Role Of Monocyte Fcγ Receptors In The Prothrombotic State Of Heparin Induced Thrombocytopenia (HIT)

HIT is an antibody-mediated disorder characterized by thrombocytopenia and thrombosis. Activation of platelet via FcγRIIA by HIT antibodies complexed to surface-bound platelet factor 4 (PF4) contributes to the prothrombotic state. However, using an in vitro microfluidic whole human blood model of HIT, we have found through depletion and repletion studies that activation of monocytes through Syk-kinase is a key step in generating a prothrombotic milieu. Here we define the Fcγ receptor(s) upstream of Syk that mediate the thrombotic signal in monocytes. Monocytes isolated from normal healthy volunteers were incubated with 50 µg/ml of blocking antibodies against FcγRIIA (CD32, clone IV.3) and/or against FcγRI (CD64, clone 10.1) or against FcγRIII (CD16, clone 3G8), washed to remove unbound antibody and added to monocyte depleted-whole blood. These monocyte-repleted samples were then stimulated with PF4 (10 µg/ml) and the HIT like monoclonal antibody KKO (50 µg/ml) for 30 min. After recalcification (5 mmol CaCl₂), samples were perfused over a von Willebrand factor-coated BioFlux microfluidic channel at a shear stress of 20 dyne/cm² at 37°C for 15 min. Platelet adhesion was quantified by fluorescence microscopy after adding Calcein-AM (3 µM), and fibrin was visualized by adding Alexa 561-labeled fibrinogen (1.5 μg/ml) to the whole blood prior to the perfusion. Selective inhibition of FcγRIIA on monocytes led to an ∼40% inhibition of fibrin deposition and comparable reduction in the formation of “coated” platelets relative to samples repleted with non-inhibited monocytes. Flow cytometry studies showed that these observations were not due to leaching of IV.3 antibody off the monocytes with subsequent blocking of platelet FcγRIIA. In contrast, selective blockade of FcγRI or FcγRIII in depletion/repletion studies or in whole blood did not significantly reduce platelet adhesion, formation of coated platelets, or fibrin accumulation. However, blocking FcγRI did decrease tissue factor (TF) activity on monocytes stimulated with PF4 and KKO and this effect was additive to FcγRIIA inhibition. In summary, our data show that monocyte-bound HIT IgG antibodies contribute to the prothrombotic state by activating FcγRIIA directly and indirectly by generating TF though FcγRI. We speculate that this activation of monocytes contributes to platelet activation and monocyte microparticle formation demonstrated previously. Drugs that target FcγRI and FcγRIIA on monocytes may help prevent thrombosis in HIT without causing platelet dysfunction or increasing the risk of bleeding.