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

Essentials 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 Background Heparin‐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. Objectives To assess the utility of dimeric recombinant soluble FcγRIIa (rsFcγRIIa) ectodomains for detecting HIT antibodies. Patients/Methods Plasma 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). Results The 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. Conclusions This 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.


Introduction
Heparin-induced thrombocytopenia (HIT) occurs when antibodies form immune complexes (ICs) with platelet factor 4 (PF4) bound to heparin or glycosaminoglycans [1][2][3]. The pathogenic ICs bind to FccRIIa, which is the only FccR on platelets, triggering their activation and aggregation, leading to thrombosis. Binding to FccRIIa on monocytes also causes both prothrombotic production of thrombin and tissue factor [4] and the clearance of platelets and thrombocytopenia [5]. Many patients treated with heparin develop antibodies against PF4-heparin, but the presence of antibody-PF4-heparin complexes does not necessarily result in clinical manifestations of thrombosis/thrombocytopenia. Antigen recognition-based methods (e.g. ELISA) detect anti-PF4-heparin antibodies, but fail to distinguish pathogenic from non-pathogenic antibodies. Thus, platelet functional assays, such as the serotonin release assay (SRA), are the most reliable for confirming HIT [2,6,7], but require access to appropriate donor platelets that are sensitive to activation, and are not easily replicated between many clinical laboratories.
The mAb KKO binds the PF4-heparin complex and activates human platelets in an FccRIIa-dependent manner [8]; it causes HIT in a human FccRIIa/PF4 transgenic mouse model [9,10]. A recent X-ray crystallography analysis showed the KKO mAb bound to a conformationdependent epitope on heparin-related pentasaccharide (fondaparinux)-bound PF4 tetramers, promoting the formation of higher-order complexes [11,12]. In contrast, a non-pathogenic antibody bound an overlapping epitope, but only in the PF4 monomer. Plate-based ELISAs present a heterogeneous mixture of PF4 forms, and so do not distinguish innocuous antibodies from those forming complexes capable of activating FccRs.
Pathological HIT antibodies engage FccRIIa, and trigger platelet activation and clearance [3] and tissue factor production [4]. The pathology depends, in part, on an R131H polymorphism within FccRIIa, which does not alter the expression levels of the receptor but does significantly alter the affinity of FccRIIa for its ligand [13]. We recently described the use of dimeric recombinant soluble FccRIIa (rsFccRIIa) to determine the proximity of pairs of IgG antibodies in immune cell-activating ICs [14]. The binding of dimeric rsFccRIIa in this assay is correlated with the capacity of IgG ICs to activate FccR-dependent cellular responses [14,15]. In this study, we tested the capacity of this unique dimeric rsFccRIIa to distinguish pathogenic antibodies, which recognize PF4-heparin complexes and are able to activate platelets, from clinically irrelevant, non-pathogenic antibodies.

Materials and methods
Plasma samples were obtained from 27 medical and surgical inpatients based at a tertiary hospital, the Royal Adelaide Hospital, in Adelaide, Australia, in whom HIT was suspected. Local ethics committee approval was obtained prior to the commencement of the study. The collection of samples conformed to institutional guidelines. Both plasma from citrated blood and sera were prepared for analysis. For the purposes of this study, and to ensure that HIT cases reflected the integration of both clinical and laboratory criteria, a diagnosis of HIT was defined as a 4T score of ≥ 4 and a positive SRA result (> 20% at 0.1 U mL À1 heparin, and suppression at 100 U mL À1 heparin) [16]. Levels of PF4-heparin autoantibodies were analyzed with an IgG-specific solid-phase ELISA (GTI, Waukesha, WI, USA) [17] and with the HemosIL AcuStar HIT IgG-specific assay (Instrumentation Laboratory, Bedford, MA, USA) [18] under standardized laboratory conditions. High specificity with the HemosIL AcuStar HIT IgG-specific assay has been previously reported [17].
The production and use of dimeric rsFccRIIa (His131 allelic form) has been described previously [14]. To assess the ability of dimeric rsFccRIIa to differentially bind pathogenic versus non-pathogenic HIT antibodies, patient plasma was used in a PF4-heparin IgG ELISA kit (Diagnostica Stago, Melbourne, Victoria, Australia) at a plasma dilution of 1 : 40, and with incubation and washing steps according to the manufacturers' instructions. The bound anti-PF4-heparin antibodies were then reacted with dimeric rsFccRIIa-biotin (0.2 lg mL À1 ) in phosphatebuffered saline (PBS) diluent containing 1 mM EDTA, 0.05% (v/v) Tween-20 and 1% (w/v) bovine serum albumin for 1 h at 37°C, and this was followed by five cycles of filling and aspirating wells with wash buffer (PBS, 0.05% v/v Tween-20). Bound receptor was then detected by the incubation with a 1 : 10 000 dilution of high-sensitivity horseradish peroxidase-streptavidin (Pierce, Rockford, IL, USA), for 1 h at 37°C, washed 10 times, and incubated with TMB Single solution (Life Technologies, Mulgrave, Victoria, Australia). The reaction was terminated with an equal volume of 1 M HCl, and absorbance at 450 nm was determined. An in-house plasma sample from a patient with confirmed HIT (4T score of 8, an SRA release of 94.2% with suppression of serotonin release with high-dose heparin, and an anti-heparin-PF4 antibody OD of > 3.0 by IgG ELISA) was used as a standard to define a nominal 100% response, to which absorbance values were normalized.
Statistical analysis was performed with GRAPHPAD PRISM version 6.05 (GraphPad Software, San Diego, CA, USA). The optimal OD cut-off for the dimeric rsFccRIIa assay (normalized value = 3) was confirmed by receiver operating characteristic analysis (not shown). Cut-off values for the PF4-heparin IgG ELISA (OD = 0.4) and for the HemosIL AcuStar HIT IgG-specific assay (1 U mL À1 ) were defined according to the manufacturers' instructions. SRA positivity was defined as > 20% serotonin release with low-dose heparin at 0.1 U mL À1 , and < 20% release with a high heparin concentration of 100 U mL À1 .

Results and discussion
A cohort of 27 patients with suspected HIT were evaluated, of whom 13 were considered to be HIT-positive because of a positive SRA finding and a 4Ts score of ≥ 4 [6]. Samples were also evaluated for anti-PF4-heparin antibodies with PF4-heparin IgG ELISA and the Hemo-sIL AcuStar HIT IgG-specific assay. The ability of a novel engineered dimeric rsFccRIIa (His131) to detect pathogenic anti-PF4-heparin antibodies was evaluated by comparison with these established assays.
The novel dimeric rsFccRIIa assay, AcuStar HIT IgGspecific assay and PF4-heparin IgG ELISA all showed significantly higher mean activity for HIT-positive samples than for HIT-negative samples (all assays, Mann-Whitney U-test P < 0.0001; Figure 1A). The performance of the assays in distinguishing HIT patients is summarized in Table 1. The dimeric rsFccRIIa assay correctly assigned 93% of the patients (25/27) and did not detect two HIT samples (i.e. two false negatives). This was similar to the performance of the AcuStar HIT IgGspecific assay, which correctly assigned 96%, with only one false negative. These two assays contrasted with the more sensitive PF4-heparin IgG ELISA, which had a lower specificity (71%), with four false positives from this cohort. Larger patient groups that include differing clinical groupings will be necessary to establish robustly the predictive value of this approach.
A comparison of the dimeric rsFccRIIa and AcuStar HIT IgG-specific assays for the HIT-positive samples showed that these two assays correlated more strongly with each other (Spearman's correlation r = 0.845, P = 0.0005) than either did with the PF4-heparin IgG ELISA (ELISA versus AcuStar assay, r = 0.813, P = 0.0015; ELISA versus dimeric rsFccRIIa assay, r = 0.673, P = 0.015).
As the SRA is a definitive functional assay for the diagnosis of HIT, and the main limitation of the PF4-heparin IgG ELISA is that it gives false positives, we examined the samples that were positive with the PF4-heparin IgG ELISA (Figure 2A) and the relationship between their activities in the SRA and in the other assays ( Figure 2B, C). Both the AcuStar HIT IgG-specific and dimeric rsFccRIIa assays appropriately assigned low, below cutoff, values to the SRA-negative samples, although some were very near the cut-off values of both assays. Furthermore, the AcuStar HIT IgG-specific and dimeric rsFccRIIa assays also correctly assigned as negative a sample (see filled blue star; Figure 2) that had~50% release at both high and low heparin doses, and was weakly positive with the PF4-heparin IgG ELISA. The AcuStar HIT IgG-specific and dimeric rsFccRIIa assays, although correlating strongly ( Figure 1B), showed some important differences ( Figure 2B,C). One HIT patient plasma sample was weakly AcuStar HIT IgG-specific assay-positive (1.78 U mL À1 ; cut-off of 1.0 U mL À1 ) and dimeric rsFccRIIa assay-negative (0.4% response; cut-off with each assay. These thresholds were 3% of the normalized signal for the dimeric rsFccRIIa assay: 1 U mL À1 for the AcuStar HIT IgG-specific assay, and an OD of 0.4 (right y-axis) for the platelet factor 4 (PF4)-heparin IgG ELISA. Two sets of parentheses mark the false negatives from the assays: two from the dimeric rsFccRIIa assay, and one from the AcuStar HIT IgG-specific assay. The third set of parentheses marks the four false positives from the PF4-heparin IgG ELISA. (B) The dimeric rsFccRIIa and AcuStar HIT IgGspecific assays were correlated for the HIT-positive (i.e. 4T score of ≥ 4 and SRA-positive) samples. Cut-off values for the two assays are shown as dotted and dashed lines, respectively. HIT samples with low antibody levels are more sensitively identified by combining both the dimeric rsFccRIIa and AcuStar HIT IgG-specific assays. When HIT positivity is defined as rsFccRII a positivity and/or AcuStar HIT IgG-specific assay positivity, sensitivity is improved over that with either assay used alone. of 3%; open triangle, Figure 2). Another HIT patient was weakly positive with the AcuStar HIT IgG-specific assay (1.05 U mL À1 ) but scored strongly in the dimeric rsFccRIIa assay (67% response; open square, Figure 2).
Thus, defining samples as HIT-positive according to AcuStar HIT IgG-specific assay-positive and/or dimeric rsFccRIIa assay-positive criteria may be an approach for improving HIT prediction with either assay alone. In HIT, pathogenic PF4-heparin IgG complexes engage and aggregate FccRIIa on platelets [1,3,5], and possibly other FccRs on monocytes [4,19]. The size and stoichiometry of IgG ICs are major determinants of both the avidity of interaction with the low-affinity FccRs [20] and the capacity of anti-PF4-heparin antibodies to activate cells. In a large proportion of laboratories, the initial clinical suspicion of HIT is followed by the performance of standard HIT IgG ELISAs. However, the diagnosis of HIT and subsequent decisions regarding patient anticoagulation and management are made challenging by the prevalence of non-pathogenic anti-PF4-heparin antibodies that are unable to activate FccRs. Although the levels of anti-PF4-heparin antibodies as detected by HIT IgG ELISA provide useful information, this measurement alone does not provide sufficient specificity for the diagnosis of HIT [7,21].
Thus, a platelet functional HIT assay, such as platelet aggregation or the SRA, is crucial for diagnosis. The SRA, however, involves handling of radioactive isotopes and requires specialized personnel, specialized equipment and rapid access to functional 'high-responder' platelets that may not be routinely available. Use of the SRA is also limited to major reference laboratories. Refinements to improve the specificity of the HIT anti-PF4-heparin antibody ELISA have included raising the OD cut-off for positive results and the addition of a high-dose heparin confirmatory step [16,22]. However, there is still a need for improved serological HIT assays.
We investigated the potential of dimeric rsFccRIIa, a probe of the FccR-mediated effector functionality of IgG complexes, to improve the specificity of a PF4-heparin IgG assay. The novel assay had superior specificity to the detection of PF4-heparin IgG by ELISA, and performed similarly to the HemosIL AcuStar HIT IgG-specific assay, a test in which high specificity has been reported previously [18]. The combined use of both the dimeric rsFccRIIa and HemosIL AcuStar HIT IgG-specific assays to assign HIT correctly identified some HIT samples that would have been false negative with the use of either assay alone. The detection of HIT antibodies with the dimeric rsFccRIIa assay adds a functional qualitative component to the assay that mechanistically underlies the possible improved specificity and utility.
Dimeric rsFccRIIa binding requires closely spaced pairs of antibodies, as occurs in ICs [14], and has been used to detect FccR functional antibodies against influenza virus and HIV envelope proteins [14,15,[23][24][25][26][27][28]. HIT pathogenesis requires Fc-mediated clustering of platelet FccRIIa, but two SRA-positive samples were not detected with the dimeric rsFccRIIa-binding assay. Studies of HIT complexes have shown that the formation of the PF4-heparin-IgG complex is dynamic and, importantly, the proximity of the IgG Fcs in the pathogenic complexes is influenced by the binding of heparin, and the epitope specificity and avidity of the bound IgG [11,12,29,30]. The failure of the dimeric rsFccRIIa assay to detect these two HIT samples suggests that the Fcs were not appropriately presented in the current ELISA format. Thus, the assay may need further optimization to a format that mimics the dynamic behavior of the PF4 complexes that cluster platelet FccRIIa in vivo. Nonetheless, our proofof-concept study has shown that the dimeric rsFccRIIa assay has the potential to be predictive of HIT and warrants further investigation. This study has established a novel FccRIIa probe as a powerful surrogate for evaluating HIT antibody functionality. The use of dimeric rsFccRIIa also offers the advantages that it is simple, scalable, and adaptable to any format, such as multiplexing [25] and flow cytometry.