Blood clotting in minimally altered whole blood

The sequences of events regulating thrombin generation during tissue factor-initiated clotting in whole blood at 37 degrees C in which the contact 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, fibrinopeptide A (FPA), thrombin-antithrombin III (TAT) complex formation, and prothrombin fragment 1.2 (F1.2) were measured via commercially available enzyme-linked immunosorbent assays (ELISAs). In a typical experiment initiated with 40 pmol/L recombinant tissue factor, visual clot time (4.5 minutes), was preceded by significant cleavage of factor V resulting in 65% factor Va heavy-chain generation but only 10% light-chain formation. At this point, 50% of the platelet factor 4 is released, suggesting that half (approximately 700 pmol/L) of the platelet prothrombinase sites available have been generated. At clot time, approximately 15 nmol/L thrombin B-chain is present; however, analyses of FPA release demonstrate that only 15% of the thrombin is acting on fibrinogen. This thrombin is produced by the action of 7 pmol/L prothrombinase. The maximum rate of thrombin production is reached well after clot time and is consistent with the presence of approximately 150 pmol/L prothrombinase (at about 7 minutes). These results suggest that factor Xa is the limiting factor for thrombin generation. After 60 minutes, 75% of the initial prothrombin (1.24 mumol/L) is consumed yielding 400 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 concentration determined by ELISA. The incomplete cleavage of prothrombin indicates both the prothrombinase complex and the formation of prothrombinase are inhibited in the reaction. TAT complex measured by ELISA is almost equivalent to B-chain concentration, but sodium dodecyl sulfate stable thrombin-antithrombin III complexes are not observed until well after clot formation and are never equivalent to ELISA-TAT values. At the point of clot formation, 80% of the fibrinogen is depleted from the fluid phase, whereas only 35% to 45% of the FPA is released, suggesting a significant incorporation of uncleaved fibrinogen into the initial clot formed.