Defining the Role of Monocytes in Heparin-Induced Thrombocytopenia (HIT): Insights from a Murine HIT Model.

Heparin-induced thrombocytopenia (HIT) is an iatrogenic disorder seen in 1–5% of patients exposed to unfractionated heparin. One unusual feature of HIT is that affected patients often have only moderate thrombocytopenia yet suffer severe, life-threatening thrombosis. We have previously defined the importance of PF4:glycosaminoglycan (GAG) antigenic complexes on the surface of circulating platelets in the development of thrombocytopenia using mice that express varying levels of human PF4 (hPF4+/+) as well as Fc γRIIA on the platelet surface after infusing the animals with a monoclonal antibody (KKO) that demonstrates HIT-like properties. Because HIT antibodies have been reported to activate monocytes to produce tissue factor in vitro, we further examined their role both in vitro and in vivo in the murine HIT model. Monocytes bind PF4 onto their surface forming antigenic PF4:GAG complexes recognized by KKO. Monocytes express antigenic complexes at low levels of PF4, a setting in which none can be demonstrated on platelets. Furthermore, monocytes express antigenic complexes at heparin concentrations that completely dissociate antigen from the surface of platelets. We then investigated the role of monocytes in a murine model of HIT. Monocytes were depleted by IV injection of 200 μL of clodronate-containing liposomes (Encapsula Nano Sciences) 12 hrs prior to IP injection of 200 μg of KKO. Over 92% of circulating monocytes were depleted for >24 hrs with a return to near baseline by 72 hrs. In co-transgenic hPF4+/+/FcγRIIA mice, clodronate did not cause a significant fall in platelet count at 4 hrs, but counts fell to 30 ± 14% of baseline by 24 hrs vs. 70 ± 23% of baseline after injection of control liposomes or no liposomes (p<0.001), thrombocytopenia persisted for 72 hrs. Nevertheless, monocyte depletion inhibited thrombosis in the HIT model. Rose Bengal (500 mg/kg) was injected and the right carotid artery injury was exposed to 3 mW green (540 nm) light for 5 min followed by an IV injection of KKO (500 μg/kg). 4 of 5 mice that did not receive liposomes and 4 of 5 mice that received control liposomes 16–24 hrs prior to KKO developed thrombosis, in contrast to 1 of 5 clodronate liposome-treated mice (p<0.01 vs. all controls) in spite of having platelet counts of 860,000 ± 185,000/μL. These studies show that antibody mediated activation of monocytes contributes to the initiation of HIT, while intravascular activation of platelets contributes to the development of thrombocytopenia. These studies also suggest that depletion of monocytes may be a novel target for therapeutic intervention in the early stages of the disease.