Expression of PS variants with EGF domains deleted or replaced and their ability to enhance APC-catalyzed factor Va inactivation.

(A) Western blotting of wtPS and variant PS expressed in CHO cells. SDS-PAGE of about 50 ng rPS under nonreducing conditions and immmunoblotting with an anti-PS polyclonal antibody was performed as described in Rezende et al.15 Molecular weight markers are shown in kilodaltons on the left. Lane 1, wild-type; lane 2, variant 123; lane 3, variant 124; lane 4, variant 134; lane 5, variant 1334. The assay described in panels B and C uses the reduction in thrombin generation measured using a chromogenic substrate to assess the enhancement by PS of APC-catalyzed factor Va inactivation.14,25 (B) The results of the initial screening of the PS variants for their ability to enhance APC-catalyzed factor Va inactivation are shown. Although each variant and the wtPS were tested at several different concentrations, the results are presented for 10 nM PS. Results (means of 3 experiments ± SEM) are expressed as a percentage of wtPS (100%). We previously showed15 that recombinant wtPS has a similar specific activity to a commercial purified human PS preparation and for both, half-maximal inhibition of thrombin generation was reached at a PS concentration of about 1 nM. Identical results were obtained for purified, semipurified, and unpurified wtPS-containing medium. (C) The ability of the variant 134 (▪) to enhance APC-catalyzed inactivation of factor Va in comparison to wtPS (♦). The results are presented for the full range of tested PS concentrations (0, 1.25, 2.5, 5, 10, 15, 20, and 25 nM) and expressed relative to the remaining activity of factor Va measured in the absence of PS (100%) as a mean of 3 experiments ± SEM for each PS concentration. In some cases the SEM cannot be seen due to its small size. There were insufficient amounts of the other variants to perform this analysis at all concentrations.