Structural and functional studies of factor V in health and disease

Sammanfattning: The homologous blood coagulation factors V (FV) and factor VIII (FVIII) are important at sites of vascular injury for the amplification of the clotting cascade. Activated FV (FVa) serves as a cofactor to the enzyme activated factor X (FXa) in the activation of prothrombin. This complex is called the prothrombinase complex. FVIII functions as a cofactor to the enzyme activated factor IX (FIXa) in the activation of factor X (FX), a complex referred to as the tenase complex. To prevent uncontrolled coagulation, the anticoagulant protein C system inactivates the cofactors FV and FVIII. Recently, it was shown that FV possesses anticoagulant properties in addition to its procoagulant function, by serving as a cofactor to the enzyme activated protein C (APC) in the down regulation of FVIIIa. As FV has a dual role in coagulation, being important in both procoagulant- and anticoagulant systems, disorders of the FV molecule can cause both bleeding tendencies and hypercoagulable states. The research presented in this thesis is based on structural and functional studies of FV under both normal and pathological conditions. The first papers (papers I-V) deal with the formation and down regulation of the prothrombinase complex, with special reference to the FVa:FXa interaction. We show that the FV activation is associated with a stepwise release of the B domain, which results in a gradual exposure of the FXa-binding site. The FXa-binding site on FVa was then outlined by targeted glycosylation to probe molecular regions involved in the FVa-FXa interaction. The FVa:FXa interaction was further defined by replacing potentially interesting residues with alanines. The last papers (papers VI-VII) focus on the elucidation of the molecular mechanisms behind two naturally occurring mutations of FV; FV New Brunswick and FV Liverpool. We show that the New Brunswick FV variant is associated with reduced stability. This finding might explain the bleeding disorder of the affected patients. The FV Liverpool mutation induces an extra sugar group in FV that appears to interfere with the two anticoagulant proteins APC and protein S. These observations can well explain the association of the Liverpool mutation with thrombosis. In the background of the thesis the biochemistry and biology of FV is described in parallel with that of FVIII as the cofactors are highly homologous both structurally and functionally.