Morphological and functional platelet abnormalities in Berkeley sickle cell mice

Abstract

Berkeley sickle cell mice are used as animal models of human sickle cell disease but there are no reports of platelet studies in this model. Since humans with sickle cell disease have platelet abnormalities, we studied platelet morphology and function in Berkeley mice (SS). We observed elevated mean platelet forward angle light scatter (FSC) values (an indirect measure of platelet volume) in SS compared to wild type (WT) (37+/-3.2 vs. 27+/-1.4, mean+/-SD; p<0.001), in association with moderate thrombocytopenia (505+/-49 x 10(3)/microl vs. 1151+/-162 x 10(3)/microl; p<0.001). Despite having marked splenomegaly, SS mice had elevated levels of Howell-Jolly bodies and "pocked" erythrocytes (p<0.001 for both) suggesting splenic dysfunction. SS mice also had elevated numbers of thiazole orange positive platelets (5+/-1% vs. 1+/-1%; p<0.001), normal to low plasma thrombopoietin levels, normal plasma glycocalicin levels, normal levels of platelet recovery, and near normal platelet life spans. Platelets from SS mice bound more fibrinogen and antibody to P-selectin following activation with a threshold concentration of a protease activated receptor (PAR)-4 peptide compared to WT mice. Enlarged platelets are associated with a predisposition to arterial thrombosis in humans and some humans with SCD have been reported to have large platelets. Thus, additional studies are needed to assess whether large platelets contribute either to pulmonary hypertension or the large vessel arterial occlusion that produces stroke in some children with sickle cell disease.

Figure 1. SS mice have markedly abnormal blood smears

Peripheral blood smears of EDTA-anticoagulated blood obtained by retrobulbar venous plexus puncture from WT, SS and β-thalassemia mice. Blood smears from SS animals show anisocytosis, poikilocytosis, sickled erythrocytes, target cells, erythrocyte fragments of varying sizes, Howell-Jolly bodies, and an increased percentage of polychromatophilic erythrocytes. In sharp contrast, smears from WT animals do not show any of these findings. Blood smears from β-thalassemia mice show anisocytosis, microcytosis, targets cells, erythrocyte fragments, and an increased percentage of polychromatophilic erythrocytes.

Fig 2. Platelets from SS mice have increased forward light scatter properties

Histogram of forward light scatter (abscissa, log scale) and number of particles positive for anti-murine αIIbβ3 (1B5) staining (ordinate) from blood of wild type and SS mice. Blood samples were reacted with Alexa⁶⁴⁷-1B5 (platelet specific mAb to murine αIIbβ3) and then the platelets were identified on the basis of light scatter properties and 1B5 staining. In the SS mice, there is a rightward shift of the entire platelet population, contributing to an elevation in the mean forward scatter value.