Characterization of Factor V activation intermediates

Abstract

Bovine Factor V was partially activated with bovine beta-thrombin and activation intermediates, and end products were isolated either by column chromatography under nondenaturing conditions or electroelution from slab gels following electrophoresis in dodecyl sulfate or both. Electrophoresis of partially activated single-chain Factor V (Mr = 330,000) revealed intermediates designated B (Mr congruent to 205,000) and C (Mr congruent to 150,000), plus end products designated C1 (Mr congruent to 120,000), D (Mr congruent to 94,000), E (Mr congruent to 74,000), F (Mr congruent to 71,000), and G (Mr congruent to 31,000). All components except C1 were visualized readily by staining with Coomassie blue. C1, however, did not stain with this dye but was readily visualized with the Schiff-periodate or silver staining procedures. Chromatography of samples of partially activated Factor V on QAE (quaternary aminoethyl)-cellulose in Ca2+ yielded fractions consisting, respectively, of B plus C and B plus D. Both were biologically active, but the former pair required further exposure to thrombin to yield activity, whereas the latter did not. Either pair, when treated with EDTA, lost activity and could be resolved by further chromatography on QAE-cellulose into isolated components B, C, and D. Activity was recovered upon reconstitution of the pairs in Ca2+, suggesting that B plus C or B plus D comprise two subunit proteins, both subunits of which are required for biological activity. Gel electrophoretic analysis of isolated B and C, after further exposure to thrombin, indicated that B is the precursor of end products C1, E, and G, whereas C is the precursor of D and F. Conventional NH2-terminal sequence analysis and amino acid composition analysis indicated that C and D share the same NH2 terminus with Factor V and that the weighted composition of B plus C is the same as Factor V. Microsequence analysis of C1 and E indicated that C1 has the NH2-terminal sequence of intermediate B, which differs from that of end product E. These results indicate that: 1) a single cleavage of Factor V yields B and C from the COOH and NH2 termini, respectively, of the parent; 2) the pair B and C constitute a Ca2+-stabilized protein of two subunits, both of which are required for subsequent expression of biological activity; 3) the precursor-product relationship between B and C and the end products of activation are: B----C1 + E + G, and C----D + F; and 4) end product D is derived from the NH2 terminus of C, and end product C1 is derived from the NH2 terminus of B.