Stabilization of thrombin-activated porcine factor VIII:C by factor IXa phospholipid.
The activation of porcine factor X by an enzymatic complex consisting of activated factor IX (factor IXa), thrombin-activated factor VIII:C (factor VIII:Ca), phospholipid vesicles, and calcium was studied in the presence of an irreversible inhibitor of factor Xa, 5-dimethylamino-naphthalene-1-sulfonyl-glutamyl-glycyl-arginyl- chloro met hyl ketone ( DEGR -CK). The formation of factor Xa was measured continuously by monitoring the increase in solution fluorescence intensity that occurs upon formation of DEGR -factor Xa. Omission of any component from the enzymatic complex reduced the reaction rate to a negligible level. In the presence of fixed excess factor IXa, the velocity of factor X activation was linearly dependent on the concentration of factor VIII:C, and thus, provided a plasma-free assay of factor VIII:C. Activation of factor VIII:C by 0.1 NIH U/ml thrombin in the presence of factor IXa, phospholipid vesicles, and calcium, followed at variable time intervals by the addition of factor X and DEGR -CK, was complete within 5 min, as judged by the fluorometric assay, and resulted in little or no loss of factor VIII:C activity over a period of 20 min; whereas, activation in the absence of either IXa or phospholipid vesicles decreased the half-life of factor VIII:C to approximately 5 min. Analysis of 125I-factor VIII:C-derived activation peptides by sodium dodecyl sulfate polyacrylamide gel radioelectrophoresis revealed identical results, regardless of whether factor IXa and/or phospholipid vesicles were included in the activation, suggesting that the lability of factor VIII:Ca is not due to a major alteration of its primary structure. We conclude that the activated porcine factor VIII:C molecule is stabilized markedly because of its interaction with factor IXa and phospholipid.
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D000941:Antigens / Q000502:physiology*; D003619:Dansyl Compounds; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D005164:Factor IX / Q000502:physiology*; D015949:Factor IXa; D005169:Factor VIII / Q000276:immunology* / Q000502:physiology; D005170:Factor X / Q000037:antagonists & inhibitors; D007457:Iodine Radioisotopes; D010713:Phosphatidylcholines / Q000494:pharmacology; D010718:Phosphatidylserines / Q000494:pharmacology; D010743:Phospholipids / Q000502:physiology*; D012967:Sodium Dodecyl Sulfate; D013552:Swine; D013917:Thrombin / Q000502:physiology
D000590:Amino Acid Chloromethyl Ketones; D000941:Antigens; D003619:Dansyl Compounds; D007457:Iodine Radioisotopes; D010713:Phosphatidylcholines; D010718:Phosphatidylserines; D010743:Phospholipids; D012967:Sodium Dodecyl Sulfate; C030824:dansylglutamyl-glycyl-arginine chloromethyl ketone; D005169:Factor VIII; D005164:Factor IX; D005170:Factor X; D015949:Factor IXa; D013917:Thrombin