Activation of bovine factor V by an activator purified from the venom of Naja naja oxiana.
The crude venom of many elapid snakes appeared to contain proteins that activated blood coagulation factor V. The factor V activator present in the venom of Naja naja oxiana was purified to homogeneity by chromatography on a mono-S column. The activator was a single chain protein with an apparent mol. wt of 48,000, as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and by gel permeation chromatography on Sephacryl S200. Activation of bovine factor V by the purified venom activator was accompanied by proteolytic cleavage of factor V and resulted in the formation of two major polypeptide chains with mol. wts of about 90,000 and 77,000. The final product obtained was compared with thrombin-activated factor V for its ability to function as cofactor in factor Xa-catalysed prothrombin activation in the presence of negatively charged phospholipid vesicles (5 mole% phosphatidylserine/95 mole% phosphatidylcholine). The Km for prothrombin obtained at a saturating amount of venom-activated factor Va was nine-fold higher than with thrombin-activated factor V (0.83 microM vs 0.09 microM, respectively) whereas both factor Va molecules stimulated the Vmax of thrombin formation some 6000-fold. Both forms of factor Va promoted the binding factor Xa to negatively charged phospholipid vesicles. However, the apparent Kd for factor Xa was less favorable in the presence of venom-activated factor V (0.67 x 10(-9) M) than in the presence of thrombin-activated factor V (0.043 x 10(-9) M). Thrombin cleaved a peptide bond in the 77,000 mol. wt polypeptide chain of venom-activated factor V, which resulted in the formation of a normal factor Va light chain. This peptide bond cleavage was, however, not associated with a change of cofactor activity. Venom treatment of thrombin-activated factor V, on the other hand, did remove a small fragment (mol. wt approximately 4000) from the heavy chain of factor Va (94,000), yielding a molecule with reduced cofactor activity. The diminished cofactor activity of venom-activated factor V is, therefore, likely due to the fact that a small peptide fragment, involved in the interaction with prothrombin and factor Xa, is missing from the heavy chain of venom-activated factor V.
Gerads
I
I
Department of Biochemistry, University of Limburg, Maastricht, The Netherlands.
Tans
G
G
Yukelson LYa
Zwaal
R F
RF
Rosing
J
J
D000818:Animals; D002417:Cattle; D004546:Elapid Venoms / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D005165:Factor V / Q000378:metabolism*; D015951:Factor Xa / Q000502:physiology; D007700:Kinetics; D008970:Molecular Weight; D010743:Phospholipids / Q000494:pharmacology; D011506:Proteins / Q000378:metabolism; D011516:Prothrombin / Q000502:physiology; D013917:Thrombin / Q000494:pharmacology
D004546:Elapid Venoms; D010743:Phospholipids; D011506:Proteins; D005165:Factor V; D011516:Prothrombin; D013917:Thrombin; D015951:Factor Xa