Antibodies with Dual Reactivity to Plasminogen and Complementary PR3 in PR3-ANCA Vasculitis

Abstract

Patients with inflammatory vascular disease caused by anti-neutrophil cytoplasmic autoantibodies (ANCA) can harbor antibodies not only to the autoantigen proteinase 3 (PR3) but also to complementary PR3 (cPR3 ), a recombinant protein translated from the antisense strand of PR3 cDNA. The purpose of this study was to identify potential endogenous targets of anti-cPR3 antibodies. Patients' plasmapheresis material was tested for the presence of antigens reactive with affinity-purified rabbit and chicken anti-cPR3105-201 polyclonal antibodies. Antigen-containing fractions were tested with patients' anti-cPR3105-201 affinity-purified IgG, and putative protein targets were sequenced by mass spectrometry. Unexpectedly, plasminogen was identified as a target of anti-cPR3 . Reactivity of affinitypurified antibodies from two patients was lost when plasminogen was converted to plasmin, indicating restricted specificity. Antiplasminogen antibodies from five patients bound plasminogen at a surfaceexposed loop structure within the protease domain. This loop contains an amino acid motif that is also found in a portion of recombinant cPR3 ; site-directed mutagenesis of this sequence decreased antibody reactivity by 30%. Functionally, antiplasminogen antibodies delayed the conversion of plasminogen to plasmin and increased the dissolution time of fibrin clots. Serologically, antiplasminogen antibody levels were higher in PR3-ANCA patients (n ϭ 72) than healthy control subjects (n ϭ 63), myeloperoxidase-ANCA patients (n ϭ 34), and patients with idiopathic thrombosis (n ϭ 57; P ϭ 0.001). Of the patients with PR3-ANCA, nine had documented deep venous thrombosis events, five of whom were positive for antiplasminogen antibodies. In summary, capitalizing on interactions with complementary proteins, specifically complementary PR3, this study identified plasminogen as a previously undescribed autoantigen in PR3-ANCA vasculitis.

Anti-Plasminogen Antibodies Compromise Fibrinolysis and Associate with Renal Histology in ANCA-Associated Vasculitis

“…The previously proposed core epitope for anti-plasminogen antibodies isolated from PR3-ANCA patients is a PHxQ motif, whereas alignment of plasminogen and tPA shows that of these three residues; only glutamic acid (Q) is conserved. 3 How might the observations in this study be reconciled with previous findings in the North American AAV cohort? If the development of anti-plasminogen autoantibodies is driven by exposure to exogenous antigens such as microbial proteins, divergent immunologic memory in patients from distinct geographic areas could influence autoantibody development.…”

“…Anti-plasminogen antibodies isolated from PR3-ANCA vasculitis patients in a North American cohort seem to recognize an epitope within the protease domain, but again, these antibodies did not bind to plasmin or thrombin for reasons that are unclear. 3 The detection of anti-tPA antibodies in AAV is intriguing. tPA is a serine protease that exhibits an overall 35% shared identity with plasminogen.…”

“…1,2 Recently, it was reported that a subset of patients with PR3-ANCA harbor antibodies against human plasminogen. 3 These anti-plasminogen antibodies recognize a structure within the protease domain, and their presence correlated with venous thromboembolic events (VTEs), suggesting functional interference with coagulation and/or fibrinolysis. 3 The relationship between anti-plasminogen antibodies and coagulation may also be directly relevant to glomerular injury and the evolution of fibrinoid necrosis, a hallmark lesion of AAV.…”

“…3 These anti-plasminogen antibodies recognize a structure within the protease domain, and their presence correlated with venous thromboembolic events (VTEs), suggesting functional interference with coagulation and/or fibrinolysis. 3 The relationship between anti-plasminogen antibodies and coagulation may also be directly relevant to glomerular injury and the evolution of fibrinoid necrosis, a hallmark lesion of AAV. In AAV, vascular injury is mediated by ANCA-induced neutrophil and monocyte activation 4 and characterized by the influx of inflammatory cells with fibrinoid necrosis affecting glomeruli and occasionally small arteries.…”

ANCA Disease

“…Moreover, this theory of autoantigen complementarity led to the discovery that protein complementary to the middle portion of PR3 is the endogenous protein plasminogen. 60 Antibodies to plasminogen are detected in patients with ANCA disease, inhibit fibrinolysis, and associate with increased risk for thrombosis.…”