Dual modulation of prothrombin activation by the cyclopentapeptide plactin

Abstract

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 α‐thrombin. Plactin inhibited the activation catalyzed by Xa in complex with Va, Ca2+ 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.

Small‐molecule modulators of zymogen activation in the fibrinolytic and coagulation systems

“…The plasma dependency is characterized in detail by Harada et al. [86], because plasminogen alone cannot substitute for plasma, and the presence of a cofactor for the plactin action is suggested. On cultured U937 cells, most of the u‐PA molecules exist in the zymogen form, scu‐PA.…”

“…Using plactin‐affinity gels, Harada et al. [86] identified prothrombin as a candidate for a cofactor from plasma. The actions of plactin and prothrombin that lead to the activation of scu‐PA are explained as follows: (a) plactin binds to prothrombin, alters its conformational status and therefore modulates prothrombin activation by factor Xa; (b) the consequence of the modulation under the conditions of the assay for cellular fibrinolytic activity is the promotion of prothrombin activation; (c) the resulting thrombin can cleave scu‐PA at Arg156–Phe157 (two residues proximal to the activation cleavage site, Lys158–Ile159) to form an inactive two‐chain u‐PA species; and (d) the tcu‐PA derivative, in turn, is processed to active tcu‐PA by dipeptidyl peptidase I‐like activity of U937 cells, possibly through the removal of Phe157–Lys158 from the newly formed N‐terminus (Fig.…”

“…The plactin‐modulation of prothrombin activation leads to different outcomes depending on the form of the catalyst factor Xa [86]. Plactin inhibits prothrombin activation by factor Xa associated with acidic phospholipid membranes, especially by the prothrombinase complex, which accounts for the physiological coagulation reaction.…”

Fibrinolytic Activation Promoted by the Cyclopentapeptide Malformin: Involvement of Cytoskeletal Reorganization

“…Plactin leads to cell-surface single-chain u-PA activation by promoting the activation of these zymogens. 20,21) However, malformin action does not involve the activation of u-PA. 4) Our results clearly demonstrate that malformin A 1 utilizes a distinct mechanism to promote fibrinolytic activity with vitronectin as a plasma cofactor. It is likely that vitronectin acts through binding to cell-surface integrins since the RGD peptide, a competitor of vitronectinintegrin interaction, abolishes the vitronectin-dependent malformin action (Fig.…”

“…4) This property of malformin A 1 is similar to that of plactin, another cyclic pentapeptide that enhances cellular fibrinolytic activity with the aid of a plasma cofactor. 3,19,20) Prothrombin and a proenzyme form of plasma hyaluronan-binding protein (factor VII-activating protease) have been identified as independent plactin cofactors in plasma. Plactin leads to cell-surface single-chain u-PA activation by promoting the activation of these zymogens.…”