Blood coagulation at the site of microvascular injury: effects of low-dose aspirin

Abstract

The sequence of coagulant reactions in vivo following vascular injury is poorly characterized. Using quantitative immunoassays, the time courses were evaluated for activation of prothrombin, factor (F)V, FXIII, fibrinogen (Fbg) cleavage, and FVa inactivation in bleeding-time blood collected at 30-second intervals from 12 healthy subjects both before and after aspirin ingestion. Prothrombin decreased at a maximum rate of 14.2 ؎ 0.6 nM per second to 10% of initial values at the end of bleeding. Significant amounts of ␣-thrombin B chain appeared rapidly at 90 seconds of bleeding and increased at a maximum rate of 0.224 ؎ 0.03 nM per second to a peak value of 38 nM. Kinetics of prethrombin 2 generation was almost identical. Prothrombinase concentration reached a peak value of 22 pM at 150 seconds and then decreased to 9 pM at the end of bleeding. Prothrombin fragment 1.2 (F1.2) was produced explosively (0.673 ؎ 0.05 nM per second), whereas thrombin-antithrombin III (TAT) complexes were generated at a much slower rate (0.11 ؎ 0.008 nM per second; P ‫؍‬ .002). FVa light chain was detectable 30 seconds later than the heavy chain (150 seconds) and was produced at a slightly slower rate (0.027 ؎ 0.001 nM per second) when compared with the heavy chain (0.032 ؎ 0.002 nM per second; P ‫؍‬ .041). The 30 000 fragment (residues 307-506) of FVa heavy chain produced by activated protein C appeared as early as at 90 seconds and increased with time. Fbg was removed from the blood shed with a high rate of 0.047 ؎ 0.02 M/s and became undetectable at approximately 180 seconds of bleeding. The velocity of FXIII activation correlated with thrombin Bchain formation. A 7-day aspirin administration (75 mg/d) resulted in significant reductions in maximum rates of (1) prothrombin removal (by 29%; P ‫؍‬ .008); generation of ␣-thrombin B-chain (by 27.2%; P ‫؍‬ .022), and prethrombin 2 (by 26%; P ‫؍‬ .014); formation of F1.2 (by 31.4%; P ‫؍‬ .009) and TAT (by 30.3%; P ‫؍‬ 0.013); (2) release of FVa heavy chain (by 25%; P ‫؍‬ .003) and FVa light chain (by 29.6%; P ‫؍‬ .007); (3) Fbg depletion from solution (by 30.5%; P ‫؍‬ .002); and (4) FXIII activation (by 28.6%; P ‫؍‬ .003). Total amounts of the proteins studied, collected at every interval, also significantly decreased following aspirin ingestion. These results indicate that low-dose aspirin impairs thrombin generation and reactions catalyzed by this enzyme at the site of the injury. IntroductionHemostasis is triggered in vivo when vascular damage exposes membrane-bound tissue factor (TF) constitutively present in the subendothelium. 1 The proteolytically active complex of TF and circulating 2-chain factor (F)VIIa, the extrinsic tenase, activates FX and FIX. FIXa and FXa, complexed with FVIIIa and FVa, respectively, form 2 enzyme-cofactor complexes-the intrinsic tenase and prothrombinase, which converts prothrombin to thrombin. The former complex activates FX at a 50-fold higher rate than the FVIIa-TF complex, whereas prothrombinase is 10 5 times more efficient in generating thrombin than ...

Aspirin Alters the Cardioprotective Effects of the Factor XIII Val34Leu Polymorphism

“…2 Thus, the differences in the total (nonactivated plus activated) FXIIIA content in the first minute of bleeding and at the end observed in the 2 groups is most likely due to a more avid absorption of FXIIIAa to the hemostatic plug as compared with that of FXIIIA and is a typical feature of the model used. 8 On the basis of in vitro studies, 3 it might be expected that FXIIIAa will appear simultaneously in the Val34 homozygotes and Leu34/Val34 heterozygotes; however, in vivo, we observed a slightly earlier appearance of FXIIIAa in the latter group, which may be due to differences in FXIIIAa binding to fibrin platelets or other components of the injured vascular wall. Also, as we do not have information regarding the synthesis, turnover, or competition for thrombin of the 2 allelic products, we do not know if the blood product concentrations are equivalent.…”

“…[7][8][9] This system describes the in vivo situation that can be contrasted with in vitro models applied to study effects of the Val34Leu polymorphism on FXIII activation. The rates of the disappearance of FXIIIA and the appearance of FXIIIAa depend on multiple processes, including the proteolytic activation of FXIII by thrombin and absorption of FXIIIA to platelets, fibrin, and other elements at the site of vascular injury, and the relative molar concentration of the products of the 2 alleles in blood.…”

“…7,8 Briefly, at venous stasis of 40 mm Hg, 2 incisions were made on the lateral aspect of a forearm using a Simplate II device (Organon Teknika), and blood was collected using heparinized capillary tubes (Kabe Labortechnik) into Eppendorf tubes, containing sodium citrate, aprotinin, peptidyl chloromethyl ketone, and heparin (Diagnostica Stago). The procedure was performed by the same investigator each time, who was blinded to the results of genotyping.…”

“…8 The time-courses of FXIII activation were performed using rabbit polyclonal antibody raised against human FXIIIA (D4679), a gift from Drs Paul Bishop and Gerry Lasser (ZymoGenetics, Seattle, Wash). FXIIIA levels were estimated by comparison with purified human FXIII standards.…”

Hyperglycemia Is Associated With Enhanced Thrombin Formation, Platelet Activation, and Fibrin Clot Resistance to Lysis in Patients With Acute Coronary Syndrome

“…Measurements were performed in blood collected at 60-s intervals from a standardized skin incision, made using a Simplate IR device (Organon Teknika, Durham, NC) at the inflation of the sphygmomanometer cuff at 40 mmHg, as described previously (13)(14)(15). Blood was collected by means of heparinized tubes (Kabe Labortechnik, NumbrechtElsenroth, Germany) into Eppendorf tubes containing anticoagulants as described previously (14,15).…”