Activation of human prothrombin by highly purified human factors V and X-a in presence of human antithrombin.

In this communication we describe the first method for isolating human Factor V. The final product contains no other coagulation components as judged by functional assays and is physically homogeneous as shown by isofocusing gel electrophoresis. In addition, we present a means for obtaining intrinsically activated human Factor X-a. This preparation is usually homogeneous as judged by isofocusing gel electrophoresis. However, on occasion, an additional minor electrophoretic species with Factor X-a activity is observed. Furthermore, we describe the use of isoelectric focusing in sucrose density gradients to free human prothrombin from contamination by coagulation factors and other components. These homogeneous human proteins are employed to examine the conversion of prothrombin to thrombin in the presence and absence of human antithrombin. The latter component is responsible for virtually all of the plasm's capacity to neutralize Factor X-a and thrombin. In the absence of antithrombin, prothrombin (67,800) is converted to the precursor P-2 (51,600) and the fragment F-a (19,500). Subsquently, P-2 is cleaved to form the precursor P-3 (37,000), and the fragment F-b (11,500). Finally, P3 IS proteolyzed to form the heavy chain T-h (29,500) and the light chain T-L (6,500) of active thrombin. In the presence of antithrombin, an additional prothrombin conversion pathway is observed in which the zymogen is directly cleaved to form P-3 and F-A + B (30,000) prior to thrombin generation. Trace amounts of free thrombin remain uninhibited by antithrombin and could bias the zymogen activation pathway. Hirudin is known to neutralized thrombin instantaneoulsly. We demonstrate that the purified leech protein also binds to P-3 and prevents thrombin formation. When hirudin is added to activation mixtures at concentrations sufficient to virtually suppress P-3 conversion to thrombin, molecular species from both activation pathways are observed. Thus two human prothrombin conversion sequences appear to be initiated by Factor X-3 and may be of physiological significance.