title: Platelets do not express the oxidized or reduced forms of tissue factor.
abstract: BACKGROUND
  Expression of tissue factor (TF) antigen and activity in platelets is controversial and dependent upon the laboratory and reagents used. Two forms of TF were described: an oxidized functional form and a reduced nonfunctional form that is converted to the active form through the formation of an allosteric disulfide. This study tests the hypothesis that the discrepancies regarding platelet TF expression are due to differential expression of the two forms.
  
  
  METHODS
  Specific reagents that recognize both oxidized and reduced TF were used in flow cytometry of unactivated and activated platelets and western blotting of whole platelet lysates. TF-dependent activity measurements were used to confirm the results.
  
  
  RESULTS
  Western blotting analyses of placental TF demonstrated that, in contrast to anti-TF#5, which is directed against the oxidized form of TF, a sheep anti-human TF polyclonal antibody recognizes both the reduced and oxidized forms. Flow cytometric analyses demonstrated that the sheep antibody did not react with the surface of unactivated platelets or platelets activated with thrombin receptor agonist peptide, PAR-1. This observation was confirmed using biotinylated active site-blocked factor (F)VIIa: no binding was observed. Likewise, neither form of TF was detected by western blotting of whole platelet lysates with sheep anti-hTF. Consistent with these observations, no FXa or FIXa generation by FVIIa was detected at the surface of these platelets. Similarly, no TF-related activity was observed in whole blood using thromboelastography.
  
  
  CONCLUSIONS
  Platelets from healthy donors do not express either oxidized (functional) or reduced (nonfunctional) forms of TF.
authors.affiliation: Department of Biochemistry, University of Vermont College of Medicine, Burlington, VT, USA.
authors.forename: Beth A
authors.initials: BA
authors.lastname: Bouchard
authors.affiliation: Department of Biochemistry, University of Vermont College of Medicine, Burlington, VT, USA.
authors.forename: Matthew T
authors.initials: MT
authors.lastname: Gissel
authors.affiliation: Department of Biochemistry, University of Vermont College of Medicine, Burlington, VT, USA.
authors.forename: Matthew F
authors.initials: MF
authors.lastname: Whelihan
authors.affiliation: Department of Biochemistry, University of Vermont College of Medicine, Burlington, VT, USA.
authors.forename: Kenneth G
authors.initials: KG
authors.lastname: Mann
authors.affiliation: Department of Biochemistry, University of Vermont College of Medicine, Burlington, VT, USA. Electronic address: sbutenas@uvm.edu.
authors.forename: Saulius
authors.initials: S
authors.lastname: Butenas
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology; D001792:Blood Platelets / Q000737:chemistry*; D015153:Blotting, Western; D005434:Flow Cytometry; D006801:Humans; D010084:Oxidation-Reduction; D012756:Sheep; D013925:Thromboplastin / Q000032:analysis* / Q000276:immunology
chemical_list: D000906:Antibodies; D013925:Thromboplastin
keywords: 312.5mM Tris, pH6.8, 10% sodium dodecyl sulfate, 50% glycerol, 0.05% bromophenol blue; 5× SPB; 80% phosphatidylcholine/20% phosphatidylserine containing vesicles; Arg-Gly-Asp-Ser; CTI; D-Phe-Pro-Arg-CH(2)Cl; F; FPRck; Factor VIIa; Flow cytometry; HRP; Hemostasis; LPS; Mr(app); PAR; PCPS; PE; PRP; Platelet; RGDS; SDS-PAGE; TAT; TEG; TF; Tissue factor; apparent molecular weight; corn trypsin inhibitor; factor; hTF; horseradish peroxidase; human TF; kDa; kilodalton; lipopolysaccharide; phycoerythrin; platelet-rich plasma; protease activated receptor; rFVIIa; recombinant FVIIa; sodium dodecyl sulfate-polyacrylamide gel electrophoresis; thrombin-antithrombin; thromboelastography; tissue factor