Indolic Uremic Solutes Enhance Procoagulant Activity of Red Blood Cells through Phosphatidylserine Exposure and Microparticle Release

Abstract

Increased accumulation of indolic uremic solutes in the blood of uremic patients contributes to the risk of thrombotic events. Red blood cells (RBCs), the most abundant blood cells in circulation, may be a privileged target of these solutes. However, the effect of uremic solutes indoxyl sulfate (IS) and indole-3-acetic acid (IAA) on procoagulant activity (PCA) of erythrocyte is unclear. Here, RBCs from healthy adults were treated with IS and IAA (mean and maximal concentrations reported in uremic patients). Phosphatidylserine (PS) exposure of RBCs and their microparticles (MPs) release were labeled with Alexa Fluor 488-lactadherin and detected by flow cytometer. Cytosolic Ca2+ ([Ca2+]) with Fluo 3/AM was analyzed by flow cytometer. PCA was assessed by clotting time and purified coagulation complex assays. We found that PS exposure, MPs generation, and consequent PCA of RBCs at mean concentrations of IS and IAA enhanced and peaked in maximal uremic concentrations. Moreover, 128 nM lactadherin, a PS inhibitor, inhibited over 90% PCA of RBCs and RMPs. Eryptosis or damage, by indolic uremic solutes was due to, at least partially, the increase of cytosolic [Ca2+]. Our results suggest that RBC eryptosis in uremic solutes IS and IAA plays an important role in thrombus formation through releasing RMPs and exposing PS. Lactadherin acts as an efficient anticoagulant in this process.

Pathophysiological aspects of red blood cells in end‐stage renal disease patients resistant to recombinant human erythropoietin therapy

“…Moreover, the current study verified the strong relation between PS exposure and R-MVs formation, 21 22 Loss of phospholipid asymmetry and additional ESRD-associated pathologies, including shape distortion, mechanical stress, chronic inflammation, oxidative stress, cellular activation, and endothelial dysfunction, also affect vesiculation of endothelial and blood cells, including RBCs. [23][24][25] The majority of previous studies have focused on MVs derived from platelets and endothelial cells for their probable prothrombotic and pro-inflammatory roles.…”

“…By eliminating uremic toxins, HD ameliorated anemia and accumulation of MVs, 22,23,45 and that endothelium damage or cardiovascular disease is more frequently appeared in rhEPO resistance, 49 our data deserve further study through methods more suited to assess issues of small particle biology.…”

“…By eliminating uremic toxins, HD ameliorated anemia and accumulation of MVs, equally in responders and non‐responders. On the other side, HD did not affect the concentration of RBC‐derived MVs or the protein composition of the RBC membrane in non‐responders, and moreover, it aggravated PS exposure on NR‐RBCs.…”

“…Moreover, the current study verified the strong relation between PS exposure and R‐MVs formation, in response to HD and rhEPO dose. Some indolic uremic solutes can induce vesiculation along with PS exposure on healthy RBCs . Loss of phospholipid asymmetry and additional ESRD‐associated pathologies, including shape distortion, mechanical stress, chronic inflammation, oxidative stress, cellular activation, and endothelial dysfunction, also affect vesiculation of endothelial and blood cells, including RBCs .…”

The Clinical Utility of Circulating Microparticles’ Measurement in Heart Failure Patients

“…Indeed, erythrocytes actively shed phospholipid-bound MPs [54]. MPs originating from erythrocytes are naturally produced in vivo during normal aging processes or they have associated with a variety of pathophysiological conditions including hematology diseases (hemolysis, sickle cell disease and thalassemia), chronic kidney disease (IgA nephropathy), uremia, stroke, acute infections, sepsis, trauma, thrombosis/embolia, allograft dysfunction [55][56][57][58][59][60]. Therefore, erythrocytes-derived MPs may secrete ex vivo during cold storage of RBCs [61].…”