Quantitative assessment of fibrinogen cross-linking by epsilon aminocaproic acid in patients with end-stage liver disease.
Analysis of the effectiveness of antifibrinolytic therapy for liver transplant recipients is hampered by lack of quantitative assays for assessing drug effects. We adapted chemical engineering tools used in polymerization studies to quantify fibrinogen cross-linking by plasma from liver transplant patients obtained before and after epsilon aminocaproic acid (EACA) therapy. A target fluorescein isothiocyanate-fibrinogen (FITC-fibrinogen) molecule was constructed; it fluoresces in a quantifiable pattern when in solution, and undergoes cross-linking in the presence of plasmin inhibitors. Cross-linking quenches the fluorescent signal, and the quenching is a quantifiable endpoint. Thus fluorescence from this reporter molecule can be used to assess functional improvement in fibrinogen cross-linking as a result of antifibrinolytic therapies, and it is sensitive to picomolar amounts of plasmin inhibitors and activators. Cross-linking of FITC-fibrinogen by patient plasma, before and after EACA therapy, was assessed using fluorescence spectrometry. Fluorescence patterns from FITC-fibrinogen indicated no significant cross-linking of the target fibrinogen as a consequence of EACA in posttreatment plasma. When the fibrinogen-FITC target was assayed without plasma in the presence of EACA at concentrations that bracket therapeutic levels (100 and 400 microg/ml), significant fluorescence quenching (target FITC-fibrinogen cross-linking) was achieved. These results suggest that fibrinogen-FITC fluorescence is sensitive enough to detect EACA activity in clinically relevant ranges, but that EACA given in usual doses is insufficient to promote fibrinogen cross-linking in patients with end-stage liver disease.
Quach
Thien
T
University of Michigan, Ann Arbor, MI, USA.
Tippens
Melissa
M
Szlam
Fania
F
Van Dyke
Rebecca
R
Levy
Jerrold H
JH
Csete
Marie
M
D000328:Adult; D015119:Aminocaproic Acid / Q000493:pharmacokinetics / Q000627:therapeutic use*; D000933:Antifibrinolytic Agents / Q000493:pharmacokinetics / Q000627:therapeutic use*; D003432:Cross-Linking Reagents; D005260:Female; D005338:Fibrin Fibrinogen Degradation Products / Q000032:analysis; D005340:Fibrinogen / Q000032:analysis*; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D005470:Fluorometry; D006801:Humans; D008107:Liver Diseases / Q000188:drug therapy*; D016031:Liver Transplantation; D008297:Male; D008875:Middle Aged
D000933:Antifibrinolytic Agents; D003432:Cross-Linking Reagents; D005338:Fibrin Fibrinogen Degradation Products; D005456:Fluorescent Dyes; C036309:fibrin fragment D; D005340:Fibrinogen; D016650:Fluorescein-5-isothiocyanate; D015119:Aminocaproic Acid