MARCKS ED Inhibits Fibrin Formation By Blocking Coagulation Protein Complex Assembly on Phosphatidylserine

Introduction: Prothrombinase is an essential enzyme of the coagulation cascade that is made up of a heterodimer of coagulation factor Xa and Va.  The complex most readily forms in the presence of lipid membranes containing the anionic lipid phosphatidylserine.  The phosphatidylserine lipid head groups provide a surface for the prothrombinase complex to assemble and significantly increase the enzyme’s turnover number.  Here, we investigate how truncation and modification of the naturally occurring myristoylated alanine-rich C-kinase substrate (MARCKS) protein into small peptide and peptidomimetic fragments (MARCKS ED) forms lipid probes that bind to phosphatidylserine, and block the assembly and enzymatic activity of the prothrombinase complex.  Compared to known phosphatidylserine binding proteins such as annexin V or lactadherin, the MARCKS ED probes are less than a tenth of the molecular weight and do not require calcium to bind to phosphatidylserine.

Methods: Using biophysical assays, we examine how the MARCKS ED probes assemble on lipid membranes containing phosphatidylserine and how this assembly alters the binding of factor Xa to these surfaces.  Effects on the enzymatic activity of the prothrombinase complex in the presence of activated platelets and other phosphatidylserine exposing membranes are explored using a modified prothrombinase assay.  The binding and anticoagulant activity of the MARCKS ED probes during whole blood coagulation are examined by quantifying fluorescently labeled MARCKS ED probe localization, platelet accumulation, and fibrin formation in microfluidic flow assays. 

Results: We find that the MARCKS ED probes can antagonize factor Xa from binding phosphatidylserine by measuring reduced association of factor Xa to lipid membranes treated with the MARCKS ED probes.  Pre-treatment of activated platelets with the MARCKS ED probes significantly reduced the assembly and enzymatic activity of prothrombinase (100% activity with vehicle control vs. 51% with 1 μM MARCKS ED peptide treatment, P < 0.05).  Similar inhibitory activity was also observed when activated platelets were replaced with other phosphatidylserine exposing lipid membranes, such as synthetic liposomes or biologic microparticles.  In the whole blood microfluidic flow assay, the MARCKS ED probes colocalized with adherent platelets and inhibited fibrin formation (100% peak fibrin intensity with vehicle control vs. 17% peak fibrin intensity with 1 μM MARCKS ED peptide treatment, P < 0.05).  The MARCKS ED probes did not alter platelet surface area coverage.  Electron microscopy images of the final clots formed in the microfluidic flow assay show an absence of fibrin formation with MARCKS ED probe treatment. 

Conclusions: We demonstrate that the MARCKS ED probes can inhibit the assembly and enzymatic activity of prothrombinase, and that this activity translates to significantly reduced fibrin formation in whole blood.  The MARCKS ED peptide and peptidomimetic probes may provide a new therapeutic tool for inhibiting the assembly of phosphatidylserine dependent coagulation factors, as well as a research tool for identifying platelet phosphatidylserine exposure.