Adhesion and spreading of transgenic mouse platelets to immobilized VWF under static conditions is unaffected by loss of filamin A interaction. (A) hGPIbα^WT and hGPIbα^FW platelets were allowed to adhere to human VWF (5 μg/mL) for 60 minutes in the presence of ristocetin (1.5 mg/mL), botrocetin (5 μg/mL), or botrocetin + Xia.B2 murine GPIbα blocking antibody (20 μg/mL). Results are mean (± SEM) from at least 4 independent experiments and demonstrate no significant difference in the total number of adherent platelets under all conditions tested. Ristocetin-dependent adhesion and botrocetin-dependent adhesion in the presence of Xia.B2 are identical and represent adhesion to human VWF mediated specifically by hGPIbα. (B) Platelet spreading on VWF (+ 1.5 mg/mL ristocetin) was analyzed from DIC images (at least 3 separate fields from 3 independent experiments) and demonstrated no significant difference between hGPIbα^WT and hGPIbα^FW platelets. Platelets treated with inhibitors of ADP and thromboxane (1 U/mL apyrase, a combination of 10μM MeSAMP and 100μM MRS2179, or MeSAMP/MRS2179 and 10μM indomethacin) demonstrated that integrin α_IIbβ₃ activation occurred directly downstream from GPIbα without a contribution from these secondary mediators. (C) Oregon Green fibrinogen binding to platelets adherent to human VWF (in the presence of 1.5 mg/mL ristocetin) was no different between hGPIbα^WT and hGPIbα^FW mice, indicating that integrin α_IIbβ₃ activation is unaffected by the loss of filamin A binding to GPIbα. DIC and fluorescent images were overlayed using Metamorph and are representative of 4 independent experiments. Scale bar represents 10 μm.