The activation of factor V by factor Xa or alpha-chymotrypsin and comparison with thrombin and RVV-V action. An improved factor V isolation procedure.
Bovine plasma factor V has been isolated by a preparative procedure involving barium sulfate adsorption, QAEC 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 molecular weight 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 are inconclusive with regard to a possible subunit structure for this factor. The purified factor V was stable for at least 1-2 weeks when stored at 4 degrees C in 0.2 M Tris-acetate, 50 mM CaCl2, 10% glycerol, pH 7.5. When stored at -20 degrees C in 50% glycerol, this preparation was stable for several months. Treatment of the purified factor V with bovine factor Xa, RVV-V, thrombin, or chymotrypsin (but not trypsin) led to a seven- to ten-fold increase in clotting activity and a concomitant decrease in apparent molecular weight. The latter was comparable for each activation system yielding the following average molecular weight values: factor VaSa, 246,000-, factor Va RVV-V, 251,500; Factor Vathr, 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 phosphatidylcholine/phosphatidylserine (1:1, w/w), the activation of factor V by factor Xa plus Ca2+ required one-third 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.
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002417:Cattle; D002918:Chymotrypsin / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D005165:Factor V / Q000302:isolation & purification / Q000378:metabolism*; D005170:Factor X / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D010713:Phosphatidylcholines / Q000494:pharmacology; D010718:Phosphatidylserines / Q000494:pharmacology; D012910:Snake Venoms; D013917:Thrombin / Q000378:metabolism
D010713:Phosphatidylcholines; D010718:Phosphatidylserines; D012910:Snake Venoms; D005165:Factor V; D005170:Factor X; D002918:Chymotrypsin; D013917:Thrombin; D002118:Calcium