Building artificial platelets

描述(由申请人提供) 摘要：在这项提案中，我们描述了一项利用生物成分构建人造血小板的计划。这一愿景基于这样一种信念，即我们关于单个生物分子的知识的关键积累将使我们能够以有意义的方式将它们整合到一个系统中，创造出蜂窝设备。我们已经确定，血小板是这种模拟生物系统的一个容易处理的目标。我们的设计策略需要了解天然血小板的功能，以便人造血小板能够赋予天然血小板的基本功能。血小板是细胞对受损血管系统的第一反应。它与暴露的胶原蛋白结合，否则胶原蛋白就会受到内皮细胞的保护。血小板通过几种细胞表面受体的表达与活化相结合。重要的是，活化的血小板通过增加外层脂膜上的磷脂酰丝氨酸(PS)来获得主要带负电的膜。然后，被激活的血小板触发一系列被称为凝血级联的生化事件，导致形成交联的纤维蛋白网络，形成堵塞受损的血管系统的塞子。人造血小板将模仿天然血小板的基本功能。它们将被制成脂泡，定义了脂类和蛋白质的组成。微流控喷射是最近开发的一种技术，类似于从肥皂膜上吹泡泡，将被用来制造囊泡。这项技术允许创建不对称的脂质双层与内叶上的PS以及膜蛋白的掺入。这些囊泡将装饰上抗胶原抗体，在血管损伤期间将其结合到暴露的胶原蛋白上。我们的血小板激活策略将利用由膜张力门控的大机械敏感通道(MSCL)。当人造血小板附着在基质上时，剪切流将激活MSCL，这将导致血清中的钙进入囊泡。扰乱酶将是人造血小板的一部分，因此一旦钙离子进入，它就会被激活，并将PS暴露在外部的脂膜上。当PS暴露时，因子V和X被激活，随后刺激凝血酶活性。凝血酶然后将纤维蛋白原裂解成纤维蛋白，纤维蛋白组装成一个持久的、交联的网络。在我们人工血小板组装的每个阶段，设计策略都可以在模拟受损血管系统的微流体平台上进行测试和验证。总之，拟议的研究描述了我们对合成生物学如何潜在地带来在医疗环境中具有巨大好处的新型细胞设备的愿景。 公共卫生相关性：血小板是受损血管系统的第一个细胞反应者，但由于其保质期短、对污染的敏感性以及储存条件的挑战，它在血库中一直处于短缺状态。利用生物成分，我们提议建造模拟天然血小板功能的人造血小板。我们设想这样的情况 蜂窝设备将在医疗环境中带来巨大的好处。

DESCRIPTION (Provided by the applicant) Abstract: In this proposal, we describe a plan to build artificial platelets from biological components. The vision is based on the belief that the critical accumulation of our knowledge about individual biomolecules will enable us to integrate them into a system in a meaningful way to create cellular devices. We have identified platelets as a tractable target of such emulated biological system. Our design strategy necessitates an understanding of the functionalities of natural platelets so that the artificial platelets can confer the essential functions of natural platelets. Platelets are the first cellular responder to injured vasculature. It binds to the exposd collagen that is otherwise protected by endothelial cells. Platelets couple binding to activation via the expression of several cell surface receptors. Importantly, activated platelets attain predominantly negatively charged membranes by increasing phosphatidylserine (PS) on the outer lipid leaflet. Activated platelets then trigger a series of biochemical events known as the coagulation cascade that results in the formation of cross-linked fibrin network that forms a plug to block the injured vasculature. The artificial platelets will mimic the essential functions of natural platelets. They will be made as lipid vesicles that have defined lipid and protein composition. Microfluidic jetting, a technique recently developed that is akin to blowing bubble from a soap film, will be used to make the vesicles. This technique allows the ability to create asymmetric lipid bilayer with PS on the inner leaflet as well as incorporation of membrane proteins. The vesicles will be decorated with anti-collagen antibody that will bind to the exposed collagen during vascular injury. Our platelet activation strategy will utilize the large mechanosensitive channel (MscL) that is gated by membrane tension. When the artificial platelets attaches to the substrate, shear flow will activate MscL, this would then causes calcium in the blood serum to enter the vesicles. Scramblase will be part of the artificial platelet such that upon calcium entry, it becomes activated and exposes PS to the outer lipid leaflet. When PS is exposed, factors V and X become activated and subsequently stimulate thrombin activity. Thrombin then cleaves fibrinogen into fibrin, which assembles into a durable and cross-linked network. At each stage of our artificial platelet assembly, the design strategy can be tested and validated in a microfluidic platform that mimics the injured vasculature. In summary, the proposed research describes our vision for how synthetic biology can potentially bring about novel cellular devices that would have tremendous benefits in medical settings. Public Health Relevance: Platelets are the first cellular responder to injured vasculature, but it is constantly under shortage in the blood bank due to its short shelf life, susceptibility to contamination, and challenges in storage condition. Using biological components, we propose to build artificial platelets that mimic the functionalities of natural platelets. We envision such cellular devices will bring tremendous benefits in medical settings.