Tissue factor expression in human leukemic cells.

Patients with acute leukemia are at increased risk for thrombotic and hemorrhagic complications, particularly those patients with acute promyelocytic leukemia (APL) undergoing induction chemotherapy. These serious complications have been attributed by some authors to the release of tissue factor (TF) procoagulant activity (PCA), particularly during cytotoxic chemotherapy. In previous studies of normal peripheral blood cells, only cells of the monocyte lineage have been found to express TF PCA. Therefore, several questions remain regarding the origin and characterization of the PCA in malignant leukemic cells, particularly those thought to be derived from granulocyte progenitor cells. We utilized a full-length cDNA probe, several monoclonal antibodies (MAbs) and a sensitive one-stage PCA assay to study the expression of TF in the human cell line, HL-60, in human peripheral blood monocytes/macrophages (Mo/Mø) and in highly purified populations of human polymorphonuclear leukocytes (PMN). In the HL-60 cells we detected low but significant levels of TF mRNA and TF antigen (TF:Ag). In unstimulated cells, coordinate increased levels of TF mRNA, TF:Ag and TF PCA expression were noted following phorbol-ester-induced macrophage differentiation of the cells, but a decreased level of TF mRNA with no change in the basal level of TF:Ag expression occurred following retinoic acid-induced granulocyte differentiation of this cell line. Long-term cultures of stimulated mature Mo/Mø demonstrated initial coordinate expression of TF mRNA, TF:Ag and TF PCA, but TF:Ag expression persisted even after 7 days (when TF PCA was undetectable). No TF PCA, TF:Ag or TF mRNA was demonstrated in highly purified populations of human PMN, regardless of culture conditions. Discordant expression of TF mRNA, TF:Ag and TF PCA in HL-60 cells suggests the possibility of novel, post-synthetic mechanisms for the regulation of TF PCA expression, which might be dependent on the phenotypic differentiation level of the cell. Such mechanisms (yet to be defined) might account for the ability of some leukemic cells, which frequently express characteristics of more than one cell line (e.g. monocytes and granulocytes), to express a TF gene product capable of activating blood coagulation.

In situ characterization of antigenic and functional tissue factor expression in human tumors utilizing monoclonal antibodies and recombinant factor VIIa as probes.

Tissue factor (TF), the primary initiator of blood coagulation in vivo, is expressed in vitro by a variety of cells. Previous efforts to localize TF in tissue and cells have been limited principally to the use of immunological techniques. In the present study, we describe a novel functional probe for TF expression, which can be utilized to localize functional TF in situ in human cells and tissues. This probe, a biotinylated phe-pro-arg-chloro-methyl-ketone-labeled rVIIa (FPR-ck-VIIa), interacts with TF via high-affinity binding sites. The binding of FPR-ck-VIIa, therefore, can be correlated with the ability of TF to activate clotting. In the described studies, TF antigen (TF:Ag) expression was examined immunohistochemically with various TF-specific monoclonal antibodies (MAbs) and was correlated with functional TF expression using our novel TF-binding probe (eg, FPR-ck-VIIa). Initial results indicate that TF:Ag expression correlates with the expression of functional TF (TF:VIIa), and the specificity of both types of probes was confirmed. Parallel antigenic and functional TF expression in situ was demonstrated in various human tumors. We believe this to be the first demonstration of functional TF in situ in human cells and tissues. We suggest that FPR-ck-VIIa should prove a useful reagent for studying the role of TF in the pathogenesis of clotting complications of human disease.

Production of modified crosslinked cell-free hemoglobin for human use: the role of quantitative determination of endotoxin contamination.

In vivo toxicity remains a major barrier to the successful use of cell-free hemoglobin (Hb) as an oxygen carrier in humans. Bacterial endotoxin (lipopolysaccharide, LPS) is known to contribute to the in vivo toxicity of Hb preparations, and the prevention of LPS contamination is a critical aspect of the effort to create an efficacious Hb blood substitute. Limulus amebocyte lysate assays for endotoxin were performed on multiple Hb samples from 26 independent production runs for the preparation of human crosslinked cell-free hemoglobin (alpha alpha Hb). High levels of LPS contamination (1- > 100 ng/mL) of alpha alpha Hb solutions were detected in multiple samples during many of the initial production runs. It was observed that LPS contamination of alpha alpha Hb solutions could occur at any step during the production sequence. Substantial enhancement by alpha alpha Hb of the biologic effects of LPS was demonstrated by two independent assays for endotoxin (the Limulus amebocyte lysate test and a mononuclear cell procoagulant assay), whereas LPS biologic activity was only slightly increased by human serum albumin and substantially diminished by IgG. These results suggest that the prevention of LPS-related toxicities in vivo may be more important to the clinical use of Hb solutions than to the use of other intravenous protein products. Therefore, it was encouraging to note that, with the careful monitoring for LPS in the production facility and in multiple samples during cell-free Hb production, sources of LPS contamination were recognized and the appropriate sites were made endotoxin-free.(ABSTRACT TRUNCATED AT 250 WORDS)