During the past 20 years contributions from many laboratories have led to the development of isolation procedures, delineation of primary structures, and more recently, to the expression of recombinant proteins associated with the coagulation cascade. In general, studies of coagulation proteins under defined conditions have demonstrated the prescience of Davie and Ratnoff and MacFarlane in their proposals of the coagulation cascade. The more recent discovery of thrombomodulin by Esmon et al has led to the identification and characterization of components of the vitamin K-dependent anticoagulant pathway. In this review we have attempted to analyze and compare the functional properties of each of the vitamin K-dependent enzyme complexes associated with the procoagulant and anticoagulant phases of blood clotting. Although dissimilarities exist, the vitamin K-dependent complexes have analogous requirements and appear to function with a common general mode of organization. Membrane-bound cofactors serve as anchoring sites for the appropriate membrane-binding enzymes. This process localizes the complex on the membrane surface and increases the catalytic efficiency for substrate utilization. Complex formation provides extraordinary improvements in the catalytic efficiency for the complexes as compared with their soluble enzyme components. Membrane- bound complexes provide a mechanism that can be regulated at a site by membrane presentation, zymogen activation, and cofactor activation or presentation. The kinetic constants obtained for the various coagulation reactions determined in vitro provide some insights into how these pathways may function in vivo. The catalytic efficiency (kcat/Km) for factor X activation by factor VIIIa/factor IXa is far in excess of the catalytic efficiency of activation of factor X by tissue factor/factor VIIa (Table 3). This may provide a rational interpretation for the observation that patients with hemophilia A and B bleed even though they appear to have an alternative pathway to factor X activation. In addition, tissue factor is not ordinarily presented by the vascular tissue that has direct access to blood. However, it appears that extravascular constitutive tissue factor is available once the blood vessel becomes disrupted. The efforts to identify the initiating reactions of the blood coagulation process have not been unambiguously successful. We conclude that factor VII is most likely a zymogen, just as are the other proenzymes of the blood clotting process. In addition, it is difficult to rationalize the importance of the intrinsic pathway of coagulation involving factor XII, prekallikrein, and high molecular weight kininogen since the congenital absence of any one of these factors does not result in abnormal bleeding.

The Saccharomyces cerevisiae CDC42 gene product, a member of the ras superfamily of low-molecularweight GTP-binding proteins, is involved in the control of cell polarity. We have analyzed the effects of three CDC42 mutations (Gly to Gin to and Asp to in the putative GTP-binding and hydrolysis domains and one mutation (Cys to in the putative isoprenylation site. The incapable of complementing the cdc42-l' mutation and was recessive to both wild-type and cdc42-1'. In double-mutant alleles, the cdc42srel188 mutation was capable of suppressing the dominant lethality associated with the three putative GTP-binding and hydrolysis mutations, suggesting that isoprenylation is necessary for the activity of the wild-type and mutant proteins.The Saccharomyces cerevisiae CDC42 gene product is involved in the control of several morphogenetic events during the cell cycle, including the generation of cell polarity, development of normal cell shape, localization of secretion, and deposition of cell-surface material (1,2,20). Its predicted amino acid sequence (20) is similar to that of members of the ras superfamily of low-molecular-weight GTP-binding proteins from S. cerevisiae and higher eukaryotes (16), especially in those domains that have been implicated in the binding and hydrolysis of GTP and in carboxylterminal modifications. CDC42 shows the greatest degree of sequence similarity to the products of two related human cDNAs, CDC42Hs (39) and G25K (27), which encode isoforms of the low-molecular-weight GTP-binding protein previously referred to as Gp or G25K (11, 33). Both of these human gene products are 80% identical (88% related amino acids) in predicted amino acid sequence to the S. cerevisiae CDC42 gene product, which we have redesignated CDC42Sc, and they are 95% identical to each other. These human genes, when expressed in S. cerevisiae under the control of a yeast promoter on a multicopy plasmid, can functionally complement the cdc42-1's mutation (27, 39). The Gp/G25K protein is an excellent in vitro phosphosubstrate for the epidermal growth factor receptor tyrosine kinase (17), but its in vivo function is unknown.In order to further elucidate the role of the CDC42Sc gene product in the control of cell polarity, we have generated new mutations in CDC42Sc by using site-directed mutagenesis (see Fig. 1). A carboxyl-terminal mutation predicted to * Corresponding author.eliminate isoprenylation results in a nonfunctional product. Mutations in the putative guanine-nucleotide-binding domain lead to a lethal phenotype that can be suppressed by combination with the carboxyl-terminal mutation. This lethality is manifested as several different and unique morphological abnormalities, which have provided clues to the function of the CDC42Sc gene product in controlling cell polarity. MATERIALS AND METHODSReagents. Enzymes, M13 dideoxy sequencing kit, mutagenesis kit, and other reagents were obtained from standard commercial sources and used according to the suppliers' Media, growth conditions, and strains. Conditions for...

Computer searches of the National Biomedical Research Foundation protein and nucleic acid sequence data bases using the NH2 terminus of the bovine factor Va 94-kilodalton heavy chain, the NH2 terminus of the 74-kilodalton factor Va light chain, and an internal 98-residue segment of porcine factor VIII revealed that both bovine factor V and porcine factor VIII are statistically homologous to human ceruloplasmin. The NH2-terminal segment of bovine factor Va heavy chain is homologous to three segments of ceruloplasmin sequence starting at residues 1, 351, and 713; the NH2-terminal sequence of bovine factor Va light chain is homologous to the same human ceruloplasmin sequence segments beginning at residues 1, 349, and 711. The longer porcine factor VIII sequence is homologous to three segments of human ceruloplasmin, residues 1-77, 400-433, and 683-191. These data indicate that factor V, factor VIII, and ceruloplasmin comprise a group of evolutionarily linked protein structures that possibly resulted from multiplication of ancestral precursor genes.Factor V and factor VIII each function as cofactors for two vitamin K-dependent serine proteases in the blood coagulation cascade (1). Bovine factor V (BFV) is a single chain glycoprotein of mass 330 kDa, which is cleaved to an active two-polypeptide form (factor Va) consisting of a 94-kDa heavy chain complexed to a 74-kDa light chain via Ca2+-dependent forces (2). The 94-kDa peptide of bovine factor Va corresponds to the NH2-terminal region of factor V; the 74-kDa peptide corresponds to the COOH-terminal region of factor V (3, 4). Factor Va functions in coagulation as a receptor on phospholipid vesicles (5) and platelets (6, 7) for the serine protease factor Xa. This complex of factor Xa, factor Va, a membrane surface, and calcium ion catalyzes the cleavage of the zymogen prothrombin to thrombin. As isolated, porcine factor VIII (PFVIII) is a two-chain structure composed of 160-and 76-kDa peptides whose association appears to depend on the presence of Ca2+ ion (8). COOHterminal cleavage of the 160-kDa heavy chain gives rise to an altered form of the two-chain cofactor consisting of a 130-kDa modified heavy chain associated with the 76-kDa light chain. Activation of factor VIII with thrombin gives rise to further loss of the COOH segment of the heavy chain, yielding a heavy chain-derived polypeptide of 82 kDa associated with a light chain-derived fragment of 69 kDa. In coagulation, activated factor VIII (factor VIIa), in the presence of Ca2+ and phospholipid, appears to function as a cofactor for the seine protease factor IXa in the activation of factor X to factor Xa. Recently EXPERIMENTAL PROCEDURES Protein sequences were determined by using an Applied Biosystems gas-phase sequencer using PFVIII isolated as described by Fass et al. (8) and using BFV isolated as described by Nesheim et al. (11). Two sequences for BFV were used for searching the Atlas of Protein Sequence and Structure (10)-namely, the 29 NH2-terminal residues of the 94-kDa heavy chain and the 25...

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Summary. Background: Inhibitory antibodies (Abs) to factor VIII (FVIII inhibitors) constitute the most significant complication in the management of hemophilia A. The analysis of FVIII inhibitors is confounded by polyclonality and the size of FVIII. Objectives: The goal of this study was to dissect the polyclonal response to human FVIII in hemophilia A mice undergoing a dosage schedule that mimics human use. Methods: Splenic B-cell hybridomas were obtained following serial i.v. injections of submicrogram doses of FVIII. Results of a novel, anti-FVIII domain-specific enzyme-linked immunosorbent assay were compared to Ab isotype and anti-FVIII inhibitory activity. Results: The robust immune response resulted in the production of 300 hybridomas per spleen. We characterized Abs from 506 hybridomas, representing the most comprehensive analysis of a protein antigen to date. Similar to the human response to FVIII, anti-A2 and anti-C2 Abs constituted the majority of inhibitors. A novel epitope was identified in the A2 domain by competition ELISA. Anti-A2 and anti-C2 Abs were significantly associated with IgG 1 and IgG 2a isotypes, respectively. Because the IgG 2a isotype is associated with enhanced Fc receptor-mediated effector mechanisms, this result suggests that anti-C2 Abs and inflammation may be linked. Additionally, we identified a novel class of Abs with dual specificity for the A1 and A3 domains. Forty per cent of the Abs had no detectable inhibitory activity, indicating that they are prominent and potentially pathologically significant. Conclusion: The expanded delineation of the humoral response to FVIII may lead to improved management of hemophilia A through mutagenesis of FVIII B-cell epitopes.