Ristocetin

Blood donations were from healthy volunteers who provided written informed consent. The pMH3 vector and CHO-S cells were purchased from AmProtein (Hangzhou, China). Restriction enzymes, T4 DNA ligase, and Taq DNA polymerase were obtained from New England BioLabs (Beverly, MA, USA). Rabbit anti-human VWF polyclonal antibody and rabbit anti-human VWF-HRP antibody were purchased from Dako Cytomation (Glostrup, Denmark). Ristocetin, tetramethylbenizidine (TMB), and collagen type III from human placenta were purchased from Sigma (St. Louis, MO, USA). Goat anti-mouse IgG and Goat anti-mouse IgG labeled with horseradish peroxidase (HRP) were purchased from Beckman-Coulter (Brea, CA, USA). SZ-123, a murine anti-human VWF A3 domain mAb (IgG1) was produced by standard hybridoma technology in our laboratory as described previously [22 (link)]. SZ-34, a murine mAb that binds to the VWF A2 domain but does not inhibit its function, was produced by standard hybridoma technology in our laboratory [25 (link)].

Ristocetin was purchased from Sigma-Aldrich (Darmstadt, Germany). Recombinant CXCL12, mouse anti-CXCR4 monoclonal antibody, mouse anti-CXCR7 monoclonal antibody, and the soluble CD40 ligand (sCD40L) ELISA kit were purchased from R & D Systems, Inc. (Minneapolis, MN, USA). NSC23766 and Y27632 were purchased from Tocris Bioscience (Bristol, UK) and Calbiochem/Novabiochem Co. (La Jolla, CA, USA), respectively. GAPDH rabbit polyclonal antibody (cat. no. SC-25778) was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Phospho-specific cofilin antibodies were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). Rac1 and Rho activation assay kits were purchased from Millipore (Billerica, MA, USA). Other materials and chemicals were obtained from commercial sources. Ristocetin was dissolved in dimethyl sulfoxide. The maximum concentration of dimethyl sulfoxide was 0.1%, which did not affect the platelet aggregation.
Y27632 and NSC23766 were dissolved in dimethyl sulfoxide. The maximum concentration of dimethyl sulfoxide was 0.3%, which did not affect the platelet aggregation, protein detection by Western blotting or ELISA for sCD40L.

Platelet agonist ADP and collagen (equine tendon) were from HELENA laboratories (USA). Ristocetin was from Sigma (R7752, USA). Anti-human GPIbα monoclonal antibody SZ2, VM16d, and AK2 were from GenTex (GTX28822, USA), YO Proteins (656, USA), and Bio-Rad (MCA740T, USA), respectively. Secondary antibody anti-human/mouse CD62P (P-selectin) APC was from Thermo Fisher scientific (17-0626, USA), and FITC-conjugated anti-human PAC-1 was from Biolegend (362803, USA). Peptides of GPIbα fragments were synthesized by GL Biochen (China) Ltd. Recombinant mouse vWF protein was from Creative BioMart (VWF-1432 M, USA), and human vWF protein was from Sino Biological (10973-H08C, USA). C57BL/6J mice, BCLB/C mice, and Wistar rat were from JSJ laboratories (China) and were bred and housed at Putuo animal care facility. Transgenic mice expressing no mouse but only human GPIbα (hTg) were described previously [19 (link)]. Animal experiments were conducted in mice or rats using protocols approved by the IACUC of Putuo Hospital. Six- to eight-week-old mice or rats were used in the study, and investigators were blinded to group allocation during data collection.