A microtiter plate assay for factor XIII A-chain-fibrin interactions.
Factor XIII A-chain-fibrin interactions regulate factor XIIIa formation and fibrin cross-linking. A microtiter plate assay was developed for studying these interactions. Microtiter plate wells were coated with fibrinogen and converted to fibrin by thrombin. After blocking the wells with bovine serum albumin, factor XIII A-chain was added and binding was monitored by incubating first with anti-factor XIII followed by anti-rabbit IgG-alkaline phosphatase. Enzymatic hydrolysis of p-nitrophenyl phosphate was quantitated by the absorbance at 405 nm. BInding was specific, sensitive, rapid, saturable, and reversible, requiring only nanograms of either factor XIII or fibrin. Binding was time- and concentration-dependent and independent of divalent cations. The bound material was identified as factor XIII A-chain by sodium dodecylsulfate-polyacrylamide gel electrophoresis and immunoblotting. Factor XIII binding was inhibited > 75% by 250 mM sodium chloride or 250 nM anti-factor XIII IgG. The method was also suitable for demonstrating binding using 0.8% plasma or with r-factor XIII expressed in Saccharomyces cerevisiae or Escherichia coli. This method is suitable for identifying the binding sites that are important for plasma factor XIII activation and factor XIIIa activity.
Achyuthan
K E
KE
Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710.
Santiago
M A
MA
Greenberg
C S
CS
D000818:Animals; D001896:Boron Compounds / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005176:Factor XIII / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism*; D005337:Fibrin / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism*; D005340:Fibrinogen / Q000378:metabolism; D006041:Goats / Q000276:immunology; D006801:Humans; D015151:Immunoblotting / Q000379:methods; D007700:Kinetics; D008163:Luminescent Measurements; D008832:Microchemistry; D008970:Molecular Weight; D009842:Oligopeptides / Q000494:pharmacology; D011485:Protein Binding; D012680:Sensitivity and Specificity; D012710:Serum Albumin, Bovine; D012965:Sodium Chloride / Q000494:pharmacology; D013917:Thrombin / Q000037:antagonists & inhibitors / Q000378:metabolism
D001896:Boron Compounds; D009842:Oligopeptides; C066061:acetylphenylalanyl-prolyl-boroarginine; D012710:Serum Albumin, Bovine; D012965:Sodium Chloride; D005337:Fibrin; D005340:Fibrinogen; D005176:Factor XIII; D013917:Thrombin