The use of phospholipid-dependent clotting assays to characterize the cell-surface procoagulant activities of T-lymphoblastoid cell lines

Abstract number: P1081

Pickering^* W., Gray† E., Goodall‡ A. H., Ran§ S., Thorpe§ P. E., Barrowcliff† T. W.

^*National Institute for Biological Standards & Control, UK; †NIBSC, UK; ‡University of Leicester, UK; §University of Texas, USA

It is accepted that leucocytes, especially monocytes, can exhibit tissue factor (TF) related procoagulant activity (PCA). Our previous studies have described a phospholipid (PL) like PCA in T-lymphoblastoid cell lines that occurs despite the presence of very low levels of TF. In this study we measured the contribution of PL to Lymphoblastoid PCA using PL-dependent assay systems (APTT, DRVVT and a purified intrinsic Xa generation system). The APTT was used in combination with inhibitory antibodies against TF and phosphatidylserine (PS). APTT and DRVVT PCA, were quantified by the ability of intact cells to shorten the buffer clotting times of platelet poor plasma, and compared against a purified bovine phospholipid standard. FXa generation was measured by incubating human FX, FIXa, FVIII with cells or different concentrations of PL and recalcified with calcium chloride. Four T-cell lines; CCRF-CEM, Jurkat, Molt-4, A3.01, as well as a control Monocytoid cell line THP-1, were cultured in RPMI under standard conditions. All cells were 90% viable before analysis, as measured by trypan blue exclusion. The PCA of the cell lines by APTT were 1.14 ± 0.13, 2.78 ± 0.8, 2.29 ± 0.49, 1.57 ± 0.8 and 4.5 ± 0.65 PLU, respectively. PCA measured by the DRVVT showed a similar pattern to the APTT; 8.49 ± 0.3, 20.8 ± 0.44, 15.24 ± 0.59, 11.49 ± 1.51 and 10.94 ± 0.98 PLU, respectively. The PL-like activity as measured by Xa generation was higher and were correspondingly found to be 14 ± 3, 18 ± 2, 34 ± 6, 40 ± 5.5 and 12 ± 2 PLU. TF antibody did not inhibit the PCA as measured by the DRVVT and FXa generation assays, and was unable to inhibit APTT activity of CCRF-CEM, Molt-4 and A3.01 (TF units mL^-1: 6.6,1.5 and 1.2 U mL^-1) which have low levels of TF, but the APTT of the higher TF activity cell lines, Jurkat (15.5 U mL^-1) and THP-1 (53.3 U mL^-1) were substantially reduced by 41 and 87%, respectively. Although the anti-PS antibody completely inhibited the APTT activity of 12.5 PLU of purified PL, it did not completely inhibit the APTT activity of the cell lines (%inhibition: 53, 48, 59, 69 for CCRF-CEM, Jurkat, Molt-4 and A3.01, respectively). The results indicate that the PCA of T-cells is mainly PL dependent, and is TF independent. The differences in activities measured by the APTT, DRVVT and intrinsic Xa generation, together with the incomplete inhibition of APTT activity by the PS antibody indicate that the PL activity of these T-cells may not be due solely to PS and merits further characterization.