Aim: Among microparticles (MP) those exposing Tissue Factor (TF) are of particular interest, for their critical role in the initiation of thrombosis. They may be a useful biomarker to identify an increased risk of thrombosis in various pathologies (e.g. cancer). We developed the Zymuphen MP-TF method, an ultra sensitive bio-immunoassay that allows the determination of MP-TF procoagulant activity in human plasma.
Method: Plasmas are prepared using a double centrifugation at Room Temperature (low and high speed) to eliminate platelets. MP-TFs are captured by a murine MoAb directed against the extracellular domain of TF, that does not inhibit TF activity. Following a washing step, FVIIa and F X are added into the reaction mixture. TF-FVIIa complexes form and activate F X into F Xa (FXa) in presence of Ca++. FXa generation is dependent on TF and MP’s anionic phospholipids concentration. Then a FXa-specific substrate (CS 11(65)) is added, reacts with FXa, releasing pNA, which absorbance is recorded at 405nm. A lyophilized calibrator, containing rec.(h) TF relipidated with synthetic phospholipids, permits the standardization of the assay. Calibration is from 0 to 5 pg/ml of TF equivalent, with a ratio of 0.1 nM PS/ 1 pg TF.
Results: Normals were < 0.2 pg/ml, while 2 pathological plasmas that were found to have high MPs concentration (9.8 and >63 nM PS) with Zymuphen MP Activity, were found at 0.95 pg/ml and > 5 pg/ml using Zymuphen MP-TF. Plasmas from LPS-stimulated whole blood following a 6 hour incubation were significantly higher than baseline (t=0h) with a MP-TF generation ranging from 5 to 22 μg/ml. Recombinant Truncated TF (1-219), when mixed at 100 pg/ml with synthetic saturating phospholipids (870nM PS), was < 0.2 pg/ml demonstrating the specificity for MP-TF.
Conclusion: Zymuphen MP-TF is a highly sensitive and specific method for the measurement of MP-TF in citrated plasma, and is a useful for assessing the clinical interest of this biomarker in pathology.