Measurement of Blood-Borne Tissue Factor Procoagulant Activity in Healthy Individuals and Cancer Patients Using a Novel Assay.

Abstract

Tissue factor (TF) is the cellular initiator of the extrinsic pathway of coagulation. Recent studies have reported circulating blood-borne TF (BBTF) antigen by methods including ELISA and flow cytometry. Whether this BBTF is active has been rarely addressed due to the lack of a simple functional assay. We have developed a new assay that measures BBTF procoagulant activity. Whole blood was collected into tubes containing citrate and corn trypsin inhibitor (FXIIa inhibitor). First, we compared the procoagulant activity obtained in detergent lyzed platelets and microparticles with peripheral blood mononuclear cells (PBMCs) following a 5 hour ex vivo stimulation of whole blood with LPS. Platelets and microparticles had 3% (23 ± 10 mU/mL) and 0.1% (1.0 ± 0.4 mU/mL), respectively, of the procoagulant activity of PBMCs (1048 ± 200 mU/mL). The procoagulant activity of PBMCs and platelets was inhibited by >90% in the presence of an anti-TF polyclonal antibody. Next, we determined the time course of TF-dependent procoagulant activity of microparticles and platelets after ex vivo LPS stimulation of whole blood. In both cases, a dramatic increase in microparticle and platelet procoagulant activity was observed after 12 hours of LPS stimulation, with a maximum activity observed at 48 hours (48 mU/mL for microparticles and 1028 mU/mL for platelets at 48 hours). The majority (>90%) of this procoagulant activity was TF-dependent. Therefore, we subsequently analyzed combined platelet and microparticle (P+MP) fractions for BBTF activity. We determined functional BBTF activity in P+MP fractions from the blood of healthy individuals with and without LPS ex vivo stimulation. In healthy individuals, very low levels of BBTF procoagulant activity was detected in the absence of LPS stimulation (0.22 ± 0.09 mU/mL) and this activity was decreased on average by 28% with an anti-TF antibody (0.16 ± 0.1 mU/mL; p = 0.01). In contrast, P+MP from LPS stimulated blood demonstrated on average 30 fold higher procoagulant activity, of which >90% was TF-dependent. Cancer is associated with an increased susceptibility to develop pathological thrombosis. Using our new assay, we demonstrate a significant elevation of the procoagulant activity of P+MP fractions from advanced (stage IV) solid tumor patients of varying histology's (n=14; 0.88 ± 0.55 mU/mL) compared with normal subjects (p <0.001). Furthermore, we showed that on average 50% of the procoagulant activity was TF-dependent (procoagulant activity was reduced to 0.57 ± 0.35 mU/mL in the presence of an anti-TF antibody; p < 0.01), suggesting that circulating BBTF in cancer patients has the potential to contribute to thrombosis in vivo. In summary, we have developed a novel assay that measures BBTF activity. This assay may be useful in the detection of a pre-thrombotic state in cancer patients.