Binding of tissue-plasminogen activator to fibrin: Effect of ultrasound

Farhan Siddiqi, Tatjana M. Odrljin, Philip J. Fay, Christopher Cox, Charles W. Francis

Research output: Contribution to journalArticlepeer-review

Abstract

Ultrasound reversibly alters the structure of polymerized fibrin, an effect that could influence tissue-plasminogen activator (t-PA) binding. We have, therefore, characterized the effects of ultrasound on binding of t-PA to fibrin using a novel system in which radiolabeled, active-site blocked, single chain tissue-plasminogen activator flowed through a fibrin gel at constant rate, and specific binding was determined by monitoring incorporation of radiolabel. Results using polymerized fibrin were compared with those using a surface of fibrin immobilized on Sepharose beads in a similar system. Interaction of t-PA with surface-immobilized fibrin involved two classes of binding sites (Kd = 31 nmol/L and 244 nmol/L) and a maximum binding ratio of 3.8 mol t-PA/mol fibrin. Ultrasound increased Kd for the high affinity site to 46 nmol/L (P < .0001), but it had no significant effects on the Kd 244 nmol/L site nor on B(max). Tissue-plasminogen activator binding to noncrosslinked fibrin involved two sites with Kds of 267 nmol/L and 952 nmol/L, while a single Kd 405 nmol/L site was identified for crosslinked fibrin. Ultrasound had no significant effect on the binding affinity for noncrosslinked fibrin, but B(max) was increased in the presence of ultrasound, from 31 μmol/L to 43 μmol/L (P < .0001). Ultrasound decreased the Kd for crosslinked fibrin to 343 nmol/L (P = .026) and also increased B(max) from 22 μmol/L to 25 μmol/L (P = .015). Ultrasound also affected the kinetics of t-PA binding to fibrin, significantly accelerating the rate of dissociation by 77% ± 5% for noncrosslinked fibrin and by 69% ± 3% for crosslinked fibrin (P < .001 for each), results indicate that ultrasound exposure accelerates t-PA binding, alters binding affinity, and increases maximum binding to polymerized fibrin, effects that may result from ultrasound-induced changes in fibrin structure.

Original languageEnglish (US)
Pages (from-to)2019-2025
Number of pages7
JournalBlood
Volume91
Issue number6
DOIs
StatePublished - Mar 15 1998
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Immunology
  • Hematology
  • Cell Biology

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