title: A novel hemostasis assay for the simultaneous measurement of coagulation and fibrinolysis.
abstract: Thrombin and plasmin are the key enzymes involved in coagulation and fibrinolysis. A novel hemostasis assay (NHA) was developed to measure thrombin and plasmin generation in a single well by a fluorimeter. The NHA uses two fluorescent substrates with non-interfering fluorescent excitation and emission spectra. The assay was tested in vitro using modulators like heparin, hirudin, epsilon-aminocaproic acid, gly-pro-arg-pro peptide and reptilase and validated by measurement of prothrombin fragment 1+2 and plasmin-alpha2-antiplasmin levels. Intra- and inter-assay coefficients of variation were < 9% and 6-25%, respectively. Interplay between coagulation and fibrinolysis was demonstrated by the effect of tissue-type plasminogen activator on thrombin generation and by the different responses of activated protein C and thrombomodulin on fibrinolysis. The last responses showed the linkage between coagulation and fibrinolysis by thrombin activatable fibrinolysis inhibitor. In conclusion, this strategy allows detection of coagulation, fibrinolysis and their interplay in a single assay.
authors.affiliation: Laboratory of Hematology, Department of Laboratory Medicine, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands.
authors.forename: Mark
authors.initials: M
authors.lastname: van Geffen
authors.forename: Arnoud
authors.initials: A
authors.lastname: Loof
authors.forename: Paul
authors.initials: P
authors.lastname: Lap
authors.forename: Jan
authors.initials: J
authors.lastname: Boezeman
authors.forename: Britta A P
authors.initials: BA
authors.lastname: Laros-van Gorkom
authors.forename: Paul
authors.initials: P
authors.lastname: Brons
authors.forename: Bert
authors.initials: B
authors.lastname: Verbruggen
authors.forename: Marian
authors.initials: M
authors.lastname: van Kraaij
authors.forename: Waander L
authors.initials: WL
authors.lastname: van Heerde
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D001777:Blood Coagulation / Q000187:drug effects / Q000502:physiology*; D001780:Blood Coagulation Tests / Q000379:methods*; D005260:Female; D005341:Fibrinolysin / Q000378:metabolism; D005342:Fibrinolysis / Q000187:drug effects / Q000502:physiology*; D006487:Hemostasis / Q000502:physiology*; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008297:Male; D008875:Middle Aged; D010714:Phosphatidylethanolamines / Q000494:pharmacology; D015203:Reproducibility of Results; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D013917:Thrombin / Q000378:metabolism; D010959:Tissue Plasminogen Activator / Q000494:pharmacology; D055815:Young Adult
chemical_list: D010714:Phosphatidylethanolamines; D006493:Heparin; D013917:Thrombin; D010959:Tissue Plasminogen Activator; D005341:Fibrinolysin