Circulating microparticles exhibit multiple discrete changes in annexin V binding and calcein‐AM cleavage in cardiovascular disease

Abstract

Cardiovascular disease is the leading cause of mortality among developed nations, and a greater understanding of disease progression is needed for improved diagnosis and therapeutic development. Cell-derived microparticles (MP) circulate and serve in cell-cell communication. Secreted MP are altered in diseased states and provide insight into pathophysiology and bio-inspired therapeutics. Our objective was to characterize the changes in circulating MP enzyme activity and surface markers in patients representing various stages of coronary artery disease (CAD). Plasma from healthy patients (n=3), patients with risk factors (RF) for CAD (n=4), CAD patients (n=5), and patients with acute myocardial infarction (AMI, n=2) were obtained and MP isolated by ultracentrifugation. MP were incubated with antibodies against cell-surface markers, annexin V, and calein-AM before analysis by flow cytometry. We found that plasma MP fell into seven discrete groups in the context of annexin V binding and calcein-AM activation. Further, the distribution of MP in these groups was a function of disease state; MP from healthy patients existed in gates 1-3, but as patients progressed toward and including CAD and AMI, MP were in gates 3-7. Platelet MP fell in gates 1 and 4; endothelial cell MP in gate 5, red blood cell MP in gates 3 and 4. Del-1, a protein mediator of uptake, was most prevalent on CAD and AMI MP. Annexin V and calcein have been used as binary MP markers, but never before in conjunction to elucidate the spectrum of circulating MP. Our results indicate that they may track disease progression. Interestingly, cell types shed MP that fell among multiple gates; cells alter secreted MP in response to disease and may serve as signals.