A Novel Connection Between Stress Erythropoiesis and Coagulation Activation in Sickle Cell Disease.

Sickle cell disease (SCD) is associated with a hypercoagulable state. Multiple studies show that plasma from these patients exhibit: 1) increased thrombin generation; 2) decreased levels of natural anticoagulant proteins; and 3) a defect in the activation of fibrinolysis. The mechanism of coagulation activation in SCD is presumed to be multi-factorial, with contributions from abnormal erythrocyte phospholipid asymmetry and induction of tissue factor (TF) following hemolysis. In addition, hemolysis in SCD leads to elevated levels of erythropoietin (EPO) in patients, increased reticulocyte counts and the presence of stress (or shift) reticulocytes in circulating blood. These stress reticulocytes retain expression of the α4b1 integrin and are demonstrably adhesive to vascular factors in SCD. We have previously reported that these stress reticulocytes bind to blood monocytes in SCD patients via the α4b1 integrin, but the effect of this interaction on either cell remained unknown in SCD.

With the increasing evidence that hemolysis and subsequent stress erythropoiesis associates with coagulation activation, we sought to evaluate the role of erythropoietin and the effect of stress reticulocyte adhesion to monocytes on coagulation activation in SCD patients.

Coagulation activation in plasma samples was examined by evaluating TF activity on microparticles derived from patients with SCD. Stress reticulocytes were visualized and enumerated from these same patients using Wright Giemsa stained blood smears counter stained with new methylene blue to detect reticulocytes. Reticulocytes were scored as a stress reticulocytes based on the amount of punctuate reticular material, cell size, and presence of nuclear material. Stress reticulocyte induction of monocyte tissue factor expression was measured by flow cytometry after incubation of THP-1 monocytic cells with purified SS RBCs or control RBCs. To determine if induced THP-1 TF expression was due stress reticulocyte binding, THP-1 TF expression was examined in the presence or absence of known inhibitors of the monocyte/stress reticulocyte interaction. TF expression on CD14+ monocytes was examined in whole blood from SCD patients using flow cytometry. Plasma erythropoietin levels were quantified by ELISA.

We found that direct binding of the stress reticulocyte increased THP-1 TF expression 2.5 fold. This increase in TF expression was completely ablated by function blocking antibodies against the α4 integrin, but not by an isotype-matched control IgG. In whole blood samples, we also found increased TF expression on CD14+ monocytes with stress reticulocytes directly bound, compared to those monocytes in the same patient without stress reticulocytes bound (p = 0.002, n =3).We noted a strong correlation between stress reticulocyte count and TF activity on plasma microparticles in SCD (r_spearman = 0.8656, CI = 0.5382 – 0.9660, p = 0.0006, n=11). Furthermore, we found that EPO induced α4b1 activation on the stress reticulocyte. This activation may promote both adhesion to the monocyte and an increase in TF expression. Consequently, we noted a strong trend towards an association of EPO with microparticle TF activity in SCD (r_spearman = 0.5740, CI=-0.06 – 0.8780, p=0.068, n= 11) suggesting that EPO, by promoting the interaction between the stress reticulocyte and the monocyte, may contribute to TF activity in SCD.

Taken together, we find that stress reticulocyte adhesion to monocytes and monocytic cells induces TF expression and may promote TF activity in patients. These data suggest a novel connection between stress erythropoiesis and coagulation activation in SCD.

No relevant conflicts of interest to declare.