Effects of separate proteolytic and high-affinity binding activities of human thrombin on rapid platelet activation. A quenched-flow study

Abstract

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.

Contributions of Glycoprotein Ib and the Seven Transmembrane Domain Receptor to Increases in Platelet Cytoplasmic [Ca2+] Induced by α-Thrombin

“…The results of the present studies are in agreement with continuous-flow kinetic studies of rapid [Ca 2+ ] i dynamics in platelets activated by R-thrombin and the analogues, γ-and diisopropyl fluorophosphate-treated R-thrombin (Jones et al, 1989). These studies demonstrated that although the high-affinity binding activity of thrombin is not required for a major increase in cytoplasmic Ca 2+ concentration, fully active R-thrombin acting at both sites induces a more rapid (e2 s) mobilization of Ca 2+ .…”

Shiga toxin 2 and lipopolysaccharide cause monocytic THP-1 cells to release factors which activate platelet function

“…After 30 seconds, the reaction wasq uenched with glutaraldehyde (1% final concentration). The stimulated platelets were pelletedi nam icrocentrifugea nd aliquots of supernatants were mixed with scintillation cocktail for analysis as described (20).…”

Shiga Toxin 2 and Lipopolysaccharide Induce Human Microvascular Endothelial Cells To Release Chemokines and Factors That Stimulate Platelet Function

“…After 3 min, the reaction was quenched with glutaraldehyde (1%), and platelets were pelleted in a microcentrifuge. Aliquots of the supernatants were then mixed with a scintillation cocktail for analysis (Beckman LS 6500), and secretion was determined by the method of Jones et al (38). P-selectin exposure.…”