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Platelet activation by HLA antibodies
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Figure 4. HLA monoclonal antibodies activate platelets in an intra-platelet-dependent manner. (A) Schematic representation of theoretically possible inter-platelet activation and intra-platelet activation induced by the HLA monoclonal antibody WIM8E5. (B) Platelet donors were selected as either “WIM8E5 matching” or “WIM8E5 nonmatching” and their platelets were stained with calcein-green or calcein-violet, respectively. Platelets from two donors were mixed in a 1:1 ratio. Platelets from a single donor or the mixed platelets were incubated with control (buffer only, no HLA antibodies) or WIM8E5. By gating for calcein-green or calcein- violet, CD62P exposure was determined for “WIM8E5 matching” and “WIM8E5 nonmatching” platelets. (C) Platelets from a “WIM8E5 matching” donor (stained with calcein green) were mixed 1:1 with platelets from a donor with either nonmatching WIM8E5 or matching WIM8E5 (stained with calcein violet). Samples were incu- bated with control (buffer only, no HLA antibodies), WIM8E5 or PAR1 activating peptide (PAR1 AP). Percentage double-positive events of calcein-green/calcein-vio- let-stained platelets are given, representing the ability of HLA antibodies to crosslink the HLA molecule with the FcγRIIa in an inter- or intra-platelet manner. Representative flow cytometry plots are shown for a mix of WIM8E5 matching + WIM8E5 nonmatching platelets and platelets from two different donors with both matching HLA typing. Paired ANOVA with the Tukey multiple comparison test. *P<0.05, **P<0.01, ***P<0.005, ****P<0.001.
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