Fibrin Specific Thrombolysis by Two-Chain Urokinase-Type Plasminogen Activator Cleaved after Arginine 156 by Thrombin

Abercrombie, David M.; Buchinski, B.; Salvato, Kathleen A.; Vovis, Gerald F.; Stump, David C.; Broeze, Robert J.

doi:10.1055/s-0038-1647331

Cited by 12 publications

(6 citation statements)

References 12 publications

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“…α‐Thrombin can specifically cleave human scu‐PA at Arg156–Phe157 [7], two residues proximal to the activation cleavage site (Lys158–Ile159). Thrombin‐cleaved tcu‐PA (tcu‐PA/T), however, showed < 1% activity of tcu‐PA (consistent with previous reports) [7,19,21]. Plactin D failed to activate tcu‐PA/T (data not shown).…”

Section: Resultssupporting

confidence: 87%

Dual modulation of prothrombin activation by the cyclopentapeptide plactin

Harada¹,

Tsuruta²,

Yamagata

et al. 2009

The FEBS Journal

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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.

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Section: Resultssupporting

confidence: 87%

Dual modulation of prothrombin activation by the cyclopentapeptide plactin

Harada¹,

Tsuruta²,

Yamagata

et al. 2009

The FEBS Journal

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“…In vitro, tcu-PA/T appears to have little amidolytic or fibrinolytic activity (11,17). How ever, it has been reported that tcu-PA/T is a potent and fibrin-specific plasminogen activator in vivo (18,19), which implies the involvement of specific factors that are able to re-activate tcu-PA/T. Liu and Gurewich have suggested that the plasminogen activating capacity of tcu-PA/T might be ascribed to fibrin fragment E-2, which can be generated in vivo via plasmin formation by endogenous tissue-type plasminogen activator (17).…”

Section: Introductionmentioning

confidence: 99%

A Sensitive Bioimmunoassay for Thrombin-cleaved Two-chain Urokinase-type Plasminogen Activator in Human Body Fluids

Braat¹,

Nauland²,

Dooijewaard³

et al. 1996

Thromb Haemost

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SummaryThrombin cleaves single-chain urokinase-type plasminogen activator (scu-PA) into a two-chain form (tcu-PA/T), which is virtually inactive in plasminogen activator assays. Little is known about the physiological importance of tcu-PA/T. To examine the occurrence of tcu-PA/T in vivo, we developed a sensitive and specific bioimmunoassay (BIA) for the assessment of tcu-PA/T in human body fluids. In this BIA, urokinase antigen was immuno-immobilized in microtiter plates and treated with cathepsin C, a specific activator of tcu-PA/T, after which plasminogen activator activity was measured. The occurrence of tcu-PA/T was examined in the plasma of 27 healthy individuals and of 17 sepsis patients, and in the synovial fluid of 16 rheumatoid arthritis patients. In addition, the concentration of urokinase antigen and scu-PA were measured in all three groups. In the plasma of the healthy individuals no measurable amounts of tcu-PA/T could be found (< detection limit of 0.2 ng/ml). In the plasma of almost all sepsis patients tcu-PA/T could be detected (median value 0.4 ng/ml). The amount of tcu-PA/T was 12% of the amount of scu-PA and accounted for about 9% of urokinase antigen. In the synovial fluid of all rheumatoid arthritis patients tcu-PA/T could be measured (median value 5.4 ng/ml) at a concentration which was twofold higher than the concentration found for scu-PA. In this group tcu-PA/T contributed to about 47% of the urokinase antigen. From these data we conclude that inactivation of scu-PA by thrombin can take place in vivo under pathological conditions which involve the production of large amounts of thrombin. This way thrombin may regulate fibrinolysis and extracellular proteolysis. The BIA for tcu-PA/T can be of use for further research on the physiological role of tcu-PA/T.

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“…This study demonstrates that, in addition to the chondroitin sulfate moiety, only EGF-like domains 5 and 6 are essential for the acceleration of the inactivation of scu-PA by thrombin. This differs from the domains that are critical for activation of protein C (EGF-like domains i4 -6) and thrombin activatable fibrinolysis inhibitor (EGF-like domains [3][4][5][6].…”

mentioning

confidence: 95%

“…In vitro, tcu-PA/T appears to have little amidolytic and fibrinolytic activity [2,3]. However, in a rabbit jugular vein thrombosis model, tcu-PA/T was found to be a potent and fibrin-specific thrombolytic agent [4,5], which may be explained by re-activation of tcu-PA/T by dipeptidyl peptidase I (cathepsin C) and by platelets [6,7], or by the promoting effect of fibrin fragment E-2 on plasminogen activation by tcu-PA/T [8]. The inactivation of scu-PA has been postulated as a mechanism for protecting a fresh blood clot from early fibrinolysis [3,9].…”

mentioning

confidence: 99%

Identification of the epidermal growth factor-like domains of thrombomodulin essential for the acceleration of thrombin-mediated inactivation of single-chain urokinase-type plasminogen activator

Schenk-Braat¹,

Morser²,

Rijken³

2001

Eur J Biochem

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Single-chain urokinase-type plasminogen activator (scu-PA) can be cleaved by thrombin into a virtually inactive form called thrombin-cleaved two-chain urokinase-type plasminogen activator (tcu-PA/T), a process accelerated by thrombomodulin, which contains six epidermal growth factor (EGF)-like domains. In this study, we identified the EGFlike domains of thrombomodulin required for the acceleration of the inactivation of scu-PA by thrombin using various forms of thrombomodulin (TM). scu-PA was treated with thrombin in the absence and presence of full-length rabbit TM (containing EGF1-6), recombinant TM comprising all of the extracellular domains including EGF1-6 (TM LEO ) and recombinant TM comprising EGF4-6 plus the interconnecting region between EGF3 and EGF4 (TM E i4-6), and the tcu-PA/T generated was quantitated in each case. Rabbit TM accelerated the inactivation of scu-PA < 35-fold, while both recombinant forms accelerated it only threefold due to the absence of a critical chondroitin sulfate moiety. Subsequently, TM E 5-6 was prepared by cyanogen bromide digestion of TM E i4-6. TM E 5-6 bound to thrombin but did not accelerate the activation of protein C. In contrast, the inactivation of scu-PA by thrombin was accelerated to the same extent as that induced by TM LEO and TM E i4-6. This study demonstrates that, in addition to the chondroitin sulfate moiety, only EGF-like domains 5 and 6 are essential for the acceleration of the inactivation of scu-PA by thrombin. This differs from the domains that are critical for activation of protein C (EGF-like domains i4 -6) and thrombin activatable fibrinolysis inhibitor (EGF-like domains 3-6).Keywords: single-chain urokinase-type plasminogen activator; thrombin; inactivation; thrombomodulin; epidermal growth factor-like domains.Single-chain urokinase-type plasminogen activator (scu-PA) is the precursor form of two-chain urokinase-type plasminogen activator (tcu-PA), which plays a role in fibrinolysis and extracellular proteolysis by converting plasminogen into plasmin [1]. Activation of scu-PA into tcu-PA occurs after cleavage at Lys158 -Ile159 by plasmin or other activators. Cleavage of scu-PA at another site can result in inactivation. Thrombin cleaves scu-PA at Arg156 -Phe157, two residues prior to the activation site, in this way generating an inactivated tcu-PA form called thrombincleaved tcu-PA (tcu-PA/T) [2]. In vitro, tcu-PA/T appears to have little amidolytic and fibrinolytic activity [2,3]. However, in a rabbit jugular vein thrombosis model, tcu-PA/T was found to be a potent and fibrin-specific thrombolytic agent [4,5], which may be explained by re-activation of tcu-PA/T by dipeptidyl peptidase I (cathepsin C) and by platelets [6,7], or by the promoting effect of fibrin fragment E-2 on plasminogen activation by tcu-PA/T [8]. The inactivation of scu-PA has been postulated as a mechanism for protecting a fresh blood clot from early fibrinolysis [3,9]. The inactivation does indeed take place in vivo, as we have demonstrated the presence of tcu-PA/T in human bod...

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Fibrin Specific Thrombolysis by Two-Chain Urokinase-Type Plasminogen Activator Cleaved after Arginine 156 by Thrombin

Cited by 12 publications

References 12 publications

Dual modulation of prothrombin activation by the cyclopentapeptide plactin

Dual modulation of prothrombin activation by the cyclopentapeptide plactin

A Sensitive Bioimmunoassay for Thrombin-cleaved Two-chain Urokinase-type Plasminogen Activator in Human Body Fluids

Identification of the epidermal growth factor-like domains of thrombomodulin essential for the acceleration of thrombin-mediated inactivation of single-chain urokinase-type plasminogen activator

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