Cellular deep proteomic profiling elucidates NLRP3 inflammasome and immunothrombosis effects of GLP-1 receptor agonist: a controlled intervention study

Background GLP-1 receptor agonists confer pleiotropic vascular protection via mechanisms other than glycemic and body weight control. Comprehensive proteomic evaluations of its immunothrombosis modulatory and vascular effects remained limited. Purpose To investigate the immunothrombosis modulatory effects of GLP-1 receptor agonist, particular NLRP3 inflammasome, through deep proteome sequencing of immune cells. Methods We obtained paired peripheral blood samples under controlled experimental settings of an obese adult newly diagnosed with type 2 diabetes mellitus (age 34 years, male, hbA1c 13.1%), naïve to and after treatment with semaglutide (0.25mg/week, subcutaneous, for 2 weeks) as the only intervention. Peripheral blood mononuclear cells (PBMC) were isolated. Samples collected from a sex-and-age-matched control subject without diabetes and who did not receive semaglutide was included in the same run (additional technical duplicate, n=2). Protein digestion was performed using standard mini kit digestion according to manufacturer’s instructions. Untargeted cellular proteomics analyses of PBMC were performed using liquid chromatography-mass spectrometry (LC-MS/MS) analysis with data-independent acquisition (DIA) approach, with NLRP3 inflammasome-related protein expression as the pre-specified primary endpoint. Results A total of 7753 proteins were identified from LC-MS/MS. Apoptosis-associated speck-like protein containing a CARD (ASC), a key component of NLRP3 inflammasome, was found elevated in diabetes compared to control (Log2FC=0.46974, P=0.0071), but significantly decreased after semaglutide therapy (Log2 FC=-0.58849, P=0.0039) and became no different to control (Log2FC= -0.11875, P=0.09, reversed direction). Similarly, capase-1 significantly reduced after semaglutide (Log2 FC=-0.30797, P=0.0093). There were also significant reductions in interleukin-1 receptor-associated kinase 4 (P=0.016), adenylyl cyclase-associated protein 1 (P=0.013), plasminogen activator inhibitor 1 (P=0.0014), prothrombin (P=0.0099), coagulation factors V (P=0.0011) and XIII A chain (P=0.00034), as well as platelet glycoprotein Ib alpha chain (P=0.00056). Significant reductions were also found in P-selectin (P=0.0026), endothelial cell-selective adhesion molecule (P=0.0034), thrombospondin-1 (P=0.000556), and the platelet-derived growth factors D (P=0.018), C (P=0.026), subunit A (P=0.0153) and subunit B (P=0.0041). Increased level was noted in expression level of Annexin A2 (P=0.032) after semaglutide therapy. Conclusions Comprehensive protein profiling of PBMCs revealed proteome landscape changes fundamentally relevant to immunothrombosis pathways including NLRP3 inflammasome, after treatment with semaglutide. These give mechanistic insights into the pleiotropic vascular protective effects of a GLP-1 receptor agonist.