Inhibition of αIIbβ3 prevents proplatelet formation on fibrinogen, vitronectin, and VWF/botrocetin, but not on laminin. (A) Bone marrow aspirates from TPO-treated mice were plated on either 100 μg/mL fibrinogen (Fg) or 50 μg/mL laminin (Lam) in the presence of 10 μM lotrafiban, an αIIbβ3 antagonist. Cells were allowed to adhere for 3 hours at 37°C prior to PBS washing and fixation. Cells were visualized with αIIb-alkaline phosphatase staining, and the total number of adherent megakaryocytes and megakaryocytes with proplatelets was assessed for each coverslip. *Significant difference between control and lotrafiban treatment; + significant difference between fibrinogen and laminin. (B) Bone marrow aspirates were plated on fibrinogen, 10 μg/mL vitronectin (Vn), or 10 μg/mL VWF plus 3 μg/mL botrocetin (to ensure megakaryocyte/VWF binding under static conditions) in the presence or absence of an anti-αIIb antibody. The total number of adherent megakaryocytes and proplatelet-bearing megakaryocytes was determined as in panel A. *Significant difference between control and anti-αIIb treatment; + significant difference between fibrinogen and the other matrices. (C) Absolute cell numbers of adherent megakaryocytes and proplatelet-bearing megakaryocytes on vitronectin, VWF/botrocetin, 100 μg/mg collagen (Col), or laminin were normalized to the absolute amounts seen on fibrinogen in each individual experiment, with equal numbers equaling 100% relative to fibrinogen (solid line). Error bars indicate SEM, n = 3.
