Impact of whole blood amustaline pathogen inactivation on plasma coagulation factor activity; Mechanism of action Free

Amustaline (S-303, 200 µM) in combination with glutathione (GSH) quencher inactivates pathogens in red blood cell concentrates with retention of RBC therapeutic function. S-303 efficiently inactivates a broad spectrum of pathogens in whole blood which may facilitate further preparation of components with a single pathogen inactivation process. However, S-303 impacts platelet function (Pongerard et al) in a dose-dependent manner. We have characterized, in detail, the effects of S-303 on plasma coagulation factor function and the mechanism by which S-303 inactivates specific coagulation factor activities.

Whole blood was treated with S-303 (100, 200 and 400 µM) and platelet-free plasma was isolated from blood by centrifugation. Global coagulation assays, prothrombin time (PT) and activated thromboplastin time (aPTT) were prolonged with 200 and 400 µM S-303 in dose-dependent fashion. This suggested that certain factors in the extrinsic, intrinsic and common pathways were impacted by higher concentrations of S-303. To understand which factors may be impacted, clotting assays with factor-deficient plasma and model systems using purified proteins were performed. Factor (F)X activation by the extrinsic Xase complex (FVIIa-TF-FX) at both low and high tissue factor concentrations was inhibited by S-303 (200 µM) in purified protein assays. Similarly, FX activation by the intrinsic Xase complex (FIXa-FVIIIa-FX) and prothrombin activation by the prothrombinase complex (PTase: FXa-FVa-FII) was also attenuated by higher concentrations of S-303. These results were in accordance with the S-303-dependent inhibition of activities of factors X, IX, VIII, VII and II, assessed by factor-deficient plasma clotting assays. Notably, FV activity from (1:1000 diluted) plasma, but not purified FV, was preserved in PTase complex and FV-deficient plasma clotting assays. Further, FV activation by thrombin was also preserved in the presence of S303. These data suggest that the inhibition of FII activation by S-303 in PTase is decreased by the presence of plasma. Plasma FVa inactivation by activated protein C (APC) anticoagulant pathway (APC-protein S-PL) was preserved in the presence of S-303. However, pre-treatment of APC and protein S with S-303 prior to inactivation of FVa in purified system assays inhibited the action of APC and protein S. These differences between the plasma assays and purified assays are likely due to other off-target effects of S-303 that are absent in purified assays.

S-303 did not inhibit either the antithrombin (AT)-dependent inactivation of thrombin in plasma or the tissue factor pathway inhibitor-dependent inactivation of thrombin generation by the FVIIa-FXa-FVa-Protein S complex at both low and high TF suggesting that the anticoagulant pathways are left intact whereas the procoagulant pathways are attenuated by S-303 treatment.

Compared with untreated controls, clot formation time was prolonged and mean clot formation was attenuated in thromboelastometry (EXTEM®) on WB treated with S-303, supporting observations above. Lysis onset time was also prolonged in the presence of added tissue plasminogen activator.

To understand the molecular bases of S-303-dependent alterations in function, S-303 was screened for off-target adduct formation in four 5-mer peptide libraries using mass spectrometry. S-303 selectively formed adducts with thiol groups on cysteine residues in these assays. Further S-303 did not break preformed disulfides in these model systems. To better understand the selective inhibition of factors X, IX, VII, prothrombin, APC and protein S that lack free thiols, proteomics was performed on a FX that was profoundly affected by S-303. S-303 formed an adduct with residue Cys³⁷⁵, spatially proximal to the catalytic triad and active site Ser³⁸⁰ in the protease domain of FX. Cys³⁷⁵ is part of a disulfide bond with Cys⁴⁰³. As a negative control, AT that was unaffected by S-303 was analyzed in the same proteomic assays in which no Cys adducts were detected, in accordance with the functional data.

Our results suggest that S-303 forms adducts with disulfide bonds in select proteins that are labile or otherwise accessible. This might suggest that functionally important disulfides exist in several coagulation factors and that S-303 and derivatives can be used to probe for these disulfides.