Plasmin-Induced Activation of Human Platelets Is Modulated by Thrombospondin-1, Bona Fide Misfolded Proteins and Thiol Isomerases.

Claudia Pielsticker,Kerstin Jurk,Lisa Raum,Martin F Brodde,Beate E Kehrel

doi:10.3390/ijms21228851

Abstract

Inflammatory processes are triggered by the fibrinolytic enzyme plasmin. Tissue-type plasminogen activator, which cleaves plasminogen to plasmin, can be activated by the cross-β-structure of misfolded proteins. Misfolded protein aggregates also represent substrates for plasmin, promoting their degradation, and are potent platelet agonists. However, the regulation of plasmin-mediated platelet activation by misfolded proteins and vice versa is incompletely understood. In this study, we hypothesize that plasmin acts as potent agonist of human platelets in vitro after short-term incubation at room temperature, and that the response to thrombospondin-1 and the bona fide misfolded proteins Eap and SCN−-denatured IgG interfere with plasmin, thereby modulating platelet activation. Plasmin dose-dependently induced CD62P surface expression on, and binding of fibrinogen to, human platelets in the absence/presence of plasma and in citrated whole blood, as analyzed by flow cytometry. Thrombospondin-1 pre-incubated with plasmin enhanced these plasmin-induced platelet responses at low concentration and diminished them at higher dose. Platelet fibrinogen binding was dose-dependently induced by the C-terminal thrombospondin-1 peptide RFYVVMWK, Eap or NaSCN-treated IgG, but diminished in the presence of plasmin. Blocking enzymatically catalyzed thiol-isomerization decreased plasmin-induced platelet responses, suggesting that plasmin activates platelets in a thiol-dependent manner. Thrombospondin-1, depending on the concentration, may act as cofactor or inhibitor of plasmin-induced platelet activation, and plasmin blocks platelet activation induced by misfolded proteins and vice versa, which might be of clinical relevance.

Highlights

The tight regulation of platelet activity is a prerequisite to maintain vascular integrity in hemostasis and inflammation
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Pthlaastmthine iinntheirbaictteidonpblaettewleetefnibprilnasomgeinn abninddtihnegriensdpuocnesde to injury protein TSP-1, extracellular adherence protein (Eap) and denatured IgG might be important in the differential modulation of platelet activation especially under inflammatory conditions

Summary

Introduction

The tight regulation of platelet activity is a prerequisite to maintain vascular integrity in hemostasis and inflammation. The central fibrinolytic serine protease plasmin is generated through the cleavage of its zymogen Glu-plasminogen physiologically by the tissue-type (tPA), with fibrin as cofactor, and urokinase-type (uPA) plasminogen activators upon vascular injury and inflammation. The homotrimeric matricellular glycoprotein thrombospondin-1 (TSP-1) serves as cofactor for plasmin generation, forming a trimolecular complex with plasminogen and tPA [6,7]. TSP-1 is released via exocytosis of α-granules by activated platelets and rebound to the platelet surface via TSP-1 receptors, e.g., CD36, CD47, αIIbβ, or indirectly, via immobilized fibrinogen or von Willebrand factor [9]. Besides TSP-1, activated platelets release the pro-fibrinolytic factors plasminogen and histidine-rich glycoprotein from α-granules, which form a trimolecular complex together with TSP-1 on the platelet surface to amplify plasmin generation [11]

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Conclusion