Functional heterogeneity of antiheparin-platelet factor 4 antibodies: Implications in the pathogenesis of the HIT syndrome

Heparin-induced thrombocytopenia represents one of the most severe drug-ind uced disorders of platelets. This syndrome is believed to be mediated throu gh antibodies generated against a heparin-platelet factor 4 complex. Comple xation of a sulfated mucopolysaccharide chain of heparin with a platelet gr anular protein (platelet factor 4) produces an allosteric modification of p latelet factor 4 resulting in neoepitope formation and the generation of an tiheparin-platelet factor 4 antibodies. These antibodies are capable of act ivating platelets by binding to heparin, platelet factor 4 and the Fc recep tor on platelets, resulting in a complex pathophysiology involving ischemic , thrombotic, and inflammatory processes. To characterize this antibody, Ig G fractions were obtained from the serum of patients with heparin-induced t hrombocytopenia using ammonium sulphate precipitation and heparin-platelet Factor 4-sepharose 4B affinity chromatography methods. With the affinity pu rification, two major components, peaks I and II, with high antiheparin-pla telet factor 4 antibody titers were eluted. The purity of all the fractiona ted immunoglobulins was established by sodium dodecylsulphate-polyacrylamid e gel electrophoretic analyses. While peak I did not induce C-14-serotonin release from platelets in the heparin-dependent assay for heparin-induced t hrombocytopenia antibodies (14C-serotonin release assay), peak II and the I gGs obtained with the ammonium sulphate precipitation method exhibited a st rong and concentration-dependent activation in the presence and absence of heparin and low molecular weight heparin. These immunoglobulins were treate d with heparinase, a cationic ion-exchange resin (Heparsorb), or dialyzed t o remove traces of heparin, and when tested in the C-14-serotonin release a ssay, showed the same high degree of activity. These data are suggestive of the generation of heparin-induced thrombocytopenia antibodies capable of a ctivating platelets directly in a nonheparin-dependent manner. These observ ations underscore the complex pathophysiology of heparin-induced thrombocyt openia syndrome and suggest that the severity of this syndrome in some pati ents may be due to the generation of "super-active" heparin-induced thrombo cytopenia antibodies capable of activating platelets without the requiremen t of heparin. This could explain why the cessation of heparin in patients d oes not necessarily correct the symptoms of heparin-induced thrombocytopeni a or associated thrombosis.