Fig. 4: The anti-mGPV antibody DOM/B interferes with thrombin-mediated cleavage of GPV and reproduces the Gp5−/− phenotype.
From: Platelet glycoprotein V spatio-temporally controls fibrin formation
a, Thrombin-mediated cleavage of GPV was assessed by flow cytometry. Mean ± s.d., n = 3, four independent experiments. b–e, Recalcified whole blood was incubated in vitro with 10 µg ml−1 anti-mGPV antibody before perfusion over collagen–TF spots. Quantification of fibrin generation during blood flow (b,d) and time to fibrin formation (c,e). b, Mean ± s.e.m., n = 8, three independent experiments, two-tailed unpaired t-test with Welch’s correction. P values: 4.5 min, P = 0.0430; 5 min, P =, 0.0029; 5.5 min, P = 0.0092; 6 min, P = 0.0186. c, Mean ± s.d., n = 8, four independent experiments, two-tailed unpaired t-test, P = 0.0010. d, Mean ± s.e.m., n = 3, three independent experiments. e, Mean ± s.d., three independent experiments, WT, n = 6; WT + DOM/C, n = 5. Quantification (f) and representative images (g) of thrombus formation upon FeCl3-induced injury of mesenteric arterioles. WT (WT + control immunoglobulin (Ig)G), n = 15 arterioles of eight mice; WT + DOM3, n = 11 arterioles of six mice; WT + DOM/C, n = 6 arterioles of four mice; WT + DOM/B, n = 9 arterioles of six mice. DOM/B versus WT, P = 0.0008. The symbol # indicates occlusive thrombus formation. Scale bar, 50 µm. h–k, Tail-bleeding time assays. h, WT (with control IgG), n = 8; WT + DOM/B, n = 11; Rhoafl/fl, Pf4-Cre, n = 9; Rhoafl/fl, Pf4-Cre + DOM/B, n = 10. Rhoafl,fl, Pf4-Cre + DOM/B versus Rhoafl,fl, Pf4-Cre, P = 0064; DOM/B versus Rhoafl,fl, Pf4-Cre, P = 0.002. i, WT (with control IgG), n = 15; Nbeal2−/−, n = 15; Nbeal2−/− + DOM/B, n = 22. Open versus occluded vessels: WT versus Nbeal2−/−, P < 0.0001; Nbeal2−/− + DOM/B versus Nbeal2−/−, P < 0.0001; comparison of occluded vessels: Nbeal2−/− + DOM/B versus WT, P < 0.0001; WT versus Nbeal2−/−, P = 0.0006. j, WT mice were injected with platelet-depletion antibody to induce thrombocytopenia (5–10% of normal counts). WT (with control IgG), n = 18; thrombocytopenia, n = 9; thrombocytopenia + DOM/B, n = 11; open versus occluded vessels: WT versus thrombocytopenic mice, P < 0.0001; thrombocytopenic mice + DOM/B versus thrombocytopenic mice, P = 0.0003; comparison of occluded vessels: thrombocytopenic mice + DOM/B versus WT, P = 0.0251. k, n = 19 (all groups). Open versus occluded vessels: WT versus WT + clopidogrel, P = 0.0031; WT + DOM/B + clopidogrel versus WT + clopidogrel, P = 0.0188; comparison of occluded vessels: WT + DOM/B + clopidogrel versus WT + clopidogrel, #P = 0.0383. f,h–k, Horizontal line represents mean. Horizontal dashed line in c, e, f and h–k indicates the end of the experiment; every dot above it did not show any occlusion/fibrin formation within the observation period. Two-tailed Kruskal–Wallis test followed by Dunn’s multiple-comparison test to compare occluded vessels. Two-tailed Fisher’s exact test was used to compare occluded versus non-occluded vessels. *,#P < 0.05, **P < 0.01, ***P < 0.001.
