Subscribe to RSS
The Intrinsic Pathway does not Contribute to Activation of Coagulation in Mice Bearing Human Pancreatic Tumors Expressing Tissue FactorFunding This work was supported by grants from Japanese Society on Thrombosis and Hemostasis (Y.H.), SENSHIN Medical Research Foundation (T.K.), National Institute of Health (Y.H. T32 HL007149 and N.M. R01 HL147149), and the John C. Parker Professorship (N.M.).
The intrinsic pathway of coagulation has been shown to contribute to thrombosis. Factor (F) XII can be activated by a variety of agents, such as polyphosphate, and promotes coagulation by converting FXI to FXIa. Thrombin can also activate FXI through a positive feedback loop. FXII−/− and FXI−/− mice had reduced thrombosis compared with wild-type mice.   Similarly, wild-type mice treated with antisense oligonucleotides (ASOs) against either FXII or FXI had reduced thrombosis compared with controls.  Importantly, reducing FXI expression or inhibiting FXIa activity significantly reduced postoperative venous thromboembolism in patients who underwent total knee arthroplasty without increasing bleeding. 
There are very few studies on the role of the intrinsic pathway in cancer-associated thrombosis. We showed that extracellular vesicles (EVs) derived from the human prostate cancer cell line PC3 contained polyphosphate. EVs are small membrane vesicles released from a variety of cells, including cancer cells. PC3-derived EVs induced a high rate of death in wild-type mice in a pulmonary embolism model whereas FXII−/− or FXI−/− mice were protected. Similarly, the administration of the anti-FXIIa antibody prevented death of wild-type mice that received PC3-derived EVs. PC3-derived EVs also expressed tissue factor (TF) and inhibition of TF also protected wild-type mice from EV-induced death. One study found that patients with nonmetastatic colorectal cancer have significantly lower plasma levels of FXII zymogen but not FXI compared with healthy controls, which suggests activation and consumption of FXII.
Pancreatic cancer is associated with a high incidence of venous thromboembolism (5–26%).  TF is expressed by pancreatic cancer cell lines and tumors.   TF-positive EVs are released from cancer cells and circulate in the blood in both patients and mouse models.   We and others showed that EV TF activity is associated with venous thromboembolism in patients with pancreatic cancer.  Importantly, in mice bearing human pancreatic BxPC-3 tumors TF derived from the tumor enhanced venous thrombosis.
In this study, we investigated the role of FXI in the activation of coagulation in a mouse model of pancreatic cancer. We used BxPC-3 cells modified to express the luciferase reporter. Tumors were grown orthotopically in Crl:NU-Foxn1nu male mice and imaged using the IVIS Lumina. Blood was collected from the inferior vena cava into citrate and platelet-poor plasma was prepared by centrifugation at 4,500 × g for 15 minutes. Mice were treated with either a nontargeted (control) ASO or an ASO selectively targeting FXI (Ionis Pharmaceuticals, 50 mg/kg, subcutaneously, single injection). Importantly, the FXI ASO reduced F11 messenger ribonucleic acid (mRNA) expression in the liver by 98%, and significantly increased the activated partial thromboplastin time (aPTT). Administration of the FXI ASO twice weekly for 3 weeks and then collecting samples 3 days later led to an approximately twofold increase in aPTT, whereas a single administration of the FXI ASO and collecting samples 3 days later led to an approximately 1.3-fold increase in aPTT. We used a single administration of FXI ASOs in control mice or in tumor-bearing mice once the tumors have reached ≥ 1.8 g. Livers and blood were collected 3 days after ASO injection. Levels of F11 mRNA expression were measured as described. Enzyme-linked immunosorbent assays were used to measure plasma levels of human TF protein (Biomedica Diagnostics, Cat#845) and thrombin antithrombin (TAT) complexes (Siemens, Cat#OWMG15).
In a previous study, we showed that BxPC-3 expresses the highest level of TF among human pancreatic cancer cell lines. In addition, BxPC-3 tumor-bearing mice had significantly increased levels of human TF protein and TAT complexes compared with controls.  Consistent with our previous studies, we observed increased plasma levels of human TF protein and TAT complexes in tumor-bearing mice compared with controls ([Fig. 1A], [B]), which indicated that tumor-bearing mice have an activated coagulation system.
We hypothesized that the intrinsic pathway would contribute to the activation of coagulation in BxPC-3 tumor-bearing mice. Therefore, we examined the effect of suppressing F11 mRNA expression using ASOs. Similar to a previous study, we observed a 92 and 97% decrease of F11 mRNA expression in control mice and tumor-bearing mice treated with the FXI ASO when compared with the level of F11 mRNA expression in control mice and tumor-bearing mice treated with control ASO, respectively ([Fig. 1C], [D]). Next, we examined if the reduction of F11 mRNA expression by treatment with the FXI ASO is associated with a reduction on functional FXI in plasma. Similar to a previous study, we observed a significantly prolonged aPTT in control mice treated with FXI ASO compared with control mice treated with control ASO (Control ASO vs. FXI ASO [mean ± standard deviation] = 27.02 ± 1.40 seconds vs. 31.5 ± 2.63 seconds, p = 0.01, unpaired t-test). Finally, we measured levels of plasma TAT complexes as a marker of activation of coagulation in control mice and tumor-bearing mice treated with control ASO or FXI ASO. We found that a reduction of F11 expression did not reduce either the basal level of TAT complexes in control mice or the elevated level of TAT complexes in tumor-bearing mice ([Fig. 1E]). Therefore, our data indicates that FXI does not contribute to the activation of coagulation in control mice or in tumor-bearing mice. A previous study showed that the extrinsic pathway mediates idling of the coagulation cascade because levels of FIX activation peptide were reduced in individuals with FVII deficiency but not in individuals with FXI deficiency.
The relative contribution of the extrinsic and intrinsic pathways to cancer-associated thrombosis may be different with various cancers. This may depend, in part, on the levels of TF and polyphosphate on the EVs. In pancreatic cancer, the high level of TF expression by EVs may drive the activation of coagulation independently of the intrinsic pathway. In contrast, in prostate cancer, in which there are lower levels of TF expression and higher levels of polyphosphate, the intrinsic pathway may amplify the coagulation cascade and contribute to venous thromboembolism.
Received: 05 August 2020
Accepted: 19 November 2020
14 January 2021 (online)
© 2021. Thieme. All rights reserved.
Georg Thieme Verlag KG
Rüdigerstraße 14, 70469 Stuttgart, Germany
- 1 Gailani D, Renné T. Intrinsic pathway of coagulation and arterial thrombosis. Arterioscler Thromb Vasc Biol 2007; 27 (12) 2507-2513
- 2 Renné T, Schmaier AH, Nickel KF, Blombäck M, Maas C. In vivo roles of factor XII. Blood 2012; 120 (22) 4296-4303
- 3 von Brühl ML, Stark K, Steinhart A. et al. Monocytes, neutrophils, and platelets cooperate to initiate and propagate venous thrombosis in mice in vivo. J Exp Med 2012; 209 (04) 819-835
- 4 Grover SP, Olson TM, Cooley BC. et al. Model-dependent contribution of FXII and FXI in venous thrombosis in mice. J Thromb Haemost 2020; 18 (11) 2899-2909
- 5 Zhang H, Löwenberg EC, Crosby JR. et al. Inhibition of the intrinsic coagulation pathway factor XI by antisense oligonucleotides: a novel antithrombotic strategy with lowered bleeding risk. Blood 2010; 116 (22) 4684-4692
- 6 Revenko AS, Gao D, Crosby JR. et al. Selective depletion of plasma prekallikrein or coagulation factor XII inhibits thrombosis in mice without increased risk of bleeding. Blood 2011; 118 (19) 5302-5311
- 7 Büller HR, Bethune C, Bhanot S. et al; FXI-ASO TKA Investigators. Factor XI antisense oligonucleotide for prevention of venous thrombosis. N Engl J Med 2015; 372 (03) 232-240
- 8 Weitz JI, Bauersachs R, Becker B. et al. Effect of osocimab in preventing venous thromboembolism among patients undergoing knee arthroplasty: the FOXTROT randomized clinical trial. JAMA 2020; 323 (02) 130-139
- 9 Nickel KF, Ronquist G, Langer F. et al. The polyphosphate-factor XII pathway drives coagulation in prostate cancer-associated thrombosis. Blood 2015; 126 (11) 1379-1389
- 10 Coumans FAW, Brisson AR, Buzas EI. et al. Methodological guidelines to study extracellular vesicles. Circ Res 2017; 120 (10) 1632-1648
- 11 Battistelli S, Stefanoni M, Lorenzi B. et al. Coagulation factor levels in non-metastatic colorectal cancer patients. Int J Biol Markers 2008; 23 (01) 36-41
- 12 Timp JF, Braekkan SK, Versteeg HH, Cannegieter SC. Epidemiology of cancer-associated venous thrombosis. Blood 2013; 122 (10) 1712-1723
- 13 Khorana AA, Connolly GC. Assessing risk of venous thromboembolism in the patient with cancer. J Clin Oncol 2009; 27 (29) 4839-4847
- 14 Wang JG, Geddings JE, Aleman MM. et al. Tumor-derived tissue factor activates coagulation and enhances thrombosis in a mouse xenograft model of human pancreatic cancer. Blood 2012; 119 (23) 5543-5552
- 15 Rosell A, Moser B, Hisada Y. et al. Evaluation of different commercial antibodies for their ability to detect human and mouse tissue factor by western blotting. Res Pract Thromb Haemost 2020; 4 (06) 1013-1023
- 16 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
- 17 Davila M, Amirkhosravi A, Coll E. et al. Tissue factor-bearing microparticles derived from tumor cells: impact on coagulation activation. J Thromb Haemost 2008; 6 (09) 1517-1524
- 18 Zwicker JI, Liebman HA, Neuberg D. et al. Tumor-derived tissue factor-bearing microparticles are associated with venous thromboembolic events in malignancy. Clin Cancer Res 2009; 15 (22) 6830-6840
- 19 Hisada Y, Alexander W, Kasthuri R. et al. Measurement of microparticle tissue factor activity in clinical samples: a summary of two tissue factor-dependent FXa generation assays. Thromb Res 2016; 139: 90-97
- 20 Kasthuri RS, Hisada Y, Ilich A, Key NS, Mackman N. Effect of chemotherapy and longitudinal analysis of circulating extracellular vesicle tissue factor activity in patients with pancreatic and colorectal cancer. Res Pract Thromb Haemost 2020; 4 (04) 636-643
- 21 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
- 22 Geddings JE, Hisada Y, Boulaftali Y. et al. Tissue factor-positive tumor microvesicles activate platelets and enhance thrombosis in mice. J Thromb Haemost 2016; 14 (01) 153-166
- 23 Bauer KA, Kass BL, ten Cate H, Hawiger JJ, Rosenberg RD. Factor IX is activated in vivo by the tissue factor mechanism. Blood 1990; 76 (04) 731-736