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. Author manuscript; available in PMC: 2022 Jan 1.
Published in final edited form as: Arterioscler Thromb Vasc Biol. 2020 Nov 12;41(1):234–249. doi: 10.1161/ATVBAHA.120.314879

Figure 4. EGTA treatment protected against cold-induced platelet activation and rapid clearance.

Figure 4.

A, Washed platelets (3 × 108/ml) from C57BL/6J mice were preincubated with vehicle or EGTA (100 μM) for 15 min at RT and then stored at RT or 4°C for indicated time. Platelets accounts were measured and shown as relative to the counts at 0 h. The statistical differences were examined by Student’s t-test. Values are means ± s.d. (n=3). * p<0.05. B, Washed platelets from C57BL/6J mice were preincubated with EGTA for 15 min at RT and then stored 4°C for 24 h. Platelets were warmed in 37°C for 15 min and added with 1 mM CaCl2. Platelet aggregation was induced by adding 0.05 U/ml thrombin. C and D, Washed platelets from C57BL/6J mice were pretreated with 100 μM EGTA or 1 mM RGDS for 15 min. Platelets were then stored at 4°C for 24 h. The amount of serotonin (C) and PF4 (D) in supernatant were measured as described in the methods. E and F, Washed platelets from C57BL/6J mice were stored at RT for indicated lengths of time. Platelets were also stored at 4°C in the presence of EGTA (100 μM). Platelets were prewarmed at 37°C for 15 min and added with 1 mM CaCl2. Aggregation was induced by addition of thrombin. G, Washed platelets from C57BL/6-Tg(CAGEGFP)1Osb/J mice were preincubated with EGTA for 15 min at RT and then stored at cold for 24 h. Pre-warmed platelets (2.5 × 108 in 0.2 ml per mouse) were injected retro-orbitally injected to 8 weeks old C57BL/6J mice. Blood samples were collected at various time points after transfusion (n=4–6). H, PRP from C57BL/6J mice were stored at RT or 4°C in the presence of absence of EGTA for 24 h, 48 h, and 72 h. Platelet counts were measured with a HEMAVET HV950FS multispecies hematology analyzer at indicated time and shown as relative platelet numbers to the basal platelet count. I, PRP from GFP mice were preincubated with vehicle or EGTA for 15 min at RT and then stored at 4°C for 24 h. Pre-warmed PRP (2.5 × 108 platelets in 0.2 ml per mouse) was injected retro-orbitally to 8 weeks old C57BL/6J mice (n=3–6).