M023 2

For platelet aggregation experiment, the washed platelets from WT and Pear1^−/− mice were diluted to 3 × 10⁸/mL with tyrode’s buffer. And the 300 µL platelet concentrates were stimulated with 1.5 µg/mL collagen (P/N385, Chrono-log) or 0.05 U/mL thrombin (HT4082A, Enzyme Research Laboratories) and measured using an aggregometer (Chrono-Log) according to the manufacturer’s protocol.
For detection of P-selectin exposure and JON/A binding in platelet, platelets (5 × 10⁷ platelets/mL) were stained with FITC-conjugated anti-mouse P-selectin antibody (553744, BD Biosciences, 1:100) or JON/A(PE) (M023-2, Emfret Analytics, 1:50) for 20 min at 37 °C in a 50 µL volume. The reaction was stopped by addition of 450 µL tyrode’s buffer and then analyzed by a flow cytometer.
For clot retraction, mouse platelets were processed as previously described^{38 (link)}. Clot size was quantified from photographs using National Institutes of Health Image J software, and retraction was expressed as retraction ratio (1-[final clot size/initial clot size]).

Platelet and preplatelet counts were determined pre-/post-splenectomy by flow cytometry as described^{28 (link)}. Preplatelets were counted as GPV⁺/GPIIbIIIa⁺ events that have larger forward/sideward scatter characteristic than platelets. Heparinised blood incubated with antibodies against activated GPIIbIIIa (M023-2) and CD62P (M130-1; both Emfret Analytics) eliminated preplatelets as microaggregates (Supplemental Figure 4D).