Treatment of Tissue Thromboplastin Membranes with Phospholipase C

E Bjørklid; A.-B Otnæss; E Storm; H Prydz; B. V Johansen; L. O Frøholm

doi:10.1055/s-0038-1649129

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 1973; 30(03): 509-518
DOI: 10.1055/s-0038-1649129

Original Article

Schattauer GmbH

Treatment of Tissue Thromboplastin Membranes with Phospholipase C

E Bjørklid

¹Institute of Medical Biology, University of Tromsø, Tromsø, Norway

²Methodology Department, National Institute of Public Health, Oslo, Norivay

,

A.-B Otnæss

¹Institute of Medical Biology, University of Tromsø, Tromsø, Norway

²Methodology Department, National Institute of Public Health, Oslo, Norivay

,

E Storm

¹Institute of Medical Biology, University of Tromsø, Tromsø, Norway

²Methodology Department, National Institute of Public Health, Oslo, Norivay

,

H Prydz

¹Institute of Medical Biology, University of Tromsø, Tromsø, Norway

²Methodology Department, National Institute of Public Health, Oslo, Norivay

,

B. V Johansen

¹Institute of Medical Biology, University of Tromsø, Tromsø, Norway

²Methodology Department, National Institute of Public Health, Oslo, Norivay

,

L. O Frøholm

¹Institute of Medical Biology, University of Tromsø, Tromsø, Norway

²Methodology Department, National Institute of Public Health, Oslo, Norivay

› Author Affiliations

Abstract

Summary

Tissue thromboplastin from human brain, partially purified by extraction with deoxycholate, gel chromatography and recombination of the protein (fraction A) and phospholipid (fraction B) fractions, was examined after treatment with phospholipase C (E.C. 3.1.4.3). Various morphological changes accompanied the loss in coagulation activity caused by the enzyme. All concentrically arranged vesicles (spherulites) disappeared. Instead, a large number of quite small vesicles and many big “blebs”, probably containing diglycerides, were seen. Fused membranes appeared after treatment with the enzyme. The phospholipid fraction (fraction B) showed similar structures as thromboplastin, but not quite the same morphological changes after enzyme treatment.

Phospholipase C treatment probably caused a splitting of the concentrically arranged tissue thromboplastin membranes, which spontaneously rearranged to form new vesicles or were fused to other membranes. The hydrophilic parts of the phospholipids are required for coagulation activity, either because they impart a certain ultrastructure to the membrane, or because they participate in the coagulation process in a more direct way, e.g. by forming complexes with factor VII or by binding factor VII through calcium bridges.

Full Text

References

References
1 Burstein C, Loyter A, and Racker E. 1971; Effect of phospholipases on the structure and function of mitochondria. Journal of Biological Chemistry 246: 4075.
2 Chen Jr. P. S, Toribara T. Y, and Warner H. 1956; Microdetermination of phosphorous. Analytical Chemistry 28: 1756.
3 Clarke N, and O’Meara A. Q. 1966; Intracellular location of thromboplastic activity in the cells of human chorion. British Journal of Haematology 12: 536.
4 Eriksson S, and Sjøvall J. 1955; The absorption spectra of bile acids in sulfuric acid. Arkiv Kemi 8: 303.
5 Finean J. B, and Martenosi A. 1965; Action of phospholipase C on muscle microsomes – A correlation of electron microscope and biochemical data. Biochimica et Biophysica acta 98: 547.
6 Gordon A. S, Wallach D. F. H, and Strauss J. H. 1969; Optical activity of plasma membranes and its modification by lysolecithin, phospholipase A and phospholipase C. Biochimica et Biophysica acta 183: 405.
7 Hvatum M, Hovig T, and Prydz H. 1969; Studies on tissue thromboplastin – electron micrography. Thrombosis et Diathesis Haemorrhagica 21: 223.
8 Hvatum M, and Prydz H. 1966; Studies on tissue thromboplastin. I. Solubilization with sodium deoxycholate. Biochimica et Biophysic acta 130: 92.
9 Hvatum M, and Prydz H. 1969; Studies on tissue thromboplastin, its splitting into two separable parts. Thrombosis et Diathesis Haemorrhagica 21: 217.
10 Kurtz S. M. 1961; A new method for embedding tissues in Vestopal. Journal of TJltrastructure Research 5: 468.
11 Lenard J, and Singer S. J. 1968; Structure of membranes. Reaction of red blood cell membranes with phospholipase C. Science 159: 738.
12 Lowry O. H, Rosebrough 1st. J, Farr A. L, and Randall R. J. 1951; Protein measurements with the folin phenol reagent. Journal of Biological Chemistry 193: 265.
13 Luft J. H. 1961; Improvements in epoxy resin embedding methods. Journal of Biophysical and Biochemical Cytology 9: 409.
14 McIlwain D. L, and Rapport M. M. 1971; The effects of phospholipase C (Clostridium perfringens) on purified myelin. Biochimica et Biophysica acta 239: 71.
15 Østerud B, Berre Å, Otnæss A.-B, Bjørklid E, and Prydz H. 1972; The activation of the coagulation factor VII by tissue thromboplastin and calcium. Biochemistry 11: 2853.
16 Pitlick F. A, Nemerson Y, Gottlieb A. J, Gordon R. G, and Williams W. J. 1971; Peptidase activity associated with the tissue factor of blood coagulation. Biochemistry 10: 2650.
17 Ray T. K, Das J, and Marinetti G. V. 1972; Alteration of the rat liver plasma membrane by phospholipase C and sodium chloride. Chemistry and Physics of Lipids 9: 35-50.
18 Reynolds E. S. 1963; The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. Journal of Cell Biology 17: 208.
19 Simpkins H, Panko E, and Tay S. 1971; Structural changes in the phospholipid regions of the axonal membrane produced by phospholipase C action. Biochemistry 10: 3851.
20 Sjøvall J. 1955; Quantitative determination of bile acids on paper chromatograms. Bile acids and steroids. Arkiv Kemi 8: 317.