The activation of factor V by factor Xa or α-chymotrypsin and comparison with thrombin and RVV-V action. An improved factor V isolation procedure

Abstract

Bovine plasma factor V was isolated by a preparative procedure involving barium sulfate adsorption, diethyl(2-hydroxypropyl)aminoethyl cellulose extraction, poly(ethylene glycol) precipitation and finally chromatography on a desulfated Sepharose 6B column. Factor V was recovered as a single peak in yields of 35-40% with a specific activity of 50-70 representing a purification of 1000-2000 fold relative to the starting plasma. The apparent MW of the purified factor V was 439,000 .+-. 5000. On sodium dodecyl sulfate gel and analytical gel electrophoresis, this factor V preparation showed multiple bands, but results were inconclusive with regard to a possible subunit structure for this factor. The purified factor V was stable for at least 1-2 wk when stored at 4.degree. C in 0.2 M Tris-acetate, 50 mM CaCl2, 10% glycerol, pH 7.5. When stored at -20.degree. C in 50% glycerol, this preparation was stable for several months. Treatment of the purified factor V with bovine factor Xa, RVV-V [factor V activator, of Russell''s viper venom], thrombin, or chymotrypsin (but not trypsin) led to a 7-10 X increase in clotting activity and a concomitant decrease in apparent MW. The latter was comparable for each activation system yielding the following average MW values: factor VaXa [factor V activated by factor Xa], 246 000; factor VaRVV-V, 251 500, Factor Vathr [factor V activated by thrombin] 239 000; .alpha.-chymotrypsin, but not trypsin, can activate plasma factor V yielding a product similar to that observed with the above activators. The molar quantities of each of the activators required varied considerably with thrombin having the highest specific activity and factor Xa the lowest. Activation by factor Xa was greatly facilitated by the addition of phospholipid. In the presence of a mixture of phosphatidycholine/phosphatidylserine (1:1, w/w), the activation of factor V by factor Xa plus Ca2+ required 1/3 the amount of factor Xa protein as that required in the absence of phospholipid. Even though each of these activators appears to act in an enzymatic manner, the chemical nature of the conversion is unknown at this time.