Prolonged Bleeding Time With Defective Platelet Filopodia Formation in the Wistar Furth Rat

Abstract

Hereditary macrothrombocytopenia is a hallmark of Wistar Furth (WF) rats. In addition, a platelet/megakaryocyte alpha granule defect, similar 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 hemostatic processes requiring cytoskeletal reorganization, such as platelet 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. Prolonged bleeding time can result from defects in platelet adhesion, aggregation, or the release reaction. Because aggregation to collagen and adenosine diphosphate were reported to be normal, we determined whether WF rat platelets are defective in their ability to adhere to substrates. 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 immunofluorescence, respectively, and scanning electron microscopy. We classified the adhered platelets according to their pattern of spreading, ie, 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 spreading within 30 seconds to 2 minutes, including many spindle-shaped forms, and forms with multiple, long filopodia. In contrast, adhered WF platelets at these early time points rarely developed long filopodia or were spindle shaped. The majority of adherent WF platelets at these early time points were either round, spread with a few short filopodia, or extensively spread with wide lamellipodial skirts. By 15 to 30 minutes, most platelets in both Wistar and WF samples were fully spread. These data show abnormal WF platelet spreading. The paucity of spindle-shaped forms and forms with long filopodia may reflect an inability of WF platelets to undergo the early stages of spreading, or, alternatively, their more rapid than normal progression through these stages. We hypothesize that this failure to spread normally may relate to prolonged bleeding times in vivo and defective clot formation in WF rats.

Antithrombotic treatment in protection against thrombogenic effects of 5‐fluorouracil on vascular endothelium: A scanning microscopy evaluation

“…Platelet adhesion and aggregation was usually associated with fibrin formation but was occasionally seen as a separate phenomenon. Platelets could be seen as small rounded or slightly irregular bodies, but also as extending long filopodia (Stenberg et al 1998) as they adhere to the underlying surface. In the 5-FU-dalteparin group, platelet accumulation was also present, with a maximum incidence on Day 14, but only occasional fibrin strands could be detected.…”

An Evaluation of the Hemostatic Effect of Externally Applied Notoginseng and Notoginseng Total Saponins

“…The bleeding time of approximately 20 minutes in the control group is roughly twice that of a previous investigation. 15 However, we modified their protocol, transecting the tail 5 mm from the tip as compared to 1 mm in the previous study, specifically to increase the bleeding time. 15 Applying notoginseng or the saponin extract externally significantly shortens the bleeding time as compared to no treatment (control).…”

“…15 However, we modified their protocol, transecting the tail 5 mm from the tip as compared to 1 mm in the previous study, specifically to increase the bleeding time. 15 Applying notoginseng or the saponin extract externally significantly shortens the bleeding time as compared to no treatment (control). The results of our study are likely due to a component of notoginseng rather than being due to the effects of the powder itself, as the bleeding times were significantly lower than the placebo group (when flour was administered) as well.…”

“…Our bleeding model was modified from that of others. 14,15 Bleeding was induced by transection of the rat tail 5 mm from the tip. The blood was blotted by the same investigator with number 3 filter paper at 15-second intervals until no blood was observed on the paper upon blotting.…”

An Evaluation of the Hemostatic Effects of Hydrophilic, Alcohol, and Lipophilic Extracts of Notoginseng

“…Our bleeding model was modified from that of others 8,9 but was the same as the model in our previous study. 5 Bleeding was induced by transection of the rat's tail 5 mm from the tip.…”