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J. Biol. Chem., Vol. 282, Issue 8, 5287-5295, February 23, 2007

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Mechanisms of Plasmin-catalyzed Inactivation of Factor VIII

A CRUCIAL ROLE FOR PROTEOLYTIC CLEAVAGE AT Arg³³⁶ RESPONSIBLE FOR PLASMIN-CATALYZED FACTOR VIII INACTIVATION^*

From the Department of Pediatrics, Nara Medical University, Kashihara, Nara 634-8522, Japan

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 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 12- and 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 Lys³⁶, Arg³³⁶, Arg³⁷², and Arg⁷⁴⁰. The 80-kDa light chain was converted into a 67-kDa subunit by cleavage at Arg¹⁶⁸⁹ and Arg¹⁷²¹, identical to the pattern induced by factor Xa. Plasmin-catalyzed cleavage at Arg³³⁶ proceeded faster than that at Arg³⁷², 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 Arg³³⁶ and Lys³⁶ occurred rapidly in the presence of A2 and A3-C1-C2 subunits, respectively. These results strongly indicated that cleavage at Arg³³⁶ was a central mechanism of plasmin-catalyzed factor VIII inactivation. Furthermore, the cleavages at Arg³³⁶ and Lys³⁶ appeared to be selectively regulated by the A2 and A3-C1-C2 domains, respectively, interacting with plasmin.

Received for publication, August 16, 2006 , and in revised form, November 30, 2006.

^* This work was supported in part by MEXT KAKENHI Grants 17390304 and 17591110 and by the Ministry of Health, Labor and Welfare of Japan for AIDS Research. A preliminary account of this work was presented at the 47th Annual Meeting of the American Society of Hematology, December 10, 2005, Atlanta, GA. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Pediatrics, Nara Medical University, 840 Shijo-cho, Kashihara City, Nara 634-8522, Japan. Tel.: 81-744-29-8881; Fax.: 81-744-24-9222; E-mail: mshima{at}naramed-u.ac.jp.

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				K. Nogami, K. Nishiya, E. L. Saenko, M. Takeyama, K. Ogiwara, A. Yoshioka, and M. Shima Identification of Plasmin-interactive Sites in the Light Chain of Factor VIII Responsible for Proteolytic Cleavage at Lys36 J. Biol. Chem., March 13, 2009; 284(11): 6934 - 6945. [Abstract] [Full Text] [PDF]