Figure 1. Botrocetin and physiological shear induce GPIb–IX signalling in human platelets.

(a) A cartoon of GPIb–IX complex illustrating botrocetin (Bc)-facilitated interaction of A1 domain of VWF with the ligand-binding domain (LBD) in GPIbα. The macroglycopeptide region (MR), the mechanosensory domain (MSD) and transmembrane domain (TMD) in GPIbα are also marked. (b) Representative flow histograms illustrating the effects of botrocetin and/or 18 dyn cm⁻² shear on the exposure of β-galactose (measured by binding of FITC-labelled ECL), intracellular calcium level (monitored by Fura-2 fluorescence), expression of P-selectin (binding of anti-P-selectin antibody) and activation of integrin αIIbβ3 (binding of PAC-1 antibody). Fresh human PRP (20 k platelets per μl) was mixed with or without 1 μg ml⁻¹ botrocetin and subjected to various uniform shear stresses. Platelets were then collected and analysed by flow cytometry for noted indicators of platelet signalling. Top row: with 1 μg ml⁻¹ botrocetin (+Bc); bottom row: without botrocetin (−Bc). Blue histogram: under no shear; red: under 18 dyn cm⁻² shear; grey: negative control. (c) Quantificative plots of platelet signalling, as either percentage of cells with noted positive signals in Supplementary Fig. 2b (top row) or median fluorescence intensity (MFI) of the entire cell population (bottom row), versus shear stress in the presence (filled squares) and absence (open squares) of botrocetin. Data are plotted as mean±s.d. (n=3). *P<0.05, **P<0.01, ***P<0.005. Plots also include data points that were obtained from mixing type 2B VWD patient plasma (pV1316M, red diamonds) or normal plasma (green) with healthy donor platelets (1:9 v/v) under no or noted shear stress.