PolyP is increased in platelet-specific XPR1-deficient mice. (A) Genotyping results of Xpr1fl/fl, Xpr1fl/+ Pf4-Cre, and Xpr1fl/fl Pf4-Cre mice. (B) Relative Xpr1 mRNA expression levels in megakaryocytes enriched from cultured bone marrow cells of Xpr1fl/fl and Xpr1fl/fl Pf4-Cre mice. (C) XPR1 signal in megakaryocyte-enriched bone marrow cell cultures of Xpr1fl/fl and Xpr1fl/fl Pf4-Cre mice measured by flow cytometry. SSC, side scatter. Percentages in the top right corner show the portion of gated cells. Megakaryocyte preparations derived from 2 (Xpr1fl/fl and Xpr1fl/fl Pf4-Cre) mice each analyzed for XPR1 content by western blot. β-Actin served as loading control (bottom). (D) Xpr1 mRNA expression in platelets of Xpr1fl/fl and Xpr1fl/fl Pf4-Cre mice. Xpr1 expression in Xpr1fl/fl platelets was set to 100%. (E) Plasma membranes isolated from 109 platelets of Xpr1fl/fl, Xpr1fl/fl Pf4-Cre, or Xpr1fl/+ Pf4-Cre mice were analyzed by western blot antibodies directed against the XPR1 N terminus. β-Actin served as the loading control. (F) Total polyP in platelets of Xpr1fl/fl, Xpr1fl/fl Pf4-Cre, or Xpr1fl/+ Pf4-Cre mice. Soluble polyP (G) and membrane-associated polyP (H) released by collagen-stimulated platelets measured as monophosphate in PPX-treated (50 μg/ml; 1 hour) platelet supernatants. Symbols represent individual mice. *P < .05, **P < .01, by 1-way analysis of variance and Tukey’s multiple comparison test. Platelet aggregation following activation by 1.5 µg/ml ristocetin (I) and 5 (J) or 10 (K) µg/ml collagen at 10 minutes. Transmission of suspended resting platelets is 0% and buffer, 100%. Student t test. n.s. nonsignificant.