Defective agonist-induced Ca²⁺ signaling and aggregate formation under flow in Stim1^−/− platelets. Fura-2–loaded wild-type (black line) or Stim1^−/− (gray line) platelets were stimulated with 0.1 U/ml thrombin, 10 μM ADP, or 10 μg/ml CRP in the presence of extracellular 1 mM EGTA or 0.5 mM Ca²⁺, and [Ca²⁺]_i was monitored. Representative measurements (A) and maximal increase in intracellular Ca²⁺ concentrations compared with baseline levels before stimulus (Δ[Ca ²⁺]_i) ± SD (n = 4 mice per group; B) are shown. (C) Impaired aggregation of Stim1^−/− platelets (gray lines) in response to CRP and collagen but not ADP and thrombin (recording time = 10 min). (D) Flow cytometric analysis of integrin αIIbβ3 activation (binding of JON/A-PE; left) and degranulation-dependent P-selectin exposure (right) in response to 0.1 U/ml thrombin, 10 μM ADP, 10 μg/ml CRP, and 1 μg/ml convulxin. Results are means ± SD (n = 6 mice per group). (E) Stim1^−/− 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 1,700 s⁻¹. (top) Representative phase-contrast images. (bottom) Mean surface coverage (left) and relative platelet deposition as measured by the integrated fluorescent intensity per square millimeter (right) ± SD (n = 4 mice). ***, P < 0.001. Bar, 100 μm.