HIT antibodies activate platelets by interacting with a negatively charged epitope on human PF4 Free

Heparin-induced thrombocytopenia (HIT) is caused by IgG antibodies (Abs) that recognize platelet factor 4 (PF4)/heparin complexes and activate platelets. However, the mere presence of anti-PF4/heparin Abs detected by ELISA does not confirm clinical relevance, as only a subset of HIT Abs can activate platelets (referred to as PA Abs). PA and non-platelet activating (NA) Abs show similar binding to PF4/heparin complexes in ELISA. Critically, only PA Abs can physically bind to PF4-pretreated platelets, suggesting that platelet surface binding is a key determinant of pathogenicity. PA Abs differ from NA Abs in that they commonly possess either an RKH or a Y5 motif and are rich in basic or tyrosin residues in an unusally long heavy chain CDR3 region. Structural analysis of human PF4 (hPF4) using PyMol reveals a distinct surface composed of clustered negatively charged residues. Each hPF4 monomer contains eight acidic amino acids (AAs), with five located at the N-terminus and two (D54 and E69) near the C-terminus, contributing to the overall negative charge. Although mouse PF4 (mPF4) shares 75% sequence identity with hPF4, it is not recognized by HIT patient sera. Unlike hPF4, the N-terminus of mPF4 bears a neutral VTSAGP tail preceding its acidic residues. This sequence may disrupt the formation of a continuous negatively charged surface, potentially explaining the lack of reactivity with human HIT Abs. Based on these observations, we hypothesize that the negatively charged surface on PF4 interacts with basic residues like in RKH motif from PA Abs, facilitating the immune complex formation and subsequent platelet activation.

To test our hypothesis, we designed three PF4 mutants: hPF4 Δ7 (deletion of seven AAs at N-terminus including five acidic ones); N-terminus-humanized mouse PF4 (h-mPF4, the twelve N-terminal AAs of mPF4 were replaced by the seven N-terminal AAs of hPF4); and hPF4 D54N/E69Q (negatively charged D54 and E69 were simultaneously mutated to neutral N and Q, respectively). We then used the P-selectin expression assay (PEA) to assess both the ability of PA Ab binding to mutant PF4-coated platelets and the extent of platelet activation induced. The assay included monoclonal human PA Abs previously cloned and expressed in our laboratory and plasma from 16 HIT patients. The Ab binding to heparin complexed with PF4 mutants was also checked using ELISA.

First, we used Cy5-labeled hPF4, hPF4 Δ7 and hPF4 D54N/E69Q to evaluate whether the PF4 mutants can bind to platelets. We found that both hPF4 mutants exhibited significantly higher extent of platelet binding compared to wild type (WT) hPF4. However, when these mutant-coated platelets were exposed to the monoclonal PA Abs and HIT patient plasmas, Ab binding was significantly reduced compared to WT hPF4-coated platelets. As a result, platelet activation by certain Abs and HIT plasmas was significantly impaired when using Δ7 or D54N/E69Q as the coating antigen. Moreover, the degree of platelet activation correlated with the binding strength of HIT Abs from patient plasma, showing a near-significant p-value. Using ELISA, we further confirmed that binding of a subset of Abs to the hPF4 Δ7/heparin complex was significantly reduced compared to their reactivity with the hPF4/heparin complex. Additionally, when human platelets were coated with mPF4, they exhibited minimal activation by all HIT Abs tested. However, platelets coated with h-mPF4 partially rescued this activity, allowing for limited HIT Ab-induced activation without a corresponding increase in Ab binding, which remained at low levels. Conclusively, HIT Abs induce platelet activation by interacting with a negatively charged epitope on hPF4. The strong correlation between HIT Ab binding to mutant PF4-coated platelets and the extent of platelet activation highlights the utility of these assays at assessing clinical relevance and identifying activation-associated epitopes.