Generation of a mouse model lacking PS expression in platelets (Pros1^lox/loxPf4-Cre⁺). (A) Genotyping results of mice with normal PS expression (Pros1^lox/loxPf4-Cre⁻), Pros1^lox/−, and Pros1^lox/loxPf4-Cre⁺ mice using genomic DNA extracted from ear tissues. The presence of lox sequences in Pros1 alleles and of Pf4-Cre transgene was evaluated by 2 independent multiplex PCRs. PCR products were then subjected to electrophoresis. The lox band had a lower molecular weight (338 bp) compared with the null band (570 bp), in accordance with Saller et al²⁵ ; the Pf4-Cre⁺ band has a higher molecular weight (450 bp) in comparison with the internal PCR control (324 bp). (B) PS antigen levels in platelet lysates from Pros1^lox/loxPf4-Cre⁻, Pros1^lox/loxPf4-Cre⁺, and Pros1^lox/− mice. Results are expressed as mean ± standard error of the mean (SEM) of percentage relative to the pooled normal platelet lysate. **P ≤ .01; ****P ≤ .0001. (C) PS antigen levels in plasma samples from Pros1^lox/loxPf4-Cre⁻, Pros1^lox/loxPf4-Cre⁺, and Pros1^lox/− mice. Results are expressed as mean ± SEM of percentage relative to the pooled normal plasma. ns, not significant; ****P ≤ .0001. (D-E) PS isoforms in plasma and platelet after activation by thrombin. Western blotting after sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions was performed using a monoclonal antibody raised against murine PS (D) and a polyclonal antibody against human PS (E). P, platelet; S, platelet releasate; Thr, thrombin. (F) Confocal microscopy of resting and thrombin-activated mouse Pros1^lox/loxPf4-Cre⁻ and Pros1^lox/loxPf4-Cre⁺ platelets, stained for -tubulin (in magenta), CD62p (in green), and PS (in red). Scale bar, 2 μm. Acquired z stacks were used for volumes and surface rendering by Imaris software.