Thrombin Provokes Degranulation of Platelet α-Granules Leading to the Release of Active Plasminogen Activator Inhibitor-1 (PAI-1).

Abstract

The balance of fibrinolytic mediators is crucial to the survival of the critically ill patient, with tissue plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) playing significant roles. While elevated levels of PAI-1 are associated with increased morbidity and mortality, the source of this PAI-1 remains elusive. Platelets contain 90% of circulating plasma PAI-1, however, their ability to release active PAI-1 is controversial. We hypothesize platelets contain active PAI-1 in α-granules capable of immediate degranulation when exposed to high concentrations of thrombin. In vitro apheresis platelets were stimulated with thrombin (1 IU/mL, 5 IU/mL) followed by the collection of supernatant (5-120 min). Supernatant and lysate PAI-1 was measured by ELISA. The experiment was repeated in the presence of t-PA followed by measurement of t-PA:PAI-1 complex measurement by ELISA. Finally, healthy whole blood underwent dilution with control and thrombin-treated platelet lysate followed by thrombelastography (TEG) in a t-PA-stimulated TEG. Thrombin provoked immediate near-complete degranulation of PAI-1 from α-granules (median 5m 5 IU/mL thrombin 125.1 ng/mL, 1 IU/mL thrombin 114.9 ng/mL, control 9.9 ng/mL). The released PAI-1 rapidly complexed with t-PA, with a 4-fold increase in complex formation in the thrombin-treated supernatant. Conversely, PAI-1 in the control lysate demonstrated a 6-fold increase in complex formation compared with thrombin lysate. Last, control platelet lysate inhibited t-PA-induced fibrinolysis by TEG (median LY30 control 15m 7.9%), while thrombin-treated platelet lysates, after PAI-1 degranulation, were unable to affect the fibrinolysis profile (median LY30 5 IU/mL 28.5%, 1 IU/mL 12.4%). Thrombin provokes rapid α-degranulation of active PAI-1, capable of complexing with t-PA and neutralizing t-PA-induced fibrinolysis by TEG.