Journal of Thrombosis and Haemostasis, 12: 1932–1934

DOI: 10.1111/jth.12718

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Evaluation of a new commercial assay to measure microparticle tissue factor activity in plasma: communication from the SSC of the ISTH K. TATSUMI,* S. ANTONIAK,* D. M. MONROE III,* A. A. KHORANA† and N. MACKMAN,* FOR THE SUBCOMMITTEE ON HEMOSTASIS AND MALIGNANCY *Department of Medicine, Division of Hematology and Oncology, UNC McAllister Heart Institute, University of North Carolina at Chapel Hill, Chapel Hill, NC; and †Taussig Cancer Institute, Cleveland Clinic Foundation, Cleveland, OH, USA

To cite this article: Tatsumi K, Antoniak S, Monroe III DM, Khorana AA, Mackman N, for the Subcommittee on Hemostasis and Malignancy. Evaluation of a new commercial assay to measure microparticle tissue factor activity in plasma: communication from the SSC of the ISTH. J Thromb Haemost 2014; 12: 1932–4.

Microparticles (MPs) are small membrane vesicles that are released by activated cells, such as monocytes, cells undergoing apoptosis, and tumor cells [1]. Full-length tissue factor (flTF) is a transmembrane receptor for factor VII (FVII)/VIIa and is essentially undetectable in the blood of healthy individuals. However, intravascular levels of flTF are increased in a variety of diseases, including cancer and endotoxemia [1]. The majority of active flTF in plasma in these disease states is present on MPs. In this report from the Hemostasis/Malignancy Subcommittee of the International Society of Thrombosis and Hemostasis, we evaluated a new commercial assay (Zymuphen MP-TF; Aniara, West Chester, OH, USA) that is reported to determine ‘the procoagulant activity of microparticles exposing TF.’ We compared this assay with our in-house assay using frozen plasma samples with or without TF-positive MPs generated ex vivo [2,3]. Measurement of TF in plasma is challenging because levels are very low even under pathological conditions [4]. TF can be measured using either antigen- or activity-based assays. Two very similar activity assays were developed independently by the Osanto and Mackman groups that involve isolating MPs through centrifugation [2,3,5]. These two assays have been used in numerous studies and measure MP TF activity in platelet-poor plasma (PPP) and platelet-free plasma (PFP) [6] and gave very similar values Correspondence: Nigel Mackman, University of North Carolina at Chapel Hill, 98 Manning Drive, Campus Box 7035, Chapel Hill, NC 27599, USA. Tel.: +1 919 843 3961; fax: +1 919 966 7639. E-mail: [email protected] Received 25 April 2014 Manuscript handled by: S. Eichinger Final decision: P. H. Reitsma, 14 August 2014

of MP TF activity (r = 0.61) using plasma samples from cancer patients [7]. We found that the activity assay was more sensitive than an in-house ELISA [2]. In plasma samples obtained from lipopolysaccharide (LPS)-stimulated blood, we could measure TF using our activity assay but not by using flow cytometry, which suggested that the level of TF-positive MPs was below the detection limit of flow cytometry [3]. Finally, there was no correlation between our in-house activity assay and an impedance-based flow cytometry assay (R. Lee, J. Zwicker, N. Mackman, Fig. S1), although the TF-positive MPs measured by impedance correlated with thrombotic risk in cancer patients [8]. In sum, these studies support the notion that activity-based assays are more sensitive and may be more reliable than antigen-based assays for measuring TF in plasma samples. In this report, we evaluated a new commercial available assay that measures MP TF activity. Method Subjects for the study were healthy volunteers who provided written informed consent in accordance with University of North Carolina Institutional Review Board protocol. The blood from 10 healthy donors was collected into citrate and divided into two equal portions. Control plasma was obtained by immediately preparing plasma [3]. TF-positive MPs were generated by incubating whole blood with bacterial LPS (10 lg mL 1, Escherichia coli serotype O111:B4; Sigma Aldrich, St. Louis, MO, USA) for 5 h at 37 °C [3]. PPP was prepared from whole blood via centrifugation at 1500 9 g for 15 min at 4 °C, snap frozen, and stored at 80 °C. Procoagulant activity (PCA) was measured using a two-step FXa generation assay. Our in-house activity assay was performed as described and uses 2.5 nmol L–1 FVIIa [2,3]. It measures total, TF-dependent, and TF-independent FXa generation because the samples are incubated with either an inhibi© 2014 International Society on Thrombosis and Haemostasis

Measurement of MP TF activity 1933

TF-dependent FXa generation in the five different LPS samples was 3.2  0.9 pg mL 1 in the Zymuphen MP-TF assay compared with 9.8  2.1 pg mL 1 in the in-house assay (n = 5, P < 0.01) (Fig. 1B). Therefore, the Zymuphen MPTF assay was significantly less sensitive than our in-house assay (only 32.4  3.7% of the in-house assay) (P < 0.01). In addition, the percentage of TF-dependent FXa generation in the Zymuphen MP-TF assay was 66.8  1.9%, which is significantly lower than the 86.5  1.0% observed in the in-house assay (n = 5, P < 0.01) (Fig. 1B). This indicates that the Zymuphen MP-TF assay is a less specific assay for TF than the in-house assay. Finally, the Zymuphen MP-TF assay had a significantly higher total FXa generation baseline value (0.47  0.03 pg mL 1) compared with the in-house assay (0.24  0.09 pg mL 1) (P < 0.05) (Fig. 1B). However, the level of TF-independent FXa generation in non-stimulated samples was similar in both assays, with 84.2  2.1% in the Zymuphen MP-TF assay and 88.2  14.6% in the in-house assay. An anti-FVII antibody abolished all FXa generation (Fig. S2). We also found that decreasing the FVIIa concentration from 12 to 2.5 nmol L 1 decreased the total FXa generation by 12.1% and increased the specificity by 4.1% in the Zymuphen assay.

tory anti-TF antibody (HTF-1) or a control IgG. The Zymuphen MP-TF assay was performed according to the manufacturer’s instructions (http://www.aniara.com/pdf/ INS-A521196.pdf) with a modification that consisted of manual washing instead of automatic washing. In addition, HTF-1 (10 lg mL 1) or mouse IgG (10 lg mL 1) were added for 15 min at room temperature before adding FVIIa (12 nmol L 1, J. Amiral, Hyphen BioMed, personal communication) and FX. We also used an inhibitory sheep anti-human FVII antibody (Affinity Biologicals, Ancaster, ON, Canada) or control IgG at 40 lg mL 1. Innovin (Dade Behring, Marburg, Germany) was used as a standard for both assays to allow direct comparison of FXa generation. This is comparable to the calibrator in the commercial assay (J. Amiral, Hyphen BioMed, personal communication). All statistical analyses were performed by using paired Student t-test. P < 0.05 was considered statistically significant. Results As expected, we observed a range of levels of PCA in the LPS samples from the different donors (Fig. 1A). A 25 Donor 2

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Fig. 1. Levels of procoagulant activity of microparticles in human plasma. Total FXa (1, black bars), TF-dependent (2, gray bars), and TFindependent FXa (3, white bars) generation in plasma samples derived from unstimulated and LPS-stimulated whole blood was measured using either an in-house assay or a Zymuphen MP-TF assay. We used samples from five different donors. Panel A shows individual samples. and panel B shows the combined data. All assays were performed in duplicate. and the data are shown as the mean  SEM. © 2014 International Society on Thrombosis and Haemostasis

1934 K. Tatsumi et al

Conclusion

Supporting Information

The strengths of the Zymuphen MP-TF assay are that it captures TF-positive MPs from plasma using a well-characterized non-inhibitory anti-human TF mouse monoclonal antibody (10H10) and uses a plate-based format. The weaknesses of the assay are its lower sensitivity, lower specificity, and higher background compared with our inhouse assay. The lower sensitivity is likely due to an inability to capture all the TF-positive MPs in the sample. The lower specificity and higher background of the Zymuphen MP-TF assay appear to be due to the use of a higher FVIIa concentration (12 nmol L 1) compared with our in-house assay (2.5 nmol L 1). Indeed, FVIIa activation of FX independent of TF is linearly dependent on the concentration of FVIIa [9]. At present, it remains to be determined if there is a clinical utility in measuring MP TF activity in patients with cancer and other patients with an increased thrombotic risk.

Additional Supporting Information may be found in the online version of this article:

Addendum N. Mackman and K. Tatsumi designed the experiments. K. Tatsumi performed the assays. N. Mackman and D. M. Monroe interpreted the data. K. Tatsumi, S. Antoniak, and A. A. Khorana wrote the manuscript. Acknowledgements We acknowledge a grant to N.M. from the National Institutes of Health (HL006350). A.A.K. acknowledges research support from the Sondra and Stephen Hardis Endowed Chair in Oncology and the Scott Hamilton CARES Initiative Grant. K.T. is the recipient of a research fellowship from the Uehara Memorial Foundation. Disclosure of Conflict of Interests The authors state that they have no conflicts of interest.

Fig. S1. Measurement of TF+ MPs by activity and impedance. Fig. S2. Effect of an FVII antibody on the levels of MP procoagulant activity. References 1 Owens AP 3rd, Mackman N. Microparticles in hemostasis and thrombosis. Circ Res 2011; 108: 1284–97. 2 Khorana AA, Francis CW, Menzies KE, Wang JG, Hyrien O, Hathcock J, Mackman N, Taubman MB. Plasma tissue factor may be predictive of venous thromboembolism in pancreatic cancer. J Thromb Hemost 2008; 6: 1983–5. 3 Lee RD, Barcel DA, Williams JC, Wang JG, Boles JC, Manly DA, Key NS, Mackman N. Pre-analytical and analytical variables affecting the measurement of plasma-derived microparticle tissue factor activity. Thromb Res 2012; 129: 80–5. 4 Key NS, Mackman N. Tissue factor and its measurment in whole blood, plasma and microparticles. Semin Thromb Hemost 2010; 36: 866–75. 5 Tesselaar ME, Romijn FP, van der Linden IK, Prins FA, Bertina RM, Osanto S. Microparticle-associated tissue factor activity: a link between cancer and thrombosis? J Thromb Hemost 2007; 5: 520–7. 6 Geddings JE, Mackman N. Tumor-derived tissue factor–positive microparticles and venous thrombosis in cancer patients. Blood 2013; 122: 1873–80. 7 Thaler J, Ay C, Mackman N, Bertina RM, Kaider A, Marosi C, Key NS, Barcel DA, Scheithauer W, Kornek G, Zielinski C, Pabinger I. Microparticle-associated tissue factor activity, venous thromboembolism and mortality in pancreatic, gastric, colorectal and brain cancer patients. J Thromb Hemost 2012; 10: 1363–70. 8 Zwicker JI, Liebman HA, Bauer KA, Caughey T, Campigotto F, Rosovsky R, Mantha S, Kessler CM, Eneman J, Raghavan V, Lenz HJ, Bullock A, Buckbinder E, Neubeg D, Furie B. Prediction and prevention of thrombotic events with enoxaparin in cancer patients with elevated tissue factor-bearing microparticles: a randomized-controlled phase II trial (the Microtec study). Br J Haematol 2013; 160: 530–7. 9 Bom VJ, Bertina RM. The contributions of Ca2+, phospholipid and tissue factor apoprotein to the activation of human blood factor X by activated factor VII. Biochem J 1990; 265: 327–36.

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Evaluation of a new commercial assay to measure microparticle tissue factor activity in plasma: communication from the SSC of the ISTH.

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