Factor VIIIa Decay Due to A2 Subunit Dissociation Is Regulated by the A1 Domain.

Abstract

FVIII contains three homologous A domains, two homologous C domains and a single B domain. The B domain and an activation peptide between the B and A3 domains are released during the activation of fVIII by thrombin. The resulting fVIIIa molecule has a heterotrimeric A1/A2/A3-C1–C2 subunit structure. FVIIIa is unstable at physiological concentrations and pH due to dissociation of the A2 subunit from the A1/A3-C1–C2 dimer, which is three-fold faster in human fVIIIa than porcine fVIIIa. Fine regulation of fVIII activity appears to be important because plasma fVIII levels are positively correlated with a risk of arterial and/or venous thrombosis. Additionally, mutations in the fVIII gene have been identified that result in fast A2 dissociation and a hemophilia phenotype. To characterize structural determinants involved in A2 subunit dissociation, we constructed and purified recombinant human, porcine and hybrid human/porcine fVIII molecules corresponding to all eight combinations of human and porcine fVIII A1–A2–A3 domains (HHH, HHP, HPH, HPP, PHH, PHP, PPH and PPP). All of the constructs contained the human C1 and C2 domains. The rate of A2 subunit dissociation was studied at pH 7.4 by measuring decay of fVIIIa activity in an assay system using purified intrinsic fXase components and displayed first-order kinetics in all cases. Constructs containing the porcine A1 domain behaved similarly to porcine fVIIIa (Table 1). In contrast, constructs containing the human A1 domain behaved similarly to human fVIII, except for the HPH construct, which displayed intermediate behavior.Table 1A domain hybrid fVIIIIa decay ratesConstructk (min− 1)aHHP0.317 ± 0.018HHH (human)0.275 ± 0.013HPP0.262 ± 0.011HPH0.193 ± 0.012PPH0.128 ± 0.005PHP0.118 ± 0.003PPP (porcine)0.109 ± 0.003PHH0.105 ± 0.002a mean and estimated s.d. from nonlinear least-squares analysisThe fVIII A domains contain two azurin-like subdomains. Additionally, the A1 domain contains a COOH-terminal 37 amino acid acidic region. To further characterize the involvement of the A1 domain in fVIIIa decay, we prepared constructs that contained the combinations hhp, phh, pph, hpp or hph of the three A1 subdomains in an otherwise human background. Comparison of their fVIIIa decay rates indicated that the acidic region is not involved in A2 dissociation (Table 2, cf. hhp versus pph). Substitution of either azurin-like porcine subdomain produced relatively stable fVIIIa molecules.Table 1A1 subdomain hybrid fVIIIIa decay ratesConstructk (min− 1)ahhp0.263 ± 0.012phh0.167 ± 0.005pph0.157 ± 0.008hpp0.133 ± 0.005hph0.134 ± 0.010a mean and estimated s.d. from nonlinear least-squares analysisThese results indicate that porcine-specific amino acid side chains in both azurin-like A1 subdomains are responsible for differential fVIIIa decay rates. A1 domain mutations may represent adaptations to differences in hemostatic requirements between species.