Effect of factor XI inhibition on tumor cell-induced coagulation activation.

BACKGROUND: Cancer-associated thrombosis is a frequent complication in patients with malignancies. While factor XI (FXI)/FXIa inhibition is efficacious in preventing postoperative venous thromboembolism, its role in tumor cell-induced coagulation is less defined. OBJECTIVES: We thus aimed to provide mechanistic insights into FXI/FXIa inhibition in tumor cell-induced coagulation activation. METHODS: Procoagulant activity (PCA) of 4 different tissue factor (TF) expressing tumor cell lines was analyzed by single-stage clotting and thrombin generation assay in the presence of a FXIa inhibitor, BMS-262084 (BMS), an inhibitory FXI antibody (anti-FXI), or peak and trough concentrations of rivaroxaban or tinzaparin. Further, tumor cell-induced platelet aggregation was recorded. Recombinant human TF served as positive control. RESULTS: Although BMS and anti-FXI potently inhibited FXIa amidolytic activity, both inhibitors efficiently mitigated recombinant human TF- and tumor cell-induced fibrin clot formation and platelet aggregation only in the presence of low TF PCA. The anticoagulant effects showed an inverse correlation with the magnitude of cellular TF PCA expression. Similarly, BMS markedly interfered with tumor cell-induced thrombin generation, with the most prominent effects on peak and total thrombin. In addition, anticoagulant effects of FXIa inhibition by 10 µM BMS were in a similar range to those obtained by 600 nM rivaroxaban and 1.6 µM tinzaparin at low TF PCA levels. However, rivaroxaban and tinzaparin also exerted marked anticoagulant activity at high TF PCA levels. CONCLUSION: Our findings indicate that FXI/FXIa inhibition interferes with tumor cell-induced coagulation activation only at low TF PCA expression levels, a finding with potential implications for future in vivo studies.

Inhibition of protein disulfide isomerase with PACMA-31 regulates monocyte tissue factor through transcriptional and posttranscriptional mechanisms.

INTRODUCTION: Protein disulfide isomerase (PDI) contributes to tissue factor (TF) regulation in monocytes. While bacitracin and quercetin-3-rutinoside mitigate myeloid TF production, the effect of PACMA-31, a more specific PDI inhibitor with distinct pharmacologic properties, remains unclear. MATERIALS AND METHODS: Lipopolysaccharide (LPS) stimulation of peripheral blood mononuclear cells (PBMCs) or citrate-anticoagulated whole blood was carried out in the presence of PACMA-31 or DMSO vehicle before monocytes were analyzed for TF expression, including antigen, procoagulant activity (PCA) and mRNA, release of IL-6 and TNFα, and LPS-induced signaling pathways. RESULTS: While PACMA-31 alone had no effect, coincubation with LPS and PACMA-31 (25 µM) enhanced LPS-induced monocyte TF production in whole blood. The effect was at least partially regulated on the transcriptional level and could not be explained by increased phosphatidylserine membrane exposure. In contrast, the same PACMA-31 concentrations were cytotoxic in isolated PBMCs. A lower dose of PACMA-31, however, restored the stimulating effect by enhancing IκB-NFκB signaling that also increased the release of IL-6 and TNFα. The protease-activated receptor 2 (PAR2) inhibitor ENMD547 but not TF antibody 10H10 or factor Xa inhibitor rivaroxaban prevented the stimulatory effect of PACMA-31 on inflammatory monocytes. In sharp contrast, short time incubation of LPS-stimulated PBMCs with 25 µM PACMA-31 was non-cytotoxic and significantly inhibited cellular TF PCA but not surface antigen expression. CONCLUSIONS: PACMA-31 regulates monocyte TF in a concentration-dependent manner by opposing transcriptional and posttranscriptional mechanisms. While low concentrations of PACMA-31 augment monocyte TF production by amplifying LPS-dependent PAR2 signaling, high concentrations convert monocyte TF into its non-coagulant state.

Bacitracin and Rutin Regulate Tissue Factor Production in Inflammatory Monocytes and Acute Myeloid Leukemia Blasts.

Aberrant expression of tissue factor (TF) by transformed myeloblasts and inflammatory monocytes drives coagulation activation in acute myeloid leukemia (AML). Although regulation of TF procoagulant activity (PCA) involves thiol-disulfide exchange reactions, the specific role of protein disulfide isomerase (PDI) and other thiol isomerases in AML-associated TF biology is unclear. THP1 cells and peripheral blood mononuclear cells (PBMCs) from healthy controls or AML patients were analyzed for thiol isomerase-dependent TF production under various experimental conditions. Total cellular and membrane TF antigen, TF PCA and TF mRNA were analyzed by ELISA, flow cytometry, clotting or Xa generation assay and qPCR, respectively. PBMCs and THP1 cells showed significant insulin reductase activity, which was inhibited by bacitracin or rutin. Co-incubation with these thiol isomerase inhibitors prevented LPS-induced TF production by CD14-positive monocytes and constitutive TF expression by THP1 cells and AML blasts. Downregulation of the TF antigen was mainly restricted to the cryptic pool of TF, efficiently preventing phosphatidylserine-dependent TF activation by daunorubicin, and at least partially regulated on the mRNA level in LPS-stimulated monocytes. Our study thus delineates a complex role of thiol isomerases in the regulation of myeloid TF PCA, with PDI being a promising therapeutic target in the management of AML-associated coagulopathies.

Myeloperoxidase has no effect on the low procoagulant activity of silica-free DNA.

Blood coagulation and innate immunity are closely interrelated. At sites of inflammation, DNA and myeloperoxidase (MPO) are released from polymorphonuclear leukocytes (PMNs) as an integral component of neutrophil extracellular traps (NETs). NETs exert pleiotropic thrombogenic effects, with DNA-mediated contact activation of factor XII (FXII) likely playing a role. We have previously shown that MPO, a highly cationic protein, regulates coagulation through heteromolecular interactions with various negatively charged structures, including membrane phospholipids and low-molecular-weight heparin. The aims of our current study were to confirm that DNA activates coagulation and to investigate whether its procoagulant activity (PCA) is regulated by PMN-derived MPO. To this end, we used thrombin generation and FXIIa amidolytic activity assays to analyze the PCA of cell-free DNA isolated with silica membrane-based (cfDNA) or silica-free procedures (PaxDNA). cfDNA potently activated FXII and promoted thrombin generation in a concentration-dependent manner, but its PCA was largely attributable to contaminating silica particles. In contrast, pure, i.e. silica-free, PaxDNA was markedly less procoagulant. Although PaxDNA amplified thrombin generation in plasma, it was devoid of any direct FXII activating activity. MPO supershifted both cfDNA and PaxDNA in gel electrophoresis, but only silica-associated PCA of cfDNA was neutralized by MPO independently of its catalytic properties. Moreover, pretreatment with DNase I abolished silica-induced thrombin generation. In summary, we show that pure DNA has rather weak PCA, which is not further inhibited by heteromolecular complex formation with exogenous MPO. Our study thus provides novel mechanistic insights into the regulation of coagulation by extracellular DNA under inflammatory conditions.

Effect of myeloperoxidase on the anticoagulant activity of low molecular weight heparin and rivaroxaban in an in vitro tumor model.

BACKGROUND: Inflammation with leukocyte activation is a hallmark of cancer-associated thrombosis (CAT), and elevated leukocytes predict venous thromboembolism in cancer outpatients. In a recent trial, rivaroxaban was more efficacious than dalteparin in preventing CAT recurrence. OBJECTIVES: In a proof-of-concept study, we aimed to provide a mechanistic basis for improved efficacy of rivaroxaban compared to low molecular weight heparin in CAT treatment. METHODS: We studied the effects of rivaroxaban, dalteparin, and tinzaparin at peak and trough levels on tumor cell-induced procoagulant activity and platelet aggregation in the presence or absence of the cationic leukocyte-derived enzyme, myeloperoxidase (MPO). Furthermore, pro-inflammatory conditions were generated by stimulating whole blood with lipopolysaccharide (LPS) or phorbol-myristate-acetate (PMA), before measuring thrombin generation in plasma supernatants. RESULTS: All three anticoagulants inhibited thrombin generation, fibrin clot formation, and platelet aggregation induced by the tissue factor-expressing prostate carcinoma cell line, 22Rv1. Pre-incubation with MPO partially attenuated the anticoagulant activity of dalteparin and tinzaparin, but not rivaroxaban, at trough levels. The effect of MPO did not involve the enzyme's catalytic properties, but required its structural integrity, as indicated by heat denaturation. In plasma obtained from LPS- or PMA-stimulated whole blood, elevated MPO antigen levels inversely correlated with the ability of tinzaparin to inhibit 22Rv1-induced thrombin generation. CONCLUSIONS: Myeloperoxidase release may partially attenuate the anticoagulant activity of trough levels of dalteparin and tinzaparin in the context of paraneoplastic leukocyte activation. However, this effect is likely not sufficient to explain the improved efficacy of rivaroxaban, and possibly other oral factor Xa inhibitors, in CAT treatment.

Monitoring multiple myeloma by next-generation sequencing of V(D)J rearrangements from circulating myeloma cells and cell-free myeloma DNA.

Recent studies suggest that circulating tumor cells and cell-free DNA may represent powerful non-invasive tools for monitoring disease in patients with solid and hematologic malignancies. Here, we conducted a pilot study in 27 myeloma patients to explore the clonotypic V(D)J rearrangement for monitoring circulating myeloma cells and cell-free myeloma DNA. Next-generation sequencing was used to define the myeloma V(D)J rearrangement and for subsequent peripheral blood tracking after treatment initiation. Positivity for circulating myeloma cells/cell-free myeloma was associated with conventional remission status (P<0.001) and 91% of non-responders/progressors versus 41% of responders had evidence of persistent circulating myeloma cells/cell-free myeloma DNA (P<0.001). About half of the partial responders showed complete clearance of circulating myeloma cells/cell-free myeloma DNA despite persistent M-protein, suggesting that these markers are less inert than the M-protein, rely more on cell turnover and, therefore, decline more rapidly after initiation of effective treatment. Positivity for circulating myeloma cells and for cell-free myeloma DNA were associated with each other (P=0.042), but discordant in 30% of cases. This indicates that cell-free myeloma DNA may not be generated entirely by circulating myeloma cells and may reflect overall tumor burden. Prospective studies need to define the predictive potential of high-sensitivity determination of circulating myeloma cells and DNA in the monitoring of multiple myeloma.