Objective-Biologically significant amounts of two procoagulant molecules, phosphatidylserine (PS) and tissue factor (TF), are transported by monocyte/macrophage-derived microvesicles (MVs). Because cellular cholesterol accumulation is an important feature of atherosclerotic vascular disease, we now examined effects of cholesterol enrichment on MV release from human monocytes and macrophages. Methods and Results-Cholesterol enrichment of human THP-1 monocytes, alone or in combination with lipopolysaccharide (LPS), tripled their total MV generation, as quantified by flow cytometry based on particle size and PS exposure. The subset of these MVs that were also TF-positive was likewise increased by cellular cholesterol enrichment, and these TF-positive MVs exhibited a striking 10-fold increase in procoagulant activity. Moreover, cholesterol enrichment of primary human monocyte-derived macrophages also increased their total as well as TF-positive MV release, and these TF-positive MVs exhibited a similar 10-fold increase in procoagulant activity. To explore the mechanisms of enhanced MV release, we found that cholesterol enrichment of monocytes caused PS exposure on the cell surface by as early as 2 hours and genomic DNA fragmentation in a minority of cells by 20 hours. Addition of a caspase inhibitor at the beginning of these incubations blunted both cholesterol-induced apoptosis and MV release.

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.

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.