FcγRIIA Mediates the Prothrombotic Role of Monocytes in HIT

Thrombosis is the most striking complication of heparin-induced thrombocytopenia (HIT). We have shown that monocytes are preferentially targeted by HIT antibodies because of the higher affinity of monocyte surface glycosaminoglycans (GAGs) for PF4 than the chondroitin sulfate GAGs on platelets. The contribution and mechanism by which monocytes promote thrombosis in HIT have not been fully elucidated. It has been reported that HIT antibodies activate monocytes through FcγRI and then by the MEK1-ERK1/2 intracellular pathway leading to the expression of tissue factor (TF) (Kasthuri et al., Blood 2012 119:5285). While activation of platelets by HIT antibodies through FcγRIIA is well established, the role of this receptor in monocyte activation in HIT is not clear. We examined the role of monocytes and their family of Fcγ receptors in HIT using several in vitro models, including a novel microfluidic system that allowed us to examine the prothrombotic pathways using human- and murine-based systems. Our studies showed that monocytes were key to the prothrombotic state; simply adding monocytes coated with PF4 and pre-activated with KKO, a HIT-like monoclonal antibody, was sufficient to form platelet-fibrin clots in reconstituted blood samples combining isolated red cells, platelets and mononuclear cells. Using three separate approaches, we also found that HIT antibodies bound to surface PF4/GAG complexes activate monocytes via the same Fc receptor that mediates platelet activation, i.e., FcγRIIA. First, using a strategy of blocking individual classes of Fcγ receptors known to be present on monocytes - FcγRI, FcγRIIA and FcγRIII - by monoclonal antibodies, we found that only anti-FcγRIIA decreased fibrin deposition (by 52 ± 8%; p<0.005 compared to non-blocked control). Second, activation of human platelets added to platelet depleted “whole blood” containing transgenic murine monocytes expressing human FcγRIIA was markedly higher than activation by monocytes lacking FcγRIIA, as measured by P selectin expression (2 ± 0.2 times higher) and annexin V binding (3 ± 2 times higher). Third, blocking the signaling pathway downstream of FcγRIIA selectively in monocytes by the Syk-specific tyrosine kinase inhibitor PRT318 abrogated the prothrombotic effect of monocytes as demonstrated by suppression of fibrin formation. These data add to our understanding of how monocyte activation promotes thrombosis in HIT. According our current model of platelet transactivation by monocytes, HIT immune complexes engage FcγRIIA both on platelets and on monocytes, leading to the activation of a common Syk-dependent pathway. In monocytes this leads to TF expression and thrombin generation. Thrombin generated by monocytes activates G protein-coupled receptors on platelets, while surface-bound HIT immune complexes activate platelets directly through FcγRIIA coupled to the immunoreceptor tyrosine-based activation motif pathway. Concurrent platelet activation via these two pathways is known to result in highly reactive COATED platelets. We believe the formation of a large population of COATED platelets contributes to the intensely prothrombotic nature of HIT. These studies highlight the importance of blocking FcγRIIA and its downstream signal pathways in monocytes as well as in platelets in order to develop rational strategies to attenuate the risk of thrombosis in patients with HIT.