Identification of myxomaviral serpin reactive site loop sequences that regulate innate immune responses.
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.
Dai
Erbin
E
Vascular Biology Research Group, Robarts Research Laboratory, London, Ontario N6A 2K8, Canada.
Viswanathan
Kasinath
K
Sun
Yun Ming
YM
Li
Xing
X
Liu
Li Ying
LY
Togonu-Bickersteth
Babajide
B
Richardson
Jakob
J
Macaulay
Colin
C
Nash
Piers
P
Turner
Peter
P
Nazarian
Steven H
SH
Moyer
Richard
R
McFadden
Grant
G
Lucas
Alexandra R
AR
D000818:Animals; D001011:Aorta / Q000378:metabolism / Q000637:transplantation; D001483:Base Sequence; D001665:Binding Sites; D001777:Blood Coagulation; D016466:CHO Cells; D002448:Cell Adhesion; D002465:Cell Movement; D002469:Cell Separation; D006224:Cricetinae; D004195:Disease Models, Animal; D005434:Flow Cytometry; D007107:Immune System; D007249:Inflammation; D007700:Kinetics; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D015233:Models, Statistical; D008969:Molecular Sequence Data; D009233:Myxoma virus / Q000378:metabolism*; D010537:Peritoneum / Q000166:cytology / Q000378:metabolism; D017395:Plasminogen Activator Inhibitor 1 / Q000378:metabolism; D015539:Platelet Activation; D011485:Protein Binding; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D015843:Serpins / Q000737:chemistry*; D013927:Thrombosis; D013997:Time Factors; D014914:Whole Blood Coagulation Time
D017395:Plasminogen Activator Inhibitor 1; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D015843:Serpins