Detection of Factor IXa Activity By the Enhanced Thrombin Generation Assay

Introduction – Factor Xa generation by the factor IXa-factor VIIIa (FIXa-FVIIIa) complex is the rate-limiting step for plasma thrombin generation (TG). Modeling of blood coagulation suggests that subnanomolar concentrations of FIXa are sufficient to support plasma TG, however, the isolated enzyme is poorly reactive with both substrate and inhibitors. Thus, most detection methods for FIXa require at least nanomolar protease concentrations, which does not allow characterization of physiologically relevant enzyme levels. Our laboratory has developed a novel, highly sensitive assay to detect FIXa activity based on TG in FIX-deficient plasma.

Method – The enhanced thrombin generation assay (ETGA) detects FIXa activity in test samples by dilution into citrated FIX-deficient plasma. Briefly, a standard curve was established by adding 10 µl of test plasma containing 0-80 pM plasma-derived FIXa (pFIXa) to 50 µl of FIX-deficient plasma. Simultaneously, human FVIII (19.2 nM) was activated with 12.8 nM thrombin for 30 sec, neutralized with a 1.25-fold molar excess of hirudin, and the resulting thrombin-activated FVIIIa was added to plasma (final plasma concentration 1.3 nM) immediately after addition of calcium and the fluorogenic substrate. Plasma TG was detected by cleavage of Z-Gly-Gly-Arg-AMC in a Biotek Synergy HT plate reader, and data exported to TECHNOTHROMBIN TGA evaluation software to generate TG curve parameters including lag time, peak thrombin concentration, time to thrombin peak and velocity index. FIXa concentration was plotted versus mean peak thrombin ± SEM (n=3) and the data fit to a parabolic function. Sample FIXa activity was obtained from this standard curve using mean peak thrombin concentration. The specificity of the TG response was confirmed by pre-incubation of test plasma with inhibitory antibodies. To prevent contact pathway-dependent FIXa generation, the monoclonal antibody (MoAb) O1A6 was added, which directly blocks FIX activation by factor XIa[PT1] . Plasma TG was ̴20-50% lower when the assay was performed in the presence of MoAb O1A6. The dependence of the remaining TG activity on FIXa was confirmed by pre-incubation of test plasma with an inhibitory anti-FIX Gla domain antibody. An inhibitory anti-TF antibody had no effect on plasma activity in this assay.

Results – The ETGA was employed to: 1) measure FIXa activity in plasma and ascites samples from patients with gynecologic malignancies (ovarian and uterine) and healthy controls, and 2) determine the half-life of FIXa in human plasma. Plasma FIXa activity from 14 patients with gynecologic malignancies, and 12 healthy controls were analyzed by the ETGA. The mean plasma FIXa activity level in the cancer group was 7.6 ± 2.1 pM, and in the control group was 8.2 ± 2.5 pM, confirmed by preincubation with MoAb as above. Coagulant activity in 6 malignant ascites samples from these patients was similarly evaluated with the ETGA assay. The citrated ascites samples demonstrated substantial TG activity, however, preincubation with inhibitory MoAb controls demonstrated that this activity was strictly TF-dependent and FIX-independent. The EGTA was also employed to detect the half-life of FIXa in FIX-deficient citrated human plasma. pFIXa (35 pM) and recombinant FIXa (50pM) was incubated in FIX-deficient plasma for 0-120 min at 37°C, and the residual FIXa activity was determined at varying timepoints. pFIXa demonstrated a remarkably long half-life in FIX-deficient plasma (36.0 ± 3.6 min). rFIXa WT demonstrated a similar half-life (40.9 ± 1.4 min), while mutation in the antithrombin (AT) binding site (R150) substantially prolonged protease half-lives (> 2 hr).

Conclusion – These results demonstrate that the ETGA is capable of detecting physiologically relevant levels of FIXa activity. Initial evaluation of plasma FIXa activity suggests no significant differences between controls and patients with gynecologic malignancies, and no significant FIXa activity in malignant ascites. While rFIXa WT and pFIXa have comparable plasma half-lives, the R150A mutation in the AT-binding exosite markedly prolonged protease half-life, consistent with the role of AT as the primary plasma FIXa inhibitor. This novel assay may have applications in the detection of physiologically relevant FIXa activity in plasma and other blood derived products, recombinant FIX preparations, and other body fluids.