Preclinical Development of a vWF Aptamer to Limit Thrombosis and Engender Arterial Recanalization of Occluded Vessels.

Endothelial surface and circulating glycoprotein von Willebrand factor (vWF) regulates platelet adhesion and is associated with thrombotic diseases, including ischemic stroke, myocardial infarction, and peripheral vascular disease. Thrombosis, as manifested in these diseases, is the leading cause of disability and death in the western world. Current parenteral antithrombotic and thrombolytic agents used to treat these conditions are limited by a short therapeutic window, irreversibility, and major risk of hemorrhage. To overcome these limitations, we developed a novel anti-vWF aptamer, called DTRI-031, that selectively binds and inhibits vWF-mediated platelet adhesion and arterial thrombosis while enabling rapid reversal of this antiplatelet activity by an antidote oligonucleotide (AO). Aptamer DTRI-031 exerts dose-dependent inhibition of platelet aggregation and thrombosis in whole blood and mice, respectively. Moreover, DTRI-031 can achieve potent vascular recanalization of platelet-rich thrombotic occlusions in murine and canine carotid arteries. Finally, DTRI-031 activity is rapidly (<5 min) and completely reversed by AO administration in a murine saphenous vein hemorrhage model, and murine toxicology studies indicate the aptamer is well tolerated. These findings suggest that targeting vWF with an antidote-controllable aptamer potentially represents an effective and safer treatment for thrombosis patients having platelet-rich arterial occlusions in the brain, heart, or periphery.

Inhalation of an RNA aptamer that selectively binds extracellular histones protects from acute lung injury.

Acute lung injury (ALI) is a syndrome of acute inflammation, barrier disruption, and hypoxemic respiratory failure associated with high morbidity and mortality. Diverse conditions lead to ALI, including inhalation of toxic substances, aspiration of gastric contents, infection, and trauma. A shared mechanism of acute lung injury is cellular toxicity from damage-associated molecular patterns (DAMPs), including extracellular histones. We recently described the selection and efficacy of a histone-binding RNA aptamer (HBA7). The current study aimed to identify the effects of extracellular histones in the lung and determine if HBA7 protected mice from ALI. Histone proteins decreased metabolic activity, induced apoptosis, promoted proinflammatory cytokine production, and caused endothelial dysfunction and platelet activation in vitro. HBA7 prevented these effects. The oropharyngeal aspiration of histone proteins increased neutrophil and albumin levels in bronchoalveolar lavage fluid (BALF) and precipitated neutrophil infiltration, interstitial edema, and barrier disruption in alveoli in mice. Similarly, inhaling wood smoke particulate matter, as a clinically relevant model, increased lung inflammation and alveolar permeability. Treatment by HBA7 alleviated lung injury in both models of ALI. These findings demonstrate the pulmonary delivery of HBA7 as a nucleic acid-based therapeutic for ALI.

Heparin cofactor II in atherosclerotic lesions from the Pathobiological Determinants of Atherosclerosis in Youth (PDAY) study.

Heparin cofactor II (HCII) is a serine protease inhibitor (serpin) that has been shown to be a predictor of decreased atherosclerosis in the elderly and protective against atherosclerosis in mice. HCII inhibits thrombin in vitro and HCII-thrombin complexes have been detected in human plasma. Moreover, the mechanism of protection against atherosclerosis in mice was determined to be the inhibition of thrombin. Despite this evidence, the presence of HCII in human atherosclerotic tissue has not been reported. In this study, using samples of coronary arteries obtained from the Pathobiological Determinants of Atherosclerosis in Youth (PDAY) study, we explore the local relationship between HCII and (pro)thrombin in atherosclerosis. We found that HCII and (pro)thrombin are co-localized in the lipid-rich necrotic core of atheromas. A significant positive correlation between each protein and the severity of the atherosclerotic lesion was present. These results suggest that HCII is in a position to inhibit thrombin in atherosclerotic lesions where thrombin can exert a proatherogenic inflammatory response. However, these results should be tempered by the additional findings from this, and other studies, that indicate the presence of other plasma proteins (antithrombin, albumin, and alpha(1)-protease inhibitor) in the same localized region of the atheroma.

Protease-activated receptor-2 signaling triggers dendritic cell development.

Dendritic cells (DC) are potent antigen-presenting cells that govern the effector cell responses of the immune system. DC are thought to continuously develop from circulating progenitors in a process that is accelerated by inflammatory stimuli. However, the physiological signals that regulate the development of DC from precursor cells have not been well defined. Here we show that a serine protease acting via protease-activated receptor-2 (PAR-2) stimulates the development of DC from bone marrow progenitor cells cultured in granulocyte-macrophage colony-stimulating factor and IL-4. DC fail to develop in bone marrow cultures treated with soy bean trypsin inhibitor, a serine protease inhibitor, but this inhibition is overcome by a PAR-2 agonist peptide. DC do not spontaneously develop from the bone marrow of PAR-2-deficient mice, but can be stimulated to do so by inflammatory mediators. These results suggest that endogenous serine proteases stimulate DC development in vitro. Thus, serine proteases may help trigger adaptive immune responses in vivo.