Tissue factor in microvesicles shed from U87MG human glioblastoma cells induces coagulation, platelet aggregation, and thrombogenesis

Abstract

Microvesicles (diameter ca 200 nm) from the cell-free supernatant of U87MG human glioblastoma cell caused platelet aggregation and coagulation in a manner identical with that previously shown for the intact cells. Both activities were inhibited by dansylarginine -N-(3- ethyl-1,5-pentanediyl) amide (DAPA), confirming the thrombin-dependent nature of both activities. The specific activities per microgram of protein were 2–10 times greater in the microvesicles than in the plasma membrane fraction, suggesting localization in specific membrane domains. Sucrose density centrifugation gave a single protein peak (density 1.14) with congruent procoagulant and platelet aggregating activities. Both activities required the extrinsic pathway, as shown by studies with factor-deficient plasmas, and both were inhibited by heating (60 min/100 degrees C), by reduction and alkylation, and by incubation of the microvesicles with rabbit anti-bovine brain tissue factor antibody. These observations were confirmed using microvesicles from the HL-60 human promyelocytic leukemia cells, which are known to contain tissue factor activity. The results suggest that both procoagulant and proaggregating activities are causally related through the presence of tissue factor in the microvesicles. Studies with the Baumgartner perfusion apparatus showed that U87MG microvesicles increased the size of adherent thrombi nearly tenfold and that these thrombi were associated with nucleated cells from the blood. The increase in adherent thrombi did not occur if perfusion was carried out in the presence of DAPA, confirming the role of thrombin in their formation.

The Pathogenetic Role of Apoptosis in Hypercoagulable States

“…Because of the co-expression of tissue factor and phosphatidylserine, the procoagulant activity of tumor cells can be inhibited by annexin V [89,90]. The procoagulant activity has also been found in shedding microvesicles released by tumor cells [91,92]. Microvesicles shedding from the primary tumor is the fust step for tumor metastasis.…”

Cancer Cell Procoagulants and Their Role in Malignant Disease

“…One form is shed membrane vesicles that appear to come from living normal and tumor cells and from lysed dead cells. 12,13,44,45 TF activity has been identified in the serum and urine of cancer patients; 116,46,47 this activity can be pelleted by centrifugation and is associated with membrane vesicles and may also play a relevant role in hypercoagulation in cancer. The second soluble form of TF is truncated TF (tTF).…”

Antimetastatic Agents. I. Role of Cellular Procoagulants in the Pathogenesis of Fibrin Deposition in Cancer and the Use of Anticoagulants and/or Antiplatelet Drugs in Cancer Treatment

“…Recent data from several laboratories suggest that the most common tumorassociated PAA, isolated from both human and murine tumor cell lines, is probably identical to a tumor-associated procoagulant activity (PCA) and therefore ultimately dependent for its PAA on thrombin generation. 10,11 Recently, Bastida and her colleagues, and others, have emphasized the adhesive properties of tumor cells rather than tumor cell PAA in studies of platelet-tumor cell interaction. This interesting new evidence, which is summarized elsewhere in this issue of Seminars, 8,9 suggests a relationship between the production of adhesive extracellular matrix proteins by tumor cells, tumor cell adherence to endothelial cells, and activation of platelets.…”

“…This evidence, which includes the histologic association of the products of clotting with growing tumors, the increased frequency of thromboembolic episodes and disseminated intravascular coagulation in patients with neoplasia, and the capacity of various hemostatically active drugs to alter the natural history of tumor growth, is reviewed extensively elsewhere in this issue of Seminars and in several recent publications. [1][2][3][4][5][6][7][8][9][10] Although the pathogenesis of this fibrin deposition in cancer is unknown, the involvement of several different coagulation pathways has been sug-gested and strategies for interruption of one or more of these pathways have been recommended as part of experimental cancer protocols.…”