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 1

TSP-1 has distinct complement regulatory roles compared with TSP-5, protects sheep erythrocytes from complement-mediated hemolysis independent of FH, and binds to central complement proteins of the alternative pathway.

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TSP-1 has distinct complement regulatory roles compared with TSP-5, prot...
(A) TSP-1 inhibits alternative complement pathway activation in contrast to TSP-5. The alternative complement pathway in NHS was activated on LPS-coated wells and with increasing concentrations of FH, eculizumab, TSP-1, or TSP-5. Platelet-derived TSP-1 (p-TSP-1) was used as an additional control to exclude artifacts caused by the histidine tag used for purification. (B) TSP-1 protects sheep erythrocytes from alternative complement pathway–mediated lysis in the absence of FH. Sheep erythrocytes were incubated with FH-depleted serum and increasing concentrations of FH, TSP-1, or TSP-5. Data are shown as mean ± SD. The alternative complement pathway and hemolytic activity were normalized against untreated control samples. Data were fitted using nonlinear regression. (C) TSP-1 binds to central proteins of the alternative pathway. Complement proteins FH, FB, C3, C5, C6, C7, C8, C9, or BSA were coated on microtiter plates and incubated with recombinant TSP-1. Bound TSP-1 was determined using specific antibodies. (D) Surface plasmon resonance (SPR) Biacore measurements demonstrating the binding of TSP-1 to key proteins of the alternative complement pathway. TSP-1 was immobilized on CM5 chips at a concentration of 0.1 μM. Binding interactions with complement proteins FH, FB, C3, C3b, C5, and C8 were assessed at various concentrations (12.3, 37.03, 111.1, 333.3, 1,000 nM). The binding data were fitted using a 1:1 Langmuir binding model to determine on and off rates, which were then used to calculate affinity constants (Kd). The graph depicts a summary of the binding of complement proteins to TSP-1 at increasing concentrations. Average Kd values, calculated from 3 repeated measurements, are presented in the accompanying table. Data are shown as mean ± SD of 3 independent experiments. ND, not detected.