Page 182 - 2018_10-Haematologica-web

M. Rijkers et al.
(Figure 4B). To further study interactions between platelets from “matching” and “nonmatching” donors, platelets from the “matching” donor were stained with calcein-green and the platelets of the “nonmatching” donor with calcein-violet (or vice versa), and double-posi- tive events were measured. Upon incubation with WIM8E5, double-positive events, although significant, barely increased from 1 to 2%, while activation induced by PAR1 activating peptide (used as the control activating agonist) led to 8% positive events (Figure 4C). When platelets from two “matching” donors were combined, which were both activated upon incubation with WIM8E5, double-positive events increased from 1 to
A
6.5% (Figure 4C). As no enhanced CD62P exposure was observed on WIM8E5 “nonmatching” platelets and WIM8E5 had a very minor effect on the percentage of double-positive events when “matching” and “nonmatch- ing” platelets were mixed, these results suggest that acti- vation of platelets is dependent on the ability of HLA anti- bodies to interact in cis (intra-platelet) with FcγRIIa.
HLA antibodies from patients’ sera can activate platelets
To establish whether the findings observed for human HLA monoclonal antibodies were physiologically rele- vant, we tested whether sera from patients, refractory to
B
C
D
Figure 5. Patients’ sera with HLA anti- bodies induce FcγRIIa-dependent acti- vation of platelets from a subset of donors. Thirteen sera containing HLA alloantibodies were incubated with platelets from two different donors. AB serum tested negative for HLA and other specific platelet antibodies was used as a control. Background CD62P and IgG binding are indicated by the gray back- ground. Activation was inhibited by pre- incubation with 10 mg/mL IV.3 or 5 mM Syk inhibitor IV. (A) CD62P exposure on platelets from donor 1 (expressing HLA A1 A2 B51). (B) IgG binding to platelets from donor 1. (C) CD62P exposure on platelets from donor 2 (expressing HLA A1 A2 B35 B62). (D) IgG binding to platelets from donor 2. Antibody speci- ficities of the sera are reported in the Online Supplementary Data.
1748
haematologica | 2018; 103(10)