Plactin D

Plactin D, a cyclic pentapeptide [cyclo(-D-Val-L-Leu-D-Leu-L-Phe-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 microM 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.

Plactin, a family of cyclopentapeptides, enhances fibrinolytic activity by elevating the activity of cellular urokinase-type plasminogen activator (u-PA), a protease involved in a variety of extracellular proteolytic events. Factor(s) in the blood plasma is an absolute requirement for this plactin activity. In this study, we found that plactin promoted plasma cofactor-dependent conversion of inactive single-chain u-PA to active two-chain u-PA on U937 cells. Using plactin-affinity chromatography, we identified prothrombin as one of the plasma cofactors. In incubations of U937 cells with prothrombin and Xa, plactin increased the formation of thrombin, which cleaved single-chain u-PA to afford the inactive two-chain form. Thrombin-cleaved two-chain u-PA was alternatively activated by cellular cystatin-sensitive peptidase activity, yielding fully active two-chain u-PA. In a purified system, plactin bound to prothrombin, altered its conformation and dually modulated factor Xa-mediated proteolytic activation of prothrombin to alpha-thrombin. Plactin inhibited the activation catalyzed by Xa in complex with Va, Ca(2+) and phospholipids (prothrombinase), whereas the activations catalyzed by nonmembrane-associated Xa were enhanced markedly by plactin. Plactin inhibited in vitro plasma coagulation, which involved prothrombinase formation. Plactin did not cause prothrombin activation or thrombosis in normal mice at doses that produced a protective effect in a thrombin-induced pulmonary embolism mouse model. Therefore, the dual modulation of prothrombin activation by plactin may be interpreted as leading to anticoagulation under physiological coagulating conditions.

Plactin, a family of cyclopentapeptides of fungal origin, enhances fibrinolytic activity by promoting of single-chain urokinase-type plasminogen activator (scu-PA) activation on the cell surface. For this activity, factor(s) in the blood plasma is absolutely required. In the previous studies, we identified prothrombin as a plasma cofactor involved in this mechanism, while the presence of another independent cofactor was suggested. The objective of this study was to identify the second cofactor and investigate the mechanism involved. Using plactin-affinity and ion-exchange chromatographies, we purified plasma hyaluronan-binding protein (PHBP) ~4,000-fold from human plasma as an independent plactin cofactor. PHBP, at ~10nM, was effective in plactin-dependent promotion of scu-PA activation by U937 cells. PHBP is a serine protease that is produced as a single-chain proenzyme (pro-PHBP) and autoproteolytically converted to an active two-chain form. Pro-PHBP was comparable to PHBP in activity to promote plactin-dependent scu-PA activation by U937 cells. Plactin enhanced both cellular binding and autoproteolytic activation of pro-PHBP. The two activities were obtained with a plactin concentration at ~30μM, which resulted in a significant increase in intrinsic fluorescence and self association of pro-PHBP. Thus, it is suggested that such changes account for both enhanced cellular binding and autoactivation of pro-PHBP, resulting in an enhancement of scu-PA activation.