Scientific Report 2005

Immunology

Tissue Factor, Coagulation Proteases, and Protease-Activated Receptors in Hemostasis, Thrombosis, and Inflammation

R. Pawlinski, G. Schabbauer, M.T. Lin, R.E. Tilley, J. Luyendyk, J. Nieva,* N. Mackman * Scripps Cancer Center, La Jolla, California

Tissue factor (TF), the initiator of blood coagulation, is expressed by cells that surround blood vessels. After vascular injury, TF is exposed to clotting factors in the blood, and coagulation is initiated. The formation of a clot stops further blood loss from the vasculature. Aberrant expression of TF also contributes to thrombosis and inflammation in a variety of disease states, such as sepsis, cancer, and atherosclerosis. We are interested in the role of TF in hemostasis, thrombosis, and inflammation.

Hemostasis

TF is essential for hemostasis because complete deficiency of TF is not compatible with life. TF-dependent clotting is counterbalanced by 3 anticoagulant molecules/pathways: the complex formed by TF and coagulation factor VIIa is inhibited by TF pathway inhibitor; the protein C pathway inactivates the clotting cofactors coagulation factors Va and VIIIa; and antithrombin inhibits all coagulation proteases, including thrombin. A complete deficiency in any of these 3 pathways leads to embryonic lethality due to thrombosis. We hypothesized that reducing the levels of TF would rescue embryos deficient in these anticoagulants.Crossbreeding of mice with low levels of TF rescued embryos deficient in TF pathway inhibitor or the protein C pathway but not those deficient in antithrombin. In adult mice, the relative levels of the 3 anticoagulant pathways were different in different tissues, indicating that the different anticoagulants acted in a tissue-specific manner.

Thrombosis

Recently, researchers have proposed a role for blood-borne TF in the propagation of a thrombus. The pool of blood-borne TF is incorporated into growing thrombi. However, different injury models have resulted in conflicting data. Using a murine laser-induced microvascular thrombosis model, we found that blood-borne TF contributes to the growth of the thrombus. In contrast, in a photochemical injury model of the common carotid artery, thrombus formation was driven primarily by TF derived from the blood vessel wall.

Currently, we are collaborating with J. Nieva, Scripps Cancer Center, to investigate the hypothesis that cancer patients have elevated levels of blood-borne TF and that this elevation contributes to the increased incidence of deep-vein thrombosis. We developed a new functional assay that measures TF activity in microparticles and platelets from the blood. We compared levels of functional blood-borne TF from healthy volunteers and from cancer patients. We found low levels of TF in healthy volunteers and elevated levels in the blood of cancer patients. Preliminary data revealed that a patient with a high level of blood-borne TF also had a deep-vein thrombosis. Our discovery that elevated levels of blood-borne TF are found in cancer patients and may correlate with the occurrence of deep-vein thrombosis suggests that these patients may benefit from anti-TF therapy.

Inflammation

Another disease in which TF expression is induced and contributes to intravascular coagulation is sepsis. In sepsis, lipopolysaccharide released by gram-negative bacteria activates the innate immune system, a situation that leads to inflammation and disseminated intravascular coagulation. The phosphatidylinositol-3´-kinase (PI3K) signaling pathway is activated by numerous stimuli and affects many cellular processes. Previously, we showed in vitro that the PI3K pathway suppresses lipopolysaccharide induction of TF and TNF-α in human monocytic cells. More recently, we showed that the PI3K pathway inhibits lipopolysaccharide-induced expression of inflammatory mediators and coagulation in a murine endotoxemia model. Insulin is a potent activator of the PI3K pathway and is used in the treatment of sepsis in humans. We found that low doses of insulin, lower than the level that affects glucose metabolism, reduce inflammation in an endotoxemia model in a PI3K-dependent manner. Further studies will determine the mechanism by which the PI3K pathway inhibits lipopolysaccharide signaling.

Publications

Chou, J., Mackman, N., Merrill-Skoloff, G., Pedersen, B., Furie, B.C., Furie, B. Hematopoietic cell-derived microparticle tissue factor contributes to fibrin formation during thrombus propagation. Blood 104:3190, 2004.

Day, S.M., Reeve, J., Pedersen, B., Farris, D.M., Myers, D.D., Im, M., Wakefield, T.W., Mackman, N., Fay, W.P. Macrovascular thrombosis is driven by tissue factor derived primarily from the blood vessel wall. Blood 105:192, 2005.

Frank, R.D., Schabbauer, G., Holscher, T., Sato, Y., Tencati, M., Pawlinski, R., Mackman, N. The synthetic pentasaccharide fondaparinux reduces coagulation, inflammation and neutrophil accumulation in kidney ischemia-reperfusion injury.
J. Thromb. Haemost. 3:531, 2005.

Mackman, N. Mouse models in haemostasis and thrombosis. Thromb. Haemost. 92:440, 2004.

Morrow, D.A., Murphy, S.A., McCabe, C.H., Mackman, N., Wong, H.C., Antman, E.M. Potent inhibition of thrombin with a monoclonal antibody against tissue factor (Sunol-cH36): results of the PROXIMATE-TIMI 27 trial. Eur. Heart J. 26:682, 2005.

Pawlinski, R., Mackman, N. Tissue factor, coagulation proteases, and protease-activated receptors in endotoxemia and sepsis. Crit. Care Med. 32(5 Suppl.):S293, 2004.

Pawlinski, R., Pedersen, B., Erlich, J., Mackman, N. Role of tissue factor in hemostasis, thrombosis, angiogenesis and inflammation: lessons from low tissue factor mice. Thromb. Haemost. 92:444, 2004.

Pedersen, B., Holscher, T., Sato, Y., Pawlinski, R., Mackman, N. A balance between tissue factor and tissue factor pathway inhibitor is required for embryonic development and hemostasis in adult mice. Blood 105:2777, 2005.

Pyo, R.T., Sato, Y., Mackman, N., Taubman, M.B. Mice deficient in tissue factor demonstrate attenuated intimal hyperplasia in response to vascular injury and decreased smooth muscle cell migration. Thromb. Haemost. 92:451, 2004.

Schabbauer, G., Tencati, M., Pedersen, B., Pawlinski, R., Mackman, N. PI3K-Akt pathway suppresses coagulation and inflammation in endotoxemic mice. Arterioscler. Thromb. Vasc. Biol. 24:1963, 2004.

Yu, J.L., May, L., Lhotak, V., Shahrzad, S., Shirasawa, S., Weitz, J.I., Coomber, B.L., Mackman, N., Rak, J.W. Oncogenic events regulate tissue factor expression in colorectal cancer cells; implications for tumor progression and angiogenesis. Blood 105:1734, 2005.