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Thromb Haemost 2019; 119(09): 1419-1432
DOI: 10.1055/s-0039-1692682
Coagulation and Fibrinolysis
Georg Thieme Verlag KG Stuttgart · New York

Apixaban Suppresses the Release of TF-Positive Microvesicles and Restrains Cancer Cell Proliferation through Directly Inhibiting TF-fVIIa Activity

Sophie Featherby‡   *
1   Biomedical Section, University of Hull, Hull, United Kingdom
,
Yahya Madkhali‡   *
1   Biomedical Section, University of Hull, Hull, United Kingdom
2   Department of Medical Laboratories, College of Applied Medical Sciences, Majmaah University, Al Majmaah, Saudi Arabia
,
Anthony Maraveyas
3   Division of Cancer, Hull York Medical School, University of Hull, Hull, United Kingdom
,
Camille Ettelaie 
1   Biomedical Section, University of Hull, Hull, United Kingdom
› Author Affiliations Funding This study was partly sponsored by Bristol-Myers Squibb (CV185-484) and pure substances were provided by Bristol Meyer Squibb (apixaban) and Bayer UK (rivaroxaban). S.F. is supported by an unrestricted grant from the Castle Hill Cancer Charity.
Further Information

Publication History

04 February 2019

13 May 2019

Publication Date:
02 July 2019 (online)

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Abstract

The activation of protease-activated receptor (PAR)-2 by factor Xa (fXa) promotes the release of tissue factor-positive microvesicles (TF+MV), and contributes to proliferation in cancer cells. This study examined the ability of direct oral anticoagulants (DOACs), apixaban and rivaroxaban, to inhibit the release of TF+MV from two cell lines (MDA-MB-231 and AsPC-1) as well as cell proliferation.

Activation of the cells with fXa (10 nM) enhanced the release of TF+MV but was suppressed in the presence of either DOAC. These MVs were found to contain fVIIa, but not fXa. Incubation of cell lines with apixaban (1.8 µM) but not rivaroxaban (1.8 µM), in the absence of fXa decreased the release of TF+MV below that of resting cells, in a PAR2-dependent manner. Furthermore, incubation with apixaban reduced the proliferation rate in both cells lines. Incubation of purified fVIIa with apixaban but not rivaroxaban resulted in complete inhibition of fVIIa proteolytic activity as measured using two fVIIa chromogenic substrates. Pre-incubation of the cells with an inhibitory anti-fVIIa antibody, with apixaban or the blocking of PAR2 suppressed the release of TF+MV to a comparable level, and reduced cell proliferation but the effect was not cumulative.

This study has established that the activation of PAR2 by TF–fVIIa complex is the principal mediator in augmenting the release of TF+MV as well as cancer cell proliferation. Importantly, for the first time we have shown that apixaban selectively inhibits the proteolytic activity of fVIIa as well as the signalling arising from the TF–fVIIa complex.

Keywords

direct oral anticoagulants - factor VIIa - microvesicles - protease-activated receptor-2 - tissue factor

* S.F. and Y.M. contributed equally to this study.


 

  • References

  • 1 Varki A. Trousseau's syndrome: multiple definitions and multiple mechanisms. Blood 2007; 110 (06) 1723-1729
    CrossrefPubMedGoogle Scholar
  • 2 Khorana AA, Francis CW, Culakova E, Kuderer NM, Lyman GH. Thromboembolism is a leading cause of death in cancer patients receiving outpatient chemotherapy. J Thromb Haemost 2007; 5 (03) 632-634
    CrossrefPubMedGoogle Scholar
  • 3 Zacharski LR, Henderson WG, Rickles FR. , et al. Effect of warfarin anticoagulation on survival in carcinoma of the lung, colon, head and neck, and prostate. Final report of VA Cooperative Study #75. Cancer 1984; 53 (10) 2046-2052
    CrossrefPubMedGoogle Scholar
  • 4 Altinbas M, Coskun HS, Er O. , et al. A randomized clinical trial of combination chemotherapy with and without low-molecular-weight heparin in small cell lung cancer. J Thromb Haemost 2004; 2 (08) 1266-1271
    CrossrefPubMedGoogle Scholar
  • 5 Seto S, Onodera H, Kaido T. , et al. Tissue factor expression in human colorectal carcinoma: correlation with hepatic metastasis and impact on prognosis. Cancer 2000; 88 (02) 295-301
    CrossrefPubMedGoogle Scholar
  • 6 Khorana AA, Ahrendt SA, Ryan CK. , et al. Tissue factor expression, angiogenesis, and thrombosis in pancreatic cancer. Clin Cancer Res 2007; 13 (10) 2870-2875
    CrossrefPubMedGoogle Scholar
  • 7 Kaido T, Oe H, Yoshikawa A, Mori A, Arii S, Imamura M. Tissue factor is a useful prognostic factor of recurrence in hepatocellular carcinoma in 5-year survivors. Hepatogastroenterology 2005; 52 (65) 1383-1387
    PubMedGoogle Scholar
  • 8 Ettelaie C, Collier MEW, Featherby S, Benelhaj NE, Greenman J, Maraveyas A. Analysis of the potential of cancer cell lines to release tissue factor-containing microvesicles: correlation with tissue factor and PAR2 expression. Thromb J 2016; 14: 2
    CrossrefPubMedGoogle Scholar
  • 9 Schaffner F, Ruf W. Tissue factor and protease-activated receptor signaling in cancer. Semin Thromb Hemost 2008; 34 (02) 147-153
    Article in Thieme ConnectPubMedGoogle Scholar
  • 10 Hron G, Kollars M, Weber H. , et al. Tissue factor-positive microparticles: cellular origin and association with coagulation activation in patients with colorectal cancer. Thromb Haemost 2007; 97 (01) 119-123
    Article in Thieme ConnectPubMedGoogle Scholar
  • 11 Thaler J, Koder S, Kornek G, Pabinger I, Ay C. Microparticle-associated tissue factor activity in patients with metastatic pancreatic cancer and its effect on fibrin clot formation. Transl Res 2014; 163 (02) 145-150
    CrossrefPubMedGoogle Scholar
  • 12 Auwerda JJ, Yuana Y, Osanto S. , et al. Microparticle-associated tissue factor activity and venous thrombosis in multiple myeloma. Thromb Haemost 2011; 105 (01) 14-20
    Article in Thieme ConnectPubMedGoogle Scholar
  • 13 Hisada Y, Ay C, Auriemma AC, Cooley BC, Mackman N. Human pancreatic tumors grown in mice release tissue factor-positive microvesicles that increase venous clot size. J Thromb Haemost 2017; 15 (11) 2208-2217
    CrossrefPubMedGoogle Scholar
  • 14 Boulanger CM, Scoazec A, Ebrahimian T. , et al. Circulating microparticles from patients with myocardial infarction cause endothelial dysfunction. Circulation 2001; 104 (22) 2649-2652
    CrossrefPubMedGoogle Scholar
  • 15 Camerer E, Huang W, Coughlin SR. Tissue factor- and factor X-dependent activation of protease-activated receptor 2 by factor VIIa. Proc Natl Acad Sci U S A 2000; 97 (10) 5255-5260
    CrossrefPubMedGoogle Scholar
  • 16 Riewald M, Ruf W. Mechanistic coupling of protease signaling and initiation of coagulation by tissue factor. Proc Natl Acad Sci U S A 2001; 98 (14) 7742-7747
    CrossrefPubMedGoogle Scholar
  • 17 Wojtukiewicz MZ, Hempel D, Sierko E, Tucker SC, Honn KV. Protease-activated receptors (PARs)--biology and role in cancer invasion and metastasis. Cancer Metastasis Rev 2015; 34 (04) 775-796
    CrossrefPubMedGoogle Scholar
  • 18 Schaffner F, Ruf W. Tissue factor and PAR2 signaling in the tumor microenvironment. Arterioscler Thromb Vasc Biol 2009; 29 (12) 1999-2004
    CrossrefPubMedGoogle Scholar
  • 19 Sedda S, Marafini I, Caruso R, Pallone F, Monteleone G. Proteinase activated-receptors-associated signaling in the control of gastric cancer. World J Gastroenterol 2014; 20 (34) 11977-11984
    CrossrefPubMedGoogle Scholar
  • 20 Ohta T, Shimizu K, Yi S. , et al. Protease-activated receptor-2 expression and the role of trypsin in cell proliferation in human pancreatic cancers. Int J Oncol 2003; 23 (01) 61-66
    PubMedGoogle Scholar
  • 21 Collier MEW, Ettelaie C. Regulation of the incorporation of tissue factor into microparticles by serine phosphorylation of the cytoplasmic domain of tissue factor. J Biol Chem 2011; 286 (14) 11977-11984
    CrossrefPubMedGoogle Scholar
  • 22 Hjortoe GM, Petersen LC, Albrektsen T. , et al. Tissue factor-factor VIIa-specific up-regulation of IL-8 expression in MDA-MB-231 cells is mediated by PAR-2 and results in increased cell migration. Blood 2004; 103 (08) 3029-3037
    CrossrefPubMedGoogle Scholar
  • 23 Su S, Li Y, Luo Y. , et al. Proteinase-activated receptor 2 expression in breast cancer and its role in breast cancer cell migration. Oncogene 2009; 28 (34) 3047-3057
    CrossrefPubMedGoogle Scholar
  • 24 Lima LG, Monteiro RQ. Activation of blood coagulation in cancer: implications for tumour progression. Biosci Rep 2013; 33 (05) e00064
    CrossrefPubMedGoogle Scholar
  • 25 Monteiro RQ, Lima LG, Gonçalves NP. , et al. Hypoxia regulates the expression of tissue factor pathway signaling elements in a rat glioma model. Oncol Lett 2016; 12 (01) 315-322
    CrossrefPubMedGoogle Scholar
  • 26 Rondon AMR, de Almeida VH, Gomes T. , et al. Tissue factor mediates microvesicles shedding from MDA-MB-231 breast cancer cells. Biochem Biophys Res Commun 2018; 502 (01) 137-144
    CrossrefPubMedGoogle Scholar
  • 27 Das K, Prasad R, Singh A. , et al. Protease-activated receptor 2 promotes actomyosin dependent transforming microvesicles generation from human breast cancer. Mol Carcinog 2018; 57 (12) 1707-1722
    CrossrefPubMedGoogle Scholar
  • 28 Grover SP, Mackman N. Tissue factor: an essential mediator of hemostasis and trigger of thrombosis. Arterioscler Thromb Vasc Biol 2018; 38 (04) 709-725
    CrossrefPubMedGoogle Scholar
  • 29 Ciurus T, Sobczak S, Cichocka-Radwan A, Lelonek M. New oral anticoagulants - a practical guide. Kardiochir Torakochirurgia Pol 2015; 12 (02) 111-118
    PubMedGoogle Scholar
  • 30 Frost C, Wang J, Nepal S. , et al. Apixaban, an oral, direct factor Xa inhibitor: single dose safety, pharmacokinetics, pharmacodynamics and food effect in healthy subjects. Br J Clin Pharmacol 2013; 75 (02) 476-487
    CrossrefPubMedGoogle Scholar
  • 31 Mueck W, Stampfuss J, Kubitza D, Becka M. Clinical pharmacokinetic and pharmacodynamic profile of rivaroxaban. Clin Pharmacokinet 2014; 53 (01) 1-16
    CrossrefPubMedGoogle Scholar
  • 32 Ettelaie C, Collier ME, Mei MP, Xiao YP, Maraveyas A. Enhanced binding of tissue factor-microparticles to collagen-IV and fibronectin leads to increased tissue factor activity in vitro. Thromb Haemost 2013; 109 (01) 61-71
    Article in Thieme ConnectPubMedGoogle Scholar
  • 33 Bonnekoh B, Wevers A, Jugert F, Merk H, Mahrle G. Colorimetric growth assay for epidermal cell cultures by their crystal violet binding capacity. Arch Dermatol Res 1989; 281 (07) 487-490
    CrossrefPubMedGoogle Scholar
  • 34 Collier ME, Ettelaie C. Induction of endothelial cell proliferation by recombinant and microparticle-tissue factor involves beta1-integrin and extracellular signal regulated kinase activation. Arterioscler Thromb Vasc Biol 2010; 30 (09) 1810-1817
    CrossrefPubMedGoogle Scholar
  • 35 Ettelaie C, Collier MEW, Maraveyas A, Ettelaie R. Characterization of physical properties of tissue factor-containing microvesicles and a comparison of ultracentrifuge-based recovery procedures. J Extracell Vesicles 2014; 3: 10
    PubMedGoogle Scholar
  • 36 Collier ME, Mah PM, Xiao YP, Maraveyas A, Ettelaie C. Microparticle-associated tissue factor is recycled by endothelial cells resulting in enhanced surface tissue factor activity. Thromb Haemost 2013; 110: 966-976
    Article in Thieme ConnectPubMedGoogle Scholar
  • 37 Ettelaie C, Collier MEW, Featherby S, Greenman J, Maraveyas A. Peptidyl-prolyl isomerase 1 (Pin1) preserves the phosphorylation state of tissue factor and prolongs its release within microvesicles. Biochim Biophys Acta Mol Cell Res 2018; 1865 (01) 12-24
    CrossrefPubMedGoogle Scholar
  • 38 Broze Jr GJ, Hickman S, Miletich JP. Monoclonal anti-human factor VII antibodies. Detection in plasma of a second protein antigenically and genetically related to factor VII. J Clin Invest 1985; 76 (03) 937-946
    CrossrefPubMedGoogle Scholar
  • 39 Böhm E, Seyfried BK, Dockal M. , et al. Differences in N-glycosylation of recombinant human coagulation factor VII derived from BHK, CHO, and HEK293 cells. BMC Biotechnol 2015; 15: 87
    CrossrefPubMedGoogle Scholar
  • 40 Tang JQ, Fan Q, Wu WH. , et al. Extrahepatic synthesis of coagulation factor VII by colorectal cancer cells promotes tumor invasion and metastasis. Chin Med J (Engl) 2010; 123 (24) 3559-3565
    PubMedGoogle Scholar
  • 41 Koizume S, Jin MS, Miyagi E. , et al. Activation of cancer cell migration and invasion by ectopic synthesis of coagulation factor VII. Cancer Res 2006; 66 (19) 9453-9460
    CrossrefPubMedGoogle Scholar
  • 42 Koizume S, Ito S, Yoshioka Y. , et al. High-level secretion of tissue factor-rich extracellular vesicles from ovarian cancer cells mediated by filamin-A and protease-activated receptors. Thromb Haemost 2016; 115 (02) 299-310
    Article in Thieme ConnectPubMedGoogle Scholar
  • 43 Madkhali Y, Featherby S, Collier MEW, Maraveyas A, Greenman J, Ettelaie C. The ratio of factor VIIa:tissue factor content within microvesicles determines the differential influence on endothelial cells. THOpen 2019; 03 (02) e132-e145
    PubMedGoogle Scholar
  • 44 Collier MEW, Ettelaie C, Goult BT, Maraveyas A, Goodall AH. Investigation of the filamin A-dependent mechanisms of tissue factor incorporation into microvesicles. Thromb Haemost 2017; 117 (11) 2034-2044
    Article in Thieme ConnectPubMedGoogle Scholar
  • 45 Guasti L, Squizzato A, Moretto P. , et al. In vitro effects of Apixaban on 5 different cancer cell lines. PLoS One 2017; 12 (10) e0185035
    CrossrefPubMedGoogle Scholar
  • 46 Wong PC, White A, Luettgen J. Inhibitory effect of apixaban compared with rivaroxaban and dabigatran on thrombin generation assay. Hosp Pract (1995) 2013; 41 (01) 19-25
    CrossrefPubMedGoogle Scholar
  • 47 Sadeghi N, Kahn D, Jeske W, Hoppensteadt D, Fareed J. Tissue factor-mediated activation of the prothrombin complex concentrate (PCC) is differently inhibited by dabigatran, rivaroxaban, and apixaban: potential clinical implication. Clin Appl Thromb Hemost 2013; 19 (06) 589-599
    CrossrefPubMedGoogle Scholar
  • 48 Adelmann D, Wiegele M, Wohlgemuth RK. , et al. Measuring the activity of apixaban and rivaroxaban with rotational thromboelastometry. Thromb Res 2014; 134 (04) 918-923
    CrossrefPubMedGoogle Scholar
  • 49 Wong PC, Crain EJ, Xin B. , et al. Apixaban, an oral, direct and highly selective factor Xa inhibitor: in vitro, antithrombotic and antihemostatic studies. J Thromb Haemost 2008; 6 (05) 820-829
    CrossrefPubMedGoogle Scholar
  • 50 Gardiner C, Kohama K, Patel I. , et al. A performance evaluation of a novel human recombinant tissue factor prothrombin time reagent (Revohem™ PT). Int J Lab Hematol 2017; 39 (05) 532-538
    CrossrefPubMedGoogle Scholar
  • 51 Pinto DJ, Orwat MJ, Koch S. , et al. Discovery of 1-(4-methoxyphenyl)-7-oxo-6-(4-(2-oxopiperidin-1-yl)phenyl)-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridine-3-carboxamide (apixaban, BMS-562247), a highly potent, selective, efficacious, and orally bioavailable inhibitor of blood coagulation factor Xa. J Med Chem 2007; 50 (22) 5339-5356
    CrossrefPubMedGoogle Scholar
  • 52 Jourdi G, Siguret V, Martin AC. , et al. Association rate constants rationalise the pharmacodynamics of apixaban and rivaroxaban. Thromb Haemost 2015; 114 (01) 78-86
    Article in Thieme ConnectPubMedGoogle Scholar
  • 53 Wong PC, Luettgen JM, Rendina AR. , et al. BMS-593214, an active site-directed factor VIIa inhibitor: enzyme kinetics, antithrombotic and antihaemostatic studies. Thromb Haemost 2010; 104 (02) 261-269
    Article in Thieme ConnectPubMedGoogle Scholar
  • 54 Wong PC, Jiang X. Apixaban, a direct factor Xa inhibitor, inhibits tissue-factor induced human platelet aggregation in vitro: comparison with direct inhibitors of factor VIIa, XIa and thrombin. Thromb Haemost 2010; 104 (02) 302-310
    Article in Thieme ConnectPubMedGoogle Scholar
  • 55 Nieman MT. Protease-activated receptors in hemostasis. Blood 2016; 128 (02) 169-177
    PubMedGoogle Scholar
  • 56 Rothmeier AS, Ruf W. Protease-activated receptor 2 signaling in inflammation. Semin Immunopathol 2012; 34 (01) 133-149
    CrossrefPubMedGoogle Scholar
  • 57 McCoy KL, Traynelis SF, Hepler JR. PAR1 and PAR2 couple to overlapping and distinct sets of G proteins and linked signaling pathways to differentially regulate cell physiology. Mol Pharmacol 2010; 77 (06) 1005-1015
    CrossrefPubMedGoogle Scholar
  • 58 Alberelli MA, De Candia E. Functional role of protease activated receptors in vascular biology. Vascul Pharmacol 2014; 62 (02) 72-81
    CrossrefPubMedGoogle Scholar
  • 59 Rousseau A, Van Dreden P, Mbemba E, Elalamy I, Larsen A, Gerotziafas GT. Cancer cells BXPC3 and MCF7 differentially reverse the inhibition of thrombin generation by apixaban, fondaparinux and enoxaparin. Thromb Res 2015; 136 (06) 1273-1279
    CrossrefPubMedGoogle Scholar