Pathogenic Role of Surface Platelet Factor 4 Complexes in Heparin-Induced Thrombocytopenia: Diagnostic and Therapeutic Implications.

Heparin-induced thrombocytopenia (HIT) is caused by antibodies that recognize complexes between high molecular weight heparin and Platelet Factor 4 (PF4). Current therapy with direct thrombin inhibitors is not effective in all cases, likely because it acts downstream of antibody-induced platelet activation. More directed therapies to the underlying pathology in HIT may be more effective. Heparin and PF4 only bind HIT antibodies over a narrow molar ratio of reactants at which ultralarge soluble complexes are formed. We asked whether similar complexes form between PF4 and endogenous platelet glycosaminoglycans (GAG) and their pathogenic role in experimental HIT. Platelet surface GAG:PF4 complexes are indeed antigenic over a narrow molar range of reactants. Heparin is not required for either HIT-IgG or a HIT-like monoclonal antibody KKO to bind to PF4 on human or mouse platelet surfaces in vitro, but enhances antigenicity when very high levels of surface PF4 are present. Antigenicity is maximal at a PF4 concentration of 50 μg/mL (well within the range that can be achieved within a thrombus) and ~25 μg/mL heparin (~0.5 U/mL, which is within the therapeutic range) optimally enhances antigenicity when surface PF4 levels were increased 4-fold. Using transgenic mice lines each with platelets expressing a different level of hPF4, ranging from 0.5 – 6 X’s human platelet levels and all expressing FcRγIIA, were given KKO. The different lines developed thrombocytopenia proportional in severity and duration to hPF4 expression. A standard subcutaneous (sq) heparininzing dose (20 U/kg, sq daily) prolonged the duration of severe thrombocytopenia in high hPF4 expressing mice. We reasoned that altering the ratio of PF4 to GAG in either direction would alter antigenicity and could block the development of thrombocytopenia. In accordance with this concept, both high concentrations of anionic heparin (100 U/kg, sq daily) and cationic protamine sulfate (2 mg/kg, sq daily) decreased KKO binding in vitro and prevented KKO-induced thrombocytopenia in vivo as a demonstration of successful therapeutic intervention. These studies affirm a central role of surface GAG:PF4 complexes in the development of HIT, suggest ways to identify patients at high risk to develop HIT even prior to heparin exposure, and offers a new and rationale therapeutic paradigm based on disrupting surface antigen formation.