Therapeutic assessment of synthetic heparin sulfates in transplantation models

尽管猪到灵长类动物器官异种移植取得了重大进展， 目前受到血栓性微血管病和血小板隔离的限制， 移植器官和/或消耗性凝血病。目前的建议测试同质 使用所述的计算建模和酶/化学工具合成硫酸肝素（HS） 在项目I（PL-I. Balagurunathan）和II（Pl-II. Desai博士）。我们假设化学合成的 被设计为优先抑制因子Xa（FXa）和/或凝血酶的HS将改善血栓形成。 在猪至灵长类动物异种移植物中观察到的微血管病和/或消耗性凝血病， 抑制凝血酶形成和血小板活化。此外，HS被优化以结合趋化因子（来自 项目III）可以作为趋化因子诱饵因子，与 抗凝这些HS将在各种异种移植模型的背景下进行评价， 来自猪的细胞和器官经遗传修饰以保护免受抗体/补体介导的， 炎症和凝血排斥机制。这些基于HS的治疗将由进行检测。- （i）在 体外测定以确定用于进一步研究的最佳HS候选药物，（ii）离体猪肺血液灌流 使用人血，（iii）狒狒中的猪动脉Tx，和（iv）狒狒中的猪肾Tx。模型（二）构成了 短期模型（以小时为单位测量移植物功能）允许快速筛选HS， 模型（iii）和（iv）构成了持续时间较长的模型（天或周）。而且 将监测HS的作用（iii）在无免疫抑制治疗的低抗原负荷模型中，或（iv） 在高抗原负荷模型中，移植受体将接受免疫抑制治疗， 抑制适应性T细胞反应。该研究的主要目的是确定一个（或多个）HS， 在抑制血栓性微血管病的发展方面更特异和更有效， 消耗性凝血病比目前临床上使用的肝素，而且，此外， 由于肝素的副作用，例如，出血，这限制了可以施用的剂量。第二 目的是研究鉴定为最佳的HS与遗传操作之间的协同作用 在器官源猪中，例如，针对抑制血栓形成的人血栓调节蛋白的表达 微血管病和消耗性凝血病。 相关性（参见说明）： 从基因工程猪身上移植器官将提供一个替代来源， 用于临床移植的人体器官，但目前因凝血障碍而复杂化， 是致命的。具体的合成硫酸乙酰肝素的管理，将设计和研究， 这一建议可能会防止这些凝血障碍，从而使异种移植， 推进临床试验。

Despite significant advances in pig-to-primate organ xenotransplantation, long-term graft survival is currently limited by the development of a thrombotic microangiopathy and platelet sequestration in the grafted organ and/or a consumptive coagulopathy in the recipient. The present proposal tests homogeneous heparin sulfates (HSs) synthesized using computational modeling and enzymatic/chemical tools as outlined in Projects I (PL-I. Dr. Balagurunathan) and II (Pl-ll. Dr. Desai). We hypothesize that chemically-synthesized HSs designed to preferentially inhibit Factor Xa (FXa) and/or thrombin will ameliorate the thrombotic microangiopathy and/or consumptive coagulopathy seen in the context of pig-to-primate xenografts by inhibiting thrombin formation and platelet activation. In addition, HSs optimized to bind chemokines (from Project III) may act as chemokine decoy factors to prevent cell sequestration synergistically with anticoagulation. These HSs will be evaluated in the context of various xenotransplantation models using cells and organs from pigs genetically modified to protect against antibody/complement-mediated, inflammatory, and coagulation rejection mechanisms. These HS-based therapies will be tested by. - (i) in vitro assays to determine the best HS candidate drugs for further studies, (ii) ex vivo pig lung hemoperfusion using human blood, (iii) pig artery Tx in baboons, and (iv) pig kidney Tx in baboons. Model (ii) constitutes a short-duration model (with graft function measured in hours) allowing rapid screening of the HSs being tested, whereas models (iii) and (iv) constitute longer-duration models (days or weeks). Furthermore, the effect of the HS will be monitored (iii) in a low-antigen load model without immunosuppressive therapy, or (iv) in a high-antigen load model in which graft recipients will receive immunosuppressive therapy aimed at inhibiting the adaptive T cell response. The prime aim of the study is to identify one (or more) HS that is more specific and more efficient at inhibiting the development of thrombotic microangiopathy and consumptive coagulopathy than is the current clinically-used heparin, and that, furthermore, is not associated with the side-effects of heparin, e.g., bleeding, that limit the dosage that can be administered. The second aim is to investigate the synergy between the HS identified as being optimal with the genetic manipulations in the organ-source pig, e.g., expression of human thrombomodulin directed towards inhibiting thrombotic microangiopathy and consumptive coagulopathy. RELEVANCE (See instructions): The transplantation of organs from genetically-engineered pigs would provide an alternative source to human organs for clinical transplantation, but is currently complicated by coagulation disturbances that can be fatal. The administration of specific synthetic heparan sulfates that will be designed and investigated in this proposal are likely to prevent these coagulation disturbances, thus enabling xenotransplantation to advance to clinical trials.