Thrombotic microangiopathies (TMA): In vivo evidence for platelet activation and endothelial cell injury, and potential for therapeutic intervention with defibrotide

Background : Endothelial cell (EC) activation and injury and platelet activation characterize thrombotic thrombocytopenic purpura (TTP) and atypical hemolytic uremic syndrome (aHUS). We found that 5 μg/ml defibrotide inhibits TMA plasma-mediated caspase 8 activation of EC, an initial step in apoptotic injury (ASH 2019, Abstract 3676), but defibrotide was reported to inhibit agonist-induced platelet activation only at clinically unachievable doses of 100-1000 μg/ ml (ASH 2019, Abstract 3614). Aims : (1) Evaluate biomarkers of platelet activation and EC injury in TMA plasmas;(2) determine whether clinically relevant defibrotide concentrations block agonist-mediated platelet activation. Methods : (1) Biomarkers for platelet activation (platelet factor 4 (PF4), β-thromboglobulin (β-TG)) and EC injury (von Willebrand factor (vWF) antigen) were measured in TMA patient plasmas (9 aHUS, 8 TTP) by ELISA. (2) Washed human platelets were incubated with the PAR-1 agonist peptide RUJL or ADP (2 μM), alone or with 5 μg/ml defibrotide. Platelet aggregation was quantified by light transmission aggregometry. Results(1) A significant increase in PF4 levels was seen in TMA patients ( n = 15) vs. healthy controls ( n = 12) (Fig. 1). A significant difference in β-TG levels was not seen in TMA patients ( n = 15) vs. controls ( n = 7). The β-TG:PF4 ratio, a marker of in vivo platelet activation (Ann Rheum Dis 2005;64:484), was >2 in TMA and control plasmas, indicating some in vitro activation, but much more highly elevated in TMA (ratio = 19.4) vs. control plasmas (ratio = 5.6) ( P = 0.0058). vWF antigen levels were not significantly different in patients vs. controls. (2) Defibrotide blocked platelet aggregation induced by both RUJL and ADP at 5 μg/ml (Fig. 2). Conclusions : FIGURE 2 : Effect of defibrotide on PAR-1 agonist and ADP induced platelet aggregation The ability of defibrotide to block TMA plasma-mediated EC injury, shown previously, and now platelet activation has implications for TMA treatment as well as in progressive COVID-19, which presents features characteristic of TMAs and vaso-occlusive disease.