In vivo hemostasis and in vitro platelet aggregate formation. (A) Tail-bleeding assay measuring blood volume loss following removal of 2 mm from the tip of the tail in both control and JAK2V617F animals. Blood volume loss was significantly less in the JAK2V617F animals (n = 6; error bars represent SD). (B) Platelet aggregate formation was assessed in a collagen-coated flow chamber at arterial shear rate (1000 s⁻¹) using whole blood anticoagulated with heparin and D-Phe-Pro-Arg-CMK. Surface coverage was increased in JAK2V617F animals as illustrated in the micrographs taken after 4 minutes of flow. Thrombi volume was assessed by protein quantification by western blot using a tubulin antibody. Densitometry results, expressed relative to control, show a statistically significant increase in thrombus formation in JAK2V617F animals (n = 5; error bars represent SD). (C) To determine whether this increase was a reflection of the raised platelet count in the JAK2V617F mice or attributable to an increase in platelet reactivity, the same experiments were performed after correcting the platelet count in JAK2V617F samples to control level by addition of mouse plasma. Mouse plasma was generated from heparinized mouse blood centrifuged at 150g to generate PRP and then 1000g to generate PPP. Blood counts pre- and postcorrection are presented in the top panel (mean and range). Surface coverage was again increased, and thrombi volume remained higher in the Jak2V617F samples compared with control (n = 4; error bars represent SD).