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. Author manuscript; available in PMC: 2019 Apr 26.
Published in final edited form as: Arterioscler Thromb Vasc Biol. 2013 Feb 28;33(5):926–934. doi: 10.1161/ATVBAHA.112.300672

Figure 5.

Figure 5

Analysis of Glycoprotein VI (GPVI) and C-type lectin-like receptor 2 (CLEC-2) double-mutant mice. A, Flow cytometric analysis of surface protein expression of Gp6−/−/Clec-2fl/fl, Pf4-Cre platelets. Platelets were stained for 15 minutes at room temperature with the indicated fluorophore-labeled antibodies and directly analyzed. Platelet count in number of platelets/µL. Platelet size is given as mean forward scatter (FSC) and was determined by FSC characteristics. Results are mean fluorescence intensities (MFI)±SD (n=5, representative of at least 3 independent measurements). *P<0.05; **P<0.01; ***P<0.001. B, Flow cytometric analysis of degranulation-dependent P-selectin exposure and integrin αIIbβ3 activation on platelets. Washed blood was incubated with the indicated agonists for 15 minutes at room temperature and analyzed on a FACSCalibur. Results are mean±SD (n=4 mice per group, representative of 3 individual experiments). **P<0.01; ***P<0.001. ADP [µmol/L]; U46619: [µmol/L]; thrombin (thr): [U/mL]; rhodocytin (RC): [µg/mL]; collagen-related peptide (CRP): [µg/mL]; convulxin (CVX): [µg/mL]. C, A 1-mm segment of the tail tip was cut, and bleeding was determined to have ceased when no blood drop was observed on the filter paper. Each symbol represents 1 individual. Fisher test: Gp6−/−/Clec-2fl/fl, Pf4-Cre vs control: 0.0351; Gp6−/−/Clec-2fl/fl, Pf4-Cre vs Clec-2fl/fl, Pf4-Cre: 0.0087. Other conditions nonsignificant. D, An 1-mm segment of the tail tip was cut, and the tail tip was immersed in saline. Each symbol represents 1 individual. Bleeding time of Gp6−/−/Clec-2fl/fl, Pf4-Cre mice is prolonged compared with control and Clec-2fl/fl, Pf4-Cre*, P<0.05.