The activation of factor X and prothrombin by recombinant factor VIIa in vivo is mediated by tissue factor.

The human coagulation system continuously generates very small quantities of Factor Xa and thrombin. Current evidence suggests that basal level activation of the hemostatic mechanism occurs via Factor VIIa-dependent activation of Factor X, but direct proof has not been available for the participation of tissue factor in this pathway. To examine this issue, we infused relatively high concentrations of recombinant Factor VIIa (approximately 50 micrograms/kg body wt) into normal chimpanzees and observed significant increases in the plasma levels of Factor IX activation peptide, Factor X activation peptide, and prothrombin activation fragment F1+2. Metabolic turnover studies with radiolabeled Factor IX activation peptide, Factor X activation peptide, and F1+2 indicate that elevated levels of the activation peptides are due to accelerated conversion of the three coagulation system zymogens into serine proteases. The administration of a potent monoclonal antibody to tissue factor, which immediately neutralizes function of the Factor VIIa-tissue factor complex in vitro, abolishes the activation of Factor X and prothrombin mediated by the infused recombinant protein, and also suppresses basal level activation of Factor IX and Factor X. The above results suggest that recombinant Factor VIIa functions as a prohemostatic agent by interacting with endogenous tissue factor sites, but definitive proof will require studies in hemophilic animals using relevant hemostatic endpoints.

Epitopes on the S1 subunit of pertussis toxin recognized by monoclonal antibodies.

To identify the neutralizing epitopes on the S1 subunit (A promoter) of pertussis toxin, we characterized anti-S1 monoclonal antibodies (MAbs) X2X5, 3CX4, and 6FX1. We confirmed by immunoblot analysis that these MAbs bind to the S1 subunit and not to the B oligomer of pertussis toxin and that they recognize different epitopes by a competitive binding enzyme-linked immunosorbent assay. These MAbs had differential abilities to neutralize the lymphocytosis-promoting factor activity of pertussis toxin in mice: 3CX4 and 6FX1 had partial neutralizing abilities, while MAb X2X5 had none. With these MAbs, the epitopes on the S1 subunit were examined by using trypsinized S1 peptides, recombinant truncated S1 molecules, and synthetic peptides. The non-neutralizing MAb X2X5 bound in immunoblots to tryptic peptides of various sizes as small as 1.5 kilodaltons; the neutralizing MAbs 3CX4 and 6FX1 bound only to a 24-kilodalton tryptic peptide band. Immunoblot studies with recombinant truncated S1 molecules demonstrated that amino acid residues 7 to 14 and 15 to 26 play an important role in the binding of neutralizing MAbs and the non-neutralizing MAb, respectively. The binding of these MAbs was not dependent upon the presence of C-terminal amino acid residues 188 to 234. To further define B-cell epitopes, the binding of the MAbs we tested to synthetic peptides representing the entire S1 subunit were examined. Neutralizing MAbs 3CX4 and 6FX1 bound to none of these peptides, further suggesting that these MAbs recognize conformational epitopes. The non-neutralizing MAb X2X5 bound to peptides 11 to 26 and 16 to 30, demonstrating that the major antigenic determinant recognized by this MAb is a linear epitope located within residues 16 to 26.

Plasmid-mediated changes in virulence of Vibrio cholerae.

The effects of several plasmids, including cloning vectors and R factors, on the virulence of Vibrio cholerae CA401R were determined by measuring the dose-related diarrheal response in orally challenged infant mice. The plasmids were also examined for their effects on the colonization ability of strain CA401R by joint infection experiments with a spectinomycin-resistant CA401 strain as an internal standard. One V. cholerae R factor, pVH2, enhanced the diarrheal response, while R factors Rts1 and pVH1 reduced it; plasmids RP4, pRK290, Sa, pSJ8, pSJ5, and pBR328 had no effect. The ability of the plasmids to affect in vitro toxin production by CA401R was variable. Cells containing large plasmids all showed a modest decrease in colonization ability. These results showed that some plasmids affected V. cholerae virulence, but that the cloning vectors pBR328, RP4, and pRK290 did not.

Transposon-facilitated recombination in classical biotypes of Vibrio cholerae.

Transposon-facilitated recombination (Tfr) donors of classical Vibrio cholerae strain 162 were constructed by introducing the ampicillin transposon Tn1 into the P conjugative plasmid and the bacterial chromosome. The improved donors mediated high-frequency, polarized transfer of chromosomal genes from origins to confirm the gene orders of the previous classical strain 162 genetic map and to establish its circularity. Significant transfer of linked genes from E1 Tor Tfr donors to classical recipients was demonstrated, and other evidence for genetic relatedness of these two V. cholerae biotypes is discussed.

Genetic mapping of toxin regulatory mutations in Vibrio cholerae.

We have mapped a regulatory site mediating the hyperproduction of cholera toxin in mutants of Vibrio cholerae strain 569B. Mutations in this locus, called htx, result in the hypertoxinogenic phenotype, as measured by the ganglioside filter assay and immunoradial diffusion. Transposon-facilitated recombination was used to construct improved genetic donors in 569B parental and hypertoxinogenic mutant strains. Subsequent mapping by conjugation indicated that the htx locus was closely linked to the rif, str, and ilv loci of V. cholerae. Analysis of recombinants from these crosses suggested the following gene order: thy str htx rif ilv arg. The close genetic linkage of htx to rif (as high as 98%) resulted in a high comutation frequency of these two loci by nitrosoguanidine mutagenesis. Transfer of the htx mutant locus from a hypertoxinogenic donor to several unrelated Tox+ strains of V. cholerae caused a detectable elevation of toxin production in the recipients. These results suggest that toxin production in diverse strains of V. cholerae is controlled by a common regulatory mechanism in which the htx gene product plays a significant role.