Carbamylation of Integrin α IIb β 3: The Mechanistic Link to Platelet Dysfunction in ESKD

Abstract

BackgroundBleeding diatheses, common among patients with ESKD, can lead to serious complications, particularly during invasive procedures. Chronic urea overload significantly increases cyanate concentrations in patients with ESKD, leading to carbamylation, an irreversible modification of proteins and peptides.MethodsTo investigate carbamylation as a potential mechanistic link between uremia and platelet dysfunction in ESKD, we used liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) to quantify total homocitrulline, and biotin-conjugated phenylglyoxal labeling and Western blot to detect carbamylated integrin αIIbβ3 (a receptor required for platelet aggregation). Flow cytometry was used to study activation of isolated platelets and platelet-rich plasma. In a transient transfection system, we tested activity and fibrinogen binding of different mutated forms of the receptor. We assessed platelet adhesion and aggregation in microplate assays.ResultsCarbamylation inhibited platelet activation, adhesion, and aggregation. Patients on hemodialysis exhibited significantly reduced activation of αIIbβ3 compared with healthy controls. We found significant carbamylation of both subunits of αIIbβ3 on platelets from patients receiving hemodialysis versus only minor modification in controls. In the transient transfection system, modification of lysine 185 in the β3 subunit was associated with loss of receptor activity and fibrinogen binding. Supplementation of free amino acids, which was shown to protect plasma proteins from carbamylation-induced damage in patients on hemodialysis, prevented loss of αIIbβ3 activity in vitro.ConclusionsCarbamylation of αIIbβ3—specifically modification of the K185 residue—might represent a mechanistic link between uremia and dysfunctional primary hemostasis in patients on hemodialysis. The observation that free amino acids prevented the carbamylation-induced loss of αIIbβ3 activity suggests amino acid administration during dialysis may help to normalize platelet function.

Protein Carbamylation and the Risk of ESKD in Patients with CKD

“…21,53,54 Recent translational work demonstrated pathologic carbamylation of a receptor required for platelet aggregation (potentially explaining some of the common bleeding diatheses seen in ESKD) could be ameliorated with amino acid supplementation in vitro . 55 In humans, amino acid administration during hemodialysis was able to lower C-Alb in a small proof-of-concept study without clinical end points. 54 In a predialysis CKD population, compared with a free diet, Di Iorio et al 56 achieved significant carbamylation reductions through dietary interventions using either a very low protein diet supplemented with ketoanalogues (nitrogen-free analogs of essential amino acids) or a Mediterranean diet.…”

Carbamylation Is Instrumental in End-Stage Kidney Disease Coagulopathies: The Impact on von Willebrand Factor and Platelet Functionality

“…These alterations contribute to the impaired function of platelets, a key factor in the abnormal clotting observed in ESKD patients. 17,18 In this study, we further explore the mechanisms by which carbamylation influences the interaction between vWF and its partner proteins, examining its impact on platelet aggregation and function. Our findings illuminate the complex ways carbamylation disrupts these essential processes, highlighting the multifaceted complications associated with ESKD.…”

“…exposure of platelets to cyanate led to structural changes in integrin αIIbβ3, a critical fibrinogen-binding protein, resulting in a fibrinogenbinding defect that impairs the aggregation of uremic platelets. 17 Furthermore, the carbamylation of fibrinogen itself influences the kinetics of fibrin clot formation, affecting the clot's structure and degradation. These changes are thought to contribute significantly to the disrupted hemostasis observed in ESKD patients.…”