Thrombospondin-1 inhibits alternative complement pathway activation in antineutrophil cytoplasmic antibody-associated vasculitis
Swagata Konwar, Sophie Schroda, Manuel Rogg, Jessika Kleindienst, Eva L. Decker, Martin Pohl, Barbara Zieger, Jens Panse, Hong Wang, Robert Grosse, Christoph Schell, Sabine Vidal, Xiaobo Liu, Christian Gorzelanny, Todor Tschongov, Karsten Häffner
Swagata Konwar, Sophie Schroda, Manuel Rogg, Jessika Kleindienst, Eva L. Decker, Martin Pohl, Barbara Zieger, Jens Panse, Hong Wang, Robert Grosse, Christoph Schell, Sabine Vidal, Xiaobo Liu, Christian Gorzelanny, Todor Tschongov, Karsten Häffner
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Research Article Immunology Inflammation Vascular biology

Thrombospondin-1 inhibits alternative complement pathway activation in antineutrophil cytoplasmic antibody-associated vasculitis

Abstract

Complement activation is a relevant driver in the pathomechanisms of vasculitis. The involved proteins in the interaction between endothelia, complement, and platelets in these conditions are only partially understood. Thrombospondin-1 (TSP-1), found in platelet α-granules and released from activated endothelial cells, interacts with factor H (FH) and vWF. However, to our knowledge, direct regulatory interaction with the complement cascade has not yet been described. Our study shows that TSP-1 is a potent, FH-independent inhibitor of the alternative complement pathway. TSP-1 binds to complement proteins and inhibits cleavage of C3 and C5 and the formation of the membrane attack complex. We validated complement-regulatory function in blood samples from patients with primary complement defects. The physiological relevance of TSP-1 was demonstrated in patients with antineutrophil cytoplasmic antibody-associated vasculitis (AAV) by significantly enhanced TSP-1 staining in glomerular lesions and increased complement activity and NETosis after TSP-1 deficiency in an in vitro and in vivo model of AAV. The complement-inhibiting function of TSP-1 represents an important mechanism in the interaction of endothelia and complement. In particular, the interplay between released TSP-1 and the complement system locally, especially on surfaces, influences the balance between complement activation and inhibition and may be relevant in various vascular diseases.

Authors

Swagata Konwar, Sophie Schroda, Manuel Rogg, Jessika Kleindienst, Eva L. Decker, Martin Pohl, Barbara Zieger, Jens Panse, Hong Wang, Robert Grosse, Christoph Schell, Sabine Vidal, Xiaobo Liu, Christian Gorzelanny, Todor Tschongov, Karsten Häffner

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Figure 2

TSP-1 modulates complement at the C3 and C5 level of the complement cascade.

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TSP-1 modulates complement at the C3 and C5 level of the complement casc...
(A) TSP-1 protects FB from cleavage by FD. FB was incubated with FD, C3b, and varying concentrations of TSP-1. Subsequently, FB and its cleavage products were visualized by Coomassie blue staining. Graph on the right illustrates band intensity of FB and its cleavage products. (B) TSP-1 inhibits cleavage of C3 by the alternative complement pathway C3 convertase. C3 convertase was generated by incubating C3(H2O) with FB and FD, followed by the addition of C3 in the presence or absence of TSP-1 or FH. As a positive control, CVF C3 convertase (CVF + FB + FD) was utilized. The graph on the right depicts the band intensity of C3-α′ chain. Lanes were run on the same gel but were noncontiguous. Data are shown as mean ± SD. (C) TSP-1 inhibits cleavage of C5. Cobra venom factor (CVF) convertase (CVFBb) was generated and added to C5 preincubated with FH, eculizumab, MFHR1, or TSP-1. The amount of released C5a was quantified by ELISA. Data are shown as mean ± SD. ANOVA was used with Dunnett’s multiple-comparison test. (D) TSP-1 inhibits the formation of MAC. Sheep erythrocytes were premixed with C7 (9 nM), C8 (7 nM), and C9 (15 nM). BSA, eculizumab, MFHR1, or TSP-1 (1.3 μM each) were preincubated with C5b-6 (0.7 nM) and then added to the erythrocytes. Data are shown as mean ± SD of 3 independent experiments; **P ≤ 0.01, ***P ≤ 0.01; 1-way ANOVA was used with Tukey’s post hoc test for comparison against control.