Peptidomimetic inhibitors for activated protein C: implications for hemophilia management.

BACKGROUND: Several clinical studies and experiments with transgenic mice have suggested that the severity of the bleeding phenotype in hemophilic patients is substantially reduced in association with impaired inactivation of factor (F) Va by activated protein C (APC) in the presence of the FV Leiden mutation. Experiments using a synthetic coagulation proteome model showed that the presence of FV Leiden significantly increased thrombin generation in the absence of FVIII or FIX. OBJECTIVE: To test the effect of APC inhibition on thrombin generation in hemophilia. METHODS: Prothrombinase and a synthetic coagulation proteome model of tissue factor-triggered thrombin generation were used. RESULTS: Peptide-based APC inhibitors, which mimic the P4-P4' residues surrounding the APC cleavage site at Arg306 of FVa, were synthesized. These compounds are specific and reversible inhibitors of APC, with Ki values as low as 1-2 microM; most have insignificant affinity for FXa or thrombin. The affinity for APC is dependent upon the location and character of the protecting groups. Representatives of this group of compounds inhibit FVa inactivation by APC and prolong FVa functional activity in the prothrombinase complex. When evaluated in a synthetic coagulation proteome model, one inhibitor partially compensated for the absence of FVIII. CONCLUSIONS: Synthetic APC inhibitors may be useful as adjuvants for hemophilia treatment.

Factor V Leiden: a genetic risk factor for thrombotic microangiopathy in patients with normal von Willebrand factor-cleaving protease activity.

Thrombotic microangiopathy (TM) is associated with abnormalities of von Willebrand factor-cleaving protease (VWCP) and other hemostatic factors. This study hypothesized that TM patients might have genetically determined thrombotic risk factors that predispose them to aberrant microvascular thrombosis. DNA samples from 30 white and 12 African American adult TM patients were analyzed for genetic alleles associated with vascular thrombosis, and plasma samples were analyzed for levels of VWCP activity. DNA was analyzed by using allele-specific polymerase chain reaction for factor V 1691A (Leiden), factor II 20 210A, methylenetetrahydrofolate reductase 667T, type 1 plasminogen activator inhibitor 4G/5G, and platelet GPIa 807T. Patients were segregated by race (white or African American) and plasma level of VWCP activity (normal or deficient). The prevalence of factor V Leiden was significantly increased among the white TM patients that had normal VWCP activity: 4 (36%) of 11 patients compared with 6 (3%) of 186 white control subjects possessed the factor V Leiden allele (P <.001; odds ratio, 17.1; 95% confidence interval, 5.4-54.0). No factor V Leiden alleles were detected in 19 white TM patients with intermediate or deficient levels of VWCP activity or in any of 12 African American patients. The prevalence of other thrombosis-associated alleles did not differ between TM patients and control subjects. These findings suggest that factor V Leiden may be a pathogenic risk factor in TM patients that have normal VWCP activity.

Tracking structural features leading to resistance of activated protein C to alpha 1-antitrypsin.

Activated protein C (APC) is a multi-modular anticoagulant serine protease, which degrades factor V/Va and factor VIIIa. Human APC (hAPC) is inhibited by human alpha 1-antitrypsin (AAT), while the bovine enzyme (bAPC) is fully resistant to this serpin. Structural features in the catalytic domains between the two species cause this difference, but detailed knowledge about the causal molecular difference is missing. To gain insight into the APC-AAT interaction and to create a human protein C resistant to AAT inhibition, we have used molecular modeling and site-directed mutagenesis. First, a structural model for bAPC based on the Gla-domainless X-ray structure of hAPC was built. Screening the molecular surface of the human and bovine APC enzymes suggested that a hAPC molecule resistant to AAT inhibition could be constructed by substituting only a few amino acids. We thus produced recombinant hAPC molecules with a single mutation (S173E, the numbering follows the chymotrypsinogen nomenclature), two mutations (E60aS/S61R) or a combination of all these substitutions (E60aS/S61R/S173E). Amidolytic and anticoagulant activities of the three mutant APC molecules were similar to those of wild-type hAPC. Inhibition of wild-type hAPC by AAT was characterized by a second-order rate constant (k2) of 2.71 M-1 s-1. The amino acid substitution at position 173 (S173E mutant) led to partial resistance to AAT (k2 = 0.84 M-1 s-1). The E60aS/S61R mutant displayed mild resistance to AAT inhibition (k2 = 1.70 M-1 s-1), whereas the E60aS/S61R/S173E mutant was inefficiently inactivated by AAT (k2 = 0.40 M-1 s-1). Inhibition of recombinant APC molecules by the serpin protein C inhibitor (PCI) in the presence and absence of heparin was also investigated.