S100A8/A9 drives the formation of procoagulant platelets through GPIbα

Abstract

S100A8/A9, also known as calprotectin or MRP8/14, is an alarmin primarily secreted by activated myeloid cells and platelets with anti-microbial, pro-inflammatory and pro-thrombotic properties. Increased plasma levels of S100A8/A9 in thrombo-inflammatory diseases are associated with thrombotic complications. We assessed the presence of S100A8/A9 in the plasma and lung autopsies from patients with COVID-19 and investigated the molecular mechanism by which S100A8/A9 affects platelet function and thrombosis. S100A8/A9 plasma levels were increased in patients with COVID-19 and sustained high levels during hospitalization correlated with poor outcomes. Heterodimeric S100A8/A9 was mainly detected in neutrophils and deposited on the vessel wall in COVID-19 lung autopsies. Immobilization of S100A8/A9 with collagen accelerated the formation of a fibrin-rich network following perfusion of recalcified blood at venous shear. In vitro, platelets adhered and partially spread on S100A8/A9 leading to the formation of distinct populations of either P-selectin or phosphatidylserine-positive platelets. Using washed platelets, soluble S100A8/A9 induced phosphatidylserine exposure but failed to induce platelet aggregation, despite GPIIb/IIIa activation and alpha-granule secretion. We identified GPIbα as the receptor for S100A8/A9 on platelets inducing the formation of procoagulant platelets with a supporting role for CD36. The effect of S100A8/A9 on platelets was abolished by recombinant GPIbα ectodomain, platelets from Bernard-Soulier Syndrome patient with GPIb-IX-V deficiency and platelets from mice deficient in the extracellular domain of GPIbα. In conclusion, we identified the S100A8/A9-GPIbα interaction as a novel targetable prothrombotic pathway inducing procoagulant platelets and fibrin formation, in particular in diseases associated with high levels of S100A8/A9, such as COVID-19.

Alterations in platelet proteome signature and impaired platelet integrin αIIbβ3activation in patients with COVID-19

“…Here, we found that S100A8/A9 protein levels were enhanced by more than 4-fold (log2 fold change of 2.25) in platelets in COVID-19 patients. As platelet transcript levels in COVID-19 patients were unchanged [21], this is likely to be the result of enhanced platelet binding and uptake of plasma S100A8/A9, potentially through GPIb/CD36 receptor interaction [66]. The enhanced basal platelet PS exposure in COVID-19 patients detected in our study would be consistent with this.…”

“…However, we cannot rule out that COVID-19 conditions may induce platelet P-selectin expression in the absence of integrin α IIb β 3 activation, such as has recently observed in a small fraction of CRP/PAR-1 activated platelets [70]. The recent study by Colicchia et al [66] is also of interest as they demonstrated that S100A8/A9 can induce P-selectin expression whilst inducing an integrin α IIb β 3 activatory state that lacked aggregatory ability and had low-fibrinogen binding capacity.…”

“…The plasma protein S100A8/A9 did not pass our selection criteria of being elevated in two out of three proteomic studies [42–44], however several studies have reported raised plasma S100A8/A9 in COVID-19 patients and plasma levels correlated to poor outcome [64–67]. S100A8/A9 can induce platelet procoagulant responses and promote platelet/neutrophil interaction by enhancing P-selectin levels [66]. Here, we found that S100A8/A9 protein levels were enhanced by more than 4-fold (log 2 fold change of 2.25) in platelets in COVID-19 patients.…”

Role of S100A8/A9 in Platelet–Neutrophil Complex Formation during Acute Inflammation

“…Additionally, we confirm the expression of the TLR4/MD2 receptor on the surface of platelets ( Supplementary Figure S2B ), as has been described previously [ 32 ]. Recent studies have shown that high concentrations of S100A8/A9 are able to induce direct platelet activation via TLR4 and GPIbα [ 28 , 33 ]. Since TLR4 is known as the counterreceptor to S100A8/A9 heterodimers, we stimulated platelets with mutated S100A8/S100A9N70AE79A heterodimers unable to form tetramers, and could confirm GPIIbIIIa—but not CD62P—upregulation ( Supplementary Figure S2C ).…”

“…Expression levels were investigated in a FACSCantoII flow cytometer (BD Bioscience). Stimulation of isolated WT platelets with S100A8/A9 tetramers was performed according to Colicchia et al with minor modifications [ 28 ]. Murine platelets were isolated via repetitive slow-speed centrifugation and resuspended in Tyrode’s buffer (20 mM HEPES, 136 mM NaCl, 2.6 mM KCl, 12 mM NaHCO 3 , 5.5 mM D-glucose, 2 mM CaCl 2 , 1 mM MgCl 2 ).…”