Current and Future Therapeutics for Treating Patients with Sickle Cell Disease.

Sickle cell disease (SCD) is the most common genetic blood disorder in the United States, with over 100,000 people suffering from this debilitating disease. SCD is caused by abnormal hemoglobin (Hb) variants that interfere with normal red blood cell (RBC) function. Research on SCD has led to the development and approval of several new SCD therapies in recent years.

Positive Allosteric Modulation of Antithrombin's Inhibitory Activity by RNA Aptamers.

The leading cause of death in adults in the United States is cardiovascular disease, with mortality and morbidity mainly attributed to thromboembolism. Heparin is the most common therapy used for treating venous and arterial thrombosis. Heparin effectively accelerates the inhibition of coagulation proteases thrombin and factor Xa through the serine protease inhibitor (serpin) antithrombin (AT).

Overview of the Therapeutic Potential of Aptamers Targeting Coagulation Factors.

Aptamers are single-stranded DNA or RNA sequences that bind target molecules with high specificity and affinity. Aptamers exhibit several notable advantages over protein-based therapeutics. Aptamers are non-immunogenic, easier to synthesize and modify, and can bind targets with greater affinity.

Identification of Aptamers That Bind to Sickle Hemoglobin and Inhibit Its Polymerization.

The pathophysiology of sickle cell disease (SCD) is dependent on the polymerization of deoxygenated sickle hemoglobin (HbS), leading to erythrocyte deformation (sickling) and vaso-occlusion within the microvasculature. Following deoxygenation, there is a delay time before polymerization is initiated, during which nucleation of HbS monomers occurs. An agent with the ability to extend this delay time or slow polymerization would therefore hold a therapeutic, possibly curative, potential.

Intracellular Expression of PAI-1 Specific Aptamers Alters Breast Cancer Cell Migration, Invasion and Angiogenesis.

Plasminogen activator inhibitor-1 (PAI-1) is elevated in various cancers, where it has been shown to effect cell migration and invasion and angiogenesis. While, PAI-1 is a secreted protein, its intercellular levels are increased in cancer cells. Consequently, intracellular PAI-1 could contribute to cancer progression.

Targeting the membrane-anchored serine protease testisin with a novel engineered anthrax toxin prodrug to kill tumor cells and reduce tumor burden.

The membrane-anchored serine proteases are a unique group of trypsin-like serine proteases that are tethered to the cell surface via transmembrane domains or glycosyl-phosphatidylinositol-anchors. Overexpressed in tumors, with pro-tumorigenic properties, they are attractive targets for protease-activated prodrug-like anti-tumor therapies. Here, we sought to engineer anthrax toxin protective antigen (PrAg), which is proteolytically activated on the cell surface by the proprotein convertase furin to instead be activated by tumor cell-expressed membrane-anchored serine proteases to function as a tumoricidal agent.

Inhibition of PAI-1 antiproteolytic activity against tPA by RNA aptamers.

Plasminogen activator inhibitor-1 (PAI-1; SERPINE1) inhibits the plasminogen activators: tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA). Elevated levels of PAI-1 have been correlated with an increased risk for cardiovascular disease. Pharmacologically suppressing PAI-1 might prevent, or successfully treat PAI-1 related vascular diseases.

Plasminogen activator inhibitor-1 inhibitors: a patent review (2006-present).

Introduction: Plasminogen activator inhibitor-1 (PAI-1), the serine protease inhibitor (serpin), binds to and inhibits the plasminogen activators-tissue-type plasminogen activator (tPA) and the urokinase-type plasminogen activator (uPA). This results in both a decrease in plasmin production and a decrease in the dissolution of fibrin clots. Elevated levels of PAI-1 are correlated with an increased risk for cardiovascular disease and have been linked to obesity and metabolic syndrome.

Effects of plasminogen activator inhibitor-1-specific RNA aptamers on cell adhesion, motility, and tube formation.

The serine protease inhibitor (serpin) plasminogen activator inhibitor-1 (PAI-1) is associated with the pathophysiology of several diseases, including cancer and cardiovascular disease. The extracellular matrix protein vitronectin increases at sites of vessel injury and is also present in fibrin clots. Integrins present on the cell surface bind to vitronectin and anchor the cell to the extracellular matrix.

Heparin cofactor II: discovery, properties, and role in controlling vascular homeostasis.

Heparin cofactor II (HCII) is a serine protease inhibitor (serpin) found in high concentrations in human plasma. Despite its discovery >30 years ago, its physiological function is still poorly understood. It is known to inhibit thrombin, the predominant coagulation protease, and HCII-thrombin complexes have been found in plasma, yet it is thought to contribute little to normal hemostasis.

A chemical genetic screen for modulators of asymmetrical 2,2'-dimeric naphthoquinones cytotoxicity in yeast.

Background: Dimeric naphthoquinones (BiQ) were originally synthesized as a new class of HIV integrase inhibitors but have shown integrase-independent cytotoxicity in acute lymphoblastic leukemia cell lines suggesting their use as potential anti-neoplastic agents. The mechanism of this cytotoxicity is unknown. In order to gain insight into the mode of action of binaphthoquinones we performed a systematic high-throughput screen in a yeast isogenic deletion mutant array for enhanced or suppressed growth in the presence of binaphthoquinones.

Protein C inhibitor regulates both cathepsin L activity and cell-mediated tumor cell migration.

Background: Protein C inhibitor (PCI) is a plasma serine protease inhibitor (serpin) that regulates several serine proteases in coagulation including thrombin and activated protein C. However, the physiological role of PCI remains under investigation. The cysteine protease, cathepsin L, has a role in many physiological processes including cardiovascular diseases, blood vessel remodeling, and cancer.

Antimetastatic potential of PAI-1-specific RNA aptamers.

The serine protease inhibitor plasminogen activator inhibitor-1 (PAI-1) is increased in several cancers, including breast, where it is associated with a poor outcome. Metastatic breast cancer has a dismal prognosis, as evidenced by treatment goals that are no longer curative but are largely palliative in nature. PAI-1 competes with integrins and the urokinase plasminogen activator receptor on the surface of breast cancer cells for binding to vitronectin.

Characterization of recombinant human protein C inhibitor expressed in Escherichia coli.

The serine protease inhibitor (serpin) protein C inhibitor (PCI; also named plasminogen activator inhibitor-3) regulates serine proteases in hemostasis, fibrinolysis, and reproduction. The biochemical activity of PCI is not fully defined partly due to the lack of a convenient expression system for active rPCI. Using pET-15b plasmid, Ni(2+)-chelate and heparin-Sepharose affinity chromatography steps, we describe here the expression, purification and characterization of wild-type recombinant (wt-rPCI) and two inactive mutants, R354A (P1 residue) and T341R (P14 residue), expressed in Escherichia coli.

Molecular mapping of the thrombin-heparin cofactor II complex.

We used 55 Ala-scanned recombinant thrombin molecules to define residues important for inhibition by the serine protease inhibitor (serpin) heparin cofactor II (HCII) in the absence and presence of glycosaminoglycans. We verified the importance of numerous basic residues in anion-binding exosite-1 (exosite-1) and found 4 additional residues, Gln24, Lys65, His66, and Tyr71 (using the thrombin numbering system), that were resistant to HCII inhibition with and without glycosaminoglycans. Inhibition rate constants for these exosite-1 (Q24A, K65A, H66A, Y71A) thrombin mutants (0.

RNA aptamer to thrombin binds anion-binding exosite-2 and alters protease inhibition by heparin-binding serpins.

We studied the RNA aptamer Toggle-25/thrombin interaction during inhibition by antithrombin (AT), heparin cofactor II (HCII) and protein C inhibitor (PCI). Thrombin inhibition was reduced 3-fold by Toggle-25 for AT and HCII, but it was slightly enhanced for PCI. In the presence of glycosaminoglycans, AT and PCI had significantly reduced thrombin inhibition with Toggle-25, but it was only reduced 3-fold for HCII.