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

通过靶向抑制中性粒细胞中的 FXII-uPAR-pAkt2 轴治疗深静脉血栓形成

• 批准号: 10044407
• 负责人: Evi X. Stavrou
• 金额: --
• 依托单位: LOUIS STOKES CLEVELAND VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-10-01 至 2023-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10044407
• 关键词: Acute; Adhesions; Affinity; Amino Acids; Anticoagulant therapy; Anticoagulants; Anticoagulation; Basic Science; Binding; Binding Sites; Biological Assay; Blood Coagulation Factor; Blood Platelets; Blood specimen; Cardiovascular system; Cessation of life; Characteristics; Chemotaxis; Clinical Research; Coagulation Process; Deep Vein Thrombosis; Development; Disease; Engineering; Event; Factor XII; Fibrin; Foundations; Future; Generations; Goals; Growth; Healthcare Systems; Hemorrhage; Hemostatic function; Human; Immune system; In Vitro; Inflammation; Interphase Cell; Investigation; Kinetics; Laboratories; Lead; Leukocyte Elastase; Leukocytes; Libraries; Ligands; Ligation; Measurement; Mediating; Microfluidics; Modeling; Morbidity - disease rate; Mus; Neutrophil Activation; Neutrophil Infiltration; Newly Diagnosed; P-Selectin; Pathology; Pathway interactions; Patients; Peptide Receptor; Peptides; Phosphorylation; Property; Proteins; Receptor Activation; Risk; Signal Transduction; Site; Specificity; Surface; System; Testing; Therapeutic; Therapeutic Effect; Thrombin; Thrombosis; Thrombus; Treatment Efficacy; Urokinase Plasminogen Activator Receptor; Veins; Venous; Venous Thrombosis; alpha 1-Antitrypsin; autocrine; base; blocking factor; cellular targeting; clinical translation; design; effectiveness evaluation; extracellular; improved; in vivo; in vivo Model; innovation; military veteran; mouse model; nanoformulation; nanomedicine; nanovesicle; neutrophil; next generation; novel; novel strategies; novel therapeutic intervention; peptide drug; platelet function; preclinical study; protein aminoacid sequence; receptor binding; screening; side effect; stoichiometry; targeted delivery; targeted treatment; technological innovation; therapeutically effective; thromboinflammation; translational approach

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.

该提案的总体目标是建立有针对性的治疗策略，以破坏 凝血因子FXII（FXII）和尿激酶纤溶酶原激活剂受体（UPAR）下调Akt2- 介导的嗜中性粒细胞激活，用于治疗深静脉血栓形成（DVT）。 DVT是主要原因 心血管死亡。已经开发了新的抗凝治疗，但是这些治疗方法 进步都与出血率提高有关。此外，这些当前的抗凝剂仅抑制 凝结终点（例如凝血酶和纤维蛋白），但不能防止上游事件，例如中性粒细胞 激活，促凝中性粒细胞外陷阱（净）形成或中性粒细胞植物的传播 聚集体，所有这些都是DVT中持续的标志性事件。在此框架中，我们建议使用独特的 基于纳米医学的治疗方法，通过下调中性粒细胞激活和净形成 FXII-UPAR-PAKT2轴的目标破坏。我们的实验室确定中性粒细胞中的FXII对于 功能。具体而言，我们已经表明，嗜中性粒细胞激活后，自分泌FXII信号通过UPAR 导致AKT2S474磷酸化（PAKT2）和净形成。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。基于这些机械性发现，我们的中心假设是针对性抑制 FXII-UPAR-PAKT2轴将在治疗中有效地处理DVT，同时最大程度地减少全身侧面 影响和出血风险。我们将通过在纳米层中包装UPAR抑制性肽来检验该假设 具有独特的表面工程，以在中性粒细胞上经历特定的异族锚固 聚合。 在此应用中，我们的目标是：1）确定候选UPAR抑制性肽，该肽破坏了FXII的约束 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AKT2 激活并执行嗜中性粒细胞和血小板功能分析； 2）使用异种装饰的纳米层 加载候选肽药物，以确定其现场选择性抑制FXII-UPAR的能力 在体外和体内相互作用并减轻DVT； 3）验证这些临床前研究，我们将确定 FXII-UPAR-PAKT2轴的组成活性及其对中性粒细胞功能的抑制作用，中性粒细胞 使用新诊断为DVT的患者的血液样本，血小板相互作用和血栓生长在体内。 最终目标是显示FXII-UPAR-PAKT2轴和下游效应子的差异丰度 DVT病理学将为未来的临床研究奠定基础，以抑制其作用。 我们的科学创新是中性粒细胞中FXII-UPAR-PAKT2信号轴的机械阐明 介导的病理。我们的技术创新是基于抑制性肽的目标的发展 纳米医学策略以阻止该轴以在DVT中为治疗益处。由于FXII是为数不多的蛋白质之一 可以保护在不增加出血风险的情况下免受血栓形成，拟议的研究有可能改善 与抑制最终共同凝结相比 目前抗凝剂的特征的途径。如果成功，拟议的治疗策略将 介绍新颖和更安全的疗法来治疗血栓性疾病，病因 退伍军人人口。