Factor XIII on activation by thrombin cross-links fibrin. A common polymorphism Val to Leu at position 34 in the FXIII A subunit is under investigation as a risk determinant of thrombosis. Because Val34Leu is close to the thrombin cleavage site, the hypothesis that it would alter the function of FXIII was tested. Analysis of FXIII subunit proteolysis by thrombin using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high-performance liquid chromatography showed that FXIII 34Leu was cleaved by thrombin more rapidly and by lower doses than 34Val. Mass spectrometry of isolated activation peptides confirmed the predicted single methyl group difference and demonstrated that the thrombin cleavage site is unaltered by Val34Leu. Kinetic analysis of activation peptide release demonstrated that the catalytic efficiency (kcat/Km) of thrombin was 0.5 for FXIII 34Leu and 0.2 (μmol/L)−1× sec−1 for 34Val. Presence of fibrin increased the catalytic efficiency to 4.8 and 2.2 (μmol/L)−1 × sec−1, respectively. Although the 34Leu peptide was released at a similar rate as fibrinopeptide A, the 34Val peptide was released more slowly than fibrinopeptide A but more quickly than fibrinopeptide B generation. Cross-linking of γ- and -chains appeared earlier when fibrin was incubated with FXIII 34Leu than with 34Val. Fully activated 34Leu and 34Val FXIII showed similar cross-linking activity. Analysis of fibrin clots prepared using plasma from FXIII 34Leu subjects by turbidity and permeability measurements showed reduced fiber mass/length ratio and porosity compared to 34Val. The structural differences were confirmed by electron microscopy. These results demonstrate that Val34Leu accelerates activation of FXIII by thrombin and consequently affects the structure of the cross-linked fibrin clot.
Although in vitro studies have been invaluable in revealing the complex biochemistry of the blood coagulation system, the mechanisms involved during the in vivo response to hypercoagulable stimuli are still unclear. We have used plasma-based enzyme-linked immunosorbent assays (ELISAs) to study the mechanisms by which the coagulation system is activated in vivo during human cardiopulmonary bypass (CPB) surgery (n = 8). A novel immunoassay for factor XIIa was used to detect activation of the contact system, factor IX activation peptide (FIXAP) was used as a marker for activation of factor IX, and prothrombin fragment F1 + 2 (F1 + 2) was used as a marker for thrombin generation. The ELISA for FIXAP is described for the first time herein. F1 + 2 levels increased early in response to surgical intervention: from a baseline of 38.7 +/- 9.7 ng/mL (mean +/-SE), levels increased rapidly during surgery and bypass to a maximum of 448.5 +/- 92.0 ng/mL. A modest yet significant increase in factor XIIa levels from 3.47 +/- 0.54 ng/mL to 4.33 +/- 0.85 ng/mL was evident during surgery before bypass, but no further significant increase was detected on establishing extracorporeal circulation. FIXAP levels demonstrated a small and late increase during surgery from 4.98 +/- 0.55 ng/mL to a maximum of 10.20 +/- 1.23 ng/mL, the increase beginning at the time of near maximal F1 + 2 levels. There was no association between activation of the contact system (factor XIIa levels) and the generation of thrombin (F1 + 2 levels). However, a strong association (r = .705) was apparent between the generation of thrombin (F1 + 2 levels) and activation of factor IX (FIXAP levels), despite the delay between the activation of prothrombin and factor IX. The data do not support the established view that contact activation resulting from exposure of blood to foreign surfaces is the major procoagulant stimulus in CPB. Instead, the results suggest that the main trigger to coagulation during CPB surgery was provided via the tissue factor-factor VIIa mechanism in response to the cutting of blood vessels, which directly activated factor X and then prothrombin. The late activation of factor IX, which presumably also contributed to maximal prothrombin activation, could have arisen due to direct tissue factor-factor VIIa action, or by secondary feedback action of thrombin on the intrinsic system.
SummaryActivation of coagulation was studied during the peri-operative period in patients undergoing cardiopulmonary bypass (CPB) surgery using activation markers which have recently become available: prothrombin fragment F1 + 2 (F1 + 2), which is a measure of total thrombin generation, and thrombin-antithrombin complex, which is a measure of inactivation of free thrombin by antithrombin. Levels of the specific marker of fibrin breakdown, D-dimer, were also determined. F1 + 2 levels were assessed using a newly developed ELISA described herein which employs a neoantigen-specific capture antibody raised using a synthetic peptide; the latter antibody has been pre-adsorbed against prothrombin to ensure high specificity for F1 + 2.Increased generation of thrombin during surgery was clearly demonstrated despite maintenance of a high concentration of heparin during the period of extracorporeal blood circulation. There was a close association (r = 0.882) between the generation of thrombin (F1 + 2 levels) and its inhibition (TAT levels). Differences were noted, however, between the information provided by F1 + 2 and TAT, which are interpreted with regard to the different in vivo fates of F1 + 2 and thrombin. The enhanced activation and inhibition of coagulation observed during CPB was suppressed once physiological blood circulation was restored, with F1 + 2 returning to pre-surgical levels within 24 h after surgery. During the post-operative period D-dimer levels, which rose in concert with F1 + 2 and TAT levels, remained highly elevated, suggesting that not all of the generated thrombin was inactivated by antithrombin. It is concluded that heparin is only partially effective as an anticoagulant during CPB surgery. F1 + 2 is an unambiguous marker of thrombin generation and its measurement may be a useful means of evaluating more effective coagulation inhibitors in CPB.
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