We explored the involvement of platelet surfaceCbound PF4 as an antigen in the pathogenesis of experimental HIT. exposure, and offers new therapeutic strategies. Introduction Heparin-induced thrombocytopenia (HIT) is an iatrogenic complication of heparin therapy caused by antibodies that recognize complexes formed between heparin and the endogenous protein platelet factor 4 (PF4).1-3 Approximately half of affected patients develop limb- or life-threatening thrombosis.4-6 Management involves careful NKH477 monitoring of platelet counts, a high index of clinical suspicion, cessation of heparin exposure, and the introduction of alternative anticoagulants.7,8 These measures have reduced the incidence of new thromboembolic complications but have had less impact on the incidence of amputations and death.9,10 Heparin remains an important anticoagulant CD133 in widespread use, and studies that help define the pathophysiology of HIT may lead to better identification of patients at risk and to more targeted intervention strategies. The antibody response in HIT is unusual in several respects. First, the major complications of HIT are related to thrombosis in contrast to other drug-induced thrombocytopenias.11 This high incidence of thrombosis may be related in part to the ability of HIT antibodies to activate platelets via FcRIIA.12,13 In a murine model of HIT, only mice with platelets that expressed both human (h) PF4 and FcRIIA developed thrombocytopenia and thrombosis when given an antiCPF4-heparin monoclonal antibody (mAb), KKO.14 A second unusual feature is the surprisingly high incidence of antiCPF4-heparin antibodies in heparinized patients, exceeding a quarter to half of all exposed patients in some settings.15-17 Why only a small portion of these patients develop HIT is not clear and no unequivocal differences between the vast majority of individuals who remain asymptomatic and the small number who develop HIT have been identified, although differences in immunoglobulin G (IgG) titers have been noted.18-21 A third characteristic of HIT antibodies (including KKO) is that they bind optimally to PF4-heparin complexes over a narrow molar ratio in vitro.1-3,22 In the case of unfractionated heparin, PF4 forms ultralarge complexes (ULCs) of larger than 670 kDa at these same molar ratios.23 These ULCs are stable, are particularly antigenic, bind multiple IgG antibodies per complex, NKH477 and promote platelet activation. It is not known whether similar complexes between PF4 and cell-surface glycosaminoglycans (GAGs) form on the surface of platelets or how heparin affects surface complex formation and antigenicity. Based on the knowledge that PF4 can bind to diverse anionic polysaccharides,24 PF4 may form similar antigenic complexes on platelets by binding to GAGs on the surface of platelets independent of heparin. The composition of these antigenic complexes and their capacity to be modulated has not been studied. We examined the effect of the anti-PF4/heparin mAb KKO (and in some studies, HIT IgG) on platelets expressing varied amounts of endogenous or exogenous PF4 on their surface both in vitro and in vivo. The results of these studies provide insight into the importance of the level of surface PF4 expression, the effect of heparin on formation of surface antigenic complexes, and potential new diagnostic and therapeutic approaches to HIT based on these new insights. Patients, materials, and methods Preparation of recombinant WT hPF4 Wild-type (WT) hPF4 in pT7-7 plasmid was NKH477 expressed in BL21DE30 pLysS bacteria, purified, and characterized as described.25 Recombinant protein was isolated from bacterial lysate supernatant by affinity chromatography using a HiTrap Heparin HP column (Amersham Bioscience, Upsala, Sweden). Proteins were purified further by fast protein liquid chromatography (FPLC) using a RESOURCE RPC column (Amersham Bioscience). Protein purity was assessed by 15% (wt/vol) sodium dodecyl sulfateCpolyacrylamide gel electrophoresis (SDS-PAGE) followed by silver staining.26 Samples were subjected to immunoblotting after electrotransfer to polyvinylidenedifluoride (PVDF) membranes using rabbit anti-hPF4 polyclonal antibody (PeproTech, Rocky Hill, NJ), followed by donkey antiCrabbit secondary antibody conjugated to horseradish peroxidase (HRP; Jackson ImmunoResearch Laboratories, West Grove, PA) and were developed using the enhanced chemiluminescence (ECL) kit (PerkinElmer Life Sciences, Boston, MA). Total protein concentrations were determined using the bicinchoninic acid assay (Pierce, Rockford, IL) as per manufacturer with BSA as standard..