Heteromutivalent Peptide-Lipid Nanoconstructs as Artificial Platelet Analogues

血小板通过损伤部位选择性多步骤机制在止血中发挥中心作用：(1)与vWF的黏附 和胶原，(2)纤维蛋白原介导的聚集形成主要止血栓，(3)生物界面 激活的血小板表面呈现阴离子磷脂酰丝氨酸(PS)用于促凝血扩增 凝血酶(因此是纤维蛋白)，以及(4)凝块局限性的血小板颗粒内容物的分泌(例如无机物 聚磷酸盐，息肉)，以提高局部纤维蛋白的稳定性。这些机制在以下方面受到严重损害 不可压缩的创伤性出血，这仍然是死亡的主要原因。中国的“黄金标准” 治疗这种出血的方法是大量输注全血或成分(血小板、血浆、红细胞)。 尤其是在创伤救治方面，血小板输注已显示出巨大的临床效益。然而， 由于以下方面的挑战，在资源有限的医院和院前无法获得血小板很少 储存方便，细菌污染风险高，保质期很短(~5天)。我们的目标是解决 这一挑战是通过设计基于生物材料的“人造血小板”纳米结构来实现的。为此，利用 在之前的R01奖(HL121212)中，我们开发了自组装脂肽纳米结构，模拟和 综合上述(1)和(2)的血小板机制。该设计在体外显示了止血能力，并 在血小板减少性小鼠尾部出血模型中，在严重创伤模型中疗效不佳。在此基础上， 我们现在建议通过设计独特的酶来在脂质体模板上模拟(3)和(4)的机制。 响应性脂肽，这将随后允许将所有四种机制整合到单个 用于卓越的人造血小板设计的纳米结构。我们的中心假设是‘模数放大 在人造血小板内模拟血小板的生物界面和分泌机制止血 构建能显著减少出血，提高创伤患者的存活率。为了测试这一点，我们的具体目标是 目的是：(1)评估刺激(纤溶酶)触发的PS在脂类纳米结构上的暴露，以获得血小板激发的 凝血酶(因此是纤维蛋白)在创伤中的局部特异性放大；(2)评估刺激(凝血酶)触发的 用于损伤部位靶向的脂质纳米结构的无机聚磷酸盐(Polyp)作为有效载荷的释放 稳定纤维蛋白凝块；以及(3)在人工血小板中整合这些独立的协同成分。 纳米结构用于评估啮齿动物创伤模型的止血效果和存活率。对…的创伤性侮辱 血管内皮导致血栓中组织纤溶酶原激活物(从而纤溶酶)的分泌增加 导致纤维蛋白迅速降解(纤溶功能亢进)，并影响凝块的稳定性。利用这一点 纤溶酶将PS暴露在人造血小板表面将增强凝血酶(从而纤维蛋白)的生成 来抵消纤溶亢进。然后，这种凝血酶也可以作为局部触发器，破坏“人造血小板”的稳定。 构建和释放被包裹的息肉，以增强纤维蛋白的稳定性和增强止血。我们的委托人 创新之处在于在单个纳米结构上独一无二地模仿了血小板的多步骤止血机制。

Platelets play a central role in hemostasis via injury site-selective multi-step mechanisms of: (1) Adhesion to vWF and collagen, (2) Fibrinogen-mediated aggregation to form the primary hemostatic plug, (3) Biointerfacial presentation of anionic phosphatidylserine (PS) on the activated platelet surface for procoagulant amplification of thrombin (hence fibrin), and (4) clot-localized secretion of platelet granule contents (e.g. inorganic polyphosphate, PolyP) to locally enhance fibrin stability. These mechanisms are significantly compromised in non-compressible traumatic hemorrhage, which remains a major cause of mortality. The `gold standard' for treating such hemorrhage is massive transfusion of whole blood or components (platelets, plasma, RBC). Especially, platelet transfusion has shown tremendous clinical benefit in saving lives in trauma. However, platelets are rarely available in resource-limited hospitals and unavailable pre-hospital, due to challenges of storage, portability, high risk of bacterial contamination and very short shelf-life (~5 days). We aim at addressing this challenge by designing biomaterials-based `artificial platelet' nanoconstructs. To this end, utilizing a previous R01 award (HL121212) we developed self-assembled lipid-peptide nanoconstructs that mimic and integrate the platelet mechanisms of (1) and (2) stated above. This design showed hemostatic ability in vitro and in thrombocytopenic mouse tail-bleeding models, and modest efficacy in severe trauma models. Building on this, we now propose to mimic the mechanisms of (3) and (4) on a liposomal template by designing unique enzyme- responsive lipopeptides, that will subsequently allow integration of all four mechanisms onto a single nanoconstruct for a superior artificial platelet design. Our central hypothesis is `Modular amplification of hemostasis via mimicry of platelet's biointerfacial and secretory mechanisms within an artificial platelet construct can significantly attenuate hemorrhage and enhance survival in trauma'. To test this, our Specific Aims are to: (1) Evaluate stimuli (plasmin)-triggered exposure of PS on lipidic nanoconstructs for platelet-inspired amplification of thrombin (hence fibrin) site-specifically in trauma; (2) Evaluate stimuli (thrombin)-triggered release of inorganic polyphosphate (PolyP) as a payload from lipidic nanoconstructs for injury site-targeted stabilization of fibrin clot; and (3) Integrate these independent synergistic components in artificial platelet nanoconstructs to evaluate hemostatic efficacy and survival in rodent trauma model. The traumatic insult to vascular endothelium results in enhanced secretion of tissue plasminogen activator (hence plasmin) at the clot site, resulting in rapid fibrin degradation (hyperfibrinolysis) and compromising clot stability. Exploiting this plasmin to expose PS on `artificial platelet' surface will allow enhanced thrombin (and hence fibrin) generation to offset hyperfibrinolysis. This thrombin can then also act as a local trigger to destabilize the `artificial platelet' constructs and release encapsulated PolyP to enhance fibrin stability and augment hemostasis. Our principal innovation is in uniquely mimicking platelet's multi-step mechanisms of hemostasis on a single nanoconstruct.