Ultralarge complexes of PF4 and heparin are central to the pathogenesis of heparin-induced thrombocytopenia

Abstract

Heparin-induced thrombocytopenia and thrombosis (HITT) is a severe complication of heparin therapy caused by antibodies to complexes between unfractionated heparin (UFH) and platelet factor 4 (PF4) that form over a narrow molar range of reactants and initiate antibody-induced platelet activation. We observed that UFH and tetrameric PF4 formed ultralarge (> 670 kDa) complexes (ULCs) only over a narrow molar range with an optimal ratio of PF4 to heparin of approximately 1:1. These ULCs were stable and visible by electron microscopy, but they could be dissociated into smaller complexes upon addition of heparin. ULCs formed inefficiently when PF4 was incubated with low-molecular-weight heparin, and none formed with the pentasaccharide fondaparinux sodium. In addition, mutation studies showed that formation of ULCs depended on the presence of PF4 tetramers. The ULCs were more reactive as determined by their capacity to bind to a HITTlike monoclonal antibody and showed greater capacity to promote platelet activation in an antibody-and Fc␥RIIA-dependent manner than were the smaller complexes. The capacity of PF4 to form ULCs composed of multiple PF4 tetramers arrayed in a lattice with several molecules of UFH may play a fundamental role in autoantibody formation, antibody-dependent platelet activation, and the propensity for thrombosis in patients with HITT.

Monocyte-bound PF4 in the pathogenesis of heparin-induced thrombocytopenia

“…4,5 In solution, formation of antigenic complexes between PF4 and heparin is critically dependent on their molar ratio, with loss of antibody binding when the optimal ratio is disrupted by an excess of either component. 6,7 Antigen formation on the platelet surface also follows a bell-shaped curve as PF4 concentration is increased, with maximal binding of antibody seen at an exogenous PF4 concentration of 50 g/mL. 8 Chondroitin sulfates (CSs) are the predominant GAG side chains expressed on platelets.…”

A case of heparin-induced thrombocytopenia (HIT) triggered by methicillin-susceptible Staphylococcus aureus (MSSA) bacteremia

“…PF4 is positively charged and may combine with administered heparin to form a PF4/heparin complex. This complex was previously reported to be formed in vitro at molar ratios of heparin to PF4 of 1:1 to 1:4 [3,4].…”

Antigen and substrate withdrawal in the management of autoimmune thrombotic disorders

“…ULCs are stable over 24 hours but are readily dissociated by small changes in heparin or PF4 concentration. 17 The in vivo relevance of these findings is supported by the relationship between the concentration of exogenous PF4 and binding of anti-PF4/H antibodies to platelets and monocytes (wherein antibody binding increases with addition of PF4 until an optimal concentration is reached above which binding falls) and the expression level of endogenous PF4 and the development of thrombocytopenia and thrombosis in an animal model. 12,14 The finding that activation of platelets and monocytes increases PF4 binding capacity may provide insight into why the risk of thrombosis is greater in settings, such as bypass surgery and trauma, among others, that are characterized by intense PF4 release, inflammation, and vascular injury.…”

“…16 At the optimal antigenic ratio of reactants, PF4 and UFH form ultra-large complexes (ULCs) in solution (molecular weight Ͼ 670 kDa). 17 The concentration of heparin required to form ULCs and the percent of total complexes they represent vary inversely with the length of the "heparin." Fondaparinux does not form ULCs, which correlates with its low risk of causing HIT.…”