The activation of plasminogen to plasmin by urokinase type plasminogen activator (uPA) is a critical step in a number of vascular processes leading resolution of fibrin thrombi, remodeling of vascular tissue matrix following injury, and sprouting and formation of new blood vessels. The initiative event of the uPA/plasmin cascade, the conversion of pro-uPA zymogen to active uPA is the limiting the therefore controlling reaction in the cascade. Once generated, plasmin can feed back and activate pro-uPA but the actual initiating event is undefined. A candidate for the initiating event is the intrinsic activity of pro-uPA itself which has been shown to be conditionally enhanced. A candidate for the initiating event is the intrinsic activity of pro-uPA itself which has been shown to be conditionally enhanced. Auto-activation is a cascade initiator until now has not been considered but will be in the present proposal. Direct control of the uPA/plasmin cascade would appear to reside in the action of the cognate serpins, PAI-1 and PAI-2. How there serpins act biochemically is known, but when and where they act physiologically is undefined. We have recently constructed a unique PAI-resistant uPA by engineering on a surface loop of the uPA molecule a replacement of the serpin binding motif, RRHR. This mutant and selected cells expressing it can be utilized to probe for where and when PAI-I acts to control the uPA/plasmin cascade. The uPA/plasmin cascade also can enhance indirectly the remodeling of tissue matrix by activating other protease cascades, mainly the matrix metalloprotease (MMP) system. We recently have shown that the uPA/plasmin cascade can converge with the activation mechanism of proMMP-9, not directly but through the intermediate action of MMP-3. Whether such cascade convergence can operate in a vascular milieu will be addressed experimentally in the present proposal.
The specific aims of the present proposal are: 1. To identify the Structural Motifs and Sub-sites on pro-uPA that Regulate its Auto-activation Potential and Intrinsic Activity. 2. To examine the Regulatory Role of PAI-I by Utilizing a Mutant Home uPA, huPA/QNIM, Missing the RRHR Serpin Binding Motif. 3. To examine the Functional Link between the uPA/Plasmin Cascade and Activation of Select Matrix Metalloproteases (MMPs).
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