Increased Platelet S100A8/S100A9 Associated with Vasculitis in Granulomatosis with Polyangiitis (GPA)
Authors: Guo Li , Berger Ben , Rowley Jesse W , Tolley Neal D , Manne Bhanu Kanth , Su Juan , Shen Sikui , Crane Paul D. , Queisser Kimberly A , Petrey Aaron C , Koening Curry L. , Rondina Matthew T. 
Published: November 2021

Abstract
Granulomatosis with polyangiitis (GPA), formerly known as Wegener's Granulomatosis, is characterized by vasculitis that predominantly affects small- and medium-sized blood vessels in the sinuses, lungs, and kidneys. In addition to vascular inflammation, GPA is also characterized by an increased risk of thrombosis. The role of platelets in GPA pathogenesis remains incompletely understood. We aimed to better understand the changes in platelet gene expression and function in patients with GPA. Forty-two patients diagnosed with GPA (n=9 with active GPA and n=33 with GPA in remission) and 25 healthy, age-, gender-, and race-matched donors were enrolled. Patients with GPA showed typical disease manifestations, with an average Birmingham Vasculitis Activity Score of 1.6 (Mean±SD 1.6±3.5). One sixth of GPA patients (7/42) had a history of thrombosis. When stimulated with thrombin receptor activating peptide (TRAP, 50nM), platelets from patients with GPA showed significantly increased expression of P-selectin as compared to healthy controls (P-selectin+% Mean±SEM: Healthy 15.50±1.84 vs GPA 25.71±16.05, P&lt;0.05). This suggests increased platelet activation in GPA, consistent with previous findings of increased platelet aggregation in vitro in GPA. In addition, released chemokines sCD40L and platelet-derived growth factor (PDGF) by activated platelets were increased in patients with GPA when we measured the cytokines in the platelet poor plasma using the Miliplex human cytokine Assay [sCD40L (ng/mL) Mean±SEM: Healthy 63.05±6.63 vs GPA 100.40±11.86, P&lt;0.05, PDGF-AA (pg/mL) Mean±SEM: Healthy 137.50±46.52 vs GPA 357.30±79.65, P=0.052]. Next, we performed RNA-sequencing on platelets from GPA patients (n=8, 3 with active GPA disease and 5 in remission) and, for comparison, 4 healthy donors. We identified 75 genes that were significantly differentially expressed between GPA patients and healthy donors. The top 30 genes are listed in Figure 1A. S100A8 and S100A9 were the top two significantly differentially expressed transcripts in patients with GPA (Fig. 1B). These two genes encode proteins that form a heterodimer S100A8/S100A9 (commonly known as calprotectin) known to be increased in the plasma of GPA patients and associated with disease activity. Interestingly, platelets have not been identified as the cellular source of plasma calprotectin in GPA previously. Significantly increased RNA and protein expression of S100A8 and S100A9 in GPA patients was independently validated by qRT-PCR and immunoblot, respectively. The mRNA expression of S100A8 and S100A9 in platelets were significantly correlated with p-ANCA and anti-MPO antibodies, indicating platelet S100A8/S100A9 promotes neutrophil activation and inflammation (Mann-Whitney nonparametric test, P&lt;0.05). As previously reported, plasma levels of calprotectin were also increased in GPA patients. To further evaluate if plateletS100A8/S100A9 mediates endothelial inflammation and vasculitis, we co-cultured platelets activated with thrombin (which increases S100A8/S100A9 secretion) with endothelial cells in the presence or absence of an anti-S100A8/S100A9 blocking antibody. Activated platelets triggered endothelial cell inflammation (e.g., increased expression of ICAM-1) that was significantly reduced when S100A8/S100A9 was blocked. In summary, the platelet transcriptome is altered in patients with GPA, with S100A8 and S100A9 being the top upregulated genes. Platelet functional responses are enhanced in patients with GPA, and our data suggests that increased plasma calprotectin levels in GPA patients may be platelet derived. Platelets and platelet S100A8/S100A9 appear to mediate vascular inflammation and thrombosis in GPA.
Figure 1 Figure 1.

Disclosures
Rondina: Novartis: Research Funding; Platelet Biogenesis: Membership on an entity's Board of Directors or advisory committees; Acticor Biotech: Membership on an entity's Board of Directors or advisory committees; Platelet Transcriptomics: Patents & Royalties.