Recombinant Factors for Hemostasis

Thrombin is the key serine protease in hemostasis because it is responsible for activating both procoagulant proteins critical for creating fibrin clots and anticoagulant proteins needed to return the system to balance. Prothrombin is converted to thrombin in primary hemostasis by activated Factor X (FXa) attached to the phospholipid membrane and secondary hemostasis by the prothrombinase complex which consists of FXa, activated Factor V (FVa), calcium ions complexed on the phospholipid surface of activated platelets. In the laboratory, prothrombin can be activated to thrombin in solutions containing high sodium citrate concentrations; however, the mechanism is not well understood. The work presented in this chapter focuses on identifying intermediates and fragments derived from prothrombin activation by sodium citrate using SDS-PAGE and N-terminal sequencing analyses. The activity of the citrate activated thrombin was analyzed by chromogenic assay and functionality evaluated by ability to cleave FXIIIa and activate fibrinogen. This research found that prothrombin could be activated in the absence of added activators. This activation was not due to autocatalytic degradation but is due to contaminating FX which alone or activated to FXa proteolyzed prothrombin. Incubating prothrombin in 25% or 35% sodium citrate also resulted in activation of prothrombin. Activation by citrate may be autocatalytic or a result of FX; however, FXa and thrombin did not play a role in citrate activation. Analysis of activation fragments indicates that the sequence of activation does not follow the primary or secondary hemostasis pathways; rather, it follows an alternative pathway with a prethrombin-1 intermediate that is subsequently proteolyzed into prethrombin-2, prethrombin-2a, thrombin, α-thrombin and β-thrombin. The activity of citrate activated thrombin was 368