Interaction of an annexin V homodimer (Diannexin) with phosphatidylserine on cell surfaces and consequent antithrombotic activity

Summary

Annexin V(AV), a protein with anticoagulant activity, exerts antithrombotic activity by binding to phosphatidylserine (PS), inhibiting activation of serine proteases important in blood coagulation. The potential use of this protein as an anticoagulant is limited as it rapidly passes from the blood into the kidneys due to its relatively small size (36 kDa). We used recombinant DNA technology to produce a homodimer of human AV (DAV, 73 kDa), which exceeds the renal filtration threshold, and has a 6.5-hour half-life in the rat circulation. Human red blood cells with externalized PS were used to show that DAV had a higher affinity for PS-exposing cells than AV. DAV labeling sensitively identifies PS-exposing cells, was found to be a potent inhibitor of the activity of the prothombinase complexes and inhibits the ability of secretory phospholipase A₂ to hydrolyze phospholipids of PS-exposing cells, reducing the formation of mediators of blood coagulation and reperfusion injury. DAV exerts dose-dependent antithrombotic activity in ratveins. This combination of activities suggests that DAV is a valuable probe to measure PS exposure and may be efficacious as a novel drug in a wide range of clinical situations.

Keywords

Phospholipids - annexins - haemostasis

This study was supported by National Institutes of Health grants HL 20985, U54 HL070583, and HL 66355.

• References

• 1 Daleke DL. Regulation of transbilayer plasma membrane phospholipid asymmetry. J Lipid Res 2003; 44: 233-242.
• 2 Devaux PF. Protein involvement in transmembrane lipid asymmetry. Annu Rev Biophys Biomol Struct 1992; 21: 417-439.
• 3 Kuypers FA, de Jong K. The role of phosphatidylserine in recognition and removal of erythrocytes. Cell Mol Biol (Noisy-le-grand) 2004; 50: 147-158.
• 4 Zwaal RF, Comfurius P, Bevers EM. Surface exposure of phosphatidylserine in pathological cells. Cell Mol Life Sci 2005; 62: 971-988.
• 5 Basse F, Stout JG, Sims PJ. et al. Isolation of an erythrocyte membrane protein that mediates Ca2+-dependent transbilayer movement of phospholipid. J Biol Chem 1996; 271: 17205-17210.
• 6 Chang CP, Zhao J, Wiedmer T. et al. Contribution of platelet microparticle formation and granule secretion to the transmembrane migration of phosphatidylserine. J Biol Chem 1993; 268: 7171-7178.
• 7 Thiagarajan P, Tait JF. Binding of annexin V/placental anticoagulant protein I to platelets. Evidence for phosphatidylserine exposure in the procoagulant response of activated platelets. J Biol Chem 1990; 265: 17420-17423.
• 8 Zwaal RFA, Schroit AJ. Pathophysiologic implications of membrane phospholipid asymmetry in blood cells. Blood 1997; 89: 1121-1132.
• 9 Fourcade O, Simon M-F, Viode C. et al. Secretory phospholipase A2 generates the novel lipid mediator lysophosphatidic acid in membrane microvesicles shed from activated cells. Cell 1995; 80: 919-927.
• 10 Koopman G, Reutelingsperger CPM, Kuijten GAM. et al. Annexin V for flow cytemetric detection of phosphatidylserine expression on B cells undergoing apoptosis. Blood 1994; 84: 1415-1420.
• 11 Ran S, Downes A, Thorpe PE. Increased exposure of anionic phospholipids on the surface of tumor blood vessels. Cancer Res 2002; 62: 6132-6140.
• 12 de Jong K, Larkin SK, Styles LA. et al. Characterization of the phosphatidylserine-exposing subpopulation of sickle cells. Blood 2001; 98: 860-867.
• 13 Kuypers FA, Lewis RA, Ernst JD. et al. Detection of altered membrane phospholipid asymmetry in subpopulations of human red cells using fluorescently labeled annexin V. Blood 1996; 87: 1179-1187.
• 14 Funakoshi T, Hendrickson LE, McMullen BA. et al. Primary structure of human placental anticoagulant protein. Biochemistry 1987; 26: 8087-8092.
• 15 Reutelingsperger CP, Hornstra G, Hemker HC. Isolation and partial purification of a novel anticoagulant from arteries of human umbilical cord. Eur J Biochem 1985; 151: 625-629.
• 16 Moss SE, Morgan RO. The annexins. Genome Biol 2004; 5: 219
• 17 Neidlinger NA, Hirvela ER, Skinner RA. et al. Postinjury serum secretory phospholipase A2 correlates with hypoxemia and clinical status at 72 hours. J Am Coll Surg 2005; 200: 173-178.
• 18 Romisch J, Grote M, Weithmann KU. et al. Annexin proteins PP4 and PP4-X. Comparative characterization of biological activities of placental and recombinant proteins. Biochem J 1990; 272: 223-229.
• 19 Neidlinger NA, Larkin SK, Bhagat A. et al. Hydrolysis of phosphatidylserine-exposing red blood cells by secretory phospholipase A2 generates lysophosphatidic acid and results in vascular dysfunction. J Biol Chem 2005; 281: 775-781.
• 20 Del Conde I, Shrimpton CN, Thiagarajan P. et al. Tissue-factor-bearing microvesicles arise from lipid rafts and fuse with activated platelets to initiate coagulation. Blood 2005; 106: 1604-1611.
• 21 Kaplun A, Ferluga J, Prydz H. et al. Protection of pregnant mice with phospholipase C and with Arvin against foetal death induced by bacterial lipopolysaccharide. Br J Exp Pathol 1979; 60: 185-192.
• 22 Romisch J, Seiffge D, Reiner G. et al. In-vivo antithrombotic potency of placenta protein 4 (annexin V). Thromb Res 1991; 61: 93-104.
• 23 Van Ryn-McKenna J, Merk H, Muller TH. et al. The effects of heparin and annexin V on fibrin accretion after injury in the jugular veins of rabbits. Thromb Haemost 1993; 69: 227-230.
• 24 Thiagarajan P, Benedict CR. Inhibition of arterial thrombosis by recombinant annexin V in a rabbit carotid artery injury model. Circulation 1997; 96: 2339-2347.
• 25 Knauf MJ, Bell DP, Hirtzer P. et al. Relationship of effective molecular size to systemic clearance in rats of recombinant interleukin-2 chemically modified with water-soluble polymers. J Biol Chem 1988; 263: 15064-15070.
• 26 Paszty C, Brion CM, Manci E. et al. Transgenic knockout mice with exclusively human sickle hemoglobin and sickle cell disease. Science 1997; 278: 876-878.
• 27 de Jong K, Emerson R, Butler H. et al. Short survival of PS exposing red blood cells in murine sickle cell anemia. Blood 2001; 98: 1577-1584.
• 28 Campos B, Mo YD, Mealy TR. et al. Mutational and crystallographic analyses of interfacial residues in annexin V suggest direct interactions with phospholipid membrane components. Biochemistry 1998; 37: 8004-8010.
• 29 Sela M. The Antigens. , Vol. 7. NewYork: Academic Press; 1987: 1-146.
• 30 De Jong K, Geldwerth D, Kuypers FA. Oxidative damage does not alter membrane phospholipid asymmetry in human erythrocytes. Biochemistry 1997; 36: 6768-6776.
• 31 Mc Farlane A. Efficient trace labeling of proteins with iodine. Nature 1958; 128: 53
• 32 Berry CN, Girard D, Lochot S. et al. Antithrombotic actions of argatroban in rat models of venous, 'mixed' and arterial thrombosis, and its effects on the tail transection bleeding time. Br J Pharmacol 1994; 113: 1209-1214.
• 33 Teoh N, Ito Y, Field J. et al. Diannexin, a novel annexin homodimer, provides prolonged microvascular protection against warm hepatic ischimia-reperfusion injury. Hepatology 2005; 42: 314A
• 34 Kuypers FA, Styles LA. The role of secretory phospholipase A2 in acute chest syndrome. Cell Mol Biol (Noisy-le-grand) 2004; 50: 87-94.
• 35 Partrick D, Moore E, Silliman C. et al. Secretory phospholipase A2 activity correlates with postinjury multiple organ failure. Crit Care Med 2001; 29: 989-993.
• 36 Abraham E, Naum C, Bandi V. et al. Efficacy and safety of LY315920Na/S-5920, a selective inhibitor of 14-kDa group IIA secretory phospholipase A2, in patients with suspected sepsis and organ failure. Crit Care Med 2003; 31: 718-728.
• 37 Aufenanger J, Samman M, Quintel M. et al. Pancreatic phospholipase A2 activity in acute pancreatitis: a prognostic marker for early identification of patients at risk. Clin Chem Lab Med 2002; 40: 293-297.
• 38 Kupiec-Weglinski J, X-D S, Busettil R. et al. Protection by Diannexin, a annexin homodimer, against cold ischemia reperfusion injury during reat liver transpalantion. Hepatology 2005; 42: 313