Microparticle-Associated Tissue Factor: A Molecular Link Between Coagulation Activation, Inflammation and Disease Progression in Early-Stage Prostate Cancer?
Authors: Haubold Katja , Rink Michael , Spath Brigitte , Amirkhosravi Ali , Francis John L. , Eifrig Barbara , Bokemeyer Carsten , Langer Florian 
Published: October 2019

Abstract
Activation of coagulation and inflammation is a characteristic finding in patients with advanced malignancies, including prostate cancer. Tissue factor (TF), a molecule involved in hemostasis, thrombosis and pro-inflammatory signaling pathways, is over-expressed on tumor cells and cells of the tumor microenvironment (i.e. endothelial cells, fibroblasts and tissue macrophages). Moreover, the enhanced release of TF into plasma in association with sub-cellular membrane vesicles, so-called plasma microparticles (MPs), has recently been associated with key events in molecular oncogenesis and cancer progression. In this study, we measured TF-specific procoagulant activity (PCA) of plasma MPs in 58 consecutive patients with clinically localized prostate cancer (mean age, 64±5 years) to explore its potential as a prognostic marker in this tumor entity. MPs were isolated from pre-operative plasma samples by sequential high-speed centrifugation for 1 h at 16,100 × g. TF-specific PCA of plasma MPs was quantified using a highly sensitive one-stage clotting assay in the presence and absence of inhibitory TF monoclonal antibody and calibration of clotting times against serial dilutions (1:10–1:105) of lipidated recombinant human full-length TF (rhTF1–263), showing a linear correlation in a log-log plot with R2>0.99. The lower detection limit of this assay for rhTF1–263 (33 kDa) was <5 pg/ml (<150 fM), and the intra- and inter-assay coefficients of variation were 7.3% and 5.4%, respectively. Total numbers of TF-positive MPs were measured by single-color flow cytometry using PE-conjugated TF monoclonal antibody (HTF-1) and microspheres for size calibration (1 μm) and sample flow standardization. TF antigen was quantified in plasma by ELISA. Calibrated automated thrombography (CAT) was used to monitor thrombin generation in platelet-free plasma samples over a 2-h period without the addition of exogenous TF or phospholipids. Intravascular coagulation activation was assessed by measuring plasma D-dimer. All assay systems were validated using MPs spontaneously shed from prostate cancer cell lines (PC-3, LNCaP and DU145) or from whole blood monocytes after challenge with endotoxin. Based on plasma fibrinogen and C-reactive protein levels, patients were stratified into those with (n=26) and those without (n=32) laboratory evidence of an acute-phase reaction (APR). Compared to healthy male controls (n=20), patients had significantly increased levels of both D-dimer (0.46±0.19 vs. 0.21±0.05 mg/l) and TF-specific PCA of plasma MPs (563±301 vs. 292±74 U/ml) (P<0.001). Among patients, laboratory evidence of an APR was associated with a significant increase in MP-associated TF PCA (699±351 vs. 452±196 U/ml) (P=0.001). Overall, we found a significant correlation between MP-associated TF PCA and plasma D-Dimer (P=0.015), suggesting that plasma MPs contributed to in-vivo coagulation activation in a TF-dependent manner. CAT also revealed significantly increased thrombin generation in patient compared to control plasmas, as indicated by a shortening in lag phase (25±4 vs. 29±5 min) and an increase in both peak thrombin generation (184±76 vs. 127±71 nM) and the endogenous thrombin potential, defined as the area under the thrombin generation curve (3576±509 vs. 2980±562 nM*min) (P<0.01). Importantly, TF-specific PCA of plasma MPs correlated neither with absolute numbers of TF-positive MPs nor with plasma TF antigen, suggesting that a substantial and variable fraction of the total plasma TF pool circulated as an inactive variant. Interestingly, systemic levels of IL-8, an inflammatory cytokine involved in TF/FVIIa-dependent, PAR-2-mediated pro-migratory signaling pathways in tumor cells and shown to be of biological relevance in advanced, hormone-refractory prostate cancer, were elevated in patients compared to controls (9±11 vs. 4±6 pg/ml) (P<0.01). In summary, our findings suggest that TF-specific PCA of plasma MPs contributes to intravascular coagulation activation in patients with early-stage prostate cancer and may represent an important molecular link between hypercoagulability, inflammation and disease progression. The above-described assay for the quantification of MP-associated TF PCA could thus be of prognostic value in the risk stratification of patients with localized prostate cancer with respect to thromboembolic complications and/or tumor recurrence.