BLOOD-CLOTTING IN MINIMALLY ALTERED WHOLE-BLOOD

The sequences of events regulating thrombin generation during tissue f actor-initiated clotting in whole blood at 37 degrees C in which the c ontact pathway was suppressed with corn trypsin inhibitor are studied using quantitative Western blotting of factor V, prothrombin, platelet factor 4, antithrombin III, and fibrinogen. In addition, fibrinopepti de A (FPA), thrombin-antithrombin III (TAT) complex formation, and pro thrombin fragment 1.2 (F1.2) were measured via commercially available enzyme-linked immunosorbent assays (ELISAs). In a typical experiment i nitiated with 40 pmol/l recombinant tissue factor, visual clot time (4 .5 minutes), was preceded by significant cleavage of factor V resultin g in 65% factor Va heavy-chain generation but only 10% light-chain for mation. At this point, 50% of the platelet factor 4 is released, sugge sting that half (approximately 700 pmol/L) of the platelet prothrombin ase sites available have been generated. At clot time, approximately 1 5 nmol/L thrombin B-chain is present; however, analyses of FPA release demonstrate that only 15% of the thrombin is acting on fibrinogen. Th is thrombin is produced by the action of 7 pmol/L prothrombinase. The maximum rate of thrombin production is reached well after clot time an d is consistent with the presence of approximately 150 pmol/L prothrom binase (at about 7 minutes). These results suggest that factor Xa is t he limiting factor for thrombin generation. After 60 minutes, 75% of t he initial prothrombin (1.24 mu mol/L) is consumed yielding 440 nmol/L prethrombin 2 and 360 nmol/L thrombin (B-chain) products. The sum of these values (800 nmol/L) is similar to the (corrected) F1.2 concentra tion determined by ELISA. The incomplete cleavage of prothrombin indic ates both the prothrombinase complex and the formation of prothrombina se are inhibited in the reaction. TAT complex measured by ELISA is alm ost equivalent to B-chain concentration, but sodium dodecyl sulfate st able thrombin-antithrombin III complexes are not observed until well a fter clot formation and are never equivalent to ELISA-TAT values. At t he point of clot formation, 80% of the fibrinogen is depleted from the fluid phase, whereas only 35% to 45% of the FPA is released, suggesti ng a significant incorporation of uncleaved fibrinogen into the initia l clot formed. (C) 1996 by The American Society of Hematology.