Platelet S1P₁is dispensable for platelet aggregation and spreading in mice. Platelets from mice in which S1pr1 was deleted in MKs (S1pr1^f/f:Pf4Cre⁺; green), littermate controls (orange), or wild-type mice (gray) were isolated, washed, and tested for their capacity to aggregate (A) and spread (B). (A, upper) Platelet aggregation in response to submaximal concentrations (25 μM) of PAR4-AP (thrombin receptor agonist) in the absence (left) or presence of exogenous S1P (0.1-10 μM; middle) or of S1P₁ agonist SEW-2871 (0.5 μM; right). (A, lower) Platelet aggregation in response to submaximal PAR4-AP in the presence of S1P (0.1 μM) in the presence or absence of S1P₁ antagonist W146 (10 μM; left) or in response to the weak platelet agonist ADP (2 μM; right), with and without exogenous S1P (10 μM) or P2Y12 antagonism (2MeSAMP, 40 μM) to address potential redundancy with P2Y12, which, similar to S1P₁, is Gαi coupled. (B) Representative scanning electron microscopy images (upper) showing the extent of platelet spreading 15 and 30 minutes after plating on fibrinogen in the presence of S1P (0.5 μM; quantification in supplemental Figure 7). Note that although S1P did not trigger aggregation (not shown), it slightly increased PAR4-AP induced aggregation. However, neither aggregation nor spreading was influenced by selective S1P₁ modulation or S1pr1 deficiency. S1P also could not compensate for the absence of functional P2Y12 by alternative engagement of Gαi. Statistical analyses by 2-way analysis of variance or the Mann-Whitney U test, as appropriate. Mean ± standard deviation is shown, symbols represent the number of mice.