-idiotypic monoclonal antibodies were produced in BALB/c mice against PMN 6, a monoclonal antibody directed against this sequence. Anti-idiotypic antibodies 6B1 and 6C4 reacted with 50% of a panel of 20 anti-LNF III monoclonal antibodies, whereas 6A3 reacted strongly only with PMN 6. This indicates that the determinants recognized by 6C4 and 6B1 represent major cross-reactive idiotopes of this family of antibodies. The binding of idiotypic antibodies to a glycolipid bearing this antigenic determinant was completely inhibited by the three anti-idiotypic antibodies, 6A3, 6B1, and 6C4. The idiotopes could be demonstrated on the heavy chain of the monoclonal antibodies by an antibody transfer technique when mild reducing conditions were employed, but a high concentration of reducing agent destroyed the idiotypic determinants. This suggests that the anti-idiotypic antibodies recognize conformational structures expressed on the heavy chain molecules. The binding of 18 monoclonal antibodies to two glycolipid antigens and to a fucosyllactosamine-bovine serum albumin conjugate was compared. Antibodies that possessed the 6C4 cross-reactive idiotope bound to fucosyllactosamine-bovine serum albumin more weakly than idiotype-negative antibodies (p = 0.001). This suggests that the 6C4-positive antibodies might represent germline structures.     

Human factor XII binding to the glycoprotein Ib-IX-V complex inhibits thrombin-induced platelet aggregation

Factor XII deficiency has been postulated to be a risk factor for thrombosis suggesting that factor XII is an antithrombotic protein. The biochemical mechanism leading to this clinical observation is unknown. We have previously reported high molecular weight kininogen (HK) inhibition of thrombin-induced platelet aggregation by binding to the platelet glycoprotein (GP) Ib-IX-V complex. Although factor XII will bind to the intact platelet through GP Ibalpha (glycocalicin) without activation, we now report that factor XIIa (0. 37 microm), but not factor XII zymogen, is required for the inhibition of thrombin-induced platelet aggregation. Factor XIIa had no significant effect on SFLLRN-induced platelet aggregation. Moreover, an antibody to the thrombin site on protease-activated receptor-1 failed to block factor XII binding to platelets. Inhibition of thrombin-induced platelet aggregation was demonstrated with factor XIIa but not with factor XII zymogen or factor XIIf, indicating that the conformational exposure of the heavy chain following proteolytic activation is required for inhibition. However, inactivation of the catalytic activity of factor XIIa did not affect the inhibition of thrombin-induced platelet aggregation. Factor XII showed displacement of biotin-labeled HK (30 nm) binding to gel-filtered platelets and, at concentrations of 50 nm, was able to block 50% of the HK binding, suggesting involvement of the GP Ib complex. Antibodies to GP Ib and GP IX, which inhibited HK binding to platelets, did not block factor XII binding. However, using a biosensor, which monitors protein-protein interactions, both HK and factor XII bind to GP Ibalpha. Factor XII may serve to regulate thrombin binding to the GP Ib receptor by co-localizing with HK, to control the extent of platelet aggregation in vivo.     

The binding of 35S-labeled recombinant factor VIII to activated and unactivated human platelets

Recombinant-derived human Factor VIII was labeled intrinsically with [35S]methionine, and its binding to washed human platelets was studied. Binding measurements were performed by incubating Factor VIII and platelets for 15 min at room temperature in Tyrode's solution supplemented with Ca2+ (5.0 mM), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (5.0 mM), 0.50% bovine serum albumin, and the Factor Xa and thrombin inhibitors 5-dimethylaminonaphthalene-1-sulfonylglutamylglycinylarginyl chloromethyl ketone and 5-dimethylaminonaphthalene-1-sulfonyl-arginine-N-(3-ethyl-1, 5-pentanediyl)amide. Separation of free from bound Factor VIII was accomplished by centrifugation through oil, and nonspecific binding was determined with excess unlabeled Factor VIII. Binding was saturable, reversible, and stimulated 20-fold after platelet activation with thrombin. Furthermore, binding was specific in that bound labeled Factor VIII could be displaced by excess unlabeled Factor VIII, but not by Factor V. Scatchard analysis indicated a single class of binding sites with Kd = 2.9 nM and 450 sites/activated platelet. The time course of displacement indicated a t1/2 of bound Factor VIII of approximately 5 min. When platelets were incubated in Ca2+, both the heavy and light chains of Factor VIII were bound, whereas exposure to EDTA resulted in the binding of the light chain only. These results demonstrate the specific reversible binding of Factor VIII to human platelets, likely mediated through the light chain.     

Clathrin structure characterized with monoclonal antibodies. I. Analysis of multiple antigenic sites

Three monoclonal antibodies that react with previously undefined antigenic determinants on the clathrin molecule have been produced and characterized. They were isolated from a fusion between myeloma cells and popliteal lymphocytes from SJL mice that had received footpad injections of human brain clathrin. This protocol was chosen to favor the production of antibodies to poorly immunogenic proteins and thereby increase the repertoire of anti-clathrin monoclonal antibodies. One antibody (X16) reacts preferentially with the heavier of the two clathrin light chains (LCa) when it is not associated with heavy chain. This specificity is different from that of the anti-LCa antibody, CVC.6, which has preferential reactivity with heavy chain-associated LCa. In addition, X16 and CVC.6 bound simultaneously to LCa, confirming that they react with different sites. The other two antibodies produced, X19 and X22, react with two different determinants on the clathrin heavy chain, based on immunoprecipitation, Western blot, and binding studies. Competitive binding studies with anti-clathrin monoclonal antibodies showed that they define a total of five distinct antigenic determinants on bovine clathrin.     

A novel approach in potential anticoagulants from peptides epitope 558–565 of A2 subunit of factor VIII

Factor VIII, a human blood plasma protein, plays an important role during the intrinsic pathway of blood coagulation cascade after its activation by thrombin. The activated form of FVIII acts as cofactor to the serine protease Factor IXa, in the conversion of the zymogen Factor X to the active enzyme Factor Xa. The Ser⁵⁵⁸–Gln⁵⁶⁵ region of the A2 subunit of Factor VIII has been shown to be crucial for FVIIIa–FIXa interaction. Based on this, a series of linear peptides, analogs of the 558–565 loop of the A2 subunit of the heavy chain of Factor VIII were synthesized using the acid labile 2-chlorotrityl chloride resin and biologically evaluated in vitro by measuring the chronic delay of activated partial thromboplastin time and the inhibition of Factor VIII activity, as potential anticoagulants.     

Monoclonal antibodies detect and stabilize conformational states of smooth muscle myosin

Antibodies with epitopes near the heavy meromyosin/light meromyosin junction distinguish the folded from the extended conformational states of smooth muscle myosin. Antibody 10S.1 has 100-fold higher avidity for folded than for extended myosin, while antibody S2.2 binds preferentially to the extended state. The properties of these antibodies provide direct evidence that the conformation of the rod is different in the folded than the extended monomeric state, and suggest that this perturbation may extend into the subfragment 2 region of the rod. Two antihead antibodies with epitopes on the heavy chain map at or near the head/rod junction. Magnesium greatly enhances the binding of these antibodies to myosin, showing that the conformation of the heavy chain in the neck region changes upon divalent cation binding to the regulatory light chain. Myosin assembly is also altered by antibody binding. Antibodies that bind to the central region of the rod block disassembly of filaments upon MgATP addition. Antibodies with epitopes near the COOH terminus of the rod, in contrast, promote filament depolymerization, suggesting that this region of the tail is important for assembly. The monoclonal antibodies described here are therefore useful both for detecting and altering conformational states of smooth muscle myosin.     

The activation of coagulation factor X. Identity of cleavage sites in the alternative activation pathways and characterization of the COOH-terminal peptide.

Bovine Factor X can be activated by two alternative pathways. The first, favored at high concentrations of the complex of tissue factor and Factor VII, is initiated by the action of Factor VII on Factor X to cleave an activation peptide from the NH2 terminus of the heavy chain, to produce alpha-Xa. This is then converted autocatalytically to another form of Factor Xa, beta-Xa, by the loss of a 17-residue glycopeptide from the COOH terminus of the heavy chain, in a lipid-dependent reaction. The alternative pathway, favored at lower activator concentrations, is initiated by the action of Factor Xa on Factor X, in the presence of lipid, to release the same COOH-terminal peptide as is produced in the conversion of alpha-Xa to beta-Xa. The intermediate produced by the loss of this peptide from Factor X,I1, can be activated directly to beta-Xa by the tissue factor-Factor VII complex, with the loss of the same NH2-terminal peptide as is produced in the conversion of Factor X to alpha-Xa. The autocatalytic activation of Factor X by Factor Xa described previously occurs to a marked extent only at very low activator concentrations, and has been shown to proceed largely by the loss of the normal NH2-terminal peptide from the heavy chain of I1-Initial experiments show that neither peptide affects the rate of coagulation by either the extrinsic or intrinsic pathways. The amino acid sequences have been determined on both sides of the peptide cleavages, and it has been shown that the cleavage sites are the same, regardless of the pathway of activation. The amino acid sequence and carbohydrate composition of the COOH-terminal peptide have been determined. The carbohydrate moiety is attached via an O-glycosidic linkage at a threonine residue, and contains galactosamine but no glucosamine.