Abstract Fri149: Acylated Toll-Like Receptor Agonists Enhance Platelet Procoagulant Activity in a CD36-Dependent Manner

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Background: Platelets express pattern recognition receptors (PRRs), including Toll-like receptors (TLRs) and the scavenger receptor CD36, linking innate immunity, inflammation, and thrombosis. Although pathogen- and damage-associated molecular patterns (PAMPs, DAMPs), as well as microbial ligands, are known to activate platelet immune signaling pathways, the mechanisms by which CD36 regulates TLR-driven platelet procoagulant activity remain unknown. Aim: Here, we tested the hypothesis that CD36 functions as a co-stimulatory receptor, amplifying platelet procoagulant responses downstream of multiple TLR-mediated pathways, including TLR2 and TLR4. Methods: Washed platelets isolated from healthy human donors (n=4) were pretreated with the CD36 inhibitor Fx-5A or vehicle and stimulated with TLR2/6 and TLR2/1 agonists (acylated lipoproteins Pam2CSK4 and Pam3CSK4), TLR4 agonist lipopolysaccharides (LPS) from distinct bacterial serotypes, classical agonists targeting glycoprotein VI (GPVI) and protease-activated receptor 4 (PAR4), or controls. Platelet procoagulant activity was assessed by flow cytometry and microscopy analysis of platelet phosphatidylserine (PS) exposure and platelet-neutrophil aggregation, as well as fibrin generation. Results: Acylated TLR2 agonists induced a robust platelet procoagulant phenotype, increasing PS exposure 10-fold compared with co-stimulation using GPVI and PAR4 agonist peptides: collagen-related peptide (CRP-XL) and AYPGKF-NH2, respectively. TLR2 activation significantly enhanced fibrin generation and platelet-neutrophil interactions. Fx-5A pretreatment reduced Pam2CSK4-induced PS exposure (lactadherin-FITC and annexin V-AlexaFluor488 fluorescence) threefold and delayed fibrin generation (P<0.05), without affecting PAR4-mediated responses. CD36 inhibition selectively attenuated TLR2-driven platelet-neutrophil aggregation. In addition, LPS stimulation promoted fibrin generation in plasma, which was delayed by CD36 inhibition in a serotype-dependent manner, indicating ligand-specific cooperation between CD36 and TLR4 signaling. Conclusion: Acylated TLR2 agonists and LPS elicit potent platelet procoagulant responses relative to classical hemostatic agonists. CD36 selectively amplifies platelet procoagulant signaling downstream of both TLR2 and TLR4, identifying CD36 as a convergent regulator of platelet-driven innate immune responses and a potential antithrombotic therapy target in systemic inflammation.

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