Pihusch Verena, Pihusch Markus, Penovici Marius, Kolb Hans J, Hiller Erhard, Pihusch Rudolf
Klinikum Grosshadern der LMU München, Medizinische Klinik III, Marchioninistrasse 15, 81377 Münich, Germany.
Thromb Res. 2005;116(3):233-40. doi: 10.1016/j.thromres.2004.12.010. Epub 2005 Jan 25.
Hepatic veno-occlusive disease (VOD) is one of the most disastrous complications after allogeneic hematopoetic stem cell transplantation (HSCT). Thrombocytopenia with refractoriness to platelet transfusions suggests an increased platelet consumption in these patients. Interactions between platelets and endothelial cells might contribute to the hypercoagulable state at the sinusoidal endothelium as a central mechanism in the pathogenesis of VOD.
The influence of activated platelets on cultured human endothelial cells was investigated in vitro. We focused on the release of plasminogen activator inhibitor-1 (PAI-1) from endothelial cells which has earlier been found to be significantly elevated in plasma of VOD patients. Endothelial cells isolated from human umbilical cords (HUVEC) were incubated with activated platelets. The release of PAI-1 in the presence or absence of specific antibodies was determined by ELISA technique. Tissue factor (TF) expression on endothelial cells was observed by flowcytometric analysis.
HUVEC incubated with activated platelets were found to release significantly more PAI-1 compared to untreated cultures. The endothelial PAI-1-secretion after incubation of HUVEC with activated platelets was completely inhibited by an IgG monoclonal antibody against human transforming growth factor beta-1 (TGF beta-1). In contrast, PAI-1 production was not suppressed after inhibition of HUVEC-platelet-interaction by an IgG monoclonal antibody against CD154 (CD40L) expressed on the surface of activated platelets. An increased release of PAI-1 and an increased expression of tissue factor (TF) on the endothelial cell surface were observed after stimulation with TGF beta-1.
TGF beta-1 released from activated platelets contributes to the hemostatic imbalance at the sinusoidal endothelium in patients with hepatic VOD by increase of endothelial cell PAI-1 production and TF expression. As a potent profibrotic cytokine, TGF beta-1 might further be involved in phlebosclerosis and sinusoidal fibrosis occurring in VOD.
肝静脉闭塞病（VOD）是异基因造血干细胞移植（HSCT）后最严重的并发症之一。血小板减少且对血小板输注难治提示这些患者血小板消耗增加。血小板与内皮细胞之间的相互作用可能导致肝血窦内皮的高凝状态，这是VOD发病机制的核心环节。
体外研究活化血小板对培养的人内皮细胞的影响。我们关注内皮细胞纤溶酶原激活物抑制剂-1（PAI-1）的释放，此前已发现VOD患者血浆中该物质显著升高。将从人脐带分离的内皮细胞（HUVEC）与活化血小板共同孵育。采用酶联免疫吸附测定（ELISA）技术测定有无特异性抗体时PAI-1的释放情况。通过流式细胞术分析观察内皮细胞上组织因子（TF）的表达。
与未处理的培养物相比，发现与活化血小板共同孵育的HUVEC释放的PAI-1显著更多。用抗人转化生长因子β-1（TGFβ-1）的IgG单克隆抗体可完全抑制HUVEC与活化血小板共同孵育后的内皮PAI-1分泌。相反，用抗活化血小板表面表达的CD154（CD40L）的IgG单克隆抗体抑制HUVEC-血小板相互作用后，PAI-1的产生未受抑制。用TGFβ-1刺激后，观察到内皮细胞表面PAI-1释放增加以及组织因子（TF）表达增加。
活化血小板释放的TGFβ-1通过增加内皮细胞PAI-1产生和TF表达，导致肝VOD患者肝血窦内皮的止血失衡。作为一种强效促纤维化细胞因子，TGFβ-1可能进一步参与VOD中发生的静脉硬化和肝血窦纤维化。
