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. Author manuscript; available in PMC: 2023 May 18.
Published in final edited form as: Nat Cardiovasc Res. 2023 Mar 23;2(4):368–382. doi: 10.1038/s44161-023-00254-6

Fig. 4. The anti-mGPV antibody DOM/B interferes with thrombin-mediated cleavage of GPV and reproduces the Gp5−/− phenotype.

Fig. 4.

(a) Thrombin-mediated cleavage of GPV was assessed by flow cytometry. Mean ± SD, n=3, 4 independent experiments. (b-e) Recalcified whole blood was incubated in vitro with 10 μg/ml anti-mGPV antibody prior to perfusion over collagen/TF spots. Quantification of fibrin generation during blood flow (b, d) and time to fibrin formation (c, e). (b) Mean ± SEM, n=8, 3 independent experiments, two-tailed unpaired t-test with Welch’s correction. 4.5 min: p=0.0430; 5 min: p=0.0029; 5.5 min: p= 0.0092; 6 min: p=0.0186. (c) Mean ± SD, n=8, 4 independent experiments, two-tailed unpaired t-test, p=0.0010. (d) Mean ± SEM, n=3, 3 independent experiments. (e) Mean ± SD, 3 independent experiments, WT: n=6, WT+DOM/C: n=5. SAC: Surface area coverage. (f) Quantification and (g) representative images of thrombus formation upon FeCl3-induced injury of mesenteric arterioles. WT (= WT + control IgG): n=15 arterioles of 8 mice, WT+DOM3: n=11 arterioles of 6 mice, WT+DOM/C: n=6 arterioles of 4 mice, WT+DOM/B: n=9 arterioles of 6 mice. DOM/B vs. WT: p=0.0008. #: 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 vs. RhoAfl,fl Pf4-cre: p=0064; DOM/B vs. RhoAfl, fl Pf4-cre: p=0.002. (i) WT (with control IgG): n=15, Nbeal2−/−: n=15, Nbeal2−/−+DOM/B: n=22. Open vs. occluded vessels: WT vs. Nbeal2−/−: p< 0.0001, Nbeal2−/− + DOM/B vs. Nbeal2−/−: p<0.0001, comparison of occluded vessels: Nbeal2−/− + DOM/B vs. WT: p<0.0001; WT vs. 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 vs. occluded vessels: WT vs. thrombocytopenic mice: p< 0.0001, thrombocytopenic mice +DOM/B vs. thrombocytopenic mice: p=0.0003, Comparison of occluded vessels: thrombocytopenic mice +DOM/B vs. WT: p=0.0251. (k) n=19 (all groups). Open vs. occluded vessels: WT vs. WT + clopidogrel: p=0.0031, WT + DOM/B + clopidogrel vs. WT + clopidogrel: p=0.0188, Comparison of occluded vessels: WT + DOM/B + clopidogrel vs. WT + clopidogrel: #p=0.0383. (f, h-k) 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 vs. non-occluded vessels. *, # p < 0.05; **p < 0.01; ***p < 0.001.