Comprehensive DIA-MS profiling of clot and plasma proteomes across immune- and coagulation-deficient mouse models

Introduction Immune checkpoint inhibitors (ICIs) targeting PD-1/PD-L1 have transformed cancer therapy but are associated with increased thromboembolic risk. Emerging evidence suggests that ICI-induced thrombosis arises through immune-mediated vascular inflammation, complement activation, and platelet hyperreactivity, rather than direct effects on coagulation factors. However, most mechanistic studies rely on short-term ex vivo assays, leaving the chronic systemic consequences of checkpoint loss poorly understood. To address this, we determine how checkpoint deficiency (PD-1–/–, B7H1–/–) alters plasma composition, clot proteome, and platelet function in vivo, and to distinguish these effects from global adaptive immune deficiency (Rag2–/–) and congenital hypocoagulability (HemA). Method We profiled plasma and clot proteomes from PD-1–/–, B7H1–/–, Rag2–/–, HemA, and wild-type mice using data-independent acquisition mass spectrometry (DIA-MS). Whole blood was analyzed for hematologic parameters (Sysmex), and platelet adhesion under arterial shear was quantified in BioFlux microfluidic chambers. Results Checkpoint loss (PD-1–/–, B7H1–/–) induced systemic complement and inflammatory activation in plasma (C3, C5, acute-phase proteins ↑1.5–3.0 fold) and enrichment of extracellular vesicle proteins. Clot proteomes were enriched in platelet α-granule proteins (P-selectin, PF4), adhesion molecules (vWF, fibronectin), and antifibrinolytic SERPINs, consistent with fibrin-dense, platelet-rich thrombi. The data from DIA-MS approach can be accessed in the PRIDE repository under the accession code: PXD068451. Conclusion The immune-regulatory roles affect coagulation pathways, via abundance of adhesion molecules, Serpins, fibrinolytic indicators such as Fibrinogen, TF, Haptoglobin and spectrins. The present dataset serves as a valuable resource to facilitate further analyses of the orchestration of coagulation-innate immunity components. Major clot constituents that quantitatively affect the unique clot structure include: anti-thrombin, calcium binders, hemoglobin modifiers, platelet adhesion, and complement factors. With significant quantitative differences in a) Pro-inflammatory cytokine regulators; b) Complement-related cytokine modulators; c) TGF-β and immune signaling and d) Cytokine stress regulators, e) Endothelial and immune response proteins, in clot proteome governing the clot fate. Publication History Article published online: 17 February 2026 © 2026. Thieme. All rights reserved. Georg Thieme Verlag KG Oswald-Hesse-Straße 50, 70469 Stuttgart, Germany