EVIDENCE THAT MEIZOTHROMBIN IS AN INTERMEDIATE PRODUCT IN THE CLOTTING OF WHOLE-BLOOD

Meizothrombin is an intermediate that is produced during the conversio n of prothrombin to thrombin in systems composed of purified factor Xa and factor Va that are quantitatively assembled on an anionic phospho lipid surface. The biological significance of this intermediate has re cently been challenged by the apparent absence of meizothrombin during clotting of sodium citrate-anticoagulated plasma. We analyzed the for mation of thrombin during coagulation of nonanticoagulated, unchilled, minimally manipulated whole blood in glass tubes. The process was sto pped at 0, 3, 5, and 7 minutes by the addition of biotinylated peptidy l chloromethylketone active-site labeling reagents. Plasma/serum was s eparated by centrifugation, and labeled species were extracted by immu noadsorption with a polyclonal anti-prothrombin antibody. The purified prothrombin-derived species were separated by SDS-polyacrylamide grad ient gel electrophoresis and visualized on a chemiluminescent avidin b lot. Meizothrombin appeared as an intermediate product of this reactio n and persisted with some increase through the it-minute time point. W e also observed incorporation of the active-site label into a species of lower molecular weight consistent with the B-1, chain of beta- and/ or gamma-thrombin. These degraded forms of thrombin have not been prev iously demonstrated in a biologically relevant preparation. Our data c learly establish the generation of meizothrombin as an intermediate pr oduct of thrombin generation during whole-blood clotting. The data als o represent the first experimental evidence for the generation of beta - and gamma-thrombin in a biologically relevant environment and time s cale.