Mechanisms of plasmin-catalyzed inactivation of factor VIII: a crucial role for proteolytic cleavage at Arg336 responsible for plasmin-catalyzed factor VIII inactivation.
Plasmin not only functions as a key enzyme in the fibrinolytic system but also directly inactivates factor VIII and other clotting factors such as factor V. However, the mechanisms of plasmin-catalyzed factor VIII inactivation are poorly understood. In this study, levels of factor VIII activity increased approximately 2-fold within 3 min in the presence of plasmin, and subsequently decreased to undetectable levels within 45 min. This time-dependent reaction was not affected by von Willebrand factor and phospholipid. The rate constant of plasmin-catalyzed factor VIIIa inactivation was approximately 12- and approximately 3.7-fold greater than those mediated by factor Xa and activated protein C, respectively. SDS-PAGE analysis showed that plasmin cleaved the heavy chain of factor VIII into two terminal products, A1(37-336) and A2 subunits, by limited proteolysis at Lys(36), Arg(336), Arg(372), and Arg(740). The 80-kDa light chain was converted into a 67-kDa subunit by cleavage at Arg(1689) and Arg(1721), identical to the pattern induced by factor Xa. Plasmin-catalyzed cleavage at Arg(336) proceeded faster than that at Arg(372), in contrast to proteolysis by factor Xa. Furthermore, breakdown was faster than that in the presence of activated protein C, consistent with rapid inactivation of factor VIII. The cleavages at Arg(336) and Lys(36) occurred rapidly in the presence of A2 and A3-C1-C2 subunits, respectively. These results strongly indicated that cleavage at Arg(336) was a central mechanism of plasmin-catalyzed factor VIII inactivation. Furthermore, the cleavages at Arg(336) and Lys(36) appeared to be selectively regulated by the A2 and A3-C1-C2 domains, respectively, interacting with plasmin.
Nogami
Keiji
K
Department of Pediatrics, Nara Medical University, Kashihara, Nara 634-8522, Japan.
Shima
Midori
M
Matsumoto
Tomoko
T
Nishiya
Katsumi
K
Tanaka
Ichiro
I
Yoshioka
Akira
A
D001120:Arginine / Q000737:chemistry / Q000378:metabolism; D002384:Catalysis; D004789:Enzyme Activation; D005165:Factor V / Q000737:chemistry / Q000378:metabolism; D005169:Factor VIII / Q000737:chemistry / Q000378:metabolism*; D005341:Fibrinolysin / Q000737:chemistry / Q000378:metabolism*; D005342:Fibrinolysis / Q000502:physiology*; D006801:Humans; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D013997:Time Factors
D021122:Protein Subunits; D005165:Factor V; D005169:Factor VIII; D001120:Arginine; D005341:Fibrinolysin