Urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA) are believed to play a role in thrombolysis as well as localized degradation of extracellular matrix proteins. There has been a keen interest in these plasminogen activators as therapeutic agents for dissolving blood clots. TPA, which possesses a strong thrombolytic specificity due to binding and potentiating effects of fibrin on the plasminogen activator, has attracted particular attention for this purpose. Although for many years uPA was believed to lack fibrin in affinity and thrombolytic specificity, recent studies suggest that Pro=urokinase or single-chain urokinase (SC-uPA), unlike the previously studied two-chain urokinase (TC-uPA), is a clot selective thrombolytic agent. These findings have raised hope that SC-uPA may be useful therapeutic agent, perhaps acting synergistically with tPA. Evaluation of full therapeutic potential of SC-uPA has, however, been hampered by the lack of understanding of the underlying molecular basis for the clot selectivity of SC-uPA and of the relative roles of uPA- versus tPA-catalyzed plasminogen activation in thrombolysis. An answer to these questions may emerge from my recent findings showing that Zn2+, a potent inhibitor of plasminogen activation by SC-uPA in the absence of fibrin, mediates a specific binding interaction between the plasminogen activator and fibrin, and potentiates activation of plasminogen in the presence of fibrin. In contrast, Zn2+ induces only rudimentary inhibition, binding and potentiation reactions with TC-uPA. Based on these preliminary findings, a hypothesis is proposed that Zn2+ plays a regulatory role in uPA- mediated thrombolysis by localizing SC-uPA on the clot and inducing fibrin-specific conversion of plasminogen to plasmin. The localized SC-uPA subsequently undergoes proteolytic conversion to TC-uPA which, relieved of both the inhibitory effect of ZN2+ and dependence on fibrin for plasminogen activation, promotes localized degradation of non-fibrin components of the clot. In order to elucidate the proposed role of Zn2+, research will focus on obtaining a detailed understanding of the mechanism by which the metal ion elicits its diverse effects on SC-uPA and on evaluating the differences in the thrombolytic action of SC-uPA with those of tPA. Towards this end, interaction of Zn2+ with SC-uPA will be investigated, effect of the metal ion on the conformation of the plasminogen activator will be examined, and the structural domains of SC-uPA and fibrin involved in their Zn2+-mediated interaction will be delineated. Effect of SC-uPA and tPA on clots with different amounts of fibrin and platelets will be investigated, local concentration of Zn2+ in these clots will be examined and the proteolytic products from the clots will be characterized. These investigations may help to elucidate the molecular mechanism involved in the action of Zn2+ on SC-uPA, providing a new understanding of the role of SC-uPA in thrombolysis and helping to design more effective thrombolytic agents than are presently possible.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL038178-02
Application #
3354251
Study Section
Biochemistry Study Section (BIO)
Project Start
1988-01-01
Project End
1990-12-31
Budget Start
1989-01-01
Budget End
1989-12-31
Support Year
2
Fiscal Year
1989
Total Cost
Indirect Cost
Name
Massachusetts General Hospital
Department
Type
DUNS #
City
Boston
State
MA
Country
United States
Zip Code
02199