Regulation of Tissue Factor Initiated Thrombin Generation by the Stoichiometric Inhibitors Tissue Factor Pathway Inhibitor, Antithrombin-III, and Heparin Cofactor-II

Abstract

The effects of the stoichiometric inhibitors tissue factor pathway inhibitor (TFPI), antithrombin-III (AT-III) and heparin cofactor-II (HC-II) on thrombin generation were evaluated in a reaction system composed of coagulation factors VIIa, X, IX, VIII, and V and prothrombin initiated by tissue factor (TF) and phospholipids. Initiation of the reaction in the absence of inhibitors resulted in explosive thrombin generation for factor VIIa⅐TF concentrations varying from 100 to 0.25 pM with the lag time or initiation phase of thrombin generation increasing from 0 to 180 s with decreasing factor VIIa⅐TF concentrations. During the propagation phase, prothrombin is quantitatively activated to 1.4 M ␣-thrombin. At normal plasma concentration (2.5 nM) full-length recombinant TFPI prolonged the initiation phase of thrombin generation 2-fold, and the rate of thrombin generation in the propagation phase of the reaction was 25-50% that of the uninhibited reaction when the reaction was initiated with 1.25-20 pM factor VIIa⅐TF. Inhibition of the reaction by TFPI is associated with a delay in factor V activation. In the presence of TFPI no explosive thrombin generation was observed when factor VIII was omitted from reactions initiated by factor VIIa⅐TF concentrations <20 pM. This indicates that in the presence of TFPI the factor IXa⅐factor VIIIa pathway becomes essential at low factor VIIa⅐TF concentrations. In the reconstituted system, AT-III (3.4 M) did not prolong the initiation phase of thrombin generation when the reaction was initiated with 1.25 pM factor VIIa⅐TF, nor did AT-III delay factor V activation. The rate of thrombin formation in the presence of AT-III was reduced to 30% that of the uninhibited reaction, and the ␣-thrombin formed was rapidly inhibited subsequent to its generation. The addition of HC-II alone at its physiological concentration (1.38 M) to the procoagulant mixture did not alter the rate or extent of thrombin generation. Subsequently, the thrombin formed was slowly inhibited by HC-II. The slow inactivation of thrombin by HC-II does not contribute to thrombin inhibition in the presence of AT-III. In contrast, the combination of physiological levels of AT-III and TFPI inhibited explosive thrombin generation initiated by 1.25 pM factor VIIa⅐TF completely.

Factor V: a combination of Dr Jekyll and Mr Hyde

“…Although factor Xa-phospholipid, thrombin, and plasmin are all capable of activating factor V to factor Va, the kinetic evaluation of tissue factor-initiated blood clotting reactions shows that the initial activator of factor V is ␣-thrombin that is initially produced by factor Xa and phospholipid during the initiation phase of the blood coagulation process. 50,52,53,[55][56][57] Although factor Xa-phospholipid is an effective activator of factor V, the concentrations of factor Xa, which exist during the initiation phase of coagulation, are insufficient to explain the activation of factor V that is observed. It is noteworthy that factor Va function is usually measured by its ability to contribute to ␣-thrombin generation.…”

“…During tissue factor-initiated blood coagulation, thrombin generation can be divided into 2 phases; an initiation phase during which very small amounts of thrombin are produced (ϳ 1%, 5 nM) and clotting is observed; during a subsequent propagation phase 700-to 900-nM thrombin is produced. 50,[55][56][57] The identity of the initial activator of factor V has been a subject of controversy. Although factor Xa-phospholipid, thrombin, and plasmin are all capable of activating factor V to factor Va, the kinetic evaluation of tissue factor-initiated blood clotting reactions shows that the initial activator of factor V is ␣-thrombin that is initially produced by factor Xa and phospholipid during the initiation phase of the blood coagulation process.…”

Demonstration of the extrinsic coagulation pathway in teleostei: Identification of zebrafish coagulation factor VII

“…Factor VIIa-tissue factor activity is regulated by tissue factor pathway inhibitor (TFPI) in a factor Xa-dependent manner (4,5). Ex vivo modeling suggests that initiation of coagulation is a threshold-mediated event related to levels of tissue factor, factor VIIa, and TFPI (6,7). However, significant questions remain regarding the initiation of coagulation, including identification of the factor(s) responsible for generation of circulating factor VIIa and the in vivo expression levels of factor VII, tissue factor, and TFPI activity.…”

Lipid Oxidation Enhances the Function of Activated Protein C

“…Effect of Oxidation on Total Thrombin Generation-A prolonged clotting time may indicate only a lengthening of the lag period before feedback loops lead to the explosive generation of thrombin, rather than a decrease in the total amount of thrombin eventually formed (46,47). These two possibilities may not have the same ultimate physiological effect.…”

“…Furthermore, small changes in the level or function of natural anticoagulants can have a major effect on potential thrombin generation. Studies of Mann and colleagues (46,47) indicate that a "threshold effect" occurs in coagulation. Thrombin must be generated to a level greater than that threshold for clotting to ensue.…”