Defective Responses of Uremic Platelets in ADP-Induced Platelet Aggregation.
Authors: Chen Chi , He Zili , Kalavar Madhumati , Khalil Maged , Arora Alka , Naik Seema , Dumlao Theresa , Chou Shyan-Yih , Steier Williams 
Published: April 2020

Abstract
Nitrogenous uremic products in the blood have been considered responsible for uremia-related platelet dysfunction. Recent studies suggest uremic platelets are defective in thromboxane A2 metabolism, independent of plasma factors. In order to gain a better view on platelet dysfunction in uremia, we generated an animal model in rat by ligating bilaterally kidney hilar structure offset with a capillary tube (16- to 18-gauge) with a success rate of 72.2%. The resulted experimental uremic rats display elevated serum BUN and creatinine levels (112.7 ± 67.29 mg/dL, n =14, and 3.27 ± 0.95 mg/dL, n =14, respectively) on Day 7 after kidney ligation, compared to those sham operated controls (BUN: 16.7 ± 5.77 mg/dL, n =14, creatinine: 0.65 ± 0.33 mg/dL, n =14). Pathological examination of kidney sections from ligated rats reveals distended renal pelvis and dilated distal tubules with limited ischemic changes of tubular wall cells, consistent with hydronephrotic features. Semi-quantitative RT-PCR of whole kidney mRNA shows dramatic increase of kidney-injury molecule 1 (KIM-1) and reduced aquaporin-2 (Aqp2) message levels, indicating renal injuries by obstruction. Functions of uremic platelets were analyzed by an optical platelet aggregometer using separated platelets supplemented with control or uremic plasma after washing, in response to various concentrations of ADP. Uremic platelets in their homogeneous plasma (PlateletUremic/PlasmaUremic) demonstrated reduced aggregation in all ADP concentrations tested, with significant reductions at 5 and 10 uM of ADP (45.8 ± 11.9%, n =12, and 48.7 ± 12.0 %, n =12, respectively), compared to those of control platelets in control plasma (PlateletControl/PlasmaControl, 56.6 ± 10.1%, n =12, and 60.8 ± 7.9%, n =12, respectively). In plasma-exchanged aggregation studies, heterogeneous uremic or control plasma were used to resuspend control or uremic platelets. When control platelets supplemented with uremic plasma (PlateletControl/PlasmaUremic) and induced to aggregate with ADP, percentage aggregation achieved were reduced significantly at low concentrations of ADP (2.5 and 1.25 uM) to levels of 35.5 ± 13.9%, n =12, and 19.3 ± 16.9%, n =12, respectively, suggesting suppressing effects of uremic plasma on platelet aggregation. When platelets from uremic rats were tested in control plasma (PlateletUremic/PlasmaControl), aggregation was also decreased significantly at lower ADP concentration (1.25uM), to 18.7 ± 12.2%, n =12, compared with that of PlateletControl/PlasmaControl (34.7 ± 14.4%, n =12), revealing a hindered response of uremic platelets when activated by lower concentrations of exogenous ADP. Since our data from RT-PCR of platelet-derived total RNA did not support any reductions in the expression levels of platelet ADP receptor P2Y12 in uremic rats, the above response pattern of uremic platelets may suggest a decreased ADP storage or release during activation and warrant further investigations.