UC Berkeley

Chronic kidney disease (CKD) patients on hemodialysis (HD) suffer elevated mortality from cardiovascular disease (CVD) linked to accumulating uremic toxicity. Recent findings in cardiac MRI indicate earlier fibrosis and hypertrophy in CKD patients, challenging the traditional belief that cardiorenal syndrome develops secondary to uremia and hypervolemia, inciting the need to re-explore CVD pathogenesis as a result of CKD. Platelets present a promising link between CKD and CVD as rich sources of tissue regulators that are abnormally stimulated during HD. However, while activation of circulating platelets by receptor binding and subsequent coagulation events are defined by a well characterized physiological response, the mechanobiology of platelet degranulation is comparatively unexplored. I hypothesized that the observed early cardiac remodeling in HD patients is in part caused by HD-aggravated platelet dysregulation leading to non-uremic circulatory imbalances. To test this, I proposed 1) characterizing the effects of HD on platelets and the circulating proteome, 2) mechanistically studying platelet activation in response to the fluidic stimulus of depressurization experienced by frictional loss along the HD flow circuit, and 3) visually observing fibrotic developments in cardiac tissues in vitro as a result of contact with platelets of varying dysregulation.

In the first study, plasma and platelets from end stage renal disease (ESRD) patients before and after HD were profiled for proteome and transcriptome growth factors (PDGF, FGF, EGF), proteolytic regulators (MMPs, TIMPs), coagulative factors (vWF, PAF, PF4), cardiac indicators (BNP, TPO, TXA2), and inflammatory markers (CRP, TNFα, IL1b) to examine HD-modulation of platelet effects beyond thrombotic activation. Elevation of plasma proteolytic regulators, fibrotic factors, and corresponding platelet RNA transcripts revealed uniquely pro-fibrotic platelet phenotypes in ESRD patients during HD. Correlations against prior time on dialysis (HD vintage) and blood flow rate (BFR) indicated worsening dysfunction with longer HD vintage and faster prescribed BFR, particularly characterized by preferentially enhanced translation and secretion of matrix metalloproteinases (MMP), tissue inhibitors of MMPs (TIMP), and inflammatory cytokines. Compensatory mechanisms of increased platelet growth factor synthesis with acute plasma MMP and TIMP increases show short-term mode-switching between dialysis sessions leading to long-term pro-fibrotic bias. Chronic pro-fibrotic adaptation of platelet synthesis was observed through changes in differential secretory kinetics of heterogeneous granule subtypes. Chronic and acute platelet responses to HD describe contributions to a pro-fibrotic milieu in ESRD.

In the second study, platelets from healthy and HD (pre- and post-treatment) donors were cyclically depressurized in static suspension to mimic the precipitous and repetitive pressure drops across the hemodialysis flow circuit. Platelet activation was measured by integrin complexing of the traditional coagulative indicator glycoprotein αIIbβ3 (PAC1) and expression of the granule surface receptor P-selectin (CD62P). The progressive increase in CD62P with no changes in PAC1 over pressure-cycling duration regardless of uremia signifies that hydrostatic depressurization involves a novel agonist-free mechanism leading to platelet degranulation as a unique case in which CD62P and PAC1 do not interchangeably indicate platelet activation. Subsequent stimulation using ADP further suggests that sustained depressurization regimens desensitize integrin αIIbβ3 activation. Variability in platelet response caused by uremia and CKD were observed by elevated baseline PAC1 in pre-dialysis samples, PAC1 retention after ADP exposure, and maximum CD62P with ADP independent of pressure. Theory for hydrostatic pressure-induced degranulation circumventing integrin-initiated signal transduction is presented based on the Starling Equation.

In the third study, induced pluripotent stem cell derived cardiomyocyte tissues were cultured with plasma and platelets from hemodialysis donors pre- and post-filtration, healthy donor plasma and platelets, and continuation media. Spontaneously contracting tissues from all test groups were visually analyzed daily for changes in troponin muscular network and functional contractile output and lysed after 28 days of culture for matricellular protein composition. Cardiac tissues developed more fibrotic phenotypes in dialysis derived media, with amplified compositional and structural changes by refreshed post-dialysis platelets over exhausted and uremic pre-dialysis platelets, illustrating a direct link bridging platelet dysfunction to the clinical outcomes of deteriorative cardiac fibrogenesis observed in the REMODELING clinical trial. Separately, healthy plasma and platelets enhanced cardiac maturity, suggesting promising implications for the tissue engineering toolbox.

Collectively, these studies depict how platelets contribute to CVD progression in CKD. The identification of unique pro-fibrotic platelet phenotypes in ESRD independent of uremic state, a novel mechanism of platelet degranulation independent of chemical stimulus, and direct visual observation of platelet-dependent cardiovascular remodeling establish a distinct pathway explaining the manifestation of uremic cardiomyopathy independent of hemodynamic overload. Future use of platelets as therapeutic agents and diagnostic tools for left ventricular remodeling, microvascular dysfunction, and myocardial interstitial fibrosis could aid the prevention of congestive heart failure.