title: Alpha-1 proteinase inhibitor M358R reduces thrombin generation when displayed on the surface of cells expressing tissue factor.
abstract: The M358R variant of alpha-1-proteinase inhibitor (API) is a potent soluble inhibitor of thrombin. Previously we engineered AR-API M358R, a membrane-bound form of this protein and showed that it inhibited exogenous thrombin when expressed on transfected cells lacking tissue factor (TF). To determine the suitability of AR-API M358R for gene transfer to vascular cells to limit thrombogenicity, we tested the ability of AR-API M358R to inhibit endogenous thrombin generated in plasma via co-expression co-expressing it on the surface of cells expressing TF. Transfected AR-API M358R formed inhibitory complexes with thrombin following exposure of recalcified, defibrinated plasma to TF on T24/83 cells, but discontinuously monitored thrombin generation was unaffected. Similarly, AR-API M358R expression did not reduce continuously monitored thrombin generation by T24/83 cell suspensions exposed to recalcified normal plasma in a Thrombogram-Thrombinoscope-type thrombin generation assay (TGA); in contrast, 1 μM hirudin variant 3 or soluble API M358R abolished thrombin generation. Gene transfer of TF to HEK 293 conferred the ability to support TF-dependent thrombin generation on HEK 293 cells. Co-transfection of HEK 293 cells with a 9:1 excess of DNA encoding AR-API M358R to that encoding TF reduced peak thrombin generation approximately 3-fold compared to controls. These in vitro results suggest that surface display of API M358R inhibits thrombin generation when the tethered serpin is expressed in excess of TF, and suggest its potential to limit thrombosis in appropriate vascular beds in animal models.
authors.affiliation: Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada.
authors.forename: Richard F
authors.initials: RF
authors.lastname: Gierczak
authors.affiliation: Department of Medicine, McMaster University, Hamilton, Ontario, Canada.
authors.forename: Laura
authors.initials: L
authors.lastname: Pepler
authors.affiliation: Department of Medicine, McMaster University, Hamilton, Ontario, Canada.
authors.forename: Vinai
authors.initials: V
authors.lastname: Bhagirath
authors.affiliation: Department of Medicine, McMaster University, Hamilton, Ontario, Canada.
authors.forename: Patricia C
authors.initials: PC
authors.lastname: Liaw
authors.affiliation: Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada; Canadian Blood Services, Centre for Innovation, Hamilton, Ontario, Canada. Electronic address: sheffiel@mcmaster.ca.
authors.forename: William P
authors.initials: WP
authors.lastname: Sheffield
mesh_terms: D019943:Amino Acid Substitution; D001777:Blood Coagulation; D045744:Cell Line, Tumor; D015870:Gene Expression; D057809:HEK293 Cells; D006801:Humans; D013917:Thrombin / Q000378:metabolism*; D013925:Thromboplastin / Q000235:genetics / Q000378:metabolism*; D013927:Thrombosis / Q000235:genetics / Q000378:metabolism; D000515:alpha 1-Antitrypsin / Q000235:genetics* / Q000378:metabolism*
chemical_list: D000515:alpha 1-Antitrypsin; D013925:Thromboplastin; D013917:Thrombin
keywords: Alpha-1 proteinase inhibitor; Coagulation; Thrombin; Thrombosis; Tissue factor