INFLUENCE OF CHRONIC KIDNEY DISEASE ON THE HAEMOSTATIC PROPERTIES, THE PLATELET TRANSCRIPTOMIC AND PLASMA PROTEOMIC PROFILES OF CORONARY ARTERY DISEASE PATIENTS

BACKGROUND - Patients with CKD present a higher incidence of coronary artery disease (CAD) and increasing evidence indicates that these patients are more likely to die of cardiovascular disease than to develop kidney failure. CKD patients show a double haemostatic profile: thrombotic tendencies, but also a bleeding diathesis. Platelets and Tissue Factor (TF), the main activator of blood coagulation, play a key role in the pathogenesis and in the thrombotic complications of CAD. TF is expressed also by circulating platelets and, of interest, platelet-associated TF expression is higher in acute coronary syndrome (ACS) patients. However, there are no studies focused on platelet-associated TF expression in CAD patients with CKD. Moreover, although it is reported that platelet transcriptome may vary in pathological conditions, no information is still available on the changes that may occur in platelet transcriptome of CAD patients with CKD. Finally, patients with end-stage renal disease (ESRD) present changes in the expression of plasma proteins associated to atherosclerosis, but no data are so far available on the plasma proteomic profile of CAD patients with mild-to-moderate CKD. AIM - The aim of this project has been to further characterize the haemostatic tendencies (assessment of TF expression and of the global haemostatic function of whole blood), the platelet transcriptome and the plasma proteome of CAD patients with and without CKD. METHODS - 139 ACS patients with (n=31) and without CKD (n=108) and 217 SA patients with (n=49) and without CKD (n=168) were enrolled. The glomerular filtration rate (GFR) has been calculated with the MDRD CKD EPI Equation and CKD is defined by an estimated GFR value < 60 ml/min. Assessment of surface and intracellular platelet TF expression has been performed by whole blood flow cytometry; the global haemostatic function by thromboelastometry (ROTEM); platelet transcriptome profiles using Illumina BeadChip Human HT-12 v4 microarray; the plasma proteomic profile by the classical gel-based proteomic approach and the results confirmed by ELISA analysis. RESULTS - The percentage of TF expression on platelet surface is significantly lower in CKD patients compared to patients without CKD in resting condition (ACS: 4.07%±1.05 and 5.23%±0.82 respectively, p<0,05; SA: 1.96%±0.8 versus 2.72%±0.41, respectively, p<0,05) and this trend is also observed after ADP stimulation (ACS: 20.32%±2.58 and 21.71%±1.52 respectively, p>0,05; SA: 12.93%±2.73 and 16.54%±1.52 respectively, p>0.05). This result is also supported by the intracellular expression of platelet TF performed on a subgroup of ACS patients: intracellular TF is significantly lower in patients with CKD compared to those without CKD (19.49%±4.04 and 27.53%±5.16 respectively, p<0,001). The haemostatic function of blood, assessed by thromboelastometry, shows that, in physiological conditions, the presence of CKD exerts different effects on the global haemostatic potential in SA and ACS patients, but in the presence of a platelet inhibitor, the formation of a pure fibrin clot is similar between these two groups of patients. Moreover, there is differential gene expression among patients with and without CKD: in particular, in CKD patients, there is the over-expression of genes involved in platelet activation and in the defence against oxidative damage, but also the down-regulation of translational elongation genes that may affect the de novo protein synthesis capacity of platelets from CKD patients. The proteomic analysis shows significant differences among patients with and without CKD (both SA and ACS) in the expression of alpha-1-Microglobulin (A1M), an anti-oxidant protein, Retinol Binding Protein 4 (RBP4), involved in the early phases of inflammation and Haptoglobin (HPT), an anti-inflammatory protein: all these proteins have a positive correlation with the severity of the renal pathology. CONCLUSIONS - In conclusion, all these data shed new light on additional mechanisms involved in CKD-associated haemostatic (thrombotic and haemorrhagic) profile of CAD patients. The peculiar platelet phenotype (lower amount of TF expression), found in CKD patients, provides information on the bleeding risk due to the platelet dysfunction responsible for the haemostatic abnormalities. The evaluation of the global haemostatic capacity of whole blood has shown that ACS patients with CKD seem to have a platelet function more “compromised” compared to SA patients with CKD, in terms of clotting time, that is slower in ACS patients. Furthermore, CKD patients present also a differential gene expression profile and differentially expressed proteins compared to patients without CKD: there is the concomitant presence of transcripts and proteins with a protective function (anti-inflammatory and anti-oxidant), but also with the role to promote the progression of CKD and the development of the thrombotic/haemorrhagic complications.