Bleeding 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.

To 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 α_IIbβ₃ (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.

Carbamylation inhibited platelet activation, adhesion, and aggregation. Patients on hemodialysis exhibited significantly reduced activation of α_IIbβ₃ compared with healthy controls. We found significant carbamylation of both subunits of α_IIbβ₃ 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 α_IIbβ₃ activity in vitro.

Carbamylation of α_IIbβ₃—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 α_IIbβ₃ activity suggests amino acid administration during dialysis may help to normalize platelet function.

出血素质在 ESKD 患者中很常见，可导致严重的并发症，特别是在侵入性手术期间。慢性尿素超载显着增加了 ESKD 患者的氰酸盐浓度，导致氨甲酰化，这是蛋白质和肽的不可逆修饰。

为了研究氨甲酰化作为 ESKD 尿毒症和血小板功能障碍之间的潜在机制联系，我们使用液相色谱-串联质谱 (HPLC-MS/MS) 来定量总高瓜氨酸，并使用生物素偶联的苯基乙二醛标记和蛋白质印迹来检测氨甲酰化整合素α _IIb β ₃（血小板聚集所需的受体）。流式细胞术用于研究分离的血小板和富含血小板的血浆的活化。在瞬时转染系统中，我们测试了受体不同突变形式的活性和纤维蛋白原结合。我们在微孔板测定中评估了血小板粘附和聚集。

氨甲酰化抑制血小板活化、粘附和聚集。与健康对照相比，血液透析患者的α _IIb β _{3活性显着降低。}我们发现接受血液透析的患者血小板上α _IIb β _{3的两个亚基均发生显着的氨甲酰化，而对照组仅发生微小的改变。}在瞬时转染系统中，β ₃亚基中赖氨酸 185 的修饰与受体活性和纤维蛋白原结合的丧失相关。补充游离氨基酸被证明可以保护血液透析患者血浆蛋白免受氨甲酰化引起的损伤，并在体外防止α _IIb β ₃活性丧失。

α _IIb β ₃的氨甲酰化（特别是 K185 残基的修饰）可能代表尿毒症与血液透析患者初次止血功能障碍之间的机制联系。观察到游离氨基酸可防止氨甲酰化诱导的α _IIb β ₃活性丧失，这表明透析期间施用氨基酸可能有助于使血小板功能正常化。