title: Design of a novel chimeric tissue plasminogen activator with favorable Vampire bat plasminogen activator properties.
abstract: Fibrinolytic agents are widely used in treatment of the thromboembolic disorders. The new generations like recombinant tissue plasminogen activator (t-PA, alteplase) are not showing promising results in clinical practice in spite of displaying specific binding to fibrin in vitro. Vampire bat plasminogen activator (b-PA) is a plasminogen activator with higher fibrin affinity and specificity in comparison to t-PA resulting in reduced probability of hemorrhage. b-PA is also resistant to plasminogen activator inhibitor-1 (PAI-1) showing higher half-life compared to other variants of t-PA. However, its non-human origin was a driving force to design a human t-PA with favorable properties of b-PA. In the present study, we designed a chimeric t-PA with desirable b-PA properties and this new molecule was called as CT-b. The construct was prepared through kringle 2 domain removal and replacement of t-PA finger domain with b-PA one. In addition, the KHRR sequence at the initial part of protease domain was replaced by four alanine residues. The novel construct was integrated in Pichia pastoris genome by electroporation. Catalytic activity was investigated in the presence and absence of fibrin. The purified protein was analyzed by western blot. Fibrin binding and PAI resistance assays were also conducted. The activity of the recombinant protein in the presence of fibrin was 1560 times more than its activity in the absence of fibrin, showing its higher specificity to fibrin. The fibrin binding of CT-b was 1.2 fold more than t-PA. In addition, it was inhibited by PAI enzyme 44% less than t-PA. Although the presented data demonstrate a promising in vitro activity, more in vivo studies are needed to confirm the therapeutic advantage of this novel plasminogen activator.
authors.affiliation: Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.
authors.forename: MohammadReza
authors.initials: M
authors.lastname: Kazemali
authors.affiliation: Cancer Genetics Research Group, Breast Cancer Research Center, ACECR, Tehran, Iran.
authors.forename: Keivan
authors.initials: K
authors.lastname: Majidzadeh-A
authors.affiliation: Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.
authors.forename: Soroush
authors.initials: S
authors.lastname: Sardari
authors.affiliation: Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.
authors.forename: Amir Hossein
authors.initials: AH
authors.lastname: Saadatirad
authors.affiliation: Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.
authors.forename: Vahid
authors.initials: V
authors.lastname: Khalaj
authors.affiliation: Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.
authors.forename: Najmeh
authors.initials: N
authors.lastname: Zarei
authors.affiliation: Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.
authors.forename: Farzaneh
authors.initials: F
authors.lastname: Barkhordari
authors.affiliation: Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.
authors.forename: Ahmad
authors.initials: A
authors.lastname: Adeli
authors.affiliation: Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran. Electronic address: mahboudi_f@pasteur.ac.ir.
authors.forename: Fereidoun
authors.initials: F
authors.lastname: Mahboudi
mesh_terms: D000818:Animals; D001709:Biotechnology; D002685:Chiroptera / Q000378:metabolism*; D017124:Conserved Sequence; D015195:Drug Design; D005343:Fibrinolytic Agents / Q000737:chemistry / Q000494:pharmacology; D006801:Humans; D008958:Models, Molecular; D010843:Pichia / Q000235:genetics; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D013923:Thromboembolism / Q000188:drug therapy; D010959:Tissue Plasminogen Activator / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*
chemical_list: D005343:Fibrinolytic Agents; D011993:Recombinant Fusion Proteins; D010959:Tissue Plasminogen Activator
keywords: Catalytic activity; Fibrin binding; Pichia pastoris; Plasminogen activator inhibitor-1 (PAI-1); Tissue plasminogen activator (t-PA); b-PA