The Importance of Platelets in the Expression of Monocyte Tissue Factor Antigen Measured by a New Whole Blood Flow Cytometric Assay

Abstract

SummaryPrevious methods for the determination of monocyte tissue factor (TF) have been technically complex, difficult to standardize, prone to spuriously elevated results and difficult to implement in a clinical laboratory environment. We report the development of a two-color whole blood cytometric technique that overcomes many of these disadvantages. The assay uses small volumes of citrated blood (1.0 ml), can be performed in under one hour (if endotoxin stimulation is not performed), is reproducible (CV = 5%) and uses methodology commonly available in clinical laboratories. Baseline (mean ± SD) expression of monocyte TF in normal subjects was very low (1.1 ± 0.95%, Mean Fluorescence [Mean FL] 0.20 ± 0.01) making relatively small increases easy to detect. Monocyte TF expression following endotoxin (LPS) stimulation for 1 h was 34.6 ± 11.2% (Mean FL 0.32 ± 0.04). LPS-stimulated activity varied between subjects (21-68%) but was remarkably consistent for individual subjects (CV = 5.4%). Stimulated monocyte TF expression was directly proportional to the platelet count and was reduced by platelet protective anticoagulants and by ingestion of aspirin. Non LPS-stimulated monocyte TF was markedly increased, in a dose-dependent manner, by adding collagen to whole blood. This was apparently associated with platelet-monocyte binding and could be abolished by anti-P-Selectin. We conclude that the whole blood flow cytometric assay of monocyte TF may be a valuable tool for clinical use and a useful model system for evaluating the humoral and cellular factors governing monocyte TF expression in a natural environment.

Role of Platelet P-Selectin and CD40 Ligand in the Induction of Monocytic Tissue Factor Expression

“…Interestingly, no TF mRNA could be detected at the earliest time point of antigen detection. Early TF antigen induction by platelets in whole blood was observed by Amirkhosravi et al, 13 who also used 15-minute incubations for their collagen stimulation experiments. Similar observations have been made by other workers.…”

“…In a recent publication, Mach et al 19 showed reduction of atherosclerosis in mice by administration of anti-CD40L antibody. Platelets, known to activate leukocytes by P-selectin tethering, 13,20 are the most recent cell type to be found capable of CD40L expression. 8 In this study, we have investigated the role of CD40-CD40L interaction in activated platelet-induced TF expression in monocytic cells, both in a cell line and in a wholeblood environment.…”

“…9 -11 Moreover, recent studies have shown that activated platelets trigger inflammatory response and procoagulant activity (PCA) in endothelial cells via the CD40-CD40L pathway. 8,12 Because platelets are also known to induce and potentiate monocyte TF expression in a P-selectin-dependent manner, 13 we have studied the possible importance of CD40-CD40L interactions in activated platelet-induced TF expression in human monocytic cells. We have used 2 systems: coculture of the vitamin D 3 -differentiated monocytic cell line U-937, which has a monocyte-like phenotype, 14,15 with purified human platelets, and stimulation of platelets in anticoagulated whole blood.…”

An Antibody–Drug Conjugate That Targets Tissue Factor Exhibits Potent Therapeutic Activity against a Broad Range of Solid Tumors

“…The impact of tissue factor HuMab (TF-011, TF-013, and TF-098) on whole-blood coagulation was assessed by TEG. Citrated whole blood, obtained from healthy donors, was incubated with LPS to induce tissue factor expression on monocytes and release of monocyte-derived tissue factorpositive microparticles (8,28). LPS treatment induced a decrease in clotting lag time compared with untreated blood (Supplementary Table S3), which, in this system, represents a measure for tissue factor activity.…”

“…Under physiologic conditions, tissue factor expression is mostly restricted to the cells of the subendothelial vessel wall, such as smooth muscle cells, pericytes, and fibroblasts, that are not in direct contact with the blood (6). In healthy individuals, blood leukocytes do not express tissue factor on the cell surface, although tissue factor expression has been described on 1% to 2% of monocytes (7,8). Activation of the coagulation cascade occurs when membrane-bound tissue factor is exposed to circulating FVII (a), for example, after disruption of the vessel wall by injury or after upregulation of tissue factor on monocytes under inflammatory conditions (9).…”