Denatured albumin is a potent ligand mediating platelet activaiton, adhesion and aggregation Free

Introduction: Serum albumin is the most abundant plasma protein, comprising approximately 50-60% of total plasma proteins. While albumin is known for regulating oncotic pressure and transporting various molecules, it is traditionally considered inert in platelet functions, with no established role in platelet-mediated hemostasis or thrombosis. Although previous studies have reported platelet adhesion to albumin-coated surfaces, such findings could be attributed to albumin impurities, albumin-fibrinogen interactions, or surface property alteration by the albumin coating, and thus were largely disregarded by the research field. Consequently, the potential platelet-albumin interactions have been considered unlikely and remained underexplored.

Methods: To investigate the specific platelet-albumin interaction, we engineered surfaces functionalized with molecular force sensors tethering albumin molecules at a surface density around 1000/µm². These force sensors emit fluorescent signals upon experiencing piconewton-level molecular forces, providing ultra-sensitive detection of platelet-albumin interaction and force transmission. This setup suspends albumin above the substrate, preserving its native structure and eliminating albumin surface adsorption. Using this system, we monitored platelet-albumin interaction, in accompany with pharmacological interventions and immunostaining to assess receptor and signaling pathways involved in platelet-albumin interaction.

Results: We confirmed that native albumin tethered to force sensors exhibited minimal fluorescence under platelets, indicating negligible platelet-albumin interaction under physiological conditions. However, when albumin was pre-denatured via heat or chemical reduction, it activated resting platelets and supported robust platelet adhesion and spreading, accompanied by strong fluorescence signals indicating force transmission from platelets to tethered albumin. These signals displayed a consistent spatial pattern: ring-like fluorescence at the platelet periphery and plaque-like signals in the central region. Inhibition of integrin α_IIbβ₃ (glycoprotein IIb/IIIa) using tirofiban or the 7E3 monoclonal antibody abolished these force signals, identifying α_IIbβ₃ as the key receptor mediating platelet binding to denatured albumin. Immunostaining further revealed that the force transmission pattern co-localized with talin and vinculin, critical components of integrin-mediated mechanotransduction. Notably, nucleated human cells (e.g., HeLa cells) did not adhere to or transmit forces on denatured albumin, suggesting that the cell-albumin interaction is specific to platelets. Furthermore, denatured albumin was also shown to be incorporated into platelet aggregates in in vitro tests, indicating a potential role in platelet aggregation.

Conclusion: Using molecular engineering and platelet force imaging, we demonstrate that denatured albumin acts as a functional ligand for integrin α_IIbβ₃, promoting platelet activation, adhesion, and aggregation. This interaction is mechanosensitive and specific to platelets.

Discussion: Given the high concentration of albumin in blood, even albumin denaturation at a low level, potentially induced by oxidative stress, alcohol consumption, or pathological conditions such as diabetes, hepatitis, or sepsis, may have significant, previously unrecognized effects on platelet function and thrombotic risk. Our findings identify denatured albumin as a novel and strong regulator of platelet activity, opening new avenues in the understanding of thrombosis and hemostasis and therapeutic targeting in the prevention of thrombosis.