Enhancement of Fibrinolysis by Plactins: Structure-activity Relationship and Effects in Human U937 Cells and in Mice

Summary

Plactin D, a cyclic pentapeptide [ cyclo (-D-Val-L-Leu-D-Leu-LPhe-D-Arg-)] produced by a fungal strain, enhances fibrinolytic activity (6). The present study deals with the structure-activity relationship of plactins and their effects in U937 cells and mice. The results obtained from 50 plactin D analogues with a single amino acid substitution demonstrated that the following substitutions were detrimental: the enantiomer for each of the five residues; a polar, an acidic or a basic residue for D-Val, L-Leu, D-Leu or L-Phe; a polar, a hydrophobic or an acidic residue for D-Arg. On the other hand, a compound with L-Leu or L-Val in place of L-Phe was seven times as active as plactin D. These results suggest an essential role of a sterically restricted arrangement of four hydrophobic residues and the adjacent basic residue. The enhancement of fibrinolysis was dependent on plasma, ranging from 2- to 3-fold when U937 cells were incubated with 15-30 μM plactin D in the presence of 6-50% plasma, while no elevation was observed when cells were incubated in the absence of plasma. Plasminogen alone could not substitute for plasma. The plactin D effect was totally abolished by anti-urokinase IgG but not by anti-tissue plasminogen activator IgG. Plactin D caused a plasma-dependent, transient increase in the cellular urokinase activity. This urokinase activation may have accounted for the increased fibrinolytic activity of plactin D-treated U937 cells. Homogenates of the lung obtained from mice 0.5 to 2 h after intravenous plactin D (5 mg/kg) showed 2- to 3-fold increased levels of fibrinolytic activity, while activities of the brain, heart, liver, spleen, kidney and aorta were not significantly affected. In conclusion, plactin D enhances fibrinolysis both in cultured mammalian cells and in experimental animals.

• References

• 1 Carmeliet P, Collen D. Role of the plasminogen/plasmin system in thrombosis, hemostasis, restenosis and atherosclerosis. Evaluation in transgenic animals. Trends Cardiovasc Med Chem 1995; 5: 117-22.
• 2 Hamsten A, Eriksson P. Fibrinolysis and atherosclerosis: an update. Fibrinolysis 1994; 8 (Suppl. 01) 253-62.
• 3 Schneiderman J, Loskutoff DJ. Plasminogen activator inhibitors. Trends Cardiovasc Med 1991; 1: 99-102.
• 4 Plow EF, Herren T, Redlitz A, Miles LA, Hoover-Plow JL. The cell biology of the plasminogen system. FASEB J 1995; 9: 939-45.
• 5 Hajjar KA. Cellular receptors in the regulation of plasmin generation. Thromb Haemost 1995; 74: 294-301.
• 6 Inoue T, Hasumi K, Kuniyasu T, Endo A. Isolation of plactins A, B, C and D, novel cyclic pentapeptides that stimulate cellular fibrinolytic activity. J Antiobiot (Tokyo) 1996; 49: 45-9.
• 7 Milton RCD, Becker E, Milton SCF, Baxter JEJ, Elsworth JF. Improved purities for Fmoc-amino acids from Fmoc-ONSu. Int J Peptide Protein Res 1987; 30: 431-2.
• 8 Goldstein JL, Basu SK, Brown MS. Receptor-mediated endocytosis of low density lipoprotein in cultured cells. Methods Enzymol 1983; 98: 241-60.
• 9 Nokihara K, Yokomizo Y. Applications of new linker for Fmoc chemistry. SynProPep Bulletin 1993; No. 10 (Shimadzu Corp).
• 10 Nobuhara M, Sakamaki M, Ohnishi H, Suzuki Y. A comparative study of high molecular weight urokinase and low molecular weight urokinase. J Biochem (Tokyo) 1981; 90: 225-32.
• 11 Wolf BB, Brown MD. Epidermal growth factor-binding protein activates soluble and receptor-bound single chain urokinase-type plasminogen activator. FEBS Lett 1995; 376: 177-80.
• 12 Stoppelli MP, Tacchetti C, Cubelis MV, Corti A, Hearing VJ, Cassani G, Appella E, Blasi F. Autocrine saturation of pro-urokinase receptors on human A431 cells. Cell 1986; 45: 675-84.
• 13 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680-5.
• 14 Hart PH, Vitti GF, Burgess DR, Whitty GA, Royston K, Hamilton JA. Activation of human monocytes by granulocyte-macrophage colony-stimulating factor: Increased urokinase-type plasminogen activator activity. Blood 1991; 77: 841-8.
• 15 Miskin R, Abramovitz R. One-phase reverse zymography after denaturing gel electrophoresis: high sensitivity detection of activity of plasminogen activator inhibitor 2 and other protease inhibitors. Fibrinolysis 1995; 9: 331-42.
• 16 Cajot J-F, Kruithof EKO, Schleuning W-D, Sordat B, Bachmann F. Plasminogen activators, plasminogen activator inhibitors and procoagulant analyzed in twenty human tumor cell lines. Int J Cancer 1986; 38: 719-27.
• 17 Stoppelli MP, Verde P, Grimaldi G, Locatelli EK, Blasi F. Increase in urokinase plasminogen activator mRNA synthesis in human carcinoma cells is a primary effect of the potent tumor promoter, phorbol myristate. J Cell Biol 1986; 102: 1235-41.
• 18 Miwa K, Yamada C, Kono T, Osada H. Retinoic acid enhances plasminogen activation on the cell surface. Thromb Res 1995; 80: 47-56.
• 19 Krätzschmar J, Haendler B, Kojima S, Rifkin DB, Schleuning W-D. Bovine urokinase-type plasminogen activator and its receptor: cloning and induction by retinoic acid. Gene 1993; 125: 177-83.
• 20 Keski-Oja J, Blasi F, Leof EB, Moses HL. Regulation of the synthesis activity of urokinase plasminogen activator in A549 human lung carcinoma cells by transforming growth factor-β. J Cell Biol 1988; 106: 451-9.
• 21 Marshall BC, Xu Q-P, Rao NV, Brown BR, Hoidal JR. Pulmonary epithelial cell urokinse-type plasminogen activator. Induction by interleukin-1β and tumor necrosis factor-α. J Biol Chem 1992; 267: 11462-9.
• 22 Pepper MS, Matsumoto K, Nakamura T, Orci L, Montesano R. Hepatocyte growth factor increases urokinase-type plasminogen activator (u-PA) and u-PA receptor expression in Madin-Darby canine kidney epithelial cells. J Biol Chem 1992; 267: 20493-6.
• 23 Hamilton JA, Piccoli DS, Leizer T, Butler DM, Croartto M, Royston KM. Transforming growth factor β stimulates urokinase-type plasminogen activator and DNA synthesis, but not prostaglandin E₂ production, in human synovial fibroblasts. Proc Natl Acad Sci USA 1991; 88: 7180-4.
• 24 Stephens RW, Pöllänen J, Tapiovaara H, Leung K-C, Sim P-S, Salonen E-M, Rønne E, Behrendt N, Danø K, Vaheri A. Activation of pro-urokinase and plasminogen on human sarcoma cells: a proteolytic system with surface-bound reactants. J Cell Biol 1989; 108: 1987-95.