Figure 2.
Platelet ERp46 is required for hemostasis, thrombosis, and platelet accumulation into a growing thrombus. (A-B) Characterization of Pf4-Cre/ERp46fl/fl mice. (A) PCR products of tail DNA from Cre− ERp46fl/fl mice and Pf4-Cre/ERp46fl/fl mice. The bands represent PCR product from the ERp46-floxed allele (upper panel, 431-bp) and the Cre gene (lower panel, 420-bp), respectively. (B) Western blots of platelet lysates using a polyclonal rabbit anti-ERp46 antibody and antibodies against PDI, ERp57, ERp72, and ERp5. Shown are the actin loading controls. Left panel representative blot; right panel, quantitative analysis of protein level by densitometry of band density of PDIs relative to the ERp46fl/fl wild-type (WT) control, which was set at 100%; mean ± SEM, ****P < .0001, n = 4, Student t test. (C) Tail bleeding times; mean ± SEM, n = 16 for each group, ****P < .001, Student t test. (D-E) Incorporation of platelets into a growing thrombus in ERp46fl/fl mice and Pf4-Cre/ERp46fl/fl mice was detected by Alexa 488 anti-CD41 using FeCl3-induced mesenteric arterial injury. Mean artery diameters were 140.2 ± 3.35 μm (SEM) in ERp46fl/fl mice, 136.0 ± 3.73 μm in Pf4-Cre/ERp46fl/fl mice (P = not significant [ns]), 136.3 ± 3.24 μm in Pf4-Cre/ERp46fl/fl mice plus rERp46 (P = ns), 132.6 ± 5.21 μm in Pf4-Cre/ERp46fl/fl mice plus rERp46(aaaaaa), P = ns, ANOVA. (D) Images at 7, 11, and 15 minutes. Dotted lines mark the vessel wall. Scale bar, 200 μm. Composite of fluorescence intensity (FI) per area analyzed (FI/μm2) in ERp46fl/fl (21 thrombi from 8 mice), Pf4-Cre/46fl/fl (20 thrombi from 8 mice), Pf4-Cre/46fl/fl plus rERp46 (24 thrombi from 8 mice), and Pf4-Cre/ERp46fl/fl plus rERp46(aaaaaa) (25 thrombi from 9 mice); mean ± SEM, *P < .05, ****P < .0001, ANOVA. (F-H) Cremaster laser injury in arterioles of Pf4-Cre/ERp46fl/fl mice and their Cre− ERp46fl/fl littermate control mice. Platelets at the site of injury were detected using anti-CD41 F(ab)2 fragments conjugated to Alexa Fluor 647. (F) Representative fluorescence images from widefield intravital microscopy for platelet accumulation (red) at the indicated time points after injury. (G) The mean ± SEM integrated FIs of anti-CD41 F(ab)2 fragments over 240 seconds from ERp46fl/fl (37 thrombi from 4 mice) and Pf4-Cre/ERp46fl/fl (26 thrombi from 4 mice). (H) The areas under the FI curves over 240 seconds were analyzed with a Mann-Whitney U test; **P < .01. (I) Time to occlusion of FeCl3-induced carotid artery thrombosis in Pf4-Cre/ERp46fl/fl mice compared with ERp46fl/fl littermate controls; ***P < .001, Student t test. ns, not significant.

Platelet ERp46 is required for hemostasis, thrombosis, and platelet accumulation into a growing thrombus. (A-B) Characterization of Pf4-Cre/ERp46fl/fl mice. (A) PCR products of tail DNA from Cre− ERp46fl/fl mice and Pf4-Cre/ERp46fl/fl mice. The bands represent PCR product from the ERp46-floxed allele (upper panel, 431-bp) and the Cre gene (lower panel, 420-bp), respectively. (B) Western blots of platelet lysates using a polyclonal rabbit anti-ERp46 antibody and antibodies against PDI, ERp57, ERp72, and ERp5. Shown are the actin loading controls. Left panel representative blot; right panel, quantitative analysis of protein level by densitometry of band density of PDIs relative to the ERp46fl/fl wild-type (WT) control, which was set at 100%; mean ± SEM, ****P < .0001, n = 4, Student t test. (C) Tail bleeding times; mean ± SEM, n = 16 for each group, ****P < .001, Student t test. (D-E) Incorporation of platelets into a growing thrombus in ERp46fl/fl mice and Pf4-Cre/ERp46fl/fl mice was detected by Alexa 488 anti-CD41 using FeCl3-induced mesenteric arterial injury. Mean artery diameters were 140.2 ± 3.35 μm (SEM) in ERp46fl/fl mice, 136.0 ± 3.73 μm in Pf4-Cre/ERp46fl/fl mice (P = not significant [ns]), 136.3 ± 3.24 μm in Pf4-Cre/ERp46fl/fl mice plus rERp46 (P = ns), 132.6 ± 5.21 μm in Pf4-Cre/ERp46fl/fl mice plus rERp46(aaaaaa), P = ns, ANOVA. (D) Images at 7, 11, and 15 minutes. Dotted lines mark the vessel wall. Scale bar, 200 μm. Composite of fluorescence intensity (FI) per area analyzed (FI/μm2) in ERp46fl/fl (21 thrombi from 8 mice), Pf4-Cre/46fl/fl (20 thrombi from 8 mice), Pf4-Cre/46fl/fl plus rERp46 (24 thrombi from 8 mice), and Pf4-Cre/ERp46fl/fl plus rERp46(aaaaaa) (25 thrombi from 9 mice); mean ± SEM, *P < .05, ****P < .0001, ANOVA. (F-H) Cremaster laser injury in arterioles of Pf4-Cre/ERp46fl/fl mice and their Cre− ERp46fl/fl littermate control mice. Platelets at the site of injury were detected using anti-CD41 F(ab)2 fragments conjugated to Alexa Fluor 647. (F) Representative fluorescence images from widefield intravital microscopy for platelet accumulation (red) at the indicated time points after injury. (G) The mean ± SEM integrated FIs of anti-CD41 F(ab)2 fragments over 240 seconds from ERp46fl/fl (37 thrombi from 4 mice) and Pf4-Cre/ERp46fl/fl (26 thrombi from 4 mice). (H) The areas under the FI curves over 240 seconds were analyzed with a Mann-Whitney U test; **P < .01. (I) Time to occlusion of FeCl3-induced carotid artery thrombosis in Pf4-Cre/ERp46fl/fl mice compared with ERp46fl/fl littermate controls; ***P < .001, Student t test. ns, not significant.

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