Treatment of Deep Vein Thrombosis via Targeted Inhibition of the FXII-uPAR-pAkt2 Axis in Neutrophils

这项提议的总体目标是建立一种有针对性的治疗策略，以扰乱 凝血因子FXII和尿激酶型纤溶酶原激活剂受体下调Akt2- 介导性中性粒细胞激活治疗深静脉血栓形成(DVT)深静脉血栓是导致 心血管疾病死亡。新的抗凝疗法已经开发出来，然而这些治疗方法 进展都与出血率的增加有关。此外，这些目前的抗凝剂只能抑制 凝血终点(如凝血酶和纤维蛋白)，但不能阻止上游事件，如中性粒细胞 中性粒细胞-血小板的激活、促凝中性粒细胞胞外陷阱(Net)的形成或增殖 聚集，所有这些都是DVT中持久的标志性事件。在此框架中，我们建议使用唯一的 基于纳米药物的治疗途径通过下调中性粒细胞激活和网络形成 有针对性地破坏FXII-uPAR-pAkt2轴。我们的实验室发现中性粒细胞中的FXII对 功能。具体地说，我们已经证明，在中性粒细胞激活后，通过uPAR自分泌FXII信号 导致Akt2S474磷酸化(PAkt2)和网络形成。抑制中性粒细胞FXII信号转导 有较小的静脉血栓。基于这些机制发现，我们的中心假设是靶向抑制 FXII-uPAR-pAkt2轴在治疗DVT方面将是有效的，同时最大限度地减少全身副作用- 影响和出血风险。我们将通过将uPAR抑制肽包装在纳米胶囊中来检验这一假说。 它们是独特的表面工程，可以在中性粒细胞-血小板上进行特定的异多价锚定 集合体。 在本申请中，我们的目标是：1)确定一种可破坏FXII结合的候选uPAR抑制肽 中性粒细胞的表面。我们将确定抑制的亲和力、化学计量比和特异性，评估Akt2 激活并进行中性粒细胞和血小板功能检测；2)使用异种多价修饰的纳米微囊 负载候选多肽药物以确定其位置选择性抑制FXII-uPAR的能力 在体外和体内相互作用和缓解DVT；3)为了验证这些临床前研究，我们将确定 FXII-uPAR-pAkt2轴的构成活性及其抑制对中性粒细胞功能的影响 血小板相互作用和血栓体外生长，使用新诊断的DVT患者的血液样本。 最终目标是显示FXII-uPAR-pAkt2轴和下游效应器在 这将为今后的临床研究抑制其作用奠定基础。 我们的科学创新是对中性粒细胞FXII-uPAR-pAkt2信号轴的机制阐明。 中介病理学。我们的技术创新是以抑制多肽为基础的有针对性的发展 纳米药物的策略，以阻断这一轴的治疗效益在深静脉血栓。因为FXII是为数不多的蛋白质之一 在不增加出血风险的情况下防止血栓形成，拟议中的研究有可能改进 抗凝剂治疗与抑制终末普通凝血的益处与风险的比较 目前的抗凝剂所特有的途径。如果成功，拟议的治疗策略将 引入新的更安全的疗法来治疗血栓性疾病，这种疾病在 退伍军人。

The overall goal of this proposal is to establish a targeted therapeutic strategy to disrupt the interaction of coagulation factor FXII (FXII) and urokinase plasminogen activator receptor (uPAR) to downregulate Akt2- mediated neutrophil activation for treatment of deep vein thrombosis (DVT). DVT is a leading cause of cardiovascular death. New anticoagulation therapies have been developed, however these therapeutic advances are all associated with increased rate of bleeding. Moreover, these current anticoagulants only inhibit coagulation end-points (e.g. thrombin and fibrin) but do not prevent upstream events such as neutrophil activation, procoagulant neutrophil extracellular trap (NET) formation, or propagation of neutrophil-platelet aggregates, all of which are persistent hallmark events in DVT. In this framework, we propose to use a unique nanomedicine-based therapeutic approach to downregulate neutrophil activation and NET formation through targeted disruption of the FXII-uPAR-pAkt2 axis. Our laboratory identified that FXII in neutrophils is critical for function. Specifically, we have shown that following neutrophil activation, autocrine FXII signals through uPAR leading to Akt2S474 phosphorylation (pAkt2) and NET formation. Inhibiting FXII signaling in neutrophils resulted in smaller venous thrombi. Based on these mechanistic findings, our central hypothesis is that targeted inhibition of the FXII-uPAR-pAkt2 axis will be therapeutically effective in treating DVT while minimizing systemic side- effects and bleeding risk. We will test this hypothesis by packaging uPAR inhibitory peptides within nanovesicles that are uniquely surface-engineered to undergo specific heteromultivalent anchorage onto neutrophil-platelet aggregates. In this application, our goals are to: 1) identify a candidate uPAR inhibitory peptide that disrupts FXII binding on the surface of neutrophils. We will determine the affinity, stoichiometry and specificity of inhibition, assess Akt2 activation and perform neutrophil and platelet function assays; 2) use heteromultivalently decorated nanovesicles loaded with the candidate peptide drug to determine their ability to site-selectively inhibit the FXII-uPAR interaction and mitigate DVT in vitro and in vivo; 3) to validate these preclinical studies, we will determine the constitutive activity of the FXII-uPAR-pAkt2 axis and the effect of its inhibition on neutrophil functions, neutrophil- platelet interactions and thrombus growth ex vivo, using blood samples from patients with newly diagnosed DVT. The end goal is to show the differential abundance of the FXII-uPAR-pAkt2 axis and downstream effectors in DVT pathology which will lay the foundation for future clinical studies to inhibit its action. Our scientific innovation is the mechanistic elucidation of the FXII-uPAR-pAkt2 signaling axis in neutrophil- mediated pathology. Our technological innovation is the development of inhibitory peptide-based targeted nanomedicine strategies to block this axis for therapeutic benefit in DVT. Since FXII is one of the few proteins that protects from thrombosis without increasing bleeding risk, the proposed studies have the potential to improve the benefit-to-risk profile of anticoagulant therapy in comparison with inhibition of the final common coagulation pathway that is characteristic of current anticoagulants. If successful, the proposed therapeutic strategy will introduce novel and safer therapies to treat thrombotic disorders, morbidities that are common among the Veteran population.