Inhibition of Src kinases, but not MAP kinases, blocks thrombus formation at high shear. (A) Heparinized mouse blood was incubated with either vehicle DMSO (i; 0.1%), the anti-GPIbα monoclonal antibody (mAb) 6D1 (ii; 10 μg/mL), the p42/44 MAP kinase inhibitors PD184161 (iii; 20 μM) and U0126 (iv; 10 μM), or the Src kinase inhibitor PD173952 (v; 25 μM) for 5 minutes at room temperature prior to being flowed through 1 × 0.1-mm microslides coated with collagen (30 μg/mL) at 800 s^-1 for 2 minutes. Excess blood was removed from microslides with modified Ca²⁺-free Tyrode buffer flowed at 800 s^-1 for 8 minutes. Thrombi were visualized by light microscope (original magnification × 630). (B) Thrombi were lysed with ice-cold lysis buffer containing NP-40 (1% [vol/vol]). Whole cell lysates (WCL) were resolved by SDS-PAGE and immunoblotted for p42/44 MAP kinase with an anti–phospho-p42/44 MAP kinase monoclonal antibody. Membranes were stripped and reprobed with anti-p42/44 MAP kinase polyclonal antibody to determine equal lane loading. Images are representative of 3 experiments performed.