Activated factor X cleaves factor VIII at arginine 562, limiting its cofactor efficiency.

BACKGROUND: Factor VIII (FVIII) and its activated form (FVIIIa) are subject to proteolysis that dampens their cofactor function. Among the proteases that attack FVIII (activated factor X (FXa), activated protein C (APC) and plasmin), only APC cleaves within the FVIII A2 domain at R562 to fully abolish FVIII activity.
OBJECTIVES: We investigated the possible involvement of the FXa cleavage at R562 within the A2 domain in the process of FVIII inactivation.
METHODS: An antibody (GMA012/R8B12) that recognizes the carboxy-terminus extremity of the A2 domain (A2C) was used to evaluate FXa action. A molecule mutated at R562 was also generated to assess the functional role of this particular residue. RESULTS AND
CONCLUSIONS: The appearance of the A2C domain as a function of time evidenced the identical cleavage within the A2 domain of FVIII and FVIIIa by FXa. This cleavage required phospholipids and occurred within minutes. In contrast, the isolated A2 domain was not cleaved by FXa. Von Willebrand factor and activated FIX inhibited the cleavage in a dose-dependent manner. Mutation R562K increased both the FVIII specific activity and the generation of FXa due to an increase in FVIII catalytic efficiency. Moreover, A2C fragment could not be identified from FVIII-R562K cleavage. In summary, this study defines a new mechanism for A2 domain-mediated FVIII degradation by FXa and implicates the bisecting of the A2 domain at R562.