AbstractCancer patients exhibit a high rate of thromboembolism (VTE). In this study, we analyzed levels of microparticle (MP) tissue factor (TF) activity in cancer patients with or without VTE. Blood was collected from cancer patients within 24 h of objectively diagnosed VTE (n=53) and from cancer patients without VTE (n=13). MPs were isolated from platelet poor plasma by centrifugation at 20,000g for 15 min. MP TF activity was measured using a two-stage chromogenic assay. Cancer patients with VTE had a significantly higher mean MP TF activity compared with cancer patients without VTE (1.7±3.8 pg/mL vs 0.6±0.4 pg/mL, 

Glover, Sam L.

Kasthuri, Raj

Key, Nigel S.

Liebman, Howard A.

Mackman, Nigel

Manly, David A.

Wang, Jianguo

PubMed

Carolina Digital Repository

Increased Microparticle Tissue Factor Activity in Cancer Patientswith Venous ThromboembolismDavid A Manly1, Jianguo Wang1, Sam L Glover1, Raj Kasthuri1, Howard A. Liebman2, NigelS Key1, and Nigel Mackman11 Division of Hematology/Oncology, Department of Medicine, Lineberger Comprehensive CancerCenter, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-70352 University of Southern California-Keck School of Medicine, Jane Anne Nohl Division ofHematology, Department of Medicine, Norris Comprehensive Cancer Center, Los Angeles, CA90033-0800Venous thromboembolism (VTE) is the second leading cause of cancer-associated mortality(1). However, cancer patients, particularly in the ambulatory setting, do not routinely receivethromboprophylaxis. This is because there are no validated biomarkers to identify patients athigh risk of VTE. A number of candidate biomarkers of thrombotic risk have been proposed,including D-dimer, soluble P-selectin, C-reactive protein, and tissue factor (TF) (2–5). TF isthe transmembrane receptor for factor VII/VIIa and functions as the primary initiator of bloodcoagulation (6). Patients with various diseases have elevated levels of TF in their plasma inthe form of membrane-associated microparticles (MPs) as well as a soluble form (7,8). MPsare submicron membrane vesicles generated from activated or apoptotic cells. Theirprocoagulant activity is increased by the presence of TF and the anionic phospholipidphosphatidylserine.TF-positive MPs are highly procoagulant and they have been linked to thrombosis in a varietyof diseases, such as cancer, sickle cell disease, and endotoxemia (9,10). Several studies havemeasured the levels of MP TF antigen and activity in cancer patients (11–13). There have beentwo retrospective studies investigating MP TF activity in cancer patients. Tesselaar andcolleagues found increased levels of MP TF activity compared with controls in pancreatic andbreast adenocarcinoma patients. In addition, Hron and colleagues reported a two-fold higherlevel of TF-positive MPs in patients with advanced colorectal cancer compared to controls(11,12). A prospective study by Khorana and colleagues reported that MP TF activity may bepredictive of VTE in patients with pancreatic cancer (13).In this study, we analyzed MP TF activity in patients with a variety of different cancers withor without acute VTE. We hypothesized that increased MP TF activity would be present inpatients with VTE, irrespective of the type of cancer. Cancer patients (n=66) were recruitedfor this study at the University of Southern California in Los Angeles. Patients were enrolledin an ongoing IRB approved treatment trial for the management of malignancy-related VTE.Blood was drawn from cancer patients without VTE (n = 13) and from cancer patients withVTE within 24 hours of diagnosis (n = 53). All patients had to have either deep vein thrombosisCorrespondence: Nigel Mackman, Ph.D., Division of Hematology/Oncology, Department of Medicine, 917 Mary Ellen Jones Building,University of North Carolina at Chapel Hill, 98 Manning Drive CB#7035, Chapel Hill NC 27599, Tel 919 843 3961, Fax 919 966 7639.Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customerswe are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resultingproof before it is published in its final citable form. Please note that during the production process errors may be discovered which couldaffect the content, and all legal disclaimers that apply to the journal pertain.NIH Public AccessAuthor ManuscriptThromb Res. Author manuscript; available in PMC 2011 June 1.Published in final edited form as:Thromb Res. 2010 June ; 125(6): 511–512. doi:10.1016/j.thromres.2009.09.019.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscriptconfirmed by compression ultrasound and/or pulmonary embolism confirmed by computedtomography (CT) angiography utilizing a 16 slice-multi-detector CT. The distribution of cancertypes in the 66 patients recruited for this study were as follows: 14 colon (10 VTE), 10 lung(7 VTE), 6 bladder (6 VTE), 5 pancreatic (3 VTE), 3 prostate (3 VTE), 3 rectal (2 VTE), 2 bileduct (2 VTE), 2 brain (2 VTE), 2 cholangio (2 VTE), 2 liver (2 VTE), 2 lymphoma (2 VTE),2 renal cell (1 VTE), 2 testis (2 VTE), and 11 other types of cancer (9 VTE). All 66 subjectswho participated in this study gave informed consent.We have recently developed an assay to measure levels of TF activity on MPs isolated fromplasma (13). Importantly, healthy individuals have very low levels of MP TF activity (0.21 ±0.11 pg/mL) (13). In this study, we found a statistically significant increase in MP TF activityin cancer patients with VTE compared to cancer patients without VTE (1.7 ± 3.8 pg/mL vs.0.5 ± 0.5 pg/mL, p < 0.05, Figure). Data is represented as mean ± standard deviation andstatistical analysis was performed using a heteroscedastic Student’s T-test. After thecompletion of our study, Tesselaar and colleagues (14) reported an increase in MP TF activityin cancer patients with VTE (n=51) compared with cancer patients without VTE (n=49). Thetwo groups were matched for age, sex, type of cancer, stage of disease and type of cancertreatment.Next, we compared levels of MP TF activity in pancreatic, lung, and colon cancer patients.One limitation of the study is that we had relatively small numbers in the three groups.Pancreatic cancer patients had the highest MP TF activity (6.6 ± 10.8 pg/mL), followed bylung (2.4 ± 2.5 pg/mL) and colon cancer (0.7 ± 0.8 pg/mL). This finding is consistent with therespective thrombosis rates in these types of cancer; it has been reported that 28.3% ofpancreatic cancer patients develop VTE within a year of metastatic malignancy, compared to7.4% for lung, and 5.7% for colon (15).Plasma D-dimer and interleukin-6 (IL-6) levels were measured using commercial enzyme-linked immunoassays (IMUCLONE D-Dimer ELISA, American Diagnostica, Stamford, CT.;Human IL-6 Quantikine HS ELISA, R & D Systems Inc, Minneapolis, MN.). Cancer patientswith VTE had a significant increase in D-dimer levels compared to cancer patients withoutVTE (4500 ± 6200 ng/mL vs. 670 ± 1800 ng/mL, p < 0.05). A recent study showed that elevatedlevels of D-dimer predict VTE in patients with cancer (16). IL-6 levels were also significantlyelevated in cancer patients with VTE compared to those without VTE (9.1 ± 4.9 pg/mL vs. 6.6± 5.3 pg/mL, p < 0.05). Recent studies indicate a link between inflammation and cancerdevelopment (17). Inflammation may also induce TF expression within the vasculature andthis would increase the risk of thrombosis in cancer patients. Interestingly, there was only aweak correlation between D-dimer and IL-6 compared to MP TF activity (r = 0.145 and r =0.283, respectively). Tesselaar and colleagues (14) also found a weak correlation between MPTF activity and levels of thrombin-antithrombin complex in cancer patients.The results of this study add to the growing body of evidence that MP TF activity may play animportant role in the development of VTE in cancer patients. Our data show that MP TF activityis significantly increased in a broad population of cancer patients with acute VTE comparedto cancer patients without VTE. However, as this was a cross sectional study, we are unableto conclude that MP TF activity can be used a biomarker of risk in these patients. Prospectivestudies are underway to test the predictive power of MP TF activity as a novel candidatebiomarker for assessing the risk of VTE in cancer patients.References1. Khorana AA, Francis CW, Culakova E, et al. Thromboembolism is a leading cause of death in cancerpatients receiving outpatient chemotherapy. Thromb Haemost 2007;5(3):632–4.Manly et al. Page 2Thromb Res. Author manuscript; available in PMC 2011 June 1.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript2. Kroger K, Weiland D, Ose C, et al. Risk factors for venous thromboembolic events in cancer patients.Ann Oncol 2006 Feb;17(2):297–303. [PubMed: 16282243]3. Ay C, Simanek R, Vormittag R, et al. High plasma levels of soluble P-selectin are predictive of venousthromboembolism in cancer patients: results from the Vienna Cancer and Thrombosis Study (CATS).Blood 2008 Oct 1;112(7):2703–8. [PubMed: 18539899]4. Cosmi B, Legnani C, Cini M, et al. The role of D-dimer and residual venous obstruction in recurrenceof venous thromboembolism after anticoagulation withdrawal in cancer patients. Haematologica 2005May;90(5):713–5. [PubMed: 15921399]5. Uno K, Homma S, Satoh T, et al. Tissue factor expression as a possible determinant ofthromboembolism in ovarian cancer. Br J Cancer 2007 Jan 29;96(2):290–5. [PubMed: 17211468]6. Mackman N, Tilley RE, Key NS. Role of the extrinsic pathway of blood coagulation in hemostasisand thrombosis. Arteriosclerosis, Thrombosis, and Vascular Biology 2007;27:1687–93.7. Bogdanov VY, Balasubramanian V, Hathcock J, et al. Alternatively spliced human tissue factor: acirculating, soluble, thrombogenic protein. Nature Med 2003;9(4):458–62. [PubMed: 12652293]8. Morel O, Toti F, Hugel B, et al. Procoagulant microparticles: disrupting the vascular homeostasisequation? Arterioscler Thromb Vasc Biol 2006;26(12):2594–604. [PubMed: 16990554]9. Shet AS, Aras O, Gupta K, et al. Sickle blood contains tissue factor-positive microparticles derivedfrom endothelial cells and monocytes. Blood 2003;102(7):2678–83. [PubMed: 12805058]10. Aras O, Shet A, Bach RR, et al. Induction of microparticle- and cell-associated intravascular tissuefactor in human endotoxemia. Blood 2004;103:4545–53. [PubMed: 14988149]11. Hron G, Kollars M, Weber H, et al. Tissue factor-positive microparticles: cellular origin andassociation with coagulation activation in patients with colorectal cancer. Thromb Haemost 2007;97(1):119–23. [PubMed: 17200778]12. Tesselaar ME, Romijn FP, van dLI, et al. Microparticle-associated tissue factor activity: a link betweencancer and thrombosis? J Thromb Haemost 2007 Mar;5(3):520–7. [PubMed: 17166244]13. Khorana AA, Francis CW, Menzies KE, et al. Plasma tissue factor may be predictive of venousthromboembolism in pancreatic cancer. J Thromb Haemost 2008 Nov;6(11):1983–5. [PubMed:18795992]14. Tesselaar MET, Romijn FP, Van Der Linden IK, et al. Microparticle-associated tissue factor activityin cancer patients with or without thrombosis. J Throm Haemost 2009;7:1421–1423.15. White RH, Chew H, Wun T. Targeting patients for anticoagulant prophylaxis trials in patients withcancer: who is at highest risk? Thromb Res 2007;120 (Suppl 2):S29–40. [PubMed: 18023711]16. Ay C, Vormittag R, Dunkler D, et al. D-dimer and prothrombin fragment 1+2 predict venousthromboembolism in patients with cancer: results from the Vienna cancer and thrombosis study. JClin Oncol 2009;27:4124–4129. [PubMed: 19636003]17. Berasain C, Castillo J, Perugorria MJ, et al. Inflammation and liver cancer: new molecular links. AnnN Y Acad Sci 2009;1155:206–221. [PubMed: 19250206]Manly et al. Page 3Thromb Res. Author manuscript; available in PMC 2011 June 1.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptFigure.Comparison of MP TF activity between cancer patients with VTE (n=53) and cancer patientswithout VTE (n=13) (Median, Inter-quartile Range, 2 Standard Deviations). Dots representMP TF activity levels beyond 2 standard deviations.Manly et al. Page 4Thromb Res. Author manuscript; available in PMC 2011 June 1.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript

Increased microparticle tissue factor activity in cancer patients with Venous Thromboembolism

http://dx.doi.org/10.1016/j.thromres.2009.09.019

Increased microparticle tissue factor activity in cancer patients with Venous Thromboembolism

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