Evaluation of Activity of Phospholipids in Blood Coagulation in Vitro^[*]

Summary

1. The effects of phospholipids, solubilized by different methods in different clotting tests are presented.

2. Complete fatty acid analysis of a phosphatidylserine fraction from beef brain is given.

3. Data relating electric charge of phospholipid particles to activity in clotting are presented.

4. A critical discussion of the factors which influence the activity of phospholipids in blood clotting terminates the paper.

^* We would like to thank P. Magidman for technical assistance with the gas chromatography of the fatty acids.

^** Part of this material was presented at the 10th Annual Symposium on Blood, Wayne State University, Detroit, 1962.

• References

• 1 Baer E, Buchnea D. Synthesis of L-α-(Dioleoyl)-Cephalin; with a Comment on the Stereochemical Designation of Glycerolphosphatides. J. Amer. chem. Soc 81: 1758 1959;
• 2 Bangham A. D. A correlation between surface charge and coagulant action of phospholipids. Nature 192: 1197 1961;
• 3 Bangham A. D. Phospholipids and sulpholipids. Nature 192: 807 1961;
• 4 Barkhan P, Newlands M. J, Wild P. Phospholipids from brain tissue as accelerators and inhibitors of blood coagulation. Bancet II: 234 1956;
• 5 Barkhan P, Silver M. J, DaCosta P. B, Tocantins B. M. Phospha-tidylserine and blood coagulation. Nature 182: 1031 1958;
• 6 Barkhan P, Silver M. J, O’Keefe B. M. The lipids of Human Erythrocytes and Platelets and Their Effects on Thromboplastin Formation. The Blood Platelets. 303 Bittle, Brown & Co; Boston: 1961
• 7 Barkhan P, Silver M. J. Biological activities of human platelets. Progr. Hemat Ill: 170 1962;
• 8 Bell W. N, Allon H. G. A brain extract as a substitute for platelets in the thromboplastin generation test. Nature 174: 880 1954;
• 9 Biggs R, Douglas A. S. The Thromboplastin Generation Test. J. clin. Path 06: 23 1953;
• 10 Ekwall P. The solubilization of Lipophilic Substances by Bile acid Salts. International Conference on Biochemical Problems of Lipids, Brussels. 1953
• 11 Esnouf M. P, Williams W. J. The Isolation and Purification of a Bovine Plasma Protein which is a Substrate for the Coagulation Fraction of Russel’s Viper Venom. Biochem. J 84: 62 1962;
• 12 Farquhar J. W. Identification and Gas-liquid Chromatographic Behavior of Plasmalogen Aldehydes and Their Acetal, Alcohol and Acetylated Alcohol Derivatives. J. Lipid Res 03: 21 1962;
• 13 Fleischer S. The Role of Lipid in Electron Transport. Abstract from Xlth Annual Symposium on Blood. Wayne State University; Detroit: 1963
• 14 Folch J. Brain Cephalin, a Mixture of Phosphatides. Separation from it of Phospha- tidylserine, Phosphatidylethanolamine and a Fraction Containing an Inositol Phosphatide. J. biol. Chem 146: 35 1942;
• 15 Folch J. The Chemical Structure of Phosphatidylserine. J. biol. Chem 174: 439 1948;
• 16 Folch J, Lees M, Sloane-Stanley G. H. A Simple Method for the Isolation and Purification of Total Lipids from Animal Tissues. J. biol. Chem 226: 497 1957;
• 17 Fullerton H. W, Anastasopoulous G. Anticoagulant Therapy. Brit, med. J 02: 1492 1949;
• 18 Grannis G, Kazal L. A, Miller O. P, Tocantins L. M. The Index of Crevicity. A Characteristic Parameter of Protein and Other Colloidal Structures. Abstract No. 37, 23A Abstracts of Papers presented at Meeting of Division of Biological Chemistry of Am. Chem. Soc. January, 1963
• 19 Gray G. M. The Separation of Long Chain Fatty Aldehydes by Gas-Liquid Chromatography. J. Chromat 04: 52 1960;
• 20 Hanahan D. J, Jayko M. E. (Dipalmitoleyl)-L-α-Lecithin. Biochem. Preparations. Vol. IV 12 John Wiley & Sons, Inc; New York: 1955
• 21 Harris W. D, Popat P. Determination of the Phosphorus Content of Lipids. J. Amer. Oil Chem. Soc 31: 126 1954;
• 22 Hecht E. On the Chemistry of Physiological Inhibitor of Blood Clotting. Fed. Proc 16: 194 1957;
• 23 Herb S. F, Magidman P, Luddy F. E, Riemenschneider R. W. Analysis of Fats and Oils by Gas-Liquid Chromatography and by Ultraviolet Spectrophotometry. J. Amer. Oil Chem. Soc 37: 127 1960;
• 24 Herb S. F, Magidman P, Barford R. A, Riemenschneider R. W. Fatty Acids of Lard. A-Identification by Gas-Liquid Chromatography. J. Amer. Oil Chem. Soc 40: 83 1963;
• 25 Hicks N. D, Pitney W. R. A Rapid Screening Test for Disorders of Thromboplastin Generation. Brit. J. Haematol 03: 227 1957;
• 26 Lea C. H. Low Temperature Research Station. Cambridge 35. HMSO; London: 1962
• 27 Luddy F. E, Barford R. A, Riemenschneider R. W. Direct Conversion of Lipid Components to their Fatty Acid Methyl Esters. J. Amer. Oil Chem. Soc 37: 447 1960;
• 28 Macfarlane R. G, Trevan J. W, Atwood A. M. P. Participation of a Fat Soluble Substance in Coagulation of the Blood. J. Physiol 99: 7 1941;
• 29 Magalhaes J. R, Venancio I. A, Cardoso H. T, Cruz W. O. Electrophoretic Studies of Dog Lymph, Arterial and Venous Plasma Fractions (Cohn Method 10). Haemat. lat 03: 235 1960;
• 30 Magidman P, Herb S. F, Barford R. A, Riemenschneider R. W. Fatty Acids of Cows’ Milk. J. Amer. Oil Chem. Soc 39: 137 1962;
• 31 Marcus A. J, Ullman H. L, Safier L. B, Ballard H. S. Platelet Phos- phatides, Their Fatty Acid and Aldehyde Composition and Activity in Different Clotting Systems. J. clin. Invest 41: 2198 1962;
• 32 Marinetti G. V, Stotz E. Chromatography of Phosphatides on Silicic Acid Impregnated Paper. Biochim.. biophys. Acta (Amst) 21: 168 1956;
• 33 Maurice P, Wallach D. F. H, Surgenor D. The Thromboplastic Properties of Ethanolamine Phosphatides. Vox Sang. (Basel). Vol. 4 91 1959
• 34 Mertz E. T, Owen C. A. Imidazole Buffer: Its use in Blood Clotting Studies. Proc. Soc. exp. Biol 43: 204 1940;
• 35 Mustard J. F, Medway W, Downie H. G, Rowsell H. C. Effects of Intravenous Phospholipid Containing Phosphatidylserine on Blood Clotting with Particular Reference to the Russell’s Viper Venom Time. Nature 196: 1063 1962;
• 36 Papahadjopoulos D, Hougie C, Hanahan D. J. Influence of Surface Charge of Phospholipids on Their Clot-Promoting Activity. Proc. Soc. exp. Biol lll: 412 1962;
• 37 Ponder E, Ponder R. V. Electrophoretic Mobility of Red Cells and Their Ghosts as Observed with an Improved Apparatus. J. exp. Biol 32: 175 1955;
• 38 Poole J. C. F, Robinson D. S. Further Observations on the Effects of Ethanolamine Phosphatide on Plasma Coagulation. Quart. J. Physiol 41: 295 1956;
• 39 Rouser G, Schloredt D. II The Fatty Acid Composition of the Active Phosphatidylethanolamines. Biochim. biophys. Acta (Amst) 28: 81 1958;
• 40 Rouser G, White S. G, Schloredt D. Phospholipid Structure and Thromboplastic Activity. I. The Phosphatide Fraction Active in Recalcified Normal, Human Plasma. Biochim. Biophys. Acta (Amst) 28: 71 1958;
• 41 Rouser G, Bauman A. J, Kritschevski G, Heller D, O’Brien J. S. Quantitative Chromatographic Fractionation of Complex Lipid Mixtures; Brain Lipids. J. Amer. Oil Chem. Soc 38: 544 1961;
• 42 Silver M. J, Rodalewicz I, Tocantins L. M. Effects on Blood Coagulation of Interaction Products of Phosphatidylserine and Plasma Proteins. Fed. Proc 21: 60 1962;
• 43 Silver M. J, Rodalewicz I, Tocantins L. M. Anticoagulant Activity of Phosphatidylserine Solubilized in Albumin Solution and Enhancement of This Activity by Heparin. Thrombos. Diathes. haemorrh. (Stuttg) 07: 204 1962;
• 44 Silver M. J, Turner D. L, Tocantins L. M. Phospholipid Antithrombo: Assay In Vitro and Evaluation in Vivo of Purified Fractions. Amer. J. Physiol 190: 8 1957;
• 45 Silver M. J, Turner D. L, Tocantins L. M. Lipid Anticoagulants. Progr. Hemat. II. 264 1959. Grüne &; Stratton, N.Y.:
• 46 Silver M. J, Turner D. L, Holburn R. R, Tocantins L. M. Anticoagulant Activity of Phosphatidlyserine Free of Lysophosphatidylserine. Proc Soc exp. Biol 100: 692 1959;
• 47 Skipski V. P, Peterson R. F, Barclay M. Separation of Phosphatidyl- ethanolamine, Phosphatidylserine and Other Phospholipids by Thin-layer Chromatography. J. Lipid Res 03: 467 1962;
• 48 Slotta K. H. Thromboplastin. I. Phospholipid Moiety of Thromboplastin. Proc. Soc. exp. Biol 103: 53 1960;
• 49 Surgenor D, Wallach D. F. H. Biophysical Aspects of Platelet Reaction Mechanisms in Clotting. The Blood Platelets, . 289 Little, Brown & Co; Boston: 1961
• 50 Surgenor D. In Progress in Coagulation. Suppl. 1 ad Vol. VII Thrombos Diathes. haemorrh. (Stuttg) 167 1962;
• 51 Therriault D. G. Phosphatidylserine-Bovine Serum Albumin interactions. Fed. Proc 20 (part I) 278 1961;
• 52 Tocantins L. M. The Coagulation of Blood. Methods of Study. 223 Grune &; Stratton, N.Y: 1955
• 53 Trappe W. Modifikation die Jodzahlbestimmungsmethode nach H.P. Kaufmann für kleine und kleinste Fettmengen. Biochem. 2 296: 180 1938;
• 54 Troup S. B, Reed C. F, Marinetti G. V, Swisher S. N. Thromboplastic Factors in Platelets and Red Blood Cells: Observations on their chemical nature and function in in vitro coagulation. J. clin. Invest 39: 342 1960;
• 55 Turner D. L, Holburn R. R, DeSipin M, Silver M. J, Tocantins L. M. Thromboplastic Activity of Phosphatidylethanolamine from Natural and Synthetic Sources. J. Lipid Res 04: 52 1963;
• 56 Turner D. L, Silver M. J. Purification of Lipid Antithromboplastin. Fed. Proc. 15. Abstr. No. 616, March 1956
• 57 Turner D. L, Silver M. J. The total Synthesis of Unsaturated Phosphatidylserine and Its Activity in Clotting Systems. Nature. In Press, 1963
• 58 Turner D. L, Silver M. J, Tocantins L. M. Phospholipid Antithromboplastin. II. Preparation and Properties. Arch. Biochem 77: 249 1958;
• 59 Wallach D. F. H, Maurice P. A, Steele B. B, Surgenor D. M. Studies on the Relationship between the Colloidal State and Clot-Promoting Activity of pure Phosphatidylethanolamines. J. biol. Chem 234: 2829 1959;
• 60 Williams W. J, Esnouf M. P. The Fractionation of Russell’s Viper Venom with Special Reference to the Coagulant Protein. Biochem. J 84: 52 1962;