Procoagulant Activity of T Lymphoblastoid Cells due to Exposure of Negatively Charged Phospholipid

Summary

We have characterised the Procoagulant activity (PCA) of six well-established cell lines by assays of tissue factor (TF), thrombin and FXa generation, flow cytometry using Annexin V, and by binding studies with Factors VIII and IXa.

The monocytic (THP-1 & U937) and promyelocytic (NB4) cells expressed high concentrations of TF antigen and activity, whereas TF in the lymphocytic cells (Molt 4, Jurkat & Nalm 6) was very low or absent. However the T-lymphoblastoid cells (Molt 4 & Jurkat) promoted the generation of large amounts of thrombin despite their low TF content, and these cells were also the most active in supporting Factor Xa generation. Molt 4 cells bound Factors VIII and IXa with high capacity and their activity was inhibited by Annexin V. These results indicate that the PCA of T-lymphoblastoid lines is due to expression of negatively charged phospholipids. Flow cytometry studies showed Annexin V binding to the major population of nonapoptotic Molt 4 cells and the PCA of Molt 4 was not increased when apoptosis was induced by staurosporine, indicating that PCA is independent of apoptotic status.

Keywords

T cells - coagulation - phospholipid

• References

• 1 Nemerson Y, Repke D. Tissue factor accelerates the activation of coagulation factor VII: the role of a bifunctional coagulation cofactor. Thrombosis Research 1985; 40: 351-8.
• 2 Østerud B, Rapaport S. Activation of factor IX by the reaction product of tissue factor and factor VII. Additional pathway for initiating blood coagulation. Proc Natl Acad Sci USA 1977; 74: 5260-4.
• 3 Rosing R, Tans G. The role of phospholipids in the activation of factor X and prothrombin. In: Zwaal RFA. ed. Coagulation and Lipids. CRC Press; Boca Raton, Florida: 1989: 159-88.
• 4 Rivers RPA, Hathaway WE, Weston WL. The endotoxin-induced coagulant activity of human monocytes. Br J Haematol 1975; 30: 311-6.
• 5 Geczy CL, Meyer PA. Leukocyte procoagulant activity in man, as in vitro correlate of delayed-type hypersensitivity. J Immunol 1982; 128: 331-6.
• 6 Edwards RL, Rickles FR. Macrophage procoagulants. Prog Haemostas Thromb 1984; 07: 183-209.
• 7 Hair GA, Padula S, Zeff R. et al. Tissue factor expression in human leukemic cells. Leukemia Res 1996; 20: 1-11.
• 8 Østerud B, Flaegstad T. Increased tissue thromboplastin activity in monocytes of patients with meningococcal infection: relation to an unfavorable prognosis. Thromb Haemost 1983; 49: 5-7.
• 9 Rothberger H, Zimmerman TS, Spiegelberg HL. et al. Leukocyte procoagulant activity. Enhancement of production in vitro by IgG and antigen-antibody complexes. Journal of Clinical Investigation 1977; 59: 549-57.
• 10 Schwager I, Jungi TW. Effect of human recombinant cytokines on the induction of macrophage procoagulant activity. Blood 1994; 83: 152-60.
• 11 Morgan D, Edwards RL, Rickles FR. Monocyte procoagulant activity as a peripheral marker of clotting activation in cancer patients. Haemostasis 1988; 18: 55-65.
• 12 Lyberg T, Prydz H. Phorbol esters induce the synthesis of thromboplastin activity in human monocytes. Biochem J 1981; 194: 699-706.
• 13 Falanga A, Gordon SG. Isolation and characterization of cancer procoagulant: a cysteine proteinase from malignant tissue. Biochemistry 1985; 24: 5558-67.
• 14 McGee MP, Li LC, Hensler M. Functional assembly of intrinisic coagulation proteases on monocytes and platelets. Comparison between cofactor activities induced by thrombin and factor Xa. J Exp Med 1992; 176: 27-35.
• 15 Tracy PB, Rothbach MS, Mann KG. Functional prothrombinase complex assembly on isolated monocytes and lymphocytes. J Biol Chem 1983; 258: 7264-67.
• 16 Barrowcliffe TW, Marshall DE, Trickett LP. et al. Procoagulant activity of T lymphoblastoid cells in extrinsic and intrinsic coagulation systems. Thromb Haemost 1989; 62 (03) 880-4.
• 17 Houbouyan L, Padilla A, Gray E, Longstaff C, Barrowcliffe TW. Inhibition of thrombin generation by heparin and LMW heparins: a comparison of chromogenic and clotting methods. Blood Coagulation & Fibrinolysis 1996; 07 (01) 24-30.
• 18 Kemball-Cook G, Tubbs JE, Dawson NJ, Barrowcliffe TW. The behavior of different factor VIII concentrates in a chromogenic factor X-activating system. Br J Haematol 1993; 84 (02) 273-8.
• 19 Thompson AR, Murphy MEP, Liu ML. et al. Loss of tolerance to exogenous and endogenous factor VIII in a mild hemophilia A patient with an Arg593 to cys mutation. Blood 1997; 90: 1902-9.
• 20 Longstaff C, Merton RE, Fabregas P, Felez J. Characterization of cellassociated plasminogen activation catalyzed by urokinase-type plasminogen activator, but independent of urokinase receptor (uPAR, CD87). Blood 1999; 3839-46.
• 21 Fadok VA, Voelker DR, Campbell PA, Cohen JJ, Bratton DL, Henson PM. Exposure of phosphatidyl serine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages. J Immunol 1992; 148: 2207-16.
• 22 McGee MP, Li LC. Functional difference between intrinisc and extrinsic coagulation pathways. Kinetics of factor X activation on human monocytes and alveolar macrophages. J Biological Chemistry 1991; 226: 8079-85.
• 23 Varadi K, Elodi S. Formation and functioning of the factor IXa-VIII complex on the surface of endothelial cells. Blood 1987; 69: 442-5.
• 24 Brinkman H-JM, Mertens K, Holthuis J, Zwart-Huinnink LA, Grijm K, Van Mourik JA. The activation of human blood coagulation factor X on the surface of endothelial cells: a comparison with various vascular cells, platelets and monocytes. Br J Haematol 1994; 87: 332-42.
• 25 Kemball-Cook G, Barrowcliffe TW. Interaction of factor VIII with phospholipids: role of composition and negative charge. Thrombosis Research 1992; 67 (01) 57-71.
• 26 Gilbert GE, Drinkwater D, Barter S, Clouse SB. Specificity of phosphatidylserine-containing binding sites for factor VIII. Studies with model membranes supported by glass microspheres (lipospheres). J Biol Chem 1992; 267 (22) 15861-8.
• 27 van Heerde WL, de Groot PG, Reutelingsperger CPM. The complexity of the phospholipid binding protein annexin V. Thromb Haemost 1995; 73: 172-9.
• 28 Ravanat C, Archipoff G, Beretz A, Freund G, Cazenave J-P, Freyssinet J-M. Use of annexin-V to demonstrate the role of phosphatidylserine exposure in the maintenance of haemostatic balance by endothelial cells. Biochem J 1992; 282: 7-13.
• 29 Seigneuret M, Deavaux PF. ATP-dependent asymmetric distribution of spin-labeled phospholipids in the erythrocyte membrane: relation to shape changes. Proc Natl Acad Sci USA 1984; 81: 3751-5.
• 30 Zwaal RFA, Schroit AJ. Pathophysiologic implications of membrane phospholipid assymmetry in blood cells. Blood 1997; 89: 1121-32.
• 31 Zhou Q, Zhao J, Stout JG, Luhm RA, Wiedmer T, Sims PJ. Molecular cloning of human plasma membrane phospholipid scramblase: a protein mediating transbilayer movement of plasma membrane phospholipids. J Biol Chem 1997; 272: 18240-4.
• 32 Hamatake M, Iguchi K, Hirano K, Ishida R. Zinc induces mixed types of cell death, necrosis, and apoptosis, in Molt-4 Cells. J Biochem 2000; 128: 933-9.
• 33 Edwards RL, Silver J, Rickles FR. Human Tumor Procoagulants: Registry of the Subcommittee on Haemostasis and Malignancy of the Scientific and Standardization Committee, International Society on Thrombosis and Haemostasis. Thromb Haemost 1993; 69 (02) 205-13.
• 34 Falanga A, Rickles FR. Pathophysiology of the thrombophilic state in the cancer patient. Semin Thromb Hemost 1999; 25 (02) 173-82.
• 35 Rickles FR, Levine MN, Dvorak HF. Abnormalities of haemostasis in malignancy. In: Haemostasis and Thrombosis: Basic Principles and Clinical Practice. Colman RW, Hirst J, Marder VJ, Clowes AW, George JN. eds. Philadelphia: Lipincott Williams & Wilkins; 2001: 1131-52.
• 36 Rickles FR, Hair GA, Zeff RA, Lee E, Bona RD. Tissue factor expression in human leukocytes and tumor cells. Thromb Haemost 1995; 74 (01) 391-5.
• 37 Nur S, Anwar M, Saleem M, Ahmad PA. Disseminated intravascular coagulation in acute leukemias at first diagnosis. Eur J Haematol 1995; 55 (02) 78-82.
• 38 Barbui T, Finazzi G, Falanga A, Battista R, Bassan R. Bleeding and thrombosis in acute lymphoblastic leukemia. Leuk Lymphoma 1993; (Suppl. 02) 43-7.
• 39 Redmond EJ, Welch M, Durrans D. et al. Acute ischaemia of the lower limb: an unusual presenting feature of acute lymphoblastic leukaemia. Eur J Vasc Surg 1993; 07 (06) 750-2.
• 40 Bilgrami S, Bernard R, Greenberg BR, Weinstein RE, Hair GA, Rickles FR. Recurrent venous thrombosis as the presenting manifestation of acute lymphocytic leukemia: Leukemic cell procoagulant activity is not responsible for the hypercoagulable state. Medical and Pediatric Oncology 1995; 24: 40-5.
• 41 Kunzelmann-Marche C, Satta N, Toti F. et al. The influence exerted by a restricted phospholipid microenvironment on the expression of tissue factor activity at the cell plasma membrane surface. Thromb Haemost 2000; 83: 282-9.
• 42 Jonasson L, Holm J, Skalli O, Bondjers G, Hansson GK. Regional accumulations of T cells, macrophages and smooth muscle cells in the human atherosclerotic plaque. Arteriosclerosis 1986; 06: 131-8.
• 43 Libby P, Mach F, Schönbeck U, Bourcier T, Aikawa M. Regulation of the thrombotic potential of atheroma. Thromb Haemost 1999; 82 (02) 736-41.