Anticoagulant and Membrane-degrading Effects of Secretory (Non-pancreatic) Phospholipase A2 Are Inhibited in Plasma

Summary

Plasma concentrations of secretory (non-pancreatic) phospholipase A2 (sPLA2) may rise 1000-fold during inflammation, and this acute phase response has been related to anticoagulant effects. In the present study this hypothesis was further investigated.

Prothrombinase activity was measured for model membranes mimicking the phospholipid composition of the outer membrane of resting and activated blood platelets. Using ellipsometry, membrane degradation by sPLA2 could be measured simultaneously with inhibition of thrombin production. The same technique was used to study clotting, by the sudden appearance of fibrin strands on the membrane. Results were compared with the effects of sPLA2 on the activation of washed platelets and platelets in plasma.

In buffer solution, model membranes were degraded by (patho)physiological concentrations of sPLA2. Even when only partially degraded, membranes rapidly lost their prothrombinase activity, indicating preferential degradation of phosphatidylserine. Addition of diluted plasma interfered with membrane degradation, and also with inhibition of prothrombinase activity. In agreement with these observations, sPLA2 inhibited thrombin production and annexin V-binding of activated washed platelets, but had no effects on platelet activation or clotting in plasma. These findings indicate that the elevated plasma sPLA2 concentrations observed in inflammatory disease will not reduce hypercoagulability in such patients.

Keywords

Phospholipase A2 - prothrombinase - anticoagulation - platelets - ellipsometry

^* Present address: Medtronic Bakken Research Center, Endepolsdomein 5, 6229 GW Maastricht, The Netherlands

• References

• 1 Heinrikson RL. Dissection and sequence analysis of phospholipases A2. Meth Enzymol 1991; 197: 201-14.
• 2 Kramer RM, Hession C, Johansen B, Hayes G, McGray P, Chow EP, Tizard R, Pepinsky RB. Structure and properties of a human non-pancreatic phospholipase A2. J Biol Chem 1989; 264: 5768-75.
• 3 Mounier C, Faili A, Vargaftig BB, Bon C, Hatmi M. Secretory phospholipase A2 is not required for arachidonic acid liberation during platelet activation. Eur J Biochem 1993; 216: 169-75.
• 4 Vadas P, Scott K, Smith G, Rajkovic I, Stefanski E, Schouten BD, Singh R, Pruzanski W. Serum phospholipase A2 activity and immunoreactivity in a prospective analysis of patients with septic shock. Life Sciences 1992; 50: 807-11.
• 5 Hack CE, Wolbink G-J, Schalkwijk C, Speijer H, Hermens WT, Van den Bosch H. A role for secretory phospholipase A2 and C-reactive protein in the removal of injured cells. Immunol Today 1997; 18: 111-5.
• 6 Nesheim ME, Taswell JB, Mann KG. The contribution of bovine factor V and factor Va to the activity of prothrombinase. J Biol Chem 1979; 254: 10952-62.
• 7 Rosing J, Speijer H, Zwaal RFA. Prothrombin activation on phospholipid membranes with positive electrostatic potential. Biochemistry 1988; 27: 8-11.
• 8 Giesen PLA, Willems GM, Hemker HC, Hermens WTh. Membrane-mediated assembly of the prothrombinase complex. J Biol Chem 1991; 266: 18720-5.
• 9 Giesen PLA, Willems GM, Hermens WTh. Production of thrombin by the prothrombinase complex is regulated by membrane-mediated transport of prothrombin. J Biol Chem 1991; 266: 1379-82.
• 10 Zwaal RFA, Comfurius P, Bevers EM. Platelet procoagulant activity and microvesicle formation. Its putative role in hemostasis and thrombosis. Biochim Biophys Acta 1992; 1180: 1-8.
• 11 Cirino G, Cicala C, Sorrentino L, Browning JL. Human recombinant non pancreatic secreted platelet phospholipase A2 has anticoagulant activity in vitro on human plasma. Thromb Res 1993; 70: 337-42.
• 12 Inada M, Crowl RM, Bekkers ACAPA, Verheij H, Weiss J. Determinants of the inhibitory action of purified 14-kDa phospholipases A2 on cell-free prothrombinase complex. J Biol Chem 1994; 269: 26338-43.
• 13 Mounier CM, Hackeng TM, Schaeffer F, Faure G, Bon C, Griffin JH. Inhibition of prothrombinase by human secretory phospholipase A2 involves binding to factor Xa. J Biol Chem 1998; 273: 23764-72.
• 14 Mounier CM, Luchetta P, Lecut C, Kodori RS, Faure G, Lambeau G, Valentin E, Singer A, Ghomashchi F, Béguin S, Gelb MH, Bon C. Basic residues of human group IIa phospholipase A2 are important for binding to factor Xa and prothrombinase inhibition. Eur J Biochem 2000; 267: 4960-9.
• 15 Boffa MC, Boffa GA. A phospholipase A2 with anticoagulant activity. Inhibition of the phospholipid activity in coagulation. Biochim Biophys Acta 1976; 429: 839-52.
• 16 Stefansson S, Kini M, Evans HJ. The basic phospholipase A2 from Naja nigricollis venom inhibits the prothrombinase complex by a novel nonenzymatic mechanism. Biochemistry 1990; 29: 7742-6.
• 17 Crowl RM, Stoller JT, Conroy RR, Stoner CR. Induction of phospholipase A2 gene expression in human hepatoma cells by mediators of the acute phase response. J Biol Chem 1991; 226: 2647-51.
• 18 Franken PA, Van den Berg L, Huang J, Gunyuzlu P, Lugtigheid RB, Verheij HM, De Haas GH. Purification and characterization of a mutant human platelet phospholipase A2 expressed in Escherichia coli. Eur J Biochem 1992; 203: 89-98.
• 19 Jain MK, Ranadive GR, Yu B-Z, Verheij HM. Interfacial catalysis by phospholipase A2: monomeric enzyme is fully catalytically active at the bilayer interface. Biochemistry 1991; 30: 7330-40.
• 20 Lindhout T, Govers-Riemslag JWP, van de Waart P, Hemker HC, Rosing J. Factor Va-factor Xa interaction. Effects of phospholipid vesicles of varying composition. Biochemistry 1982; 21: 5494-502.
• 21 Rosing J, Bakker HM, Thomassen CLGD, Hemker HC, Tans G. Characterization of two forms of human factor Va with different cofactor activities. J Biol Chem 1993; 268: 21130-6.
• 22 Wolbink G-J, Schalkwijk C, Baars JW, Wagstaff J, Van den Bosch H, Hack CE. Therapy with interleukin-2 induces the systemic release of phospholipase A2. Canc Immunol Immunother 1995; 41: 287-93.
• 23 Bevers EM, Comfurius P, Zwaal RFA. Changes in membrane phospholipid distribution during platelet activation. Biochim Biophys Acta 1983; 736: 57-66.
• 24 Speijer H, Giesen PLA, Zwaal RFA, Hack CE, Hermens W Th. Critical micelle concentrations and stirring are rate limiting in the loss of lipid mass during membrane degradation by phospholipase A2. Biophys J 1996; 70: 2239-47.
• 25 Kugiyama K, Ota Y, Takazoe K, Moriyama Y, Kawano H, Miyao Y, Sakamoto T, Soejima H, Ogawa H, Doi H, Sugiyama S, Yasue H. Circulating levels of secretory type II phospholipase A2 predict coronary events in patients with coronary artery disease. Circulation 1999; 100: 1280-4.
• 26 Wolbink G-J, Bossink AWJ, Groeneveld ABJ, de Groot MCM, Thijs LG, Hack CE. Complement activation in patients with sepsis is in part mediated by C-reactive protein. J Infect Dis 1998; 177: 81-7.
• 27 Grandbois M, Clausen-Schaumann H, Gaub H. Atomic force microscope imaging of phospholipid bilayer degradation by phospholipase A2. Biophys J 1998; 74: 2398-404.
• 28 Reviakine I, Brisson A. Formation of supported phospholipid bilayers from unilamellar vesicles investigated by atomic force microscopy. Langmuir 2000; 16: 1806-15.
• 29 Barenholz Y, Thompson TE. Sphyngomyelins in bilayer and biological membranes. Biochim Biophys Acta 1980; 604: 129-58.
• 30 Cullis PR, De Kruijff B. Lipid polymorphism and the functional roles of lipids in biological membranes. Biochim Biophys Acta 1979; 559: 399-420.
• 31 Kerns RT, Kini RM, Stefansson S, Evans HJ. Targeting of venom phospholipases: The strongly anticoagulant phospholipase A2 from Naja nigricollis venom binds to coagulation factor Xa to inhibit the prothrombinase complex. Arch Biochem Biophys 1999; 369: 107-13.
• 32 Demel RA, Geurts van Kessel WSM, Zwaal RFA, Roelofsen B, Van Deenen LLM. Relation between various phospholipases actions on human red cell membranes and the interfacial phospholipid pressure in monolayers. Biochim Biophys Acta 1975; 406: 97-107.
• 33 Polgar J, Kramer RM, Um SL, Jakubowski JA, Clemetson KJ. Human group II 14 kDa phospholipase A2 activates human platelets. Biochem J 1997; 327: 259-65.
• 34 Bevers EM, Comfurius P, Zwaal RFA. The nature of the binding site for prothrombinase at the platelet surface as revealed by lipolytic enzymes. Eur J Biochem 1982; 122: 81-5.
• 35 Fourcade O, Simon M-F, Viodé C, Rugani N, Leballe F, Ragab A, Fournié B, Sarda L, Chap H. Secretory phospholipase A2 generates the novel lipid mediator lysophosphatidic acid in membrane microvesicles shed from activated cells. Cell 1995; 80: 919-27.
• 36 Okumura K, Ohkura N, Inoue S, Ikeda K, Hayashi K. A novel phospholipase A2 inhibitor with leucine-rich repeats from the blood plasma of Agkistrodon blomhoffi siniticus . J Biol Chem 1998; 273: 19469-75.
• 37 Lindahl M, de Wit C, Westrom B, von Schenck H, Tagesson C. Demonstration of a phospholipase A2 inhibitor in human plasma and in plasma from the European hedgehog (Erinaceus europaeus) . Int J Biochem 1991; 23: 287-92.
• 38 Kim SH, Park JM, Ryu CK, Lee MG. Determination of a new naphthoquinone derivative, NQ12, in human plasma and urine by high-performance liquid chromatography. Res Commun Mol Pathol Pharmacol 1999; 103: 101-7.
• 39 Yuk D-Y, Ryu C-K, Hong J-T, Chung K-H, Kang W-S, Kim Y, Yoo H-S, Lee M-K, Lee C-K, Yun Y-P. Antithrombotic and antiplatelet activities of 2-chloro-3-[4-(ethylcarboxy)-phenyl]-amino-1,4-naphthoquinone (NQ12), a newly synthetized 1,4-naphthoquinone derivative. Biochem Pharmacol 2000; 60: 1001-8.
• 40 Deguchi H, Fernandez JA, Hackeng TM, Banka CL, Griffin JH. Cardiolipin is a normal component of human plasma lipoproteins. Proc Natl Acad Sci USA 2000; 97: 1743-8.