Thrombin-catalyzed activation of factor VIII with His substituted for Arg372 at the P1 site

Abstract

Thrombin-catalyzed proteolysis at Arg372 of factor VIII is essential for procofactor activation. However, hemophilia A patients with the missense mutation Arg372 to His possess a mild to moderate phenotype yet show no detectable cleavage at this bond. To evaluate this discrepancy, we prepared and stably expressed a recombinant, B-domainless factor VIII mutant (R372H) that possessed approximately 1% the specific activity of wild type. Cleavage at R372H by thrombin occurred with an approximately 80-fold decreased rate compared with wild type. N-terminal sequence analysis of the derived A2 subunit confirmed that cleavage occurred at the His372-Ser373 bond. Factor VIII R372H was activated slowly, attained lower activity levels, and exhibited an apparent reduced inactivation rate compared with factor VIII wild type. These observations were attributed to a reduced cleavage rate at His372. Factor Xa generation assays showed similar Michaelis-Menten constant (K(m), apparent) values for thrombin-catalyzed activation for either factor VIII form, but suggested an approximately 70-fold reduced maximum velocity (V(max)) for factor VIII R372H. However, prolonged reaction with thrombin yielded similar activity and stability values for the mutant and wild-type factor VIIIa forms. These results indicate a markedly reduced rate of cleavage following substitution at the P(1)Arg, and this property likely reflects the severity of the hemophilia A phenotype.

Figure 1.

Time course of cleavage of recombinant factor VIII by thrombin. Recombinant factor VIII wild type (WT) and R372H (100 nM) were reacted with thrombin (2.5 nM) for the indicated times as described in “Materials and methods.” Samples were run on 8% gel followed by Western blotting using an anti-A2 (A), anti-A1 (C), or anti–light chain (D) monoclonal antibody. Panel B shows quantitative densitometry of the ratio of A2 subunit/A1-A2 subunit from blotting data obtained from panel A. ○ indicates wild type; •, R372H. The data were fitted to a straight line. SC and LCh represent a single chain and the intact light chain, respectively.

Figure 2.

Time course of thrombin activation of recombinant factor VIII. Recombinant factor VIII wild type, R372H, and R372Q (50 nM) were reacted with thrombin (20 nM) for the indicated times, after which activation was terminated by hirudin and each sample was tested immediately for factor VIIIa activity in a one-stage clotting assay. The inset shows an expanded activation time course during the initial 10 minutes of the reaction. ○ indicates wild type; •, R372H; and □, R372Q. The zero point was taken prior to addition of thrombin.

Figure 3.

Time-dependent decay of factor Xa generating activity by factor Xase composed of R372H and wild-type factor VIIIa forms. Factor VIII (300 nM) was reacted with thrombin (50 nM) for 18 hours at 37°C. After the addition of hirudin and a 30-fold dilution of reactant, aliquots were removed at the indicated times and reacted with factor IXa (20 nM) and phospholipid vesicles (10 μM), and factor Xa generation was initiated with the addition of factor X (300 nM) as described in “Materials and methods.” ○ indicates wild type; •, R372H. The initial activity of factor Xa generated (100% level) for factor VIII wild type and R372H was 13.0 and 6.1 nM/min, respectively. Initial rates of factor Xa generation were plotted as a function of incubation time and fitted to a single exponential decay curve. Experiments were performed at least 3 separate times and average values are shown.

Figure 4.

Kinetics of recombinant factor VIII activation by thrombin. Variable amounts of factor VIII were reacted with thrombin (0.04 nM) for 1 minute. Thrombin was inactivated by addition of hirudin, and factor VIIIa was reacted with factor IXa (20 nM) in the presence of phospholipid vesicles (10 μM). Factor Xa generation was initiated by addition of factor X (300 nM) as described in “Materials and methods.” ○ indicates wild type; •, R372H. Initial rates of factor Xa generation are plotted as a function of factor VIII concentration and fitted to the Michaelis-Menten equation by nonlinear least squares regression. The inset shows the rate of factor Xa generation using factor VIII R372H activated by thrombin. All experiments were performed at least 3 separate times and average values are shown.

Figure 5.

Competition of thrombin activation of factor VIII wild type by factor VIII R372H. Mixtures of factor VIII wild type (10 nM) and variable concentrations of R372H were reacted with thrombin (0.04 nM) for 1 minute. After the addition of hirudin, factor VIIIa was reacted with factor IXa (20 nM) in the presence of phospholipid vesicles (10 μM). Factor Xa generation was initiated by addition of factor X (300 nM). Factor Xa generated in the absence of R372H (100% level) was approximately 160 nM/min. Data were corrected for the amount of factor Xa generated from factor VIII R372H. Initial rates of factor Xa generation were plotted as a function of R372H and fitted to a noncompetitive inhibition equation by nonlinear least squares regression. Experiments were performed at least 3 separate times and average values are shown.