Signaling Pathways in HIT Immune Complex Stimulation of Monocyte Tissue Factor.

In heparin-induced thrombocytopenia (HIT), platelet-fibrin thrombi form in vivo due to generation of thrombin initiated by intravascular tissue factor (TF). Stimulation of monocytic cells in vitro by the HIT immune complex (IC), consisting of heparin/PF4 complexes and IgG anti-hep/PF4 antibodies, triggers TF expression. The HIT IC binds to Fcγ receptors on monocytic cells to initiate a signal cascade which results in new TF transcription. Though much is known about monocyte TF transcription as a consequence of LPS stimulation of TLR4, the mechanism by which HIT IC engagement of FcγR leads to TF transcription is unknown. Transcription of pro-inflammatory and anti-inflammatory cytokines as a consequence of IC engagement of FcγRs on monocytes has been reported to involve the ras/Erk and/or PI3K/Akt pathways downstream of syk. We tested the hypothesis that HIT IC- FcγR engagement signals via a PI3K/Akt pathway to trigger TF transcription. We created a model HIT IC consisting of an optimal ratio of heparin and recombinant human PF4, bound by well-characterized monoclonal anti-hep/hPF4 antibody KKO. Using human monocytic cell line THP-1, we observed that the HIT IC induction of new TF mRNA, assessed by qRT-PCR after treatment for 2 h at 37°C, was reduced approximately 80% by pretreatment of the cells with PI3K inhibitor wortmannin (100 nM; n = 3, p < 0.05). In addition, the same effect was observed with another IC, heat-aggregated human IgG, namely 80% inhibition of stimulated TF expression by wortmannin (pretreatment with 100 nM, 37°C, 30 min, n = 4, p< 0.05). Of note, the LPS-mediated increase in TF expression was enhanced over 2-fold (1 ug/ml, 2 h, p <0.05) by the same wortmannin pretreatment, in direct contrast to the result with IC stimulation. In preliminary studies with Akt inhibitor SH5, we have observed ~80% inhibition of IC-stimulated TF expression. We have verified the effects of PI3K inhibition of IC-stimulation of TF (75–80% inhibition by wortmannin pretreatment) by using primary peritoneal macrophages derived from FcγRIIa-transgenic mice, which express a similar FcγR pattern as human cells and our HIT mouse model. Immunoblot analysis of phospho-Akt using anti-Ser473 Akt antibody(generously provided by Donna Woulfe, PhD, Thomas Jefferson University) verified the phosphorylation of Akt by HIT IC treatment and marked reduction when PI3K was inhibited. In summary, HIT IC stimulation of monocytic cell TF involves a PI3K/Akt pathway in a way clearly distinct from LPS-stimulated TF expression. Further studies are in progress to elucidate the upstream activators and downstream effectors, the role of other signaling pathways and the identity of the responsible transcription factor complexes.