Bovine thrombin preparations are highly immunogenic and appear to be associated with an increased risk for adverse clinical outcomes during subsequent surgical procedures. The clinical safety of these commonly used preparations needs to be reassessed, and reexposure to these agents should likely be avoided.

Activated factor V (Va) serves as an essential protein cofactor for the conversion of prothrombin to thrombin by factor Xa. Analysis of the factor V cDNA indicates that the protein contains several types of internal repeats with the following domain structure: A1-A2-B-A3-C1-C2. In this report we describe the isolation and characterization of genomic DNA coding for human factor V. The factor V gene contains 25 exons which range in size from 72 to 2820 bp. The structure of the gene for factor V is similar to the previously characterized gene for factor VIII. Based on the aligned amino acid sequences of the two proteins, 21 of the 24 intron-exon boundaries in the factor V gene occur at the same location as in the factor VIII gene. In both genes, the junctions of the A1-A2 and A2-A3 domains are each encoded by a single exon. In contrast, the boundaries between domains A3-C1 and C1-C2 occur at intron-exon boundaries, which is consistent with evolution through domain duplication and exon shuffling. The connecting region or B domain of factor V is encoded by a single large exon of 2820 bp. The corresponding exon of the factor VIII gene contains 3106 bp. The 5' and 3' ends of both of these exons encode sequences homologous to the carboxyl-terminal end of domain A2 and the amino-terminal end of domain A3 in ceruloplasmin. There is otherwise no homology between the B domain exons.(ABSTRACT TRUNCATED AT 250 WORDS)

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Factor V inhibitors may develop as spontaneous autoantibodies, as alloantibodies after exposure to bovine thrombin preparations, or in factor V–deficient patients after plasma therapy. Clinical manifestations range from asymptomatic laboratory abnormalities to life-threatening hemorrhage. We have characterized the anti–factor V antibodies from 12 patients diagnosed with factor V inhibitors. In 8 patients, hemorrhagic complications (5 autoantibodies and 3 bovine thrombin-induced alloantibodies) developed, and 4 were asymptomatic (2 autoantibodies and 2 alloantibodies). The IgG fractions from all 12 patients immunoprecipitated the factor Va light chain, but only the 8 IgG fractions associated with hemorrhage inhibited factor V activity in a prothrombinase assay. Nine IgG fractions, including the 8 patients with hemorrhage, immunoprecipitated the isolated second C-type domain (C2). The 8 IgG fractions from the symptomatic patients also immunoprecipitated recombinant chimeras containing only the N-terminal third of the factor V C2 domain, and isolated recombinant C2 domain abrogated the inhibitory effect of the antibodies. Five of the inhibitory IgG fractions blocked binding of factor V to phosphatidylserine. These results suggest that inhibitory anti–factor V antibodies are associated with hemorrhagic manifestations and frequently bind to a common region within the C2 domain, whether originating spontaneously or after exposure to bovine thrombin.

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We studied 88 hemodialysis patients for the presence of antibodies to human factor II (hFII), bovine factor V (bFV), and human beta2-glycoprotein 1 (beta2GPI). Forty-one patients had elevated anti-hFII antibodies, 17 had elevated anti-bFV antibodies, and 9 had elevated anti-beta2GPI antibodies. Fifty-two patients had elevated antibodies to one or more protein. Patients with PTFE grafts had elevated antibodies most frequently (21 [75%] vs. 20 fistulas [45%; p = 0.016 compared with PTFE] and 11 tunneled catheters [68.8%]). Twelve of 13 patients (92.3%) with PTFE grafts and thrombosis had elevated antibody levels, compared with 9 of 15 without thrombosis (60%; p = 0.049). The number of thromboses and mean thrombosis rates were significantly higher in PTFE patients with antibodies (1.24 vs. 0.14 thromboses, p < 0.01; 42.67 vs. 6.44 thromboses/100 patient years, p < 0.05). When analyzed individually, thrombotic complications occurred more frequently in patients with PTFE grafts and elevated anti-bFV antibodies (p = 0.016), but did not correlate with anti-hFII or anti-beta2GPI antibodies. Thrombotic complications did not correlate with elevated antibody levels in patients with AV fistulas or cuffed catheters. In conclusion, hemodialysis patients with PTFE grafts frequently have elevated antibodies to FII, FV, and beta2GPI, and the presence of elevated antibody levels to one or more of these proteins is associated with an increased thrombotic risk. Further studies are necessary to determine whether limiting exposure to bovine thrombin preparations will decrease the incidence of these antibodies and PTFE graft thrombosis.

SummaryFour factor VIII light chain constructs containing hemophilia A mutations at R2304 and R2307 were prepared and expressed in mammalian cells. These mutations are located in a putative phosphatidylserine binding site identified by peptide studies (spanning amino acids 2303-2332). The levels of all four mutants in conditioned medium were significantly less than wild type by immunoprecipitation and ELISA. R2304H and wild type factor VIII light chains were concentrated by cation exchange chromatography from medium. R2304H and wild type factor VIII light chains bound immobilized phosphatidylserine similarly. The reconstituted cofactor activity of R2304H factor VIII light chain was slightly greater than wild type factor VIII light chain. These results are consistent with the recently reported crystal structure of factor VIII C2 domain that suggests R2304H is not directly involved in phospholipid binding. The observed clinical phenotype is probably due to decreased circulating levels of a functional protein.

SummaryFactor VIII and factor V share a repetitive domain structure of A1-A2-B-A3-C1-C2. To define the region(s) within the factor VIII heavy chain that result in inefficient expression of the recombinant protein, we expressed a series of factor VIH/factor V chimeras that contained heterologous sequences from the A1 and/or A2 domains. Substitution of the factor VIIIA1 domain dramatically reduced secretion of factor V ~ 500-fold, whereas substitution of the factor VIII A2 domain had minimal effect on secretion. Conversely, substitution of the factor V A1 domain increased secretion of factor VIII ~3-fold, whereas substitution of the factor V A2 domain actually reduced secretion ~4-fold. Pulse chase experiments confirmed that reduced expression levels were due to decreased secretion rather than instability of secreted protein. Smaller substitutions did not further localize within the A1 domain the regions responsible for inefficient secretion.