PROLONGED BLEEDING-TIME WITH DEFECTIVE PLATELET FILOPODIA FORMATION IN THE WISTAR FURTH RAT

Hereditary macrothrombocytopenia is a hallmark of Wistar Furth (WF) ra ts. In addition, a platelet/megakaryocyte alpha granule defect, simila r to that of patients with gray platelet syndrome, is present. Several observations indicate cytoskeletal abnormalities in WF platelets and megakaryocytes, suggesting the potential for functional defects in hem ostatic processes requiring cytoskeletal reorganization, such as plate let adhesion and spreading. However, no bleeding abnormality has been noted. Here, we report a prolonged bleeding time (>30 minutes in 10 of 11 rats tested) with defective clot formation in the WF strain. Prolo nged bleeding time can result from defects in platelet adhesion, aggre gation, or the release reaction. Because aggregation to collagen and a denosine diphosphate were reported to be normal, we determined whether WF rat platelets are defective in their ability to adhere to substrat es. Platelet adherence and spreading was evaluated from 30 seconds to 30 minutes on Formvar-coated, carbon-stabilized grids or poly-L-lysine -coated glass coverslips by transmission electron microscopy or immuno fluorescence, respectively, and scanning electron microscopy. We class ified the adhered platelets according to their pattern of spreading, i e, rounded, rounded or spreading with short filopodia, spindle-shaped, spreading with long filopodia, spreading with lamellipodia, and fully spread. Adherent normal rat platelets displayed all stages of spreadi ng within 30 seconds to 2 minutes, including many spindle-shaped forms , and forms with multiple, long filopodia. In contrast, adhered WF pla telets at these early time points rarely developed long filopodia or w ere spindle shaped. The majority of adherent WF platelets at these ear ly time points were either round, spread with a few short filopodia, o r extensively spread with wide lamellipodial skirts. By 15 to 30 minut es, most platelets in both Wistar and WF samples were fully spread. Th ese data show abnormal WF platelet spreading. The paucity of spindle-s haped forms and forms with long filopodia may reflect an inability of WF platelets to undergo the early stages of spreading, or, alternative ly, their more rapid than normal progression through these stages. We hypothesize that this failure to spread normally may relate to prolong ed bleeding times in vivo and defective clot formation in WF rats. (C) 1998 by The American Society of Hematology.