Detection of endogenous tissue factor levels in plasma using the calibrated automated thrombogram assay

Ollivier, Véronique; Jian-guo, Wang; Manly, David A.; Machlus, Kellie R.; Wolberg, Alisa S.; Jandrot‐Perrus, Martine; Mackman, Nigel

doi:10.1016/j.thromres.2009.03.003

Cited by 55 publications

(67 citation statements)

References 47 publications

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“…During this step, known as the propagation phase, 10 TF becomes less important, because the reaction is mainly driven by the FVIIa/FXa and FVa/FXa complexes. 18,24 To further understand to what extent tumor cell TF contributes to thrombin generation, two different strategies were used: (i) inhibiting cell-associated TF with a specific anti-TF antibody, and (ii) performing the assay in FVIIdeficient plasma. In both cases, the thrombin generation NB4 K562 HEL H69 MCF7 NB4 K562 HEL H69 MCF7 NB4 K562 HEL H69 MCF7 NB4 K562 HEL H69 MCF7 NB4 K562 HEL H69 MCF7 NB4 K562 HEL H69 MCF7 NB4 K562 HEL H69 MCF7 NB4 K562 HEL © F e r r a t a S t o r t i F o u n d a t i o n capacity of all cell lines was significantly reduced, and the decrease was proportional to cell TF content.…”

Section: Discussionmentioning

confidence: 99%

Characterization of the thrombin generation potential of leukemic and solid tumor cells by calibrated automated thrombography

Marchetti¹,

Diani²,

Cate³

et al. 2012

Haematologica

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BackgroundThrombin, the final enzyme of blood coagulation, is a multifunctional serine protease also involved in the progression of cancer. Tumor cells may activate blood coagulation proteases through the expression of procoagulant activities. However, specific information about the thrombin generation potential of malignant tissues is lacking. In this study we applied a single global coagulation test, the calibrated automated thrombogram assay, to characterize the specific procoagulant phenotypes of different tumor cells. Design and MethodsMalignant hematologic cells (i.e. NB4, HEL, and K562) or solid tumor cells (i.e. MCF-7 breast cancer and H69 small cell lung cells) were selected for the study. The calibrated automated thrombogram assay was performed in normal plasma and in plasma samples selectively deficient in factor VII, XII, IX or X, in the absence or presence of a specific anti-tissue factor antibody. Furthermore, cell tissue factor levels were characterized by measuring antigen, activity and mRNA expression. ResultsIn normal plasma, NB4 induced the highest thrombin generation, followed by MCF-7, H69, HEL, and K562 cells. The anti-tissue factor antibody, as well as deficiencies of factors VII, IX and XII affected the thrombin generation potential of malignant cells to different degrees, allowing differentiation of the two different pathways of blood clotting activation -by tissue factor or contact activation. The thrombin generation capacity of NB4 and MCF-7 cells was tissue factor-dependent, as it was highly sensitive to inhibition by anti-tissue factor antibody and factor VII deficiency, while the thrombin generation capacity of H69, HEL and K562 was contact activation-dependent, as no thrombin was generated by these cells in factor XII-deficient plasma. ConclusionsThis study demonstrates that the calibrated automated thrombogram assay is capable of quantifying, characterizing, and comparing the thrombin generation capacity of different tumor cells. This provides a useful tool for understanding the key factors determining the global procoagulant profile of tumors, which is important for addressing specific targeted therapy for the prevention of thrombosis and for cancer.Key words: leukemic cells, thrombin generation, tissue factor, tumor, thrombosis. 2012;97(8):1173-1180. doi:10.3324/haematol.2011 This is an open-access paper. Citation: Marchetti M, Diani E, ten Cate H, and Falanga A. Characterization of the thrombin generation potential of leukemic and solid tumor cells by calibrated automated thrombography. Haematologica Characterization of the thrombin generation potential of leukemic and solid tumor cells by calibrated automated thrombography ABSTRACT© F e r r a t a S t o r t i F o u n d a t i o n

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Section: Discussionmentioning

confidence: 99%

Characterization of the thrombin generation potential of leukemic and solid tumor cells by calibrated automated thrombography

Marchetti¹,

Diani²,

Cate³

et al. 2012

Haematologica

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“…Antibodies may recognize ''encrypted'' nonfunctional TF or degraded forms of TF protein that are not active, leading to poor correlation between levels of TF-protein and TF activity (13). Osterud and Bjorklid suggested that antibodies do not meet the stringent criteria of monospecificity (26), this may explain the discrepancy between TF activity and TF protein.…”

Section: Discussionmentioning

confidence: 99%

“…TF-positive MPs can be analyzed by immunological assays [flow cytometry, impedance flow cytometry, and enzyme-linked immunosorbent assay (ELISA)] or functional assays (FXa-generating activity, thrombin generation assay, and clot formation assay), (10,11,(13)(14)(15). Some reports have demonstrated discrepancies between TF-protein and TF activity levels, which have been explained by antibody binding to ''encrypted'' or degraded forms of inactive TF-protein (13,16).…”

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confidence: 99%

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Fluorescent particles in the antibody solution result in false TF- and CD14-positive microparticles in flow cytometric analysis

et al. 2011

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Tissue factor (TF)-positive microparticles (MPs) are highly procoagulant, and linked to thrombosis in sepsis and cancer. MP-associated TF may be assayed by immunological or functional methods. Several reports have demonstrated discrepancies between TFprotein and TF-activity, which have been explained by antibody binding to ''encrypted'' or degraded forms of inactive TF-protein. Our goal was to evaluate the possible interference of fluorescent antibody aggregates in solutions containing antibodies against TF and CD14 in flow cytometric analysis. Using monocyte-derived microparticles (MPs) released from human monocytes, incubated with or without lipopolysaccharides (LPS) in vitro, we measured MP-associated TF-protein (flow cytometry) and TF-activity (clot formation assay). MPs released from monocytes exposed to LPS (1 ng mL 21 ) had $14 times higher TF-activity than MPs originated from monocytes exposed to only culture medium. However, using untreated anti-TF antibodies (American Diagnostica and BD) in the flow cytometric analysis, MPs released from unstimulated monocytes had a similar number of TF-positive events as MPs secernated from LPS-stimulated monocytes [$45,000 events mL 21 (American Diagnostica); $15,000 events mL 21 (BD)]. These TF-positive events did not exert any TF-activity, and centrifugation (17,000g, 30 min, 48C) of the antibody solutions prior to use effectively removed the interfering fluorescent events. Removal of fluorescent interference, probably in the form of fluorescent antibody aggregates, from the antibody solutions by centrifugation is essential to prevent the occurrence of false positive flow cytometric events. The events can be mistaken as MP-associated TF-protein, and interpreted as a discrepancy between TF-protein and TF-activity. ' 2011 International Society for Advancement of Cytometry

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“…Recent publications have highlighted how low-grade contact activation is greatly amplified by the presence of phospholipids simulating the procoagulant membranes of activated platelets. 7,8 Thus, the dramatic effects on recalcification times observed with preactivation of platelets in their study 2 likely stem from platelet-dependent amplification of material-induced contact activation and not from FXIIa generation by platelets per se. In Figure 1B, we show that the Amelung instrument they used 2 generates far more FXIIa than comparable assays.…”

mentioning

confidence: 81%

Response: Platelets do not generate activated factor XII—how inappropriate experimental models have led to misleading conclusions

Boknäs

Faxälv

Ström

et al. 2014

Blood

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Detection of endogenous tissue factor levels in plasma using the calibrated automated thrombogram assay

Abstract: SummaryBackground-The calibrated automated thrombogram (CAT) assay measures thrombin generation in plasma.

Cited by 55 publications

References 47 publications

Characterization of the thrombin generation potential of leukemic and solid tumor cells by calibrated automated thrombography

Characterization of the thrombin generation potential of leukemic and solid tumor cells by calibrated automated thrombography

Fluorescent particles in the antibody solution result in false TF- and CD14-positive microparticles in flow cytometric analysis

Response: Platelets do not generate activated factor XII—how inappropriate experimental models have led to misleading conclusions

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