We and others have shown that at this molar ratio, PF4 and UFH form high molecular weight complexes (>670 kDa), with each complex capable of binding as many as three antibody molecules [10]. are dynamic and mutable. Heparin removes cell surface-bound PF4 in most individuals, but removal is incomplete in those with high pre-exposure surface-bound PF4 levels. Such individuals retain critically localized cellular antigenic complexes at the time antibodies develop and are at risk to develop HIT. This article reviews the scientific basis for this model and its clinical implications. Keywords: glycosaminoglycans, heparin, immune thrombocytopenia, Platelet Factor 4, thrombocytopenia, thrombosis Background Heparin-induced thrombocytopenia (HIT) is an iatrogenic autoimmune disorder associated with a high risk of limb- and life-threatening thrombosis, especially in those exposed to unfractionated heparin (UFH), which contains high molecular weight glycosaminoglycans (GAGs).The salient clinical features were first described in the 1970s [1], and the immune basis Uridine 5′-monophosphate was Uridine 5′-monophosphate appreciated soon thereafter [2]. However, it was not until 1992 that the platelet chemokine, Platelet Factor 4 (PF4) complexed to heparin or other high molecular negatively-charged molecules, such as other GAGs, was identified as the inciting antigen and target Mouse monoclonal to CD40 of HIT antibodies [3]. The pathogenesis had been thought to involve circulating PF4/heparin immune complexes that bind to the FcRIIA receptor on platelets and related Fc receptors on monocytes, endothelial cells and other vascular cells that promote platelet activation and generation of thrombin, setting up an explosive feed forward, prothrombotic loop [4]. Identification of PF4 as the target antigen led to the development of ELISA kits to detect HIT antibody, which are used to supplement functional assays, such as the serotonin-release assay, which in turn, vary in sensitivity and are not widely available [5,6]. It was hoped that antibody Uridine 5′-monophosphate detection would facilitate rapid diagnosis in complex medical situations where co-existing causes of thrombocytopenia and thrombosis are common. However, subsequent studies have shown that up to 50% of patients exposed to UFH in settings of intense platelet activation, such as cardiopulmonary bypass surgery, already have and/or develop anti-PF4/heparin antibodies [4], but only 1%C3% of such patients would develop HIT. The unanticipated prevalence of HIT antibodies not only has limited the diagnostic value of ELISA-based diagnosis, but it also opened the question of why only a subset of patients with such antibodies develops HIT. Recent studies show an incomplete correlation with IgG isotype and titer [6] and no correlation with FcRIIA polymorphisms [7]. The contribution of antigenic site specificity [4] has not been explored. The rapid onset of HIT, as early as day 5 after initial UFH exposure, is unusual for an IgG-mediated primary immune response [4]. Other patients develop thrombosis 1C2 weeks after the last identified exposure to UFH, which has been called delayed HIT, and its mechanistic basis remains unclear. and mechanistic studies PF4 and surface GAGs form HIT antigenic complexes Several groups have reported that the molar ratio of UFH to PF4 modulates antigenicity. Binding of HIT antibodies is optimized at a 1:1 molar ratio of UFH and tetrameric PF4 [8,9]. We and others have shown that at this molar ratio, PF4 and UFH form high molecular weight complexes (>670 kDa), with each complex capable of binding as many as three antibody molecules [10]. An excess of either PF4 or UFH reduces both formation of these large complexes and antibody-binding. Such antigenic complexes also develop on the platelet surface in the presence of Uridine 5′-monophosphate free PF4. When PF4 is added to human or mouse platelets at increasing concentrations, binding of the murine monoclonal HIT-like antibody, KKO, [11] and activation of platelets by human HIT antibodies, follow a bell-shaped curve. Using human or mouse platelets, binding of human antibody and KKO peak at 50 g mL?1 PF4 [12]. Platelets differ from other vascular cells in that the surface GAGs are composed almost exclusively of proteoglycans with chondroitin sulfate (CS) side chains [13]. PF4 binds to heparin with higher affinity than it does to cell surface GAGs, especially CS [14]. The effect of heparin on the development of platelet antigenicity depends on the amount of PF4 present. At low levels of added PF4 (e.g. 12 g mL?1), even low concentrations of UFH markedly reduce antibody-binding. In the presence of concentrations of PF4 optimal for antigenicity (50 g mL?1), higher UFH concentrations are required to disrupt antibody-binding. At yet higher concentrations of PF4 (e.g. 200 g mL?1), antigenicity actually increases when UFH is added at concentrations that span the therapeutic range (1C4 g mL?1), and then falls as the UFH concentration is increased further. The initial increase in antibody-binding may be attributable to the UFH removing PF4 from the platelet surface until the optimal PF4/GAG ratio is reached at which point Uridine 5′-monophosphate large antigenic complexes predominate; antibody-binding falls once these.