Formation of thrombin is triggered when membrane-localized tissue factor (TF) is exposed to blood. In closed models of this process, thrombin formation displays an initiation phase (low rates of thrombin production cause platelet activation and fibrinogen clotting), a propagation phase (>95% of thrombin production occurs), and a termination phase (prothrombin activation ceases and free thrombin is inactivated). A current controversy centers on whether the TF stimulus requires supplementation from a circulating pool of blood TF to sustain an adequate procoagulant response. We have evaluated the requirement for TF during the progress of the blood coagulation reaction and have extended these analyses to assess the requirement for TF during resupply ("flow replacement"). Elimination of TF activity at various times during the initiation phase indicated: a period of absolute dependence (<10 s); a transitional period in which the dependence on TF is partial and decreases as the reaction proceeds (10 -240 s); and a period in which the progress of the reaction is TF independent (>240 s). Resupply of reactions late during the termination phase with fresh reactants, but no TF, yielded immediate bursts of thrombin formation similar in magnitude to the original propagation phases. Our data show that independence from the initial TF stimulus is achieved by the onset of the propagation phase and that the ensemble of coagulation products and intermediates that yield this TF independence maintain their prothrombin activating potential for considerable time. These observations support the hypothesis that the transient, localized expression of TF is sufficient to sustain a TF-independent procoagulant response as long as flow persists.
Tissue factor (TF)2 is a 263-amino acid glycoprotein with three major domains: 1) an extracellular domain (residues 1-219) that binds with high affinity to factor VIIa; 2) a transmembrane domain (residues 220 -242) that anchors TF to the membrane surface; and 3) a cytoplasmic domain (residues 243-263) (1-3). Binding of plasma factor VIIa to membrane-bound TF results in an ϳ2 ϫ 10 7 -fold increase in the enzymatic activity of factor VIIa toward its natural substrates factor IX and factor X (4). Most studies have concluded that membrane-bound TF, expressed by inflammatory cells and cells outside the vasculature, are the key initiators of the blood coagulation process (5-7).The generation of thrombin, the enzyme responsible for clot formation as well as other procoagulant and anticoagulant functions during the blood coagulation process occurs in a nonlinear fashion (8). During an initiation phase, tiny amounts of thrombin are generated, platelets, zymogens, and procofactors are activated, and complex enzymes assembled (9, 10). Subsequently, a propagation phase of thrombin generation occurs, characterized by dramatic increases in both the rate of thrombin generation and levels of thrombin. The duration of the initiation phase, which roughly corresponds to the clotting time of blood and plasma, is predomi...