Title : Defining the clinical contribution of platelet-derived FXIII-A to regulation of fibrinolysis
Abstract:
Background: Platelets contain an abundance of cellular FXIII-A in their cytoplasm which is released upon stimulation and participates in extracellular cross-linking reactions. As FXIII-A lacks a signal release peptide, the mechanisms involved in its externalisation remain unclear.
Aims: Defining the clinical contribution of platelet-derived FXIII-A to regulation of fibrinolysis and identify the signalling mechanisms of their exposure on the platelet surface following stimulation.
Methods: Washed human platelets were activated with (thrombin 100 nM + convulxin 100 ng/ml) ± α???β? inhibitor (tirofiban 1 µg/ml), pan-caspase inhibitor (ZVAD 100 µM), caspase-3 inhibitor (Z-DEVD 100 µM), Src family kinases (SFKs) inhibitor (dasatinib 4 µM) or PTP1B inhibitor (MSI-1436 2 µM). FXIII-A and phosphatidylserine (PS) exposure were detected by flow cytometry and confocal microscopy. Model thrombi formed from FXIII deficient plasma + FITC-labelled fibrinogen ± platelets, transglutaminase inhibitor (1mM) or the inhibitors described above. Thrombi were bathed in tissue plasminogen activator (1 µM) and lysis rate determined as fluorescence release.
Results: The number of platelets expressing FXIII-A and PS was significantly increased following strong dual activation with thrombin + convulxin (PS 79.6 ± 6.6 vs. 3.4 ± 1.1 %; FXIII-A 74.3 ± 6.3 vs. 5.4 ± 1.6 %) compared to resting platelets. Flow cytometry and confocal microscopy revealed a significant reduction in FXIII-A and PS exposure after inhibition of α???β?, caspases, caspase-3 and SFKs. Inhibition of PTP1B had no effect on FXIII-A or PS exposure. Platelet FXIII-A stabilized FXIII- depleted thrombi however, this stabilising effect was reduced upon inhibition of α???β?, caspases, caspase-3 and SFKs.
Conclusions: Platelet-derived FXIII-A promote the thrombus stability against the degradation and the intracellular signalling of the integrin α???β?, caspases and SFKs are required for their externalisation and activity to enhance the extracellular cross-lined functions.
