Structure, Biology, and Genetics of Factor VIII. Factor VIII Function, страница 6

Numerous studies correlated the appearance of 90,000, 50,000, 43,000, and 73,000 MW polypeptides with peak factor VIII activity. MLID83154239 MLID86216230  109,112,113 Mutagenesis studies showed that cleavages after residues 740 and 1,648 were not required for cofactor activity. MLID88190085  114 By contrast, mutation at either Arg 372 or Arg 1,689 yielded molecules that were not cleaved by thrombin at the mutated site and were not susceptible to thrombin activation. MLID88190085 MLID89318011  114,115 Resistance to thrombin cleavage at one site did not alter susceptibility to thrombin cleavage at the other cleavage sites. The importance of cleavage at residues 372 and 1,689 for activation of factor VIII was also elucidated when missense mutations were identified in hemophilia A patients at either residue 372 or residue 1,689. MLID88327107 MLID89274393  116,117 In contrast to most hemophilia A patients, these patients have normal levels of circulating factor VIII antigen but no detectable factor VIII activity. These findings indicate that activation requires cleavage at both residues 372 and 1,689, but does not appear to require a specific sequential order for cleavage at these sites. Cleavage at 1,689 releases factor VIII from the inhibitory influence of vWF and accounts for a portion of the increase in factor VIII activity. MLID89367278  118 However, cleavage at 1,689 appears additionally to increase the activity of factor VIII in the absence of vWF. MLID92235250  119

The B domain, delimited by amino acid residues 740 and 1,648, is cleaved from factor VIII during and activation. Comparison of the deduced amino acid sequence of porcine, murine, and human factor VIII showed a striking divergence within the B domains, whereas the bordering A2 and A3 domains exhibit 80–85% homology. MLID85061550 MLID93300511  18,26 Factor VIII molecules constructed to lack most or all of the B domain have specific activities, thrombin cleavage products (except for the B domain), and thrombin activation coefficients similar to the wild-type molecule. MLID86287369  120,121 The B domain deletion molecules exhibit no detectable difference in vWF binding, survival in plasma, and ability to normalize the cuticle bleeding time after infusion into a factor VIII-deficient dog, compared with the wild-type factor VIII. MLID93271477  54 By these analyses, removal of the B domain did not affect in vitro or in vivo procoagulant activity. One notable difference between wild-type and B-domain deleted factor VIII is that the B domain deleted molecule is expressed at 5–10-fold higher levels, exhibits a reduced association with BiP in the endoplasmic reticulum, and is secreted more efficiently. MLID88087401 MLID93271477  36,54 This suggests that the B domain may regulate factor VIII biosynthesis. It is also possible that the B domain may have procoagulant, anticoagulant, or vasoactive properties heretofore unknown. For example, a portion of the B domains of factor V and factor VIII can serve as a substrate for transglutaminase activity of factor XIII. MLID86278011  122,123

Inactivation of Factor VIII

The activity of thrombin-activated factor VIII requires all polypeptides of the heterotrimer composed of the 50,000, 43,000, and 73,000 MW species. MLID89229064 MLID91224994  124–126 The 53,000-MW A1 domain is in a metal ion-dependent association with the 73,000-MW light chain. The 43,000-MW A2 domain polypeptide is associated with the A1 domain and 73,000-MW light chain by electrostatic interactions that likely involve residues 336–372 between the A1 and A2 domains. MLID92317036 MLID93352596  127,128 A detailed characterization of thrombin-activated factor VIIIa was hampered due to its marked instability. Protein concentration and pH are important factors for isolation of a stable thrombin-activated factor VIIIa. MLID90110237  129 Decay of factor VIII activity after thrombin activation in vitro does not correlate with any specific proteolytic event. MLID81110682  130,131 Loss of procoagulant activity in vitro is due to reversible dissociation of the 43,000-kd A2 domain polypeptide from the heterotrimer that occurs at physiologic pH. MLID92317036 MLID91286275  127,132 The specific activity of porcine factor VIII is approximately fivefold greater than human factor VIIIa, which correlates with a lower Kd of the A2 domain polypeptide compared with the thrombin-activated heterotrimer. MLID91286275 MLID93054719  132,133 Once activated by thrombin, factor VIIIa is stabilized by the addition of factor X and phospholipid. MLID84204051  134