Using Platelet Proteomics as a Marker of Cardiac Failure in a Pacing-Induced Pig Heart Failure Model
Authors: Rothwell Stephen W. , Haigney Mark C. , Wi Shao-kui , Clark Madelaine , Eidelman Ofer , Jozwik Catherine , Srivastava Meera , Mueller Gregory P. , Shou Matie , Pollard Harvey B. 
Published: April 2020

Cardiac failure is a leading cause of morbidity and mortality in the United States today. There is also a well-described gender bias to this disease, with the occurrence of cardiac failure higher in men than women. Anti-platelet drugs are the mainstay for prevention and therapy of cardiovascular disease in humans, and we have therefore conjectured that blood platelets could function as essential biomarkers of these processes. Thus, by studying the protein expression of platelets using proteomic tools, we anticipated that a proteomic profile of the “cardiac platelet” could be defined that would permit mechanistic discrimination of the development of this process. For this study, we hypothesized that signal transduction pathways, specifically including inflammatory or pro-coagulatory pathways, are key mediators of platelet contributions to the cardiac failure process. To test this hypothesis, ventricular dysfunction was induced in male and female swine by the placement of an atrial active-fixation pacing lead and active pacing initiated at 200 bpm. Ventricular performance was defined by Fractional Shortening (Fractional Shortening = [(left ventricular end-diastolic diameter - left ventricular end-systolic diameter)]/left ventricular end-diastolic diameter). When the fractional shortening (normal 32%) dropped below 16%, the animals were considered to be in terminal cardiac failure, and were euthanized. Blood samples were collected throughout the experiment and platelets isolated for analysis. Proteomic analysis of platelet lysates was performed using antibody microarrays (Clontech 507 AB arrays; n = 2 males and 2 females, replicates = 2), reverse capture microarrays (replicates = 6) and Western blotting (n ≥2). Platelets samples were compared as pairs of platelet lysates from initial and terminal bleeds. Samples of ventricular muscle tissue collected following termination of the experiments were run in parallel studies and were compared against gender matched untreated pigs. Results of the analysis demonstrated that the expression of many proteins was altered both in platelets and heart tissue in a gender-dependent manner following cardiac stress. The top 5 proteins up-regulated in platelets isolated from males were p57, integrin b1, GCIP, K channel a and NF-ATc1. Three of these proteins, p57, GCIP and NF-ATc1, are associated with cell proliferation and may reflect increased megakaryocyte production of platelets. In a recent study with Sanqi (Yao et al., Phytomedicine 2008), a traditional Chinese medicine with cardiovascular effects, k channel subfamily V was also shown to be increased in platelets. In contrast, in female platelets, the top 5 proteins were GAP-43 and IL1beta which are proteins associated with inflammation, beta-catenin which is a protein with wide-spread effects mediated through the cytoskeleton, STAT3 and COMT (catechol-O-methyl transferase). None of these proteins were in the top 5 up-regulated proteins in heart tissue. The top 5 down-regulated proteins in the platelets were, for the males: GIT1, Bog, IL1beta, TIAR and Ku70 and for the females: SCAR-1, crp-2, NTF, SMN and MGMT. GIT1 is involved in cytoskeletal changes and is normally phosphorylated by src kinase following stimulation by platelet activation. TIAR, an mRNA-binding protein, regulates expression of diverse proteins such as MYC, IL-6 and Fas. It has been implicated in cellular processes ranging from proliferation to apoptosis. In the female samples, SCAR-1 interacts with GPVI to form the bridge to collagen binding. Down regulation of this protein may inhibit platelet binding to extracellular substrates. The proteins SMN and MGMT in the hematopoietic system are commonly associated with stem cell differentiation and may also relate to levels or rates of differentiation of megakaryocytes into platelets. Confirmatory experiments with reverse capture methodology supported our findings for Bog, GIT, catenin, SCAR-1 and crp2 as well as caspase 6 and TRAF2, proteins that were not in the top 5 group. Western blots confirmed changes in caspases 6 and PKCa. In conclusion, our preliminary data support the hypothesis that that signal transduction pathways in platelets may be key mediators of platelet contributions to cardiac failure. Network analysis of the many proteins altered during this process will be necessary to understand the varied and subtle changes that may be occurring during this process.