Chen D, Riesbeck K, Kemball-Cook G, McVey J H, Tuddenham E G, Lechler R I, Dorling A
Department of Immunology, Imperial College School of Medicine, Hammersmith Hospital, London, UK.
Transplantation. 1999 Feb 15;67(3):467-74. doi: 10.1097/00007890-199902150-00021.
Thrombotic vascular occlusion occurs in disorders of diverse etiology, including atherosclerosis, vasculitis, and disseminated intravascular coagulation. The same process results in hyperacute rejection of renal allografts transplanted into sensitized patients and remains a major problem in experimental xenotransplantation.
We have previously described the design and expression of several genetic constructs encoding novel fusion proteins with anticoagulant properties. They are based on two naturally occurring soluble anticoagulant proteins, human tissue factor pathway inhibitor (hTFPI) and the leech protein hirudin, which act early and late in the clotting cascade, respectively. We report the expression of human hTFPI-CD4 on the surface of immortalized porcine endothelial cells (IPEC), and show that it functions across the species divide as evidenced by the binding of membrane-expressed porcine tissue factor (pTF)-human factor VIIa complexes.
Using a human plasma recalcification clotting assay, we distinguished between pTF-dependent and pTF-independent fibrin generation, and we have demonstrated that expression of hTFPI-CD4 on IPEC effectively prevented pTF-dependent clotting. Moreover, we show that when hTFPI-CD4 was co-expressed with the hirudin construct, the procoagulant properties of in vitro cultured, activated IPEC were almost completely abolished.
These results suggest that these novel anticoagulant molecules may prove useful therapeutic agents for gene therapy or for transgenic expression in animals whose organs may be used for cliniCal xenotransplantation.
血栓性血管闭塞发生于多种病因的疾病中，包括动脉粥样硬化、血管炎和弥散性血管内凝血。同样的过程导致移植到致敏患者体内的肾移植发生超急性排斥反应，并且在实验性异种移植中仍然是一个主要问题。
我们之前描述了几种编码具有抗凝特性的新型融合蛋白的基因构建体的设计和表达。它们基于两种天然存在的可溶性抗凝蛋白，即人组织因子途径抑制剂（hTFPI）和水蛭蛋白水蛭素，它们分别在凝血级联反应的早期和晚期起作用。我们报道了人hTFPI-CD4在永生化猪内皮细胞（IPEC）表面的表达，并表明它能跨越物种屏障发挥作用，膜表达的猪组织因子（pTF）-人因子VIIa复合物的结合证明了这一点。
使用人血浆复钙凝血试验，我们区分了pTF依赖性和pTF非依赖性纤维蛋白生成，并且我们证明了IPEC上hTFPI-CD4的表达有效地阻止了pTF依赖性凝血。此外，我们表明，当hTFPI-CD4与水蛭素构建体共表达时，体外培养的活化IPEC的促凝特性几乎完全被消除。
这些结果表明这些新型抗凝分子可能被证明是用于基因治疗或用于其器官可用于临床异种移植的动物的转基因表达的有用治疗剂。
