GPVI blockade via intervention with JAQ1 antibody mitigates elastase AAA expansion. (A) Study schematic. Male Ldlr−/− mice (aged 8-10 weeks) underwent laparotomy and elastase (5 μL of 10 mg/mL porcine pancreatic elastase; closed circles) or HI elastase (open circles) as sham was applied to the adventitia of infrarenal aorta for 5 minutes to initiate AAA. At 14 days after laparotomy, mice were blindly randomized while normalized for aortic diameter and given either a 1-time IP injection of 50-μg JAQ1 antibody (n = 16, blue) or 50-μg IgG control (n = 13, red). After another 14 days, mice were euthanized, final aortic diameter was measured ex vivo, and tissues were harvested for further analysis. (B) Intraluminal aortic diameters measured by weekly ultrasound (∗∗∗P < .001, elastase-IgG vs elastase-JAQ1 by 3-way ANOVA with Holm-Sidak post hoc analysis). (C) Aortic diameters in JAQ1-treated vs control-treated mice measured ex vivo 28 days after elastase surgery (∗∗∗P < .001, elastase vs HI and elastase-IgG vs elastase-JAQ1 by 2-way ANOVA with Holm-Sidak post hoc analysis). (D) Representative images of aortas ex vivo from the indicated groups. Platelet activation measured 14 days after IgG or JAQ1 antibody intervention indicated by surface P-selectin (FITC) levels in response to varying doses of the GPVI specific agonist convulxin (E) or thrombin (F) in JAQ1 vs IgG-treated mice measured by flow cytometry (n = 4-6 per group). As expected, a significant reduction in convulxin-mediated (E) activation was observed in platelets isolated from mice treated with JAQ1 antibody (P < .001, for the interaction between intervention and platelet treatment assessed by 3-way ANOVA; ∗∗∗P < .001, between JAQ1 and IgG by Holm-Sidak post hoc analysis). (F) In response to thrombin, a significant increase in platelet activation was observed in mice with a AAA (closed circles) compared with mice without a AAA (open circles) (P = .024, for the interaction between AAA status and platelet treatment assessed by 3-way ANOVA; ∗∗∗P <.001, for comparison of surgery within 1 U/mL thrombin treatment by Holm-Sidak post hoc analysis). No significant effect of JAQ1 intervention on thrombin mediate platelet activation was noted (P = .140, for the interaction between intervention and platelet treatment). (G) Representative flow cytometry scatterplots from the indicated conditions. Data are represented as individual data points or mean ± SEM. FITC, fluorescein isothiocyanate; IP, intraperitoneal.
Figure 5.

GPVI blockade via intervention with JAQ1 antibody mitigates elastase AAA expansion. (A) Study schematic. Male Ldlr−/− mice (aged 8-10 weeks) underwent laparotomy and elastase (5 μL of 10 mg/mL porcine pancreatic elastase; closed circles) or HI elastase (open circles) as sham was applied to the adventitia of infrarenal aorta for 5 minutes to initiate AAA. At 14 days after laparotomy, mice were blindly randomized while normalized for aortic diameter and given either a 1-time IP injection of 50-μg JAQ1 antibody (n = 16, blue) or 50-μg IgG control (n = 13, red). After another 14 days, mice were euthanized, final aortic diameter was measured ex vivo, and tissues were harvested for further analysis. (B) Intraluminal aortic diameters measured by weekly ultrasound (∗∗∗P < .001, elastase-IgG vs elastase-JAQ1 by 3-way ANOVA with Holm-Sidak post hoc analysis). (C) Aortic diameters in JAQ1-treated vs control-treated mice measured ex vivo 28 days after elastase surgery (∗∗∗P < .001, elastase vs HI and elastase-IgG vs elastase-JAQ1 by 2-way ANOVA with Holm-Sidak post hoc analysis). (D) Representative images of aortas ex vivo from the indicated groups. Platelet activation measured 14 days after IgG or JAQ1 antibody intervention indicated by surface P-selectin (FITC) levels in response to varying doses of the GPVI specific agonist convulxin (E) or thrombin (F) in JAQ1 vs IgG-treated mice measured by flow cytometry (n = 4-6 per group). As expected, a significant reduction in convulxin-mediated (E) activation was observed in platelets isolated from mice treated with JAQ1 antibody (P < .001, for the interaction between intervention and platelet treatment assessed by 3-way ANOVA; ∗∗∗P < .001, between JAQ1 and IgG by Holm-Sidak post hoc analysis). (F) In response to thrombin, a significant increase in platelet activation was observed in mice with a AAA (closed circles) compared with mice without a AAA (open circles) (P = .024, for the interaction between AAA status and platelet treatment assessed by 3-way ANOVA; ∗∗∗P <.001, for comparison of surgery within 1 U/mL thrombin treatment by Holm-Sidak post hoc analysis). No significant effect of JAQ1 intervention on thrombin mediate platelet activation was noted (P = .140, for the interaction between intervention and platelet treatment). (G) Representative flow cytometry scatterplots from the indicated conditions. Data are represented as individual data points or mean ± SEM. FITC, fluorescein isothiocyanate; IP, intraperitoneal.

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