A Novel Assay for Tissue Factor Pathway Inhibitor (TFPI) Anticoagulant Activity in Plasma.

Introduction. Several studies have shown that full-length free TFPI is a much stronger anticoagulant in diluted prothrombin time (dPT) assays than truncated and lipoprotein associated TFPI. Although it has been suggested that TFPI anticoagulant activity is of importance for the effect of TFPI in vivo, no assays have so far been established and validated to assay TFPI anticoagulant activity in patient samples. Aims. To develop and validate an assay of TFPI anticoagulant activity and study the relationship between TFPI anticoagulant activity and TFPI free and total antigen and TFPI chromogenic substrate activity. Materials and Methods. TFPI anticoagulant assay: 32 μL of plasma was first incubated for 5 min with either 8 μL TBS or anti-TFPI immunoglobulin, which completely blocked TFPI activity. Then, 30 μL recombinant, relipidated tissue factor (Innovin®) at 1/200 fold dilution was added and incubated for 3 min. Clotting was initiated by the addition of 30 μL CaCl2 (35 mM). Results were expressed as a ratio, with clotting times in the presence of anti-TFPI as the denominator. This ratio was normalized against the ratio obtained with a reference plasma within each run to minimize day to day variation in clotting times. After normalization, high ratios reflect high functional TFPI anticoagulant activity, whereas low ratios reflect low activity. TFPI antigen assays: TFPI free and total antigen were assayed with commercial kits from Stago, France. TFPI free antigen is specific for circulating full-length TFPI, whereas TFPI total antigen detects both full-length and truncated forms of TFPI. Study population: Control subjects (n=436) of the Leiden Thrombophila Study (LETS). Results. Mean TFPI anticoagulant ratio was 1.03 (SD 0.0066, range 0.87–1.22). The assay coefficient of variation was 2.8% between days and from 0.6% to 1.5% within days. Linear regression showed that the n-TFPIac ratio was strongly associated to the levels of TFPI free antigen, but weakly associated to bound TFPI (table 1). Conclusions. We have established a novel and reproducible assay for detection of TFPI anticoagulant activity in plasma. TFPI anticoagulant ratio was strongly associated to free antigen, and may be considered an activity assay of full-length TFPI in plasma. Table 1 - The association of normalized TFPI anticoagulant ratio to other TFPI assays TFPI assay Normalized TFPI anticoagulant ratio- b (95% CI) Correlation coefficient- r TFPI free antigen 0.049 (0.044 to 0.053) 0.73 Total TFPI 0.033 (0.027 to 0.038) 0.48 TFPI chrom. subst. activity 0.031 (0.025 to 0.037) 0.46 Bound TFPI 0.020 (0.014 to 0.026) 0.30 Standardized regression coefficient (b), 95% confidence interval (CI) and correlation coefficient (r). (b) shows the change in n-TFPIac ratio per increase of one standard deviation in the TFPI assay studied.