Altered Red Cell and Platelet Adhesion in the Hemolytic Diseases: Hereditary Spherocytosis, Paroxysmal Nocturnal Hemoglobinuria and Sickle Cell Anemia.

Increasing evidence exists to suggest that intravascular hemolysis may have important pathophysiological consequences resulting from reduced vascular nitric oxide (NO) bioavailability due to hemoglobin-mediated NO scavenging; such consequences may include endothelial dysfunction and vasculopathy. Hemolytic diseases such as hereditary spherocytosis (HS), paroxysmal nocturnal hemoglobinuria (PNH) and sickle cell anemia (SCA), despite having diverse etiologies, share a number of complications that include pulmonary and systolic arterial hypertension, cutaneous leg ulcerations and, in PNH and SCA, thrombosis and possibly stroke. Since reduced vascular NO bioavailability may affect cellular adhesion molecule function, we sought to compare the adhesive properties of red cells and platelets in these diseases. Blood samples were collected from healthy control subjects and from patients with HS (non-splenectomized), PNH (with hemolysis) or SCA (in steady state and not on hydroxyurea therapy); all patients presented reticulocytosis. Red blood cells (RBC) and platelets were washed and resuspended in RPMI medium (RBC) or Krebs solution (platelets). RBC suspensions were then allowed to adhere to fibronectin (FN)-coated plates (20mg/ml) and platelets to fibrinogen (FB)-coated plates (50mg/ml), employing previously standardized static adhesion assays. Cell adhesion was expressed as the percentage of cells adhered. RBC adhesion to FN was slightly, but significantly, increased for cells from HS and PNH patients compared to control RBC (13.8±1.1 %, n=7, P<0.05; 13.7±0.8 %, n=6, P<0.05 for HS and PNH, respectively, compared to 10.5±0.7 % of cells adhered, n=7 for control; Mann-Whitney Test). SCA RBC adhesion to FN, however, was markedly increased when compared to control RBC adhesion (25.7±4.6 %, n=9, P<0.01). With regard to platelet adhesion, HS platelets did not demonstrate any significant alteration in adhesion to FB (14.78±2.91 % compared to 10.94±1.31 % for HS and control, respectively; n=10 and 11, P>0.05). In contrast, PNH and SCA platelet adhesions were significantly increased (23.1±4.7 %, n=8, P<0.05 and 29.5±6.1 %, n=6, P<0.001 for HPN and SCA, respectively). Interestingly, whilst control platelet adhesion was much lower than hemolytic disease platelet adhesion, co-incubation of platelets with a thrombin stimulus (100 U/ml) significantly increased the adhesion of control platelets only (241.2±21.0 %, n=10, P<0.002; Wilcoxon Matched-Pairs test), indicating that hemolytic disease platelets appear to be in an already activated state. Significant alterations in platelet adhesive properties were seen, therefore, in PNH and SCA, but not HS. Such alterations may contribute to the pathophysiology of these diseases, particularly in thrombotic events. Marked alterations in RBC adhesion were, however, seen only in SCA. Thus, whilst hemolysis is common to HS, PNH and SCA, we found cell adhesive functions to vary between diseases. Although it is possible that the rate of intravascular hemolysis is greater in SCA than HS, it is unlikely that the rate of SCA intravascular hemolysis is much greater than that occurring in PNH, where the bulk of hemolysis occurs intravascularly. Thus, the markedly increased cellular adhesive properties observed in SCA appear not to be mediated by hemolysis alone, but probably involve the participation of other mechanisms peculiar to the disease.