Defective agonist-induced Ca²⁺ response and aggregate formation under flow in Orai1^−/− platelets. Fura-2–loaded wild-type (black line) or Orai1^−/− (gray line) platelets were stimulated with thrombin (0.1 U/mL), ADP (10 μM), or CRP (10 μg/mL) in calcium-free medium or in the presence of extracellular Ca²⁺ (1 mM), and [Ca²⁺]_i was monitored. Representative measurements (A) and maximal Δ[Ca ²⁺]_i plus or minus SD (n = 4 per group; B) are shown. (C) Impaired aggregation of Orai1^−/− platelets (gray lines) in response to collagen, but not ADP and thrombin. (D) Flow cytometric analysis of αIIbβ3 integrin activation (left panel) and degranulation-dependent P-selectin exposure (right panel) in response to thrombin (0.1 U/mL), ADP (10 μM), CRP (10 μg/mL), and CVX (1 μg/mL). Results are means plus or minus SD of 6 mice per group. (E) Orai1^−/− platelets in whole blood fail to form stable thrombi when perfused over a collagen-coated (0.2 mg/mL) surface at a shear rate of 1700 s⁻¹. (Top) Representative phase contrast images. (Bottom) Mean surface coverage (left) and relative platelet deposition as measured by the integrated fluorescent intensity (IFI) per square millimeter (right) plus or minus SD (n = 4). Bar represents 30 μm. *P < .05; ***P < .001.