KINETICS OF FACTOR-VIII LIGHT-CHAIN CLEAVAGE BY THROMBIN AND FACTOR XA - A REGULATORY ROLE OF THE FACTOR-VIII HEAVY-CHAIN REGION LYS713-ARG740

Activation and limited proteolysis of factor VIII have been investigat ed with respect to the role of the heavy-chain region Lys713-Arg740. T he kinetics of factor VIII activation have been analyzed in a system c onsisting of human factor VIII, factor IXa, factor X, phospholipids, a nd thrombin or factor Xa. Plasma-derived factor VIII is activated by t hrombin with a second-order rate constant of 3.3+/-0.3X10(6) M(-1) s(- 1), which proved to be slightly higher than for activation by factor X a. The second-order rate constant of activation by thrombin of plasma- derived factor VIII in the presence of a monoclonal antibody against t he sequence Lys713-Arg740 is markedly reduced. The same result was obt ained for activation by thrombin and factor Xa of factor VIII with a d eletion including the sequence Lys713-Arg740, des-(713-1637)-factor VI II. This suggests that the region Lys713-Arg740 promotes factor VIII a ctivation by both thrombin and factor Xa. Since factor VIII activation is associated with proteolysis, cleavage of factor VIII heavy and lig ht chains was analyzed quantitatively. These studies indicated that he avy-chain cleavage of des-(713-1637)-factor VIII is similar to that of plasma-derived factor VIII. In contrast, cleavage of the light chain of des-(713-1637)-factor VIII is clearly reduced. Furthermore, the sec ond-order rate constant (0.2+/-0.1X10(6) M(-1) s(-1)) of des-(713-1637 )-factor VIII light-chain cleavage by thrombin was reduced tenfold com pared with that of plasma-derived factor VIII. Proteolysis by factor X a yielded similar results. The rate of des-(713-1637)-factor VIII ligh t-chain cleavage by thrombin is similar to that of isolated light-chai n, but isolated light-chain is cleaved by factor Xa 20-fold more effic iently than the light chain in des-(713-1637)-factor VIII. We conclude that activation of factor VIII by both thrombin and factor Xa is clos ely associated with light-chain cleavage. Furthermore, within the fact or VIII heterodimer, the heavy-chain sequence Lys713-Arg740 promotes b oth activation and light-chain proteolysis.