title: Thrombolysis by chemically modified coagulation factor Xa.
abstract: Essentials Factor Xa (FXa) acquires cleavage-mediated tissue plasminogen activator (tPA) cofactor activity. Recombinant (r) tPA is the predominant thrombolytic drug, but it may cause systemic side effects. Chemically modified, non-enzymatic FXa was produced (Xai-K), which rapidly lysed thrombi in mice. Unlike rtPA, Xai-K had no systemic fibrinolysis activation markers, indicating improved safety.
  
  
  
  Background Enzymatic thrombolysis carries the risk of hemorrhage and re-occlusion must be evaded by co-administration with an anticoagulant. Toward further improving these shortcomings, we report a novel dual-functioning molecule, Xai-K, which is both a non-enzymatic thrombolytic agent and an anticoagulant. Xai-K is based on clotting factor Xa, whose sequential plasmin-mediated fragments, FXaβ and Xa33/13, accelerate the principal thrombolytic agent, tissue plasminogen activator (tPA), but only when localized to anionic phospholipid. Methods The effect of Xai-K on fibrinolysis was measured in vitro by turbidity, thromboelastography and chromogenic assays, and measured in a murine model of occlusive carotid thrombosis by Doppler ultrasound. The anticoagulant properties of Xai-K were evaluated by normal plasma clotting assays, and in murine liver laceration and tail amputation hemostatic models. Results Xa33/13, which participates in fibrinolysis of purified fibrin, was rapidly inhibited in plasma. Cleavage was blocked at FXaβ by modifying residues at the active site. The resultant Xai-K (1 nm) enhanced plasma clot dissolution by ~7-fold in vitro and was dependent on tPA. Xai-K alone (2.0 μg g(-1) body weight) achieved therapeutic patency in mice. The minimum primary dose of the tPA variant, Tenecteplase (TNK; 17 μg g(-1) ), could be reduced by > 30-fold to restore blood flow with adjunctive Xai-K (0.5 μg g(-1) ). TNK-induced systemic markers of fibrinolysis were not detected with Xai-K (2.0 μg g(-1) ). Xai-K had anticoagulant activity that was somewhat attenuated compared with a previously reported analogue. Conclusion These results suggest that Xai-K may ameliorate the safety profile of therapeutic thrombolysis, either as a primary or tPA/TNK-adjunctive agent.
authors.affiliation: Centre for Blood Research and Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC, Canada. ed.pryzdial@blood.ca.|Centre for Innovation, Canadian Blood Services, Ottawa, ON, Canada. ed.pryzdial@blood.ca.
authors.forename: E L G
authors.initials: EL
authors.lastname: Pryzdial
authors.affiliation: Centre for Blood Research and Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC, Canada.|Centre for Innovation, Canadian Blood Services, Ottawa, ON, Canada.
authors.forename: S C
authors.initials: SC
authors.lastname: Meixner
authors.affiliation: Centre for Blood Research and Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC, Canada.|Centre for Innovation, Canadian Blood Services, Ottawa, ON, Canada.
authors.forename: K
authors.initials: K
authors.lastname: Talbot
authors.affiliation: Centre for Innovation, Canadian Blood Services, Ottawa, ON, Canada.|Department of Pathology and Molecular Medicine, McMaster University, Hamilton, ON, Canada.
authors.forename: L J
authors.initials: LJ
authors.lastname: Eltringham-Smith
authors.affiliation: Centre for Blood Research and Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC, Canada.|Michael Smith Laboratories and Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC, Canada.
authors.forename: J R
authors.initials: JR
authors.lastname: Baylis
authors.affiliation: Centre for Blood Research and Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC, Canada.|Centre for Innovation, Canadian Blood Services, Ottawa, ON, Canada.
authors.forename: F M H
authors.initials: FM
authors.lastname: Lee
authors.affiliation: Centre for Blood Research and Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC, Canada.|Michael Smith Laboratories and Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC, Canada.
authors.forename: C J
authors.initials: CJ
authors.lastname: Kastrup
authors.affiliation: Centre for Innovation, Canadian Blood Services, Ottawa, ON, Canada.|Department of Pathology and Molecular Medicine, McMaster University, Hamilton, ON, Canada.
authors.forename: W P
authors.identifier: 0000-0002-5870-8189
authors.initials: WP
authors.lastname: Sheffield
mesh_terms: D000818:Animals; D000925:Anticoagulants / Q000737:chemistry; D015951:Factor Xa / Q000008:administration & dosage* / Q000031:analogs & derivatives*; D005260:Female; D005342:Fibrinolysis; D006487:Hemostasis; D006801:Humans; D008099:Liver / Q000378:metabolism; D051379:Mice; D061214:Patient Safety; D010743:Phospholipids / Q000737:chemistry; D010958:Plasminogen / Q000737:chemistry; D011994:Recombinant Proteins / Q000008:administration & dosage / Q000737:chemistry; D000077785:Tenecteplase; D013916:Thrombelastography; D015912:Thrombolytic Therapy; D013927:Thrombosis / Q000628:therapy; D010959:Tissue Plasminogen Activator / Q000008:administration & dosage* / Q000378:metabolism; D016896:Treatment Outcome; D018608:Ultrasonography, Doppler
chemical_list: D000925:Anticoagulants; D010743:Phospholipids; D011994:Recombinant Proteins; C000625832:Xai-K protein; D010958:Plasminogen; D015951:Factor Xa; C067262:PLAT protein, human; C104096:TNK-tissue plasminogen activator; D010959:Tissue Plasminogen Activator; D000077785:Tenecteplase
keywords: factor Xa; fibrinolysis; plasmin; therapeutic thrombolysis; tissue plasminogen activator