P150	Thrombin/ADP-stimulated platelets release coagulation factor XIIIA in microvesicles.
P.Cordell, P.Grant, R.Pease
Academic Unit of Molecular Vascular Medicine, LIGHT Laboratories, University of Leeds, Leeds, GB.

FXIIIA is a transglutaminase present in plasma and platelet cytosol that stabilizes blood clots by covalent crosslinking of fibrin. Whilst translocation of FXIIIA to the platelet periphery has been observed externalization of the protein has not been extensively investigated. The protein lacks a signal sequence but could nonetheless be secreted in microvesicles, which are released from platelets upon agonist stimulated exocytosis. We investigated the release of FXIIIA from platelets purified from citrated platelet rich plasma by gel filtration. Platelets were either activated with 2.5U/ml thrombin, 5mM Calcium and 10microM ADP or were treated with apyrase and prostaglandin E1 to act as a control sample. Whole platelets were pelleted at 1000 g with supernatants further fractionated into vesicles pelleting at 15,000 g and 100,000 g and a vesicle depleted supernatant. Sequential immunoprecipitation immunoblotting was used to measure FXIIIA in these fractions. A substantial proportion of FXIIIA (~40%) was pelleted at 15,000 g from the supernatant of activated platelets whilst only 5% was found in the corresponding control fraction. Some FXIIIA (~2%) was found in the 100,000 g pellet from activated platelet supernatant but none was detectable in the control. Traces of FXIIIA were also present in vesicle depleted supernatant from both resting and activated platelets and where thrombin was present this FXIIIA was cleaved (79 KDa). In contrast FXIIIA in the 15,000 g and 100,000 g pellets (83 KDa) remained uncleaved by thrombin supporting an intravesicular localisation. LDH measurements showed only ~7% of total platelet LDH activity present in the 15,000 g pellet from activated platelet supernatant giving a ~6 fold enrichment of FXIIIA. LDH activity in other supernatant fractions was below detectable levels. Recovery of FXIIIA was ~90%. Analysis of supernatants stained with the membrane dye FM 1 43 by confocal microscopy showed that these were essentially free of whole platelets but contained membranous particles of <15% the area of intact platelets. Thus FXIIIA is released in considerable quantities in membrane bound vesicles upon thrombin/ADP activation. Elucidation of the mechanism of microvesicle release may uncover new targets for antithrombotic agents.

Copyright © 2005 S. Karger AG, Basel. Any further use of this abstract requires written permission from the publisher.