A novel fibrin(ogen)olytic trypsin-like protease from Chinese oak silkworm (Antheraea pernyi): Purification and characterization

A novel fibrin(ogen)olytic protease from Antheraea pernyi (important economically insect), named cocoonase, was isolated by a combination of ion-exchange chromatography and gel filtration. Furthermore, the characterization of cocoonase was investigated using fibrin(ogen)olytic, thrombolysis, and hemorrhagic assays. The NH₂-terminal sequence (IVGGY SVTID KAPYQ) was established by Edman degradation. Based on the N-terminal sequencing, cocoonase cDNA has been cloned by means of RT-PCR and 5′RACE. It is composed of 261 amino acid residues and possesses the structural features of trypsin-like serine protease. The purified cocoonase showed specific esterase activity on N-β-benzoyl-l-arginine ethyl (BAEE), and the kinetic constants, Km and Vmax were 2.577 × 10⁻³ mol/L and 4.09 × 10⁻³ μmol/L/s, respectively. Cocoonase showed strong activities on both fibrin and fibrinogen, preferentially hydrolyzed Aα and Bβ chains followed by γ-chains of fibrinogen. Cocoonase exhibited a thrombolysis activity both in vitro (blood-clot lysis activity assay) and in vivo (carrageenan-induced thrombosis model). These findings indicate that A. pernyi cocoonase ia a novel fibrin(ogen)olytic enzyme and may have a potential clinical application as an antithrombotic agent.     

Solubilized phenyl-pyrazole ureas as potent, selective 5-HT2A inverse-agonists and their application as antiplatelet agents

Potent 5-HT_2A inverse-agonists containing phenyl-pyrazole ureas with an amino side chain were identified. Optimization of this series resulted in selective compounds that proved effective in modulating 5HT-induced amplification of ADP-stimulated human platelet aggregation.     

Identification and characterization of Harobin, a novel fibrino(geno)lytic serine protease from a sea snake (Lapemis hardwickii)

A gene encoding a novel serine protease designated as Harobin is cloned and identified from a sea snake venom gland bacteriophage T7 library. It has 265 amino acids and shares 50-70% similarity to terrestrial snake serine proteases. In addition to the 12 conservative Cys, it has three more Cys residues that may contribute to its higher enzymatic stability. Harobin is expressed in Pichia pastoris and purified. Recombinant Harobin exhibits an amidolytic activity, and specifically degrades Aalpha, Bbeta-chain of fibrinogen. It functions as a defibrase both in vitro and in vivo, and reduces thrombosis. Harobin prolongs the coagulation time and the bleeding time of mice and reduces the fibrinogen levels of rats as well. Meanwhile, intravenous injection of Harobin leads to the reduction of blood pressure in SHR rats. It results from the ability of Harobin that cleaves angiotensin I and release bradykinin from plasma kininogen in vitro and in vivo. These data suggest that Harobin is a novel defibrase and has a potential to be an agent for the therapy of thrombosis and hypertension.     

Hybrid molecules of scutellarein and tertramethylpyrazine’s active metabolites for ischemic stroke

A series of hybrid molecules of scutellarein and tertramethylpyrazine’s active metabolites have been synthesized. Compared to the original compound, these prepared compounds exhibited higher water solubility, more appropriate logP and better stability. Importantly, compounds 11b, 11d and 11e showed improved neuroprotective activity against the H₂O₂-induced cell death in PC12 cells, and better antithrombosis activity. The optimized compound 11b was further evaluated by cerebral ischemia/ reperfusion in the middle cerebral artery occlusion (MCAO) model, the results showed that the compound could significantly reduce the infarct area and decrease the neuronal cell damage in CA1 pyramidal neurons. Overall, we demonstrated that the twin drug strategy could be applied in the development of agents for the treatment of ischemic stroke.