Extended Data Fig. 7. DOM/B does not alter platelet responses upon stimulation with collagen or thrombin in vitro, despite interfering with thrombin-mediated cleavage of GPV.

(a-c) Washed platelets were incubated in vitro with 10 μg/ml of the indicated antibodies for 5 min and stimulated with Horm or soluble collagen. Light transmission was recorded on an Apact four-channel aggregometer over 20 min. Representative aggregation curves of n=3, 2 independent experiments. LEN/B: anti-α2 integrin antibody⁵¹. (d, e) Flow cytometry reveals unaltered reactivity of DOM/B-treated WT platelets upon thrombin stimulation compared to WT controls. Mean ± SD. n=3 mice, 4 independent experiments, two-tailed unpaired t-test with Welch’s correction. (f) Aggregation upon thrombin-stimulation is not affected in the presence of DOM/B. Light transmission was recorded on an Apact four-channel aggregometer over 10 min. Representative curves of n=3, 3 individual experiments. (g) Thrombin exosites I and II were blocked by the aptamers HD1 and HD22, respectively. Platelets were incubated with the indicated antibody (10 μg/ml) prior to thrombin stimulation. Thrombin-mediated cleavage of GPV was assessed by flow cytometry. Mean ± SD. n=3 mice, 3 independent experiments. (h) Thrombin clotting time was assessed using a ball coagulometer in GPV mutant or anti-mGPV mAb treated PRP in the presence or absence of HD22. Antibody concentration: 10 μg/ml; aptamer concentration: 1.5 μM f.c., thrombin: 17 nM. Mean ± SD. WT, WT+DOM/B, WT+DOM/C: n=5, Gp5^−/−: n=4, Gp5^dThr, Gp5^−/−+HD22, Gp5^dThr+HD22: n=3, WT+DOM/B+HD22, WT+DOM/C+HD22: n=6, WT+HD22: n=8. One-way ANOVA followed by Dunn’s test for multiple comparisons.