Conservation of Critical Functional Domains in Murine Plasminogen Activator Inhibitor-1

Victoria A Ploplis,Rashna D Balsara,Zhi Xu,Francis J Castellino,Daniel A Lawrence,Natalia V Gorlatova

doi:10.1074/jbc.m314197200

Abstract

Plasminogen activator inhibitor-1 is the main physiological regulator of tissue-type plasminogen activator in normal plasma. In addition to its critical function in fibrinolysis, plasminogen activator inhibitor-1 has been implicated in roles in other physiological and pathophysiological processes. To investigate structure-function aspects of mouse plasminogen activator inhibitor-1, the recombinant protein was expressed in Escherichia coli and purified. Five variant recombinant murine proteins (R76E, Q123K, R346A, R101A, and Q123K/R101A) were also generated using site-directed mutagenesis. The variant (R346A) was found to be defective in its inhibitory activity against tissue plasminogen activator relative to its wild-type counterpart. Enzyme-linked immunosorbent assay and surface plasmon resonance experiments demonstrated reduced vitronectin-binding affinity of the (Q123K) variant (K(D) = 1800 nm) relative to the wild-type protein (K(D) = 5.4 nm). Kinetic analyses indicated that the (Q123K) variant had a slower association (k(on) = 2.92 x 10(4) m(-1) s(-1)) to, and a faster dissociation from, vitronectin (k(off) = 5.3 x 10(-2) s(-1)), (wild-type k(on) = 1.03 x 10(6) m(-1) s(-1) and k(off) = 5.27 x 10(-3) s(-1)). The Q123K/R101A variant demonstrated an even lower vitronectin-binding ability. Low density lipoprotein receptor-related protein binding was decreased for the (R76E) variant. It was also demonstrated that the plasminogen activator inhibitor-1/vitronectin complex decreased the interaction of plasminogen activator inhibitor-1 with low density lipoprotein receptor-related protein. These results indicate that the complex interactions traditionally associated with different plasminogen activator inhibitor-1 functions apply to the murine system, thus showing a commonality of subtle functions among different species and evolutionary conservation of this protein. Further, this study provides additional evidence that the human hemostasis system can be studied effectively in the mouse, which is a great asset for investigations with gene-altered mice.

Highlights

Plasminogen activator inhibitor-1 (PAI-1)1 is a member of the serine protease inhibitor (SERPIN) superfamily and is the Previous in vitro studies have elaborated structure/function relationships of human PAI-1, resulting in the delineation of functional domains that play a significant role in regulating its pathophysiology
VN Binding by ELISA—The VN-binding capacity of the Q123K, R101A, and Q123K/R101A variants of PAI-1 were compared with the WT protein and the other two PAI-1 variants (R76E and R346A) by ELISA. (Q123K)-PAI-1 demonstrated impaired binding capacity to VN relative to the WT protein and the other two variants (Fig. 3), which is similar to the human protein studies
Unlike the common reversible “lock and key” model between inhibitors and serine proteases, the inhibition of PAI-1 toward its protease substrates has been described as a suicidal process following a branched pathway mechanism, with the majority of products being kinetically trapped as a covalently linked complex [16]

Summary

Introduction

Plasminogen activator inhibitor-1 (PAI-1) is a member of the serine protease inhibitor (SERPIN) superfamily and is the Previous in vitro studies have elaborated structure/function relationships of human PAI-1, resulting in the delineation of functional domains that play a significant role in regulating its pathophysiology. An R 3 E mutation at amino acid position 76 of PAI-1 within this domain results in a protein with preserved inhibitor activity but diminished affinity for LRP. These targeted mutations of human PAI-1 indicate that functional changes at sites other than those targeted are minor and, are ideal for delineating specific functional domains in select biological processes involving PAI-1. One practical value of this work is to evaluate the commonality of these subtle functions of PAI-1 across species, with special emphasis on the murine system, which is so widely employed for in vivo studies of PAI-1 The results of this investigation are described

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Conclusion