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Platelet activation by HLA antibodies
FcγRIIa-dependent platelet activation has been firmly implicated in the pathogenesis of heparin-induced throm- bocytopenia.17 In vitro experiments have demonstrated that platelet activation by heparin-platelet factor 4 can be inhibited by intravenous immunoglobulin.18 To study whether the mechanism of platelet activation by mono- clonal antibodies is similar to that described for heparin- induced thrombocytopenia, platelets were pre-incubated with intravenous immunoglobulin and subsequently incu- bated with the HLA monoclonal antibodies WIM8E5 or SN607D8. Intravenous immunoglobulin diminished CD62P surface exposure in a dose-dependent manner and completely blocked α-granule release at a concentration of 2 mg/mL (Online Supplementary Figure S5A). These results suggest that high levels of IgG can compete with HLA monoclonal antibodies for binding to FcγRIIa.
are crosslinked by the antibody). Rubinstein and co-work- ers showed that antibodies directed to beta-2-microglobu- lin can bind to FcγRIIa on other platelets (inter-platelet binding) resulting in their activation.32 Activation of platelets in patients with heparin-induced thrombocy- topenia is considered to occur both in an inter- and intra- platelet manner.18 To elucidate whether platelet activation by HLA monoclonal antibodies occurs in an inter-and/or intra-platelet manner, we studied whether platelets miss- ing the binding epitope of the activating HLA monoclonal antibody WIM8E5 (“nonmatching”) could be activated in the presence of platelets which were able to bind WIM8E5 (“matching”). In the case of inter-platelet activation, the HLA of the “matching” platelets can theoretically be crosslinked with the FcγRIIa on the “nonmatching” platelets (Figure 4A). Platelets from a donor with an HLA type not matching with WIM8E5 did not show increased levels of CD62P upon incubation with WIM8E5. When these platelets were mixed with platelets from a donor with an HLA type matched for WIM8E5, CD62P surface expression remained unaltered (Figure 4B). Platelets from a WIM8E5 “matching” donor did show enhanced CD62P surface exposure, and levels did not change when platelets were mixed with platelets from a “nonmatching” donor
Figure 2. Integrin αIIbb3 activation and platelet agglutina- tion are induced by HLA monoclonal antibodies. A) Integrin αIIbb3 activation, derived from PAC-1 binding, upon incuba- tion with 10 mg/mL WIM8E5, SN607D8 or SN230G6 com- pared to control (buffer only, no HLA antibodies). Flow cytometry plots are representative of more than eight inde- pendent experiments with different donors. (B) Platelet agglutination upon addition of HLA monoclonal antibodies (mAbs), measured by light transmission aggregometry. Mean ± SD of percentage maximum aggregation. Paired t- tests (A) or paired ANOVA with the Tukey multiple compari- son test (B). *P<0.05, **P<0.01, ***P<0.005, ****P<0.001.
Platelet activation by HLA monoclonal antibodies occurs through intra-platelet binding to FcγRIIa
FcγRIIa-dependent platelet activation can theoretically occur in either an inter-platelet-dependent manner (the HLA molecule of one platelet is crosslinked with the FcγRIIa of another platelet) or intra-platelet-dependent manner (an HLA molecule and FcγRIIa on a single platelet
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