Heparin-Induced Thrombocytopenia Antibodies Inhibit PF4-Dependent Enhancement of Activated Protein C Formation by Binding to Antigenic Complexes Formed with the Chondroitin Sulfate Side-Chain of Thrombomodulin

Abstract 721

Previous studies have shown that platelet factor 4 (PF4) increases activated protein C (aPC) generation both in vitro and in vivo. PF4 increase of aPC generation by thrombin (IIa) and thrombomodulin (TM) complex followed a bell-shaped curve when tested in solution, on human TM expressing HEK293 and on endothelial cells. PF4 failed to enhance aPC in the presence of chondroitin sulfate (CS)-free TM. These results were consistent with PF4 binding to the CS on the TM glycosaminoglycan (GAG) domain and forming complexes that are similar to PF4/GAG antigenic complexes seen in heparin-induced thrombocytopenia (HIT). We tested the hypothesis that PF4 forms a HIT-like antigenic complex with the TM-CS using the HIT-like monoclonal antibody KKO. KKO abolished the potentiating effects of PF4 on aPC formation measured with TM in solution or with a TM-expressing cell line. To further address the nature of complexes formed between PF4 and TM, we used a mutant of PF4, PF4^T38Q, which forms complexes with GAGs that are not recognized by KKO and a subgroup of HIT antibodies. Similar to PF4, PF4^T38Q potentiated TM-dependent aPC generation in a bell-shaped manner, but this potentiation was not blocked by KKO. Moreover, KKO did not have any effect when PF4 was replaced with protamine sulfate (PS), which can also form macromolecular complexes with heparin/GAGs and can also enhance aPC generation. We also tested HIT antibodies isolated from patients that developed HIT with thrombocytopenia and thromboembolism developing >4 days after the last exposure to heparin. Patient IgGs specific for PF4/GAG complex were purified using PF4 bound to heparin columns. Specific binding of antibodies to PF4/heparin complexes was checked by ELISA. Complex-specific antibodies were then tested in an aPC generation assay in the presence IIa and TM and near peak concentration of PF4 and compared to a control human IgG. Three of four patient‘s antibodies significantly inhibited the increase in aPC generation in the presence of PF4. These studies provide evidence that HIT-like PF4/GAG complexes develop naturally in vivo. In this case, the ability of HIT or HIT-like antibodies to specifically inhibit the PF4-dependent increase in aPC formation may contribute to the prothrombotic state in HIT.