Proteomic study of extracellular vesicles reveals novel biomarkers for pathogenesis of immune thrombotic thrombocytopenic purpura

Abstract

Abstract Background: Immune thrombotic thrombocytopenic purpura (iTTP) is a potentially fatal blood disorder, resulting from autoantibodies against a plasma metalloprotease ADAMTS13. However, the exact pathogenic mechanism remains elusive. There is no specific biomarker that predicts disease relapse and adverse outcomes. Extracellular vesicles (EVs), membrane-enclosed particles released by cells into the extracellular environment, may serve as excellent biomarkers for disease severity and may also play a crucial role in disease pathogenesis by transporting biomolecules, such as DNA, RNA, proteins, and lipids, to other cells. Here, we present the first comprehensive examination of the number, size, and contents of small EVs isolated from plasma of patients with acute iTTP and those in the healthy controls. Methods: Plasma-derived small EVs were isolated by ,differential centrifugation followed by size exclusion chromatography. The number and sizes of EVs were determined by Nanoparticle tracking analysis and transmission electron microscopy (TEM). The protein contents of small EVs were determined by unbiased proteomics using a Q Executive HF hybrid quadrupole-Orbitrap benchtop mass spectrometer that combines ultra-high field Orbitrap mass analyzer. Results: NanoSight and TEM revealed that the number of small EVs (e.g., particle size from 50 nm to 200 nm) in acute iTTP was significantly higher than that in the healthy controls. Western blotting analysis demonstrated that the isolated small EVs were positive for CD9 and Flotillin 1, but negative for GM130. Proteomic results showed that compared with the healthy controls, the small EVs from acute iTTP patients exhibited a clear signature of sterile inflammation, with 5-10-fold increased expression of S100A8/A9 (calprotectin), a damage-associated molecular pattern (DAMP). Also, a striking coordinated decrease of complement components, including C3, C4A, C4B, C5, and C9 (50-70% reduction), as well as C1-inhibitor, was observed. The small EVs from acute iTTP also surprisingly showed a decrease in several platelet-derived adhesion molecules, including ITGB3 (integrin 3 subunit) (16-fold lower. Similarly, the expression of GP1BB (platelet glycoprotein 1b) was also down by ~12-fold, suggesting that platelet contents in these small EVs are reduced during acute iTTP, which is consistent with the disease of platelet activation and consumption. Moreover, the cargo of small EVs in acute iTTP also suggested altered immune signaling pathways with decreased CD14 (a co-receptor for TLR4 on monocytes and an increased IL-6 receptor subunit gp130 (IL6ST), IL-1 receptor antagonist (IL1RN), and leukocyte immunoglobulin-like receptors (LILRB1). Given widespread microvascular thromboses, the hallmark of iTTP, it is not surprising that markers of endothelial cell interaction and vascular stress were perturbed as demonstrated by the downregulation of surface glycoproteins, like ICAM-1 and ICAM-2 on small EVs (2-3-fold lower). ICAM-3, and PECAM-1 were 4 and 5-fold lower. The small EVs in acute iTTP also exhibited changes in proteins related to cellular metabolism and oxidative stress management. Among those were glucose-6-phosphate dehydrogenase (G6PD), a key enzyme that generates NADPH for reductive stress responses, glutathione peroxidase (GPX1), a critical antioxidant enzyme that reduces hydrogen peroxide, and glutathione peroxidase 3 and 4, and superoxide dismutases (SOD1/2), suggestive of a broad reduction in oxidative stress defenses in acute iTTP. Conclusions: Our results demonstrate for the first time the enriched inflammatory DAMPs and the depletion of complement components and regulatory proteins, consistent with sterile inflammation and complement consumption in the small EVs from acute iTTP. Additionally, our results reveal that platelet adhesion machinery is shifted out of the small EVs with altered immune signaling and resulted endothelial dysfunction and perturbed metabolic/oxidative balance, and loss of protective intercellular communications in acute iTTP. Together, our findings suggest that small EVs in acute iTTP are not bystanders, but potential active participants in the disease's vicious cycle.