The subunit structure of thrombin-activated factor V. Isolation of activated factor V, separation of subunits, and reconstitution of biological activity

Abstract

Activated Factor V (Va) was prepared by treating a high molecular weight form of Factor V with thrombin. The activated Factor V was isolated by ion exchange chromatography and was composed of two polypeptide chains (Mr = 115,000 and 73,000). These chains were separated by ion exchange chromatography in the presence of EDTA. Biologically active Factor Va was restored from the inactive chains by incubation of the two chains in buffers containing MnCl2. Restoration of biological activity was correlated with formation of a complex between the chains as monitored by either disc gel electrophoresis or gel filtration chromatography. The apparent molecular weight of the activated Factor V was 290,000. Factor V was not dissociated in EDTA. However, this protein was split by thrombin to yield an activation intermediate composed of two chains (Mr = 210,000 and 115,000). Like activated Factor V, the two chains of the intermediate can be dissociated in EDTA and separated by gel filtration chromatography. The Factor V activity was restored by incubation of the two inactive chains in buffers containing MnCl2. Like Factor Va, restoration of the biological activity corresponds to formation of a complex between the chains with a higher molcular weight than either of the isolated chains. Incubation of the activation intermediate with thrombin increased the specific activity 3- to 4-fold. This increase in specific activity resulted from cleavage of the heavy chain of the Factor V intermediate by thrombin.