Dimeric FcγR ectodomains detect pathogenic anti‐platelet factor 4–heparin antibodies in heparin‐induced thromobocytopenia

Abstract

FcγRIIa mediates life‐threatening heparin‐induced thrombocytopenia (HIT). Most anti‐platelet factor (PF)4‐heparin IgGs are not pathogenic so diagnosis of HIT is challenging. Dimeric rsFcγRIIa was used to quantify receptor‐binding activity of anti‐PF4‐heparin antibodies. Dimeric rsFcγRIIa binding specifically correlated with occurrence of HIT. Summary BackgroundHeparin‐induced thrombocytopenia (HIT) is a major and potentially fatal consequence of antibodies produced against platelet factor 4 (PF4)–heparin complexes following heparin exposure. Not all anti‐PF4–heparin antibodies are pathogenic, so overdiagnosis can occur, with resulting inappropriate use of alternative anticoagulation therapies that have associated risks of bleeding. However, definitive platelet functional assays are not widely available for routine analysis. ObjectivesTo assess the utility of dimeric recombinant soluble FcγRIIa (rsFcγRIIa) ectodomains for detecting HIT antibodies. Patients/MethodsPlasma from 27 suspected HIT patients were tested for pathogenic anti‐PF4–heparin antibodies by binding of a novel dimeric FcγRIIa ectodomain probe. Plasmas were also tested by the use of PF4–heparin IgG ELISA, the HemosIL AcuStar HIT IgG‐specific assay, and a serotonin release assay (SRA). ResultsThe dimeric rsFcγRIIa test produced no false positives and excluded four samples that were positive by IgG ELISA. In this small patient cohort, the novel assay correctly assigned 93% of the suspected HIT patients, with two of the HIT patients being scored as false negatives. The improved discrimination of the novel assay over the IgG ELISA, which scored four false positives, supports the mechanistic interpretation that binding of dimeric rsFcγRIIa detects pairs of closely spaced IgG antibodies in PF4–heparin immune complexes. ConclusionsThis study found the cell‐free, function‐based dimeric rsFcγRIIa assay to be convenient, simple, and potentially predictive of HIT. The assay had improved specificity over the IgG ELISA, and correlated strongly with the AcuStar HIT IgG‐specific assay, warranting further evaluation of its potential to identify HIT in larger patient cohorts.

The Human FcγRII (CD32) Family of Leukocyte FcR in Health and Disease

“…Other factors that affect interactions between IgG and the FcγRII are the size of the IgG immune complex ( 112 ), the distribution of epitopes ( 111 , 113 ), the geometry of the Fc in the complex, and receptor localization in membrane domains ( 114 ) which may also influence the avidity of immune complex binding. The state of the cell expressing the receptor ( 115 ) can also influence interaction with IgG.…”

Concern About the Adverse Effects of Thrombocytopenia and Thrombosis After Adenovirus-Vectored COVID-19 Vaccination

“…According to current research results, we have known that the adenovirus-vector vaccine activates platelets and produce PF4 antibodies, but only a subset of sensitized patients progress to life-threatening complications of thrombocytopenia and thrombosis, [22][23][24] the reasons and mechanisms need to be further explored.…”

“…22,23 PF4/heparin antibody binds and activates FcγRIIA on platelets and monocytes and mediates HIT and thrombosis. 24 It is precise because the clinical manifestations of thrombocytopenia and thrombosis after adenovirus-vectored COVID-19 vaccination and the production of peculiar auto anti-PF4 antibodies are very similar to HIT. This syndrome is named vaccine-induced immune thrombotic thrombocytopenia (VITT).…”

“…22,23 PF4/heparin antibody binds and activates FcγRIIA on platelets and monocytes and mediates HIT and thrombosis. 24…”

Fc Binding by FcγRIIa Is Essential for Cellular Activation by the Anti-FcγRIIa mAbs 8.26 and 8.2

“…FcγRIIa is the most widely expressed FcγR of human leukocytes and the only FcγR on platelets ( 18 ). FcγRIIa potently activates platelets when engaged by the Fc of anti-platelet antibodies ( 19 , 20 ), including anti-platelet factor 4 (PF4) antibodies which trigger heparin induced thrombocytopenia ( 21 , 22 ). FcγRIIa also triggers a rare thrombotic thrombocytopenia induced by anti-PF4 antibodies after vaccination for SARS CoV-2 spike vectored by a recombinant adenovirus (ChAdOx1, AstraZeneca) ( 23 , 24 ).…”