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The Extracellular Protease EpiP from S. aureus Triggers Blood Coagulation by Proteolytically Activating Prothrombin and Platelet Protease-Activated Receptor 1

1Department of Pharmaceutical and Pharmacological Sciences, University of Padua, Padova, Italy, 2Center for Thrombosis and Hemostasis (CTH), University Medical Center Mainz, Mainz, Germany, 32Department o Medicine, University of Padua, Padua, Italy, 4GSK Vaccines, Siena, Italy, 5Department of Molecular Medicine, Unit of Biochemistry, University of Pavia, Pavia, Italy

Abstract Number: LPB0039

Meeting: ISTH 2021 Congress

Theme: Vascular Biology » Inflammation and Sepsis

Background: Staphylococcus aureus is a Gram-positive bacterium known for its pathogenicity in humans, responsible for both mild and systemic infections, i.e. bacteremia and sepsis. During the last decades, clinical evidence provide new insights for a positive relation between S. aureus infections and dramatic thrombotic complications, such as DIC. Among the huge arsenal of virulence factors, extracellular proteases might play a role in triggering thrombotic events in infectious diseases, whereby bacterial proteases could activate the coagulation cascade by proteolytically converting ProT zymogen into thrombin species and stimulating platelet aggregation.

Aims: In this work, we investigated if the subtilisin-like protease EpiP from S. aureus is involved in thrombus formation during staphylococcal infections, directly activating Prothrombin (ProT) and platelets.

Methods: Biochemical techniques: limited proteolysis, enzymatic chromogenic assay, mass spectrometry; coagulation assays: fibrin generation and platelet aggregation; fluorescence microscopy.

Results: Staphylococcal EpiP converts ProT into an active species which is able to hydrolyze the thrombin-specific substrate S2238. The time-course analysis of ProT activation allowed to identify the EpiP cleavage sites (Arg155-Ser156, Arg271-Thr272, and Arg320-Ile321), identical to those hydrolyzed by factor Xa under physiological conditions. The activation products of ProT by EpiP can induce fibrin clot formation, either from a fibrinogen solution or platelets-free plasma (PFP), and platelets aggregation. Surprisingly, EpiP can proteolyze PAR1(38-60) peptide at the same site of thrombin cleavage (Arg41-Ser42) and electrostatically interact with GpIbα(268-282) peptide, as demonstrated by SPR. Ultimately, we directly observed EpiP-mediated platelets agglutination by fluorescence microscopy.

Conclusions: The extracellular protease EpiP from S. aureus, can proteolyze the inactive ProT into an active thrombin species which is able to trigger blood coagulation. Moreover, EpiP directly induces platelet aggregation activating PAR1 receptor after binding to GpIbα on platelet surface. These results widen our understanding of biochemical mechanism whereby S. aureus proteases can initiate coagulation, establishing a direct link between infections and higher thrombotic risk.

To cite this abstract in AMA style:

De Filippis V, Artusi I, Pontarollo G, Acquasaliente L, Pagotto A, Radu CM, Bagnoli F, Pietrocola G, Speziale P. The Extracellular Protease EpiP from S. aureus Triggers Blood Coagulation by Proteolytically Activating Prothrombin and Platelet Protease-Activated Receptor 1 [abstract]. Res Pract Thromb Haemost. 2021; 5 (Suppl 2). https://abstracts.isth.org/abstract/the-extracellular-protease-epip-from-s-aureus-triggers-blood-coagulation-by-proteolytically-activating-prothrombin-and-platelet-protease-activated-receptor-1/. Accessed August 16, 2022.

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/the-extracellular-protease-epip-from-s-aureus-triggers-blood-coagulation-by-proteolytically-activating-prothrombin-and-platelet-protease-activated-receptor-1/