Heteromutivalent Peptide-Lipid Nanoconstructs as Artificial Platelet Analogues

血小板通过损伤部位选择性的多步骤机制在止血中发挥核心作用：（1）粘附vWF 和胶原蛋白，（2）纤维蛋白原介导的聚集以形成初级止血栓，（3）生物界面 阴离子磷脂酰丝氨酸（PS）在活化血小板表面的呈递用于促凝血放大 凝血酶（因此是纤维蛋白）的凝块定位分泌，和（4）血小板颗粒内容物（如无机 聚磷酸盐（PolyP）以局部增强纤维蛋白稳定性。这些机制受到严重损害， 不可压缩的创伤性出血，这仍然是死亡的主要原因。"黄金标准" 治疗这种出血是大量输注全血或成分（血小板、血浆、RBC）。 尤其是血小板输注在创伤救治中显示出巨大的临床效益。然而，在这方面， 血小板很少在资源有限的医院和不可用的院前，由于挑战， 储存、便携性、细菌污染的高风险和非常短的保质期（~5天）。我们旨在解决 通过设计基于生物材料的“人造血小板”纳米结构来应对这一挑战。为此，利用 先前的R 01奖（HL 121212），我们开发了自组装的脂质-肽纳米结构， 整合上述（1）和（2）的血小板机制。该设计显示了体外止血能力， 在血小板减少的小鼠尾部出血模型中，以及在严重创伤模型中的适度功效。在此基础上， 我们现在建议通过设计独特的酶- 响应性脂肽，随后将允许所有四种机制整合到单个 纳米构建体用于上级人造血小板设计。我们的中心假设是“模块放大"， 通过模拟人工血小板内血小板生物界面和分泌机制实现止血 构建物可以显著减少出血并提高创伤中的存活率。为了验证这一点，我们的具体目标 （1）评价刺激物（纤溶酶）触发的PS暴露于血小板激发的 创伤中凝血酶（因此纤维蛋白）位点特异性扩增;（2）评估刺激（凝血酶）触发 无机多磷酸盐（PolyP）作为有效载荷从用于损伤位点靶向的生物纳米构建体中释放 稳定纤维蛋白凝块;和（3）将这些独立协同成分整合到人工血小板中 在啮齿动物创伤模型中评估止血功效和存活的纳米构建物。创伤性的侮辱 血管内皮导致凝块处组织纤溶酶原激活物（因此纤溶酶）的分泌增强 这导致快速纤维蛋白降解（纤维蛋白溶解亢进）并损害凝块稳定性。利用这一 纤溶酶将PS暴露在“人造血小板”表面上将允许增强凝血酶（和因此纤维蛋白）的生成 抵消纤维蛋白溶解亢进这种凝血酶也可以作为局部触发剂，使“人造血小板”不稳定。 构建并释放包封的PolyP，以增强纤维蛋白稳定性并增强止血。我们的主要 创新在于在单个纳米结构上独特地模拟血小板的多步止血机制。

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.