Peptides derived from MARCKS block coagulation complex assembly on phosphatidylserine

Noah Kastelowitz,Ormacinda R White,Jorge Di Paola,Lawrence F Brass,Timothy J Stalker,Peter N Brown,Ryo Tamura,Abimbola Onasoga,Gary L Brodsky,Brian R Branchford,Hang Yin

doi:10.1038/s41598-017-04494-y

Abstract

Blood coagulation involves activation of platelets and coagulation factors. At the interface of these two processes resides the lipid phosphatidylserine. Activated platelets expose phosphatidylserine on their outer membrane leaflet and activated clotting factors assemble into enzymatically active complexes on the exposed lipid, ultimately leading to the formation of fibrin. Here, we describe how small peptide and peptidomimetic probes derived from the lipid binding domain of the protein myristoylated alanine-rich C-kinase substrate (MARCKS) bind to phosphatidylserine exposed on activated platelets and thereby inhibit fibrin formation. The MARCKS peptides antagonize the binding of factor Xa to phosphatidylserine and inhibit the enzymatic activity of prothrombinase. In whole blood under flow, the MARCKS peptides colocalize with, and inhibit fibrin cross-linking, of adherent platelets. In vivo, we find that the MARCKS peptides circulate to remote injuries and bind to activated platelets in the inner core of developing thrombi.

Highlights

Phosphatidylserine (PS) is an essential cofactor of the coagulation proteins involved in hemostasis and thrombosis
We found that D-myristoylated alanine-rich C kinase substrate (MARCKS) ED could antagonize binding of factor Xa (FXa) to PS, and that this activity inhibited the enzymatic activity of coagulation factor complex prothrombinase in the presence of a variety of PS-containing membranes
We observed D-MARCKS ED binding to platelets in the thrombus core, but we did not see a significant effect on fibrin formation

Summary

Introduction

Phosphatidylserine (PS) is an essential cofactor of the coagulation proteins involved in hemostasis and thrombosis. PS is an anionic lipid that is normally sequestered to the inner leaflet of the plasma membranes and only exposed on the outer membrane surface during activated processes like apoptosis or platelet activation. During blood clotting, activated platelets undergo changes in membrane shape and expose PS on their outer membrane leaflet via calcium-dependent phospholipid scramblases[1,2,3]. We have previously demonstrated that peptides based on the effector domain of MARCKS (MARCKS ED) bind with increasing affinity to lipid membranes that are both highly curved and enriched in PS22. We establish that a peptide can antagonize a lipid-protein interaction, and that this activity can have significant effects in a complex multicomponent pathway like blood coagulation

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Conclusion