Abstract: SA-PO592
Revealing Complement Regulatory Functions of Thrombospondin-1 as New Potential Mechanism in Renal Disease
Session Information
- Pediatric Nephrology - II
November 05, 2022 | Location: Exhibit Hall, Orange County Convention Center‚ West Building
Abstract Time: 10:00 AM - 12:00 PM
Category: Pediatric Nephrology
- 1800 Pediatric Nephrology
Authors
- Konwar, Swagata, Universitatsklinikum Freiburg, Freiburg, Baden-Württemberg, Germany
- Kleindienst, Jessika, Universitatsklinikum Freiburg, Freiburg, Baden-Württemberg, Germany
- Decker, Eva L., Albert-Ludwigs-Universitat Freiburg, Freiburg im Breisgau, Baden-Württemberg, Germany
- Pohl, Martin, Universitatsklinikum Freiburg, Freiburg, Baden-Württemberg, Germany
- Gorzelanny, Christian, Universitatsklinikum Hamburg-Eppendorf, Hamburg, Hamburg, Germany
- Tschongov, Todor Alexandrov, Universitatsklinikum Freiburg, Freiburg, Baden-Württemberg, Germany
- Häffner, Karsten, Universitatsklinikum Freiburg, Freiburg, Baden-Württemberg, Germany
Group or Team Name
- AG Pediatric Nephrology
Background
Overactivation of the complement system can lead to severe kidney and vascular diseases like atypical hemolytic uremic syndrome (aHUS) and ANCA-associated vasculitis. In recent years it is becoming clearer that coagulation, thrombosis and extracellular matrix are deeply connected with the complement system.
Thrombospondin-1 (TSP-1) is a major compound of α-granules in platelets and also present in Weibel-Palade bodies of endothelial cells. TSP-1 is rapidly released after injury and involved in the regulation of clot formation and platelet aggregation. Binding of complement factor H (FH) to TSP-1 has been reported before, however, no research has examined possible intrinsic TSP-1 complement regulation.
Methods
Alternative pathway ELISA, Hemolysis, cofactor, decay acceleration and terminal complement complex (TCC) formation assay were performed as described previously (Michelfelder et. al, JASN, 2018).
Factor D inhibition assay was performed as described previously (Edwards et. al, J Biol Chem, 1999)
Binding assays: TSP-1 was coated on 96 well plates and incubated with increasing amounts of C3 or C5. Binding was analysed using HRP-coupled antibodies.
Cell culture: HUVEC were transfected with TSP-1 siRNA using lipofectamine as instructed. Immune fluorescence staining was performed with specific antibodies against TSP-1 and C3.
Results
TSP-1 is able to strongly inhibit the alternative pathway (AP) in normal human and aHUS patient serum. The inhibition is only partly dependent on FH. TSP-1 binds to central complement proteins of the alternative pathway. It has no intrinsic cofactor or decay acceleration activity, but inhibits cleavage of FB and C3. Additionally, TSP-1 prevents formation of TCC. Knockdown of TSP-1 in endothelial cells leads to an increase in C3 deposition on HUVEC. This increase can be ameliorated by adding recombinant TSP-1 back into the system.
Conclusion
In several in vitro experiments we have found that TSP-1 directly inhibits the activation of the AP in multiple ways. We believe that TSP-1 can act as a bridge between platelets, extracellular matrix, coagulation and complement system and thereby contribute to pathomechanisms of renal diseases. This knowledge could lead to the development of new treatment strategies for complement mediated diseases in the future.
Funding
- Commercial Support –