Subscribe to RSS
DOI: 10.1055/s-0038-1650159
Human Spleen Insoluble Fibrinolytic Proteinase Acting at Neutral pH: Its Partial Purification and Characterization
Publication History
Received 04 July 1980
Accepted after revision 25 February 1981
Publication Date:
05 July 2018 (online)

Summary
An insoluble fibrinolytic enzyme with a molecular weight of approximately 30000, was purified from the human spleen. A single protein band possessing fibrinolytic activity was obtained on polyacrylamide gel disk electrophoresis at pH 4.5.
The enzyme, tentatively termed spleen fibrinolytic proteinase (SFP), degraded fibrinogen at neutral pH following Michaelis-Menten kinetics. This fibrinogenolytic activity was not inhibited by t-AMCHA, a specific plasmin inhibitor. SFP barely degraded certain synthetic ester or polypeptide substrates for trypsin, chymotrypsin, plasmin, Xa, elastase and collagenase. These results indicate a different nature for SFP compared to other enzymes examined. SFP was found to digest no elastin and its fibrinogenolytic activity was strongly inhibited by STI, indicating that it was not an elastase. SFP required neither Zn++ nor Ca+ + for its fibrinogenolytic activity, indicating that it differed from metal-dependent proteinases such as collagenase. SFP was inhibited by DFP but not by TLCK, suggesting that it contains an active serine residue, but no trypsin type histidine at its active center. These results appear to show that SFP is a unique proteinase in the spleen, which is capable of degrading fibrin and fibrinogen at neutral pH.
-
References
- 1 Bleyl U, Kuhn W, Graeff H. Retikuloendotheliale Clearance intravasaler Fibrinmonomere in der Milz. Thromb Diath Haemorrh 1969; 22: 87-100
- 2 Lee L, McCluskey RT. Immunohistochemical demonstration of the reticuloendothelial clearance of circulating fibrin aggregates. J Exp Med 1962; 116: 611-618
- 3 Heyes H, Mohr W, Theiss W. Fibrin deposition and removal in disseminated intravascular coagulation induced by endotoxin and saline loading: Radioautographic findings in rats. Thromb Haemostas 1978; 40: 499-511
- 4 Okamoto U, Yamamoto J, Nagamatsu Y, Horie N. Fibrinolytic activity of lung and spleen extracts observed in conventional but not in germ-free rats. Thromb Haemostas 1979; 42: 726-733
- 5 Nagamatsu Y. A human spleen factor having capability to degrade fibrin. – Partial purification and some properties. J Physiol Soc Jpn 1978; 40: 463-471 (in Japanese with English abstract).
- 6 Schumacher GFB, Schill WB. Radial diffusion in gel for microdetermination of enzymes. II Plasminogen activator, elastase and nonspecific proteases. Anal Biochem 1972; 48: 9-26
- 7 Werb Z, Gordon S. Elastase secretion by stimulated macrophages. Characterization and regulation. J Exp Med 1975; 142: 361-377
- 8 Hesterin S. The reaction of acetylcholine and other carboxylic acid derivatives with hydroxylamine, and its analytical-application. J Biol Chem 1949; 180: 249-261
- 9 Erlanger BF, Kokowsky N, Cohen W. The preparation and properties of two new chromogenic substrates of trypsin. Arch Biochem Biophys 1961; 95: 271-278
- 10 Masui Y, Takemoto T, Sakakibara S, Hori H, Nagai Y. Synthetic substrates for vertebrate collagenase. Biochem Med 1977; 17: 215-221
- 11 KABI Diagnostica. Synthetic chromogenic substrates. Data sheet 1978
- 12 Andrews P. Estimation of the molecular weights of proteins by Sephadex gel-filtration. Biochem J 1963; 91: 222-233
- 13 Reisfeld RA, Lewis UJ, Williams DE. Disc electrophoresis of basic proteins and peptides on polyacrylamide gel. Nature 1962; 195: 281-283
- 14 Lineweaver H, Burk D. Determination of enzyme dissociation constants. J Am Chem Soc 1934; 56: 658-666
- 15 Bieth J, Spiess B, Wermuth CG. The synthesis and analytical use of a highly sensitive and convenient substrate of elastase. Biochem Med 1974; 11: 350-357
- 16 Schaffer NK, May SC, Summerson WH. Serine phosphoric acid from diisopropylphosphoryl chymotrypsin. J Biol Chem 1953; 202: 67-76
- 17 Petra PH, Cohen W, Shaw E. Isolation and characterization of the alkylated histidine from TLCK inhibited trypsin. Biochem Biophys Res Commun 1965; 21: 612-618
- 18 Okamoto S, Sato S, Takada Y, Okamoto U. An active stereoisomer (trans form) of AMCHA and its antifibrinolytic (antiplasminic) action in vitro and in vivo. Keio J Med 1964; 12: 177-185
- 19 Starkey PM, Barrett AJ. Neutral proteinases of human spleen. Purification and criteria for homogeneity of elastase and cathepsin G. Biochem J 1976; 155: 255-263
- 20 Starkey PM, Barrett AJ. Human lysosomal elastase. Catalytic and immunological properties. Biochem J 1976; 155: 265-271
- 21 Starkey PM, Barrett AJ. Human cathepsin G. Catalytic and immunological properties. Biochem J 1976; 155: 273-278
- 22 LoSpalluto JJ, Fehr K, Ziff M. Degradation of immunoglobulins by intracellular proteases in the range of neutral pH. J Immunol 1970; 105: 886-897
- 23 Harris ED, Cartwright EC. Mammalian collagenases. Proteinases in mammalian cells and tissues. North-Holland; Amsterdam: 1977: 249-283
- 24 Lebez D, Kopitar M. Leucocyte proteinases.I. Low molecular weight cathepsins of F and G type. Enzymol 1970; 39: 271-283
- 25 Prose PH, Lee L, Balk SD. Electron microscopic study of the phagocytic fibrin-clearing mechanism. Am J Pathol 1965; 47: 403-417
- 26 Gans H, Lowman JT. The uptake of fibrin and fibrin-degradation products by the isolated perfused rat liver. Blood 1967; 29: 526-539
- 27 Sherman LA, Harwig S, Lee J. In vitro formation and in vivo clearance of fibrinogen: fibrin complexes. J Lab Clin Med 1975; 86: 100-111
- 28 Ahlgren T, Berghem H, Lagergren H, Lahnborg G, Schildt B. Phagocytic and catabolic function of the reticuloendothelial system in dogs subjected to defibrinogenation. Thromb Res 1976; 8: 819-828
- 29 Lahnborg G, Berghem L, Lagergren H, Schildt B. Influence of thrombin induced disseminated intravascular coagulation on RES function in rabbits. Thromb Res 1976; 9: 653-656
- 30 Kunz F, Constantini R, Seminitz E, Mikuz G, Schmalzl F, Holzknecht F. The production of disseminated intravascular coagulation (DIC) by spaced injections of endotoxin in nonpregnant, normolipemic rats. Thromb Res 1977; 12: 119-130