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J. Biol. Chem., Vol. 281, Issue 12, 8041-8050, March 24, 2006

This Article Full Text Full Text (PDF) All Versions of this Article: 281/12/8041    most recent M509454200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Dai, E. Articles by Lucas, A. R. Search for Related Content PubMed PubMed Citation Articles by Dai, E. Articles by Lucas, A. R. Social Bookmarking                      What's this?

Identification of Myxomaviral Serpin Reactive Site Loop Sequences That Regulate Innate Immune Responses^*

Erbin Dai, Kasinath Viswanathan, Yun Ming Sun, Xing Li^¶, Li Ying Liu, Babajide Togonu-Bickersteth, Jakob Richardson, Colin Macaulay^¶, Piers Nash, Peter Turner^||, Steven H. Nazarian, Richard Moyer^||, Grant McFadden, and Alexandra R. Lucas¹

From the Vascular Biology Research Group, Robarts Research Laboratory, London, Ontario N6A 2K8, Canada, the Department of Microbiology and Immunology, University of Western Ontario, London, Ontario N6A 2K8, Canada, ^¶Research and Development, Viron Therapeutics, Inc., London, Ontario N6G 4X8, Canada, and the ^||Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, Florida 32610-0266

The thrombolytic serine protease cascade is intricately involved in activation of innate immune responses. The urokinase-type plasminogen activator and receptor form complexes that aid inflammatory cell invasion at sites of arterial injury. Plasminogen activator inhibitor-1 is a mammalian serpin that binds and regulates the urokinase receptor complex. Serp-1, a myxomaviral serpin, also targets the urokinase receptor, displaying profound anti-inflammatory and anti-atherogenic activity in a wide range of animal models. Serp-1 reactive center site mutations, mimicking known mammalian and viral serpins, were constructed in order to define sequences responsible for regulation of inflammation. Thrombosis, inflammation, and plaque growth were assessed after treatment with Serp-1, Serp-1 chimeras, plasminogen activator inhibitor-1, or unrelated viral serpins in plasminogen activator inhibitor or urokinase receptor-deficient mouse aortic transplants. Altering the P1-P1' Arg-Asn sequence compromised Serp-1 protease-inhibitory activity and anti-inflammatory activity in animal models; P1-P1' Ala-Ala mutants were inactive, P1 Met increased remodeling, and P1' Thr increased thrombosis. Substitution of Serp-1 P2–P7 with Ala₆ allowed for inhibition of urokinase but lost plasmin inhibition, unexpectedly inducing a diametrically opposed, proinflammatory response with mononuclear cell activation, thrombosis, and aneurysm formation (p < 0.03). Other serpins did not reproduce Serp-1 activity; plasminogen activator inhibitor-1 increased thrombosis (p < 0.0001), and unrelated viral serpin, CrmA, increased inflammation. Deficiency of urokinase receptor in mouse transplants blocked Serp-1 and chimera activity, in some cases increasing inflammation. In summary, 1) Serp-1 anti-inflammatory activity is highly dependent upon the reactive center loop sequence, and 2) plasmin inhibition is central to anti-inflammatory activity.

Received for publication, August 26, 2005 , and in revised form, October 27, 2005.

^* This work was supported by the Heart and Stroke Foundation of Ontario, Canada Grant T5605 and the Canadian Institutes of Health Research Grant MOP 77578. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Robarts Research Institute, Vascular Biology Research Group, University of Western Ontario, 100 Perth Dr., P.O. Box 5015, London, Ontario N6A 2K8, Canada. Tel.: 519-685-8500 (ext. 34071); Fax: 519-663-3446; E-mail: arl{at}robarts.ca.

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