Platelet-targeted thromboprophylaxis with a human serum albumin fusion drug: Preventing thrombosis and reducing cardiac ischemia/reperfusion injurywithout bleeding complications.

Background: Myocardial infarction (MI) as a consequence of atherosclerosis-associated acute thrombosis is a leading cause of death and disability globally. Antiplatelet and anticoagulant drugs are standard therapies in preventing and treating MI. However, all clinically used drugs are associated with bleeding complications, which ultimately limits their use in patients with a high risk of bleeding. We have developed a new recombinant drug, targ-HSA-TAP, that combines targeting and specific inhibition of activated platelets as well as anticoagulation. This drug is designed and tested for a prolonged circulating half-life, enabling unique thromboprophylaxis without bleeding complications. Methods: Targ-HSA-TAP combines a single-chain antibody (scFv) that targets activated glycoprotein IIb/IIIa on activated platelets, human serum albumin (HSA) for prolonged circulation, and tick anticoagulant peptide (TAP) for coagulation FX inhibition. A non-binding scFv is employed as a non-targeting control (non-targ-HSA-TAP). Its efficacy was investigated in vivo using murine models of acute thrombosis and cardiac ischemia-reperfusion (I/R) injury. Results: Our experiments confirmed the targeting specificity of targ-HSA-TAP to activated platelets and demonstrated effective prevention of platelet aggregation and thrombus formation, as well as FXa inhibition in vitro. Thromboprophylactic administration of targ-HSA-TAP subcutaneously in mice prevented occlusion of the carotid artery after ferric chloride injury as compared to non-targ-HSA-TAP and PBS-control treated mice. By comparing the therapeutic outcomes between targ-TAP and targ-HSA-TAP, we demonstrate the significant improvements brought by the HSA fusion in extending the drug's half-life and enhancing its therapeutic window for up to 16 h post-administration. Importantly, tail bleeding time was not prolonged with targ-HSA-TAP in contrast to the clinically used anticoagulant enoxaparin. Furthermore, in a murine model of cardiac I/R injury, mice administered targ-HSA-TAP 10 h before injury demonstrated preserved cardiac function, with significantly higher ejection fraction and fractional shortening, as compared to the non-targ-HSA-TAP and PBS control groups. Advanced strain analysis revealed reduced myocardial deformation and histology confirmed a reduced infarct size in targ-HSA-TAP treated mice compared to control groups. Conclusion: The inclusion of HSA represents a significant advancement in the design of targeted therapeutic agents for thromboprophylaxis. Our activated platelet-targeted targ-HSA-TAP is a highly effective antithrombotic drug with both anticoagulant and antiplatelet effects while retaining normal hemostasis. The long half-life of targ-HSA-TAP provides the unique opportunity to use this antithrombotic drug for more effective, long-lasting and safer anti-thrombotic prophylaxis. In cases where MI occurs, this prophylactic strategy reduces thrombus burden and effectively reduces cardiac I/R injury.

Immunization with a whole cell vaccine reduces pneumococcal nasopharyngeal density and shedding, and middle ear infection in mice.

Pneumococcal Conjugate Vaccines (PCVs) have substantially reduced the burden of disease caused by Streptococcus pneumoniae (the pneumococcus). However, protection is limited to vaccine serotypes, and when administered to children who are colonized with pneumococci at the time of vaccination, immune responses to the vaccine are blunted. Here, we investigate the potential of a killed whole cell pneumococcal vaccine (WCV) to reduce existing pneumococcal carriage and mucosal disease when given therapeutically to infant mice colonized with pneumococci. We show that a single dose of WCV reduced pneumococcal carriage density in an antibody-dependent manner. Therapeutic vaccination induced robust immune responses to pneumococcal surface antigens CbpA, PspA (family 1) and PiaA. In a co-infection model of otitis media, a single dose of WCV reduced pneumococcal middle ear infection. Lastly, in a two-dose model, therapeutic administration of WCV reduced nasal shedding of pneumococci. Taken together, our data demonstrate that WCV administered in colonized mice reduced pneumococcal density in the nasopharynx and the middle ear, and decreased shedding. WCVs would be beneficial in low and middle-income settings where pneumococcal carriage in children is high.

Activated Coagulation FXII: A Unique Target for In Vivo Molecular Imaging.

BACKGROUND: Current clinical imaging of thromboembolic diseases often relies on indirect detection of thrombi, which may delay diagnosis and ultimately the institution of beneficial, potentially lifesaving treatment. Therefore, the development of targeting tools that facilitate the rapid, specific, and direct imaging of thrombi using molecular imaging is highly sought after. One potential molecular target is FXIIa (factor XIIa), which initiates the intrinsic coagulation pathway but also activates the kallikrein-kinin system, thereby initiating coagulation and inflammatory/immune responses. As FXII (factor XII) is dispensable for normal hemostasis, its activated form (FXIIa) represents an ideal molecular target for diagnostic and therapeutic approaches, the latter combining diagnosis/identification of thrombi and effective antithrombotic therapy. METHODS: We conjugated an FXIIa-specific antibody, 3F7, to a near-infrared (NIR) fluorophore and demonstrated binding to FeCl3-induced carotid thrombosis with 3-dimensional fluorescence emission computed tomography/computed tomography and 2-dimensional fluorescence imaging. We further demonstrated ex vivo imaging of thromboplastin-induced pulmonary embolism and detection of FXIIa in human thrombi produced in vitro. RESULTS: We demonstrated imaging of carotid thrombosis by fluorescence emission computed tomography/computed tomography and measured a significant fold increase in signal between healthy and control vessels from mice injected with 3F7-NIR compared with mice injected with nontargeted probe (P=0.002) ex vivo. In a model of pulmonary embolism, we measured increased NIR signal in lungs from mice injected with 3F7-NIR compared with mice injected with nontargeted probe (P=0.0008) and healthy lungs from mice injected with 3F7-NIR (P=0.021). CONCLUSIONS: Overall, we demonstrate that FXIIa targeting is highly suitable for the specific detection of venous and arterial thrombi. This approach will allow direct, specific, and early imaging of thrombosis in preclinical imaging modalities and may facilitate monitoring of antithrombotic treatment in vivo.

An Ultrasound-Responsive Theranostic Cyclodextrin-Loaded Nanoparticle for Multimodal Imaging and Therapy for Atherosclerosis.

Atherosclerosis is a major cause of mortality and morbidity worldwide. Left undiagnosed and untreated, atherosclerotic plaques can rupture and cause cardiovascular complications such as myocardial infarction and stroke. Atherosclerotic plaques are composed of lipids, including oxidized low-density lipoproteins and cholesterol crystals, and immune cells, including macrophages. 2-Hydroxypropyl-beta-cyclodextrin (CD) is FDA-approved for capturing, solubilizing, and delivering lipophilic drugs in humans. It is also known to dissolve cholesterol crystals and decrease atherosclerotic plaque size. However, its low retention time necessitates high dosages for successful therapy. This study reports CD delivery via air-trapped polybutylcyanoacrylate nanoparticles (with diameters of 388 ± 34 nm) loaded with CD (CDNPs). The multimodal contrast ability of these nanoparticles after being loaded with IR780 dye in mice is demonstrated using ultrasound and near-infrared imaging. It is shown that CDNPs enhance the cellular uptake of CD in murine cells. In an ApoE-/- mouse model of atherosclerosis, treatment with CDNPs significantly improves the anti-atherosclerotic efficacy of CD. Ultrasound triggering further improves CD uptake, highlighting that CDNPs can be used for ultrasound imaging and ultrasound-responsive CD delivery. Thus, CDNPs represent a theranostic nanocarrier for potential application in patients with atherosclerosis.

A Recombinant Fusion Construct between Human Serum Albumin and NTPDase CD39 Allows Anti-Inflammatory and Anti-Thrombotic Coating of Medical Devices.

Medical devices directly exposed to blood are commonly used to treat cardiovascular diseases. However, these devices are associated with inflammatory reactions leading to delayed healing, rejection of foreign material or device-associated thrombus formation. We developed a novel recombinant fusion protein as a new biocompatible coating strategy for medical devices with direct blood contact. We genetically fused human serum albumin (HSA) with ectonucleoside triphosphate diphosphohydrolase-1 (CD39), a promising anti-thrombotic and anti-inflammatory drug candidate. The HSA-CD39 fusion protein is highly functional in degrading ATP and ADP, major pro-inflammatory reagents and platelet agonists. Their enzymatic properties result in the generation of AMP, which is further degraded by CD73 to adenosine, an anti-inflammatory and anti-platelet reagent. HSA-CD39 is functional after lyophilisation, coating and storage of coated materials for up to 8 weeks. HSA-CD39 coating shows promising and stable functionality even after sterilisation and does not hinder endothelialisation of primary human endothelial cells. It shows a high level of haemocompatibility and diminished blood cell adhesion when coated on nitinol stents or polyvinylchloride tubes. In conclusion, we developed a new recombinant fusion protein combining HSA and CD39, and demonstrated that it has potential to reduce thrombotic and inflammatory complications often associated with medical devices directly exposed to blood.