Fig. 3
From: Endoplasmic reticulum protein 29 negatively regulates platelet functions and thrombosis in mice
ERp29 deficiency shortens the bleeding time and potentiates thrombosis. (A-B) Characterization of Pf4-Cre/ERp29fl/fl mice. (A) PCR products of tail DNA from ERp29fl/fl and Pf4-Cre/ERp29fl/fl mice. The ERp29-floxed allele yielded a 614 bp product and Pf4-Cre recombinase was 420 bp. (B) Western blot analysis of ERp29 expression in platelets, white blood cells (WBCs) and endothelial cells (ECs) from ERp29fl/fl and Pf4-Cre/ERp29fl/fl mice. GAPDH serves as loading control. (C) Tail bleeding time in ERp29fl/fl and Pf4-Cre/ERp29fl/fl mice; Mean ± SEM. *P <.05, Student’s t test. (D-E) Incorporation of platelets into a growing thrombus in ERp29fl/fl and Pf4-Cre/ERp29fl/fl mice was detected by Dylight-488-conjugated anti-CD42c after FeCl3 (4%)–induced mesenteric arterial injury. Images at 7, 11, and 15 min. The dotted lines mark the vessel wall. Scale bar, 200 μm. The mean artery diameter was 168.1 ± 6.316 μm in ERp29fl/fl mice and 175.8 ± 6.959 μm in Pf4-Cre/ERp29fl/fl mice. (P = not significant, NS). (E) Composite of fluorescence intensity (FI) per area analyzed (FI/µm2) in ERp29fl/fl (32 thrombi from 5 mice), Pf4-Cre/ERp29fl/fl (26 thrombi from 4 mice); Mean ± SEM, **P <.01, ***P <.001, ANOVA. ns, not significant. (F) Platelet adhesion on collagen under flow conditions. Whole blood was labeled with Dylight-488-conjugated anti-CD42c and perfused through collagen–coated BioFlux plates at 40 dynes/cm2 for 5 min. Scale bar, 100 μm. Platelet adhesion (covered area) was quantified using Image J; Mean ± SEM, ****P <.0001, Student’s t test