The aim of this study was to investigate the relation between platelet function and kidney failure in patients with end stage renal disease and cardiorenal syndrome using a flow cytometer based, functional platelet assay. We show that P-selectin expression after stimulation with ADP, CRP and TRAP is lower in patients with chronic kidney disease as compared to healthy controls. Furthermore, we demonstrate that the EC50 was not different between groups. This means that platelet sensitivity itself is not affected for the different agonists, but the maximal platelet response is significantly lower.
Platelets were activated with 3 different stimuli. We chose for the agonists TRAP, ADP and CRP, which stimulate the three major physiological platelet activation pathways. TRAP activates the thrombin receptor Proteinase Activated Receptor 1 (PAR-1) on platelets. ADP is normally present in platelet dense granules. Upon platelet activation, ADP is released to activate nearby platelets via the P2Y receptors. CRP activates the receptor glycoprotein VI, the major collagen receptor on platelets. So, all 3 stimuli are of physiological importance.
P-selectin is found in the α-granula of platelets. A deficiency in α-granula could lead to ineffective haemostasis. Two different explanations are possible for the deficient platelet α-granula release found in chronic kidney disease. This could be due to depletion of α-granula itself, or due to a deficiency in the release of α-granula. An impaired α-granule release has already been reported in uraemia
]. This is further supported by the recent observations of Schoorl et al. of an increase in platelets depleted from granules in patients undergoing chronic haemodialysis
]. Nevertheless, we cannot exclude an impairment of release.
The clinical bleeding tendency in uremic patients received much attention
]. Haemo- or peritoneal dialysis was found to improve haemostasis without correcting platelet aggregation defects. In contrast, compensatory mechanisms in the form of high von Willebrand factor (VWF) levels preserved relatively normal adhesion of uremic platelets to injured vessel wall models
]. Moreover, as soon as the hematocrit - determining the red blood cell-mediated radial transport of blood platelets towards the vessel wall - was corrected by recombinant erythropoietin, clinical bleeding became less prominent. In contrast, thromboembolic/cardiovascular morbidity remains an important problem in these patients
]. Especially in cardiorenal failure, there seems no or little benefit from treatment with antiplatelet agents, whilst risk of bleeding may increase
]. Even though reports on platelet reactivity in patient with chronic kidney disease show conflicting results
], it has been attributed to an acquired thrombocytopathy characterized by decreased aggregation of platelets in response to stimuli. There are only a few studies that used functional tests to study platelet function in chronic kidney disease. Aggarwal et. al. found a higher P-selectin expression in patients with end stage renal disease receiving haemodialysis compared to healthy controls after stimulation with a single concentration of ADP (0.2 μM). This suggests an increased reactivity
]. Moal et. al performed a similar study in which ADP (200 μM) and TRAP (50 μM) in a single concentration were used to stimulate platelets in healthy controls and end stage renal disease patients receiving haemodialysis. They found a lower P-selectin expression in patients compared to controls, indicating reduced platelet reactivity in patients with chronic kidney disease
]. Most assays used previously are influenced by in vitro
artifacts, platelet isolation and are dependent on VWF or hematocrit. In our functional assay there is no role for VWF and hematocrit. Moreover, since fresh whole blood was used, and samples were fixated subsequent to stimulation, in vitro
platelet activation was negligible. We couldn’t find an immediate effect of haemodialysis, but long term dialysis and classical risk factors for athero-vascular disease could all be studied with this assay. Larger populations should be studied in order to find the factors influencing platelet reactivity in vivo
In our study, aspirin did not suppress the expression of P-selectin on platelets. Considering the fact that aspirin function is related to inhibition of tromboxane formation and does not influences release of granules, this is an expected result. Moreover, our finding is in accordance with a study by Stumpf et. al., in which P-selectin expression on platelets did not show a difference between patients taking aspirin compared to non-aspirin users
]. Further investigation is required to determine the influence of different drugs on platelet reactivity.
We here demonstrate that the flow cytometry based platelet activation assay can be used in clinical practice to study variables influencing platelet response in patients with renal failure. The assay is based on incubation with different agonists in whole blood and subsequent fixation. Platelets are sensitive for in vitro artifacts (especially platelet isolation, shear stress, pH and temperature). In our assay platelets are not isolated. The incubation in whole blood under standardized condition and the subsequent fixation step makes in vitro artifacts unlikely. When repeating the assay within healthy subjects at different blood collection moments, we find high reproducibility. Moreover, this assay can be used in routine clinical practice.