Effects of separate proteolytic and high-affinity binding activities of human thrombin on rapid platelet activation. A quenched-flow study.
We have used a general quenched-flow approach to study platelet function as early as 0.3 s after stimulation with three types of human thrombin: alpha-thrombin, gamma-thrombin, which is proteolytically active but does not bind to the high-affinity sites, and di-isopropyl fluorophosphate-derivatized (DIP)-alpha-thrombin, an active site-inhibited analogue that does bind to the high-affinity site. Large doses of gamma-thrombin evoked moderate aggregation and serotonin release, but minimal phosphorylation of the 20 and 47 kDa proteins. The initial (1.5-3.0 s) increase in cystolic free calcium concentration ([Ca2+]i) indicated by Indo-1 was also diminished, but by 5 s was nearly as high (1.0 microM) as with alpha-thrombin. A large dose of DIP-alpha-thrombin, on the other hand, induced minimal aggregation, serotonin secretion and [Ca2+]i response within 6 s. There was, however, a transient dephosphorylation of the 20 kDa protein. When combined, gamma- and DIP-alpha-thrombin were approximately additive in their ability to induce aggregation and serotonin secretion, but strongly synergistic in phosphorylating the 20 and 47 kDa proteins. The [Ca2+]i increase was not, however, enhanced over that induced by gamma-thrombin alone. These results demonstrate that phosphorylation of either the 20 or 47 kDa proteins is not correlated with [Ca2+]i dynamics and is neither required nor directly involved in platelet aggregation and secretion induced by thrombin. The high-affinity binding activity of thrombin is not necessary for rapid platelet Ca2+ influx, aggregation and serotonin release within the first critical seconds of activation.
Jones
G D
GD
Department of Biochemistry, University of Virginia, Charlottesville 22908.
Carty
D J
DJ
Freas
D L
DL
Spears
J T
JT
Gear
A R
AR
D001792:Blood Platelets / Q000187:drug effects* / Q000378:metabolism; D002118:Calcium / Q000097:blood; D003600:Cytosol / Q000378:metabolism; D006801:Humans; D007531:Isoflurophate / Q000031:analogs & derivatives / Q000494:pharmacology; D007700:Kinetics; D010766:Phosphorylation; D010974:Platelet Aggregation / Q000187:drug effects; D012701:Serotonin / Q000097:blood; D013917:Thrombin / Q000378:metabolism / Q000494:pharmacology*
D007531:Isoflurophate; D012701:Serotonin; C027798:DFP-alpha-thrombin; D013917:Thrombin; D002118:Calcium