Binding of tissue-plasminogen activator to fibrin: effect of ultrasound.
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 Bmax. 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 Bmax was increased in the presence of ultrasound, from 31 mumol/L to 43 mumol/L (P < .0001). Ultrasound decreased the Kd for crosslinked fibrin to 343 nmol/L (P = .026) and also increased Bmax from 22 mumol/L to 25 mumol/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). These 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.
Siddiqi
F
F
Vascular Medicine Unit, Department of Medicine, University of Rochester School of Medicine & Dentistry, Rochester, NY 14642, USA.
Odrljin
T M
TM
Fay
P J
PJ
Cox
C
C
Francis
C W
CW
D001665:Binding Sites; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D005337:Fibrin / Q000737:chemistry / Q000378:metabolism*; D005782:Gels; D006801:Humans; D007700:Kinetics; D010477:Perfusion; D011108:Polymers; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D010959:Tissue Plasminogen Activator / Q000378:metabolism*; D014465:Ultrasonics
D005782:Gels; D011108:Polymers; D011994:Recombinant Proteins; D005337:Fibrin; D010959:Tissue Plasminogen Activator