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

该提案的总体目标是建立一种靶向治疗策略，以破坏 凝血因子FXII（FXII）和尿激酶纤溶酶原激活物受体（uPAR）下调Akt 2- 介导的中性粒细胞活化用于治疗深静脉血栓形成（DVT）。DVT是导致 心血管死亡已经开发了新的抗凝治疗，但是这些治疗 所有进展都与出血率增加有关。此外，这些目前的抗凝剂仅抑制 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 活化、促凝血中性粒细胞胞外陷阱（NET）形成或嗜中性粒细胞-血小板增殖 聚集体，所有这些都是DVT中的持续标志性事件。在这个框架中，我们建议使用一个独特的 一种基于纳米医学的治疗方法，通过下调中性粒细胞活化和NET形成， 靶向破坏FXII-uPAR-pAkt 2轴。我们的实验室发现，中性粒细胞中的FXII对于 功能具体来说，我们已经表明，中性粒细胞活化后，自分泌FXII信号通过uPAR 导致Akt 2S 474磷酸化（pAkt 2）和NET形成。抑制中性粒细胞中的FXII信号传导导致 在较小的静脉血栓中。基于这些机制的发现，我们的中心假设是， 的FXII-uPAR-pAkt 2轴将在治疗DVT中是治疗有效的，同时最小化全身性副作用。 影响和出血风险。我们将通过在纳米囊泡内包装uPAR抑制肽来检验这一假设 其被独特地表面工程化以进行特异性异多价锚定到嗜血小板-血小板上 集料. 在本申请中，我们的目标是：1）鉴定破坏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、化学计量和特异性，评估Akt 2 活化并进行中性粒细胞和血小板功能测定; 2）使用异多价修饰的纳米囊泡 以测定它们位点选择性抑制FXII-uPAR的能力 相互作用并减轻体外和体内DVT; 3）为了验证这些临床前研究，我们将确定 FXII-uPAR-pAkt 2轴的组成性活性及其抑制对中性粒细胞功能、中性粒细胞- 血小板相互作用和血栓生长，使用新诊断DVT患者的血液样本。 最终目标是显示FXII-uPAR-pAkt 2轴和下游效应子在细胞中的差异丰度。 这将为今后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中FXII-uPAR-pAkt 2信号传导轴的机制阐明， 介导的病理学我们的技术创新是开发基于抑制肽的靶向 纳米医学策略来阻断这一轴，以获得DVT的治疗益处。因为FXII是少数几种 在不增加出血风险的情况下防止血栓形成，拟议的研究有可能改善 与抑制最终常见凝血相比，抗凝治疗的获益-风险特征 这是目前抗凝剂的特征。如果成功，所提出的治疗策略将 引入新的和更安全的疗法来治疗血栓性疾病， 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.