The Role of Fcγ Receptor Signaling, Tissue Factor Expression and Monocyte-Platelet Cross-Talk in the Initiation of Thrombosis by Immune Complexes.

Thrombus formation in response to physical disruption of the vascular endothelium is an essential response to vessel injury. In contrast, thrombus formation is pathologic when the endothelium is physically intact but blood and endothelial cells are activated by inflammation. Thrombosis secondary to immune complexes is a major cause of morbidity and mortality in hospitalized patients. We recently generated and characterized the first transgenic mouse model of heparin-induced thrombocytopenia/thrombosis (HIT/T) to recapitulate the salient features of the disease and confirmed that complexes of heparin and platelet factor 4, antibodies to the complex, and FcγRIIa-dependent platelet activation are both necessary and sufficient to model the disease in vivo. It is also likely that immune complex activation of monocytes and endothelial cells occurs in HIT/T. However, the interaction between activated blood and endothelial cells and tissue factor positive microparticles (TF⁺-MP) that may result in thrombin generation is not clear. Recent studies (del Conde et al., Blood 2005) showed that phosphatidylserine and PSGL-1 on the surface of monocyte-derived TF⁺-MP enables their fusion with activated platelets. Collagen-activated platelets incubated with TF⁺-MP were reported to cause increased TF-VIIa procoagulant activity (PCA) compared to non-activated platelets. We hypothesized that platelets activated by HIT/T immune complexes would also result in increased TF-PCA when incubated with monocyte-derived TF⁺-MP. To test this hypothesis we generated TF⁺-MP from THP-1 cells, a human monocytic cell line, stimulated with LPS (6 hr) and A23187 (subsequent 15 min). TF⁺-MPs were co-incubated with untreated or agonist-treated platelets. The HIT/T immune complex was prepared by incubating optimal ratios of heparin and recombinant human PF4 with KKO, a mouse monoclonal anti-heparin-PF4 antibody. Other agonists included anti-CD9 (producing a particulate immune complex) and collagen. Using a chromogenic assay of Xa generation we found that TF⁺-MPs were necessary to detect TF-PCA. PCA increased by 12–25% when TF⁺-MPs were incubated with platelets stimulated by collagen or anti-CD9 as compared to untreated platelets. When TF⁺-MPs were incubated with platelets stimulated by the HIT/T immune complex, there was a 2-fold increase in the PCA. The increase in TF-PCA was observed to be proportional to the concentration of microparticles added. The results suggest an important role in platelet-monocyte cross-talk in initiating and increasing TF procoagulant activity upon immune complex stimulation.