Skip to main content
. Author manuscript; available in PMC: 2014 Dec 1.
Published in final edited form as: J Thromb Haemost. 2013 Dec;11(12):2190–2193. doi: 10.1111/jth.12428

Figure 1. Platelet responses to rhodocytin in the absence of Sema4D.

Figure 1

(A&B) Platelets from Sema4D(+/+) (WT) and Sema4D(−/−) mice were stimulated with rhodocytin at time zero. (C) Mean ± SEM, N≥5. N.S. = not significant. (D&E) Sema4D(−/−) platelets were preincubated with 80 µg/ml recombinant Sema4D (rS4D) for 10 min at room temperature and stimulated with 4.3 nM rhodocytin at time zero. Mean ± SEM, N=6. The dashed line indicates the time to half-maximal aggregation for WT platelets rhodocytin for comparison (from Figure 1C). (F) Platelets were stimulated with 8.9 nM rhodocytin with stirring. The extent of aggregation is indicated at the bottom of each lane. * refers to platelets that have changed shape but have no measurable aggregation; ** platelets that have completed shape change and just begun to aggregate. Proteins were precipitated with anti-Clec-2 and immunoblotted with phosphotyrosine antibody, 4G10P. Lysates were also probed for Syk phosphorylated on Y519/520. (G) WT platelets were incubated with 10 µM Integrilin for 10 min followed by 8.9 nM rhodocytin for 150 sec with or without stirring as indicated. Lysates were probed for phospho-Syk. N=3. A representative immunoblot is shown. (H) Platelets were activated with 8.9 nM rhodocytin under stirred conditions. Syk phosphorylation was detected with anti-pSyk. Mean ± SEM, N=3. (I) Platelets were activated under stirred conditions with 8.9 nM rhodocytin. Phosphorylation of Clec-2 was detected by immunoprecipitating with anti-Clec-2 and blotting with 4G10P (N=3).