Background

Severe leptospirosis is frequently complicated by a hemorrhagic diathesis, of which the pathogenesis is still largely unknown. Thrombocytopenia is common, but often not to the degree that spontaneous bleeding is expected. We hypothesized that the hemorrhagic complications are not only related to thrombocytopenia, but also to platelet dysfunction, and that increased binding of von Willebrand factor (VWF) to platelets is involved in both platelet dysfunction and increased platelet clearance.

Methodology/Principal findings

A prospective study was carried out in Semarang, Indonesia, enrolling 33 hospitalized patients with probable leptospirosis, of whom 15 developed clinical bleeding, and 25 healthy controls. Platelet activation and reactivity were determined using flow cytometry by measuring the expression of P-selectin and activation of the α_IIbβ₃ integrin by the binding of fibrinogen in unstimulated samples and after ex vivo stimulation by the platelet agonists adenosine-diphosphate (ADP) and thrombin-receptor activating peptide (TRAP). Platelet-VWF binding, before and after VWF stimulation by ristocetin, as well as plasma levels of VWF, active VWF, the VWF-inactivating enzyme ADAMTS13, thrombin-antithrombin complexes (TAT) and P-selectin were also measured. Bleeding complications were graded using the WHO bleeding scale. Our study revealed that platelet activation, with a secondary platelet dysfunction, is a feature of patients with probable leptospirosis, especially in those with bleeding manifestations. There was a significant inverse correlation of bleeding score with TRAP-stimulated P-selectin and platelet-fibrinogen binding (R = -0.72, P = 0.003 and R = -0.66, P = 0.01, respectively) but not with platelet count. Patients with bleeding also had a significantly higher platelet-VWF binding. Platelet counts were inversely correlated with platelet-VWF binding (R = -0.74; P = 0.0009. There were no correlations between platelet-VWF binding and the degree of platelet dysfunction, suggesting that increased platelet-VWF binding does not directly interfere with the platelet α_IIbβ₃ signaling pathway in patients with probable leptospirosis.

Conclusion/Significance

Platelet dysfunction is common in probable leptospirosis patients with manifest bleeding. Increased VWF-platelet binding may contribute to the activation and clearance of platelets.

中文翻译：

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血小板功能障碍导致疑似钩端螺旋体病患者出血并发症

背景

严重的钩端螺旋体病常常并发出血素质，其发病机制仍然很大程度上未知。血小板减少症很常见，但通常没有达到预期的自发出血程度。我们假设出血并发症不仅与血小板减少有关，还与血小板功能障碍有关，而血管性血友病因子（VWF）与血小板的结合增加与血小板功能障碍和血小板清除率增加有关。

方法/主要发现

在印度尼西亚三宝垄进行了一项前瞻性研究，招募了 33 名疑似钩端螺旋体病住院患者，其中 15 名出现临床出血，另外 25 名健康对照。使用流式细胞术测定 P-选择素的表达和通过未刺激样品中纤维蛋白原的结合以及血小板激动剂二磷酸腺苷 (ADP)和体外刺激后的 α _IIb β ₃整联蛋白的激活来测定血小板活化和反应性。凝血酶受体激活肽（TRAP）。还测量了瑞斯托菌素刺激 VWF 之前和之后的血小板-VWF 结合，以及 VWF、活性 VWF、VWF 失活酶 ADAMTS13、凝血酶-抗凝血酶复合物 (TAT) 和 P-选择素的血浆水平。使用世界卫生组织出血量表对出血并发症进行分级。我们的研究表明，血小板活化以及继发性血小板功能障碍是疑似钩端螺旋体病患者的一个特征，尤其是那些有出血表现的患者。出血评分与 TRAP 刺激的 P-选择素和血小板纤维蛋白原结合呈显着负相关（分别为 R = -0.72，P = 0.003 和 R = -0.66，P = 0.01），但与血小板计数无关。出血患者的血小板-VWF 结合也显着升高。血小板计数与血小板-VWF 结合呈负相关（R = -0.74；P = 0.0009。血小板-VWF 结合与血小板功能障碍程度之间没有相关性，表明血小板-VWF 结合增加不会直接干扰血小板疑似钩端螺旋体病患者的α _IIb β _{3信号通路。}

结论/意义

血小板功能障碍在可能患有明显出血的钩端螺旋体病患者中很常见。VWF-血小板结合的增加可能有助于血小板的激活和清除。