TY - JOUR
T1 - Binding of tissue-plasminogen activator to fibrin
T2 - Effect of ultrasound
AU - Siddiqi, Farhan
AU - Odrljin, Tatjana M.
AU - Fay, Philip J.
AU - Cox, Christopher
AU - Francis, Charles W.
PY - 1998/3/15
Y1 - 1998/3/15
N2 - 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.
AB - 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.
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U2 - 10.1182/blood.v91.6.2019
DO - 10.1182/blood.v91.6.2019
M3 - Article
C2 - 9490686
AN - SCOPUS:0032521423
SN - 0006-4971
VL - 91
SP - 2019
EP - 2025
JO - Blood
JF - Blood
IS - 6
ER -