[Ca²⁺]_i elevations and thrombus formation in mouse platelets adhering to acid-soluble type I collagen under flow. Mice were anesthetized and blood was drawn from the retro-orbital plexus, using heparin-coated capillary tubes, or by cardiac puncture. Platelets were washed, loaded with FLUO 3-AM (16 μM), suspended (5 × 10⁷/mL) with washed human erythrocytes (40% hematocrit) in HEPES–Tyrode buffer, pH 7.4, containing 2 mM Ca²⁺ and Mg²⁺ and 1.75 mM probenecid, and perfused over immobilized acid-soluble type I collagen at the shear rate of 400 (A-F) or 1500 seconds (G). (A,C) Platelets from WT mice exhibited α-like (short-lasting) and γ-like (long-lasting) [Ca²⁺]_i peaks. (B) Platelets from GPVI^−/− mice exhibited only α-like peaks. (D) Platelets from α2^−/− mice exhibited no [Ca²⁺]_i oscillations and only transient contact with the surface. (E) Number of platelets exhibiting α-like and γ-like [Ca²⁺]_i elevations (activated platelets) calculated relative to total adhering platelets. (F) Percentage of firmly adherent platelets calculated as described in “Measurement of Ca²⁺ mobilization in adhering platelets.” Data in panels B and C are the mean ± 95% CI of 3 different experiments. (G) Volume of thrombi over a 0.38 mm² collagen surface after perfusion for 3 minutes at 37°C of blood containing 40 U/mL heparin and 10 μM mepacrine. Data are the mean ± SEM of measurements in 5 different positions of the chamber. The 3 images show the thrombi formed by fluorescent platelets at the end of perfusion; only single platelets are seen in the case of GPVI^−/− and α2^−/− mice. Significant difference from the corresponding control (WT): *P < .05; **P < .01; ***P < .001.