Development of pharmacoproteomic platforms for monitoring changes in the thrombin mediated signaling and aggregation of human platelets treated with direct thrombin inhibitors
Authors: Clement Cristina 
Published: April 2020

Thrombin is a serine‐protease known to induce the activation of platelets to aggregate by binding to and cleaving the extracellular N‐terminal domains of protease‐activated receptors 1 and 4 (PAR1 and PAR4). Dabigatran is an oral anticoagulant and a reversible direct inhibitor of thrombin (DTI), thereby inactivating both fibrin‐bound and unbound (ie, free) thrombin. Recently, it has been shown that dabigatran can inhibit platelet aggregations through a direct effect on platelet thrombin receptors (PAR‐4), which is mainly correlated with a reduced expression of GPIIb/IIIa, CD63, and P‐selectin on platelets after dabigatran treatment. Herein, we engaged on further exploring the human platelets proteome changes in response to thrombin activation in presence of the FDA approved DTI, dabigatran. We hypothesized that a global proteomics analysis of human platelets treated with dabigatran would reveal the integrin‐linked kinase (ILK) and integrin mediated signaling as major pathways which would be down‐regulated in the thrombin‐activated platelets treated with dabigatran. The downregulation of such pathways would explain the dabigatran mediated inhibition of thrombin activated platelets aggregation. To test this hypothesis, we developed a pharmacoproteomics platform that monitor changes in the protein expression profiles of platelets, purified from healthy human donors and ex‐vivo activated with thrombin in presence or absence of dabigatran. The proteomics platform employed the nanoLC‐ESI MS/MS sequencing of tryptic/Glu‐C/Lys‐C generated peptides from platelets releasates, as well as a Q‐Exactive quadrupole orbitrap mass spectrometer coupled with the label free quantification (LFQ) module provided by PEAKS X (Bioinformatics Solutions Inc.). The proteomic analysis retrieved about 300 proteins from the platelets treated with dabigatran as compared with 500 proteins from the platelets control, untreated (FDR <1.0% for proteins and <0.5% for peptides). The quantitative analysis of the biochemical pathways was accomplished with ingenuity pathway analysis (IPA; Ingenuity Systems) using the protein MS1 and MS2 exclusive spectra counts ratios extracted from LFQ analyses. The bioinformatics analysis predicted that many proteins involved in the actin‐mediated cell signaling and movement pathways, including rhoA, rhoGDI, clathrin mediated‐endocytosis, and ILK and integrin signaling pathways, were at least two‐fold statistically significant down‐regulated (p<0.05) in the dabigatran‐treated platelets. Remarkably, biomarkers from the integrin mediated signaling pathways (such as of GPIIb/IIIa) were validated to be down‐regulated by the dabigatran treatment of thrombin activated platelets. In addition, the pharmacoproteomic platform was further validated with novel DTI tetrapeptides lead compounds shown to completely inhibit the thrombin‐induced platelet aggregation at concentrations of 0.8–20 uM. These data further advocate for the use of the LFQ proteomics profiling as a reliable assay for monitoring the efficacy of selected drug treatment during ACS management.

Support or Funding Information

Funding: Radiation Oncology Department of Weill Cornell Medicine

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