Using a newly developed
thrombin-dependent platelet
thrombin generation assay, we observed that the feedback activation of
thrombin generation on the platelet surface does not depend on TF, as anti-TF
antibodies inhibiting TF-induced
thrombin formation in platelet-depleted plasma had no effect compared with vehicle-treated controls. Feedback activation of
thrombin generation in the presence of platelets was significantly diminished by membrane impermeant
thiol blockers or by the
thiol isomerase-inhibitors
bacitracin and anti-PDI antibody RL90, respectively. Platelet
thrombin formation depends on binding of
coagulation factors to the platelet surface. Therefore, involvement of
thiol isomerases in this binding was investigated. As shown by confocal microscopy and flow cytometry,
thrombin-stimulated platelets exhibited increased surface-associated PDI as well as extracellular
disulfide reductase activity compared with unstimulated platelets. Flow cytometric analysis revealed that membrane impermeant
thiol blockers or PDI inhibitors, which had been added after platelet stimulation and after
phosphatidylserine exposure to exclude their influence on primary platelet activation, significantly inhibited binding of all
coagulation factors to
thrombin-stimulated platelets.
CONCLUSIONS: