Importance of factor Xa in determining the procoagulant activity of whole-blood clots.

Abstract

The binding of thrombin to fibrin is thought to be an important mechanism by which thrombi exhibit procoagulant activity; however, the extent to which other procoagulants are associated with thrombi has not been previously defined. This study was designed to determine whether clotting factors other than thrombin are bound to whole-blood clots and can thereby contribute to significant procoagulant activity. Clots formed in vitro from human blood exhibited minimal thrombin activity when incubated in plasma depleted of vitamin K-dependent factors by barium-citrate adsorption, as indicated by increases in the concentration of fibrinopeptide A (FPA), a marker of fibrin formation, to 72 nM after 30 min. Incubation of clots in barium-absorbed plasma repleted with 0.9 ,uM human prothrombin under the same conditions resulted in marked increases in the concentration of FPA (> 1,000 nM) and clotting by 30 min. The increases in FPA were attributable to activation of the added prothrombin by clot-associated Factor Xa, judging from concomitant increases in the concentration of prothrombin fragment 1.2. Similar results were obtained with thrombi induced in the axillary arteries of dogs by vascular injury and incubated with plasma in vitro. Activation of prothrombin was inhibited in a dose-dependent manner by tick anticoagulant peptide, a direct inhibitor of Factor Xa, at concentrations of 0.5-5.0 gM. Clot-associated Factor Xa activity was resistant to inhibition by anti-thrombin III, judging from the lack of inhibition of prothrombin activation during incubation of clots in plasma containing heparin pentasaccharide, an anti-thrombin 111-mediated inhibitor of Factor Xa. Thus, the activity of Factor Xa appears to be an important determinant of the procoagulant activity of whole-blood clots and arterial thrombi, and is resistant to inhibition by anti-thrombin III-dependent inhibi-

Prolonged Activation of Prothrombin on the Vascular Wall After Arterial Injury

“…Abdominal aortic injury was induced in rabbits by overinflation and multiple passages of a 4F embolectomy catheter. Rabbits were killed 15 minutes or 4,8,24,48, 72, 96, or 120 hours after injury. Aortic segments were incubated ex vivo to define bound procoagulant activity.…”

“…7 Accumulation of platelets and fibrin at the site of arterial injury, even when thrombus is not grossly apparent, also mediates the progression of thrombosis by binding activated coagulation factors. 8,9 We have shown that arterial thrombi induce marked activation of the coagulation system attributable to platelet-associated Xa/Va activity and thrombin bound to fibrin. 8 However, the extent to which procoagulant factors bind to the injured vessel wall, particularly in the absence of significant mural thrombus, and the duration of procoagulant activity associated with the vascular wall after arterial injury are not well defined.…”

“…We hypothesized that the Xa/Va complex expressed by platelets that bind to the vascular wall after injury may be an important determinant of thrombin elaboration. 8,10,11 Accordingly, the objectives of this study were to characterize the functional activity of Xa/Va and thrombin associated with the vascular wall after balloon-induced injury to the rabbit aorta and the extent to which the activity of these factors contributes to procoagulant activity over time. In addition, we characterized the extent to which aspirin and heparin attenuate vascular wall-associated procoagulant activity after injury.…”

“…factor X were isolated from pooled citrated plasma and were purified by affinity chromatography against the other vitamin K-dependent proteins, as previously described. 8,12 Prothrombin and factor X preparations were incubated with 5 mmol/L D-Phe-D-Pro-L-Arg chloromethyl ketone (PPACK; Calbiochem) to inhibit trace amounts of contaminating active proteases. These preparations were then extensively dialyzed against 0.02 mol/L HEPES, 0.15 mol/L NaCl, pH 7.4 (HEPES saline), to remove free D-Phe-D-Pro-L-Arg chloromethyl ketone.…”

Characterization of the Binding of Factor Xa to Fibrinogen/Fibrin Derivatives and Localization of the Factor Xa Binding Site on Fibrinogen

“…Recently, coagulation factor Xa has been found to be adsorbed to fibrin clots and this clot-bound factor Xa may be involved in thrombin generation and be resistant to the heparindependent AT 111 inhibition [16]. In the present study, we studied the characteristics of the binding of factor Xa to fibrinogedfibrin derivatives, in a comparative evaluation with thrombin binding.…”