Phosphatidylserine-Positive Erythrocytes Modulate Tissue Factor Activity In Vivo and In Vitro in Sickle Cell Disease.

Evaluation of fluid phase markers reveals activated blood coagulation in sickle cell disease (SCD), with enhanced in vivo thrombin generation. Studies have shown that whole blood tissue factor (WTBF) activity is elevated, and that both monocytes and endothelial cells (ECs) contribute to WBTF. In a recent preliminary study, we have found an unexpected and interesting positive correlation between WBTF and the levels of both reticulocytes (R=0.6) and phosphatidylserine (PS)-positive red cells (R=0.4) in SCD and an inverse correlation with hemoglobin (R=-0.4). We have therefore evaluated potential mechanisms for erythroid modulation of WBTF. Reticulocytes being PS-positive, we initially evaluated the effects of PS-positive red cells on endothelial TF using human lung microvascular ECs and ionophore-treated PS-exposing control erythrocytes, since sickle erythrocytes also express other adhesion markers. Following incubation with appropriate treatments for 24 hours, ECs were analyzed for TF expression (with E-selectin as a positive control for EC activation). Co-incubation of PS-positive red cells (15% ±) with ECs increased endothelial expression of TF in a hematocrit-dependent manner. PS-positive cells increased TF expression by 140, 200, 260 and 320% at hematocrits of 2, 5, 10 and 20%, respectively, compared to media controls. Results similar to those observed with TF were also noted with E-selectin. PS-positive sickle erythrocytes have been classified as either type I or type II cells. Type I express low levels of PS (reticulocytes), while type II are highly PS-positive and include sickle dense cells. We next evaluated whether both type I and type II PS-positive red cells triggered endothelial TF expression. Type II cells were prepared by incubating control erythrocytes with calcium ionophore, and type I cells were generated by incubating type II cells with predetermined amounts of annexin V (100 μg annexin V per 25X10⁶ PS-positive cells). FACS analyses demonstrated appropriate PS levels in each PS positive category. We show that type I cells had no effect. TF expression occurred following incubation of ECs with type II erythrocytes (0.21±0.06 units TF with type II vs 0.10±0.06 with PS-negative cells at a 2% hematocrit, n=5, P<0.01). In final patient related studies we have observed a marked correlation between WBTF and type II PS-positive red cells (R=0.5, P<0.00001, n=81). No such correlation was noted with type I. We conclude that while the reticulocyte component of the hemolytic anemia does not appear to affect WBTF, type II PS cells (including potentially dense cells) modulate WBTF activity in SCD with implications for both the thrombophilic and adhesive pathophysiologic complications of this disease entity. These studies in addition have potential implications for other disease states where erythrocyte PS exposure occurs.