Binding of plasma factor XIII to thrombin-receptor activated human platelets Béla Nagy Jr 1 ; Zsuzsa Simon 1 ; Zsuzsa Bagoly 2 ; László Muszbek 2 ; János Kappelmayer 1 1 Department of Clinical Biochemistry and Molecular Pathology, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary; 2 Clinical Research Center and Thrombosis, Haemostasis and Vascular Biology Research Group, Hungarian Academy of Sciences, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary Summary Platelet-bound coagulation factor XIII (FXIII) is targeted and concentrated at the site where platelet-rich thrombi are form- ed. Previous studies were in disagreement about the nature of FXIII binding to platelets. In this study, thrombin-receptor acti- vating peptide (TRAP)-stimulated human whole blood and washed platelets were analysed by flow cytometry for the bind- ing of FXIII using a monoclonal antibody against the A subunit of FXIII (FXIII-A). Here, we demonstrate that FXIII-A positivity sig- nificantly increased on activated platelets in whole blood com- pared to unstimulated sample, but not in washed platelets. GPIIb/IIIa receptor plays an essential role in FXIII binding, as fi- brinogen receptor antagonist eptifibatide and fibrinogen binding inhibitor RGDS tetrapeptide significantly prevented the binding Keywords Factor XIII, platelet activation, TRAP, fibrinogen of FXIII. Furthermore, stimulated platelets from a patient with severe type I Glanzmann thrombasthenia showed insignificant FXIII-A positivity versus healthy controls. In addition, basal neg- ligible amount of FXIII on washed platelets was only slightly in- creased when highly purified plasma FXIII (FXIII-A 2 B 2 ) was added upon platelet activation by TRAP. Similarly, no remarkable FXIII-A positivity was observed when we used FXIII-A 2 B 2 with γA/γA fibrinogen. However, γA/γ' fibrinogen significantly aug- mented FXIII binding on TRAP-stimulated platelets in the pres- ence of non-activated FXIII-A 2 B 2 . We conclude that FXIII-A 2 B 2 of plasma origin binds to thrombin-receptor activated platelets via GPIIb/IIIa receptor-bound fibrinogen with γ'-chain and is not capable of direct platelet binding. Thromb Haemost 2009; 101: 83–89 Platelets and Blood Cells Correspondence to: János Kappelmayer, MD, PhD, Dsc Department of Clinical Biochemistry and Molecular Pathology Medical and Health Science Center, University of Debrecen Nagyerdei krt. 98. H-4032, Debrecen, Hungary Tel.: +36 52 340 006, Fax: +36 52 417 631 E-mail: kappelmayer@med.unideb.hu Received: January 22, 2009 Accepted after minor revision: March 14, 2009 Prepublished online: May 11, 2009 doi:10.1160/TH09-01-0054 © 2009 Schattauer GmbH, Stuttgart 83 Introduction Blood coagulation factor XIII (FXIII) is a protransglutaminase that is essential for maintaining haemostasis as a key regulator of fibrinolysis (1). Plasma FXIII is a heterotetrameric zymogen (FXIII-A 2 B 2 ) that consists of two potentially active A subunits (FXIII-A) and two carrier/inhibitory B subunits (FXIII-B) (2). The cellular form of FXIII (FXIII-A 2 ) that is a dimer of two A subunits, is present in monocytes, macrophages and the platelet cytosol (reviewed in [2, 3]). FXIII is targeted and concentrated at the site where platelet clots are formed (2). FXIII increases the fibrinolytic resistance of platelet-rich thrombi through the cross-linking of α 2 -antiplas- min to fibrin (4, 5) and the presence of platelets accelerates the cross-linking process (5). The interaction between activated FXIII (FXIIIa) and platelets has been investigated in a few for- mer studies (6–8), and a saturable and specific binding of FXIIIa to thrombin-stimulated, but not to resting platelets was demon- strated (6–8). However, data on the binding site of FXIIIa were contradictory. In the first study, GPIIb/IIIa receptor and platelet- bound fibrin(ogen) were excluded as binding site for FXIIIa; RGD peptide did not influence the interaction between FXIIIa and platelets, and FXIIIa binding to platelets from two patients with severe Glanzmann thrombasthenia (GT) was normal (6). The binding of FXIIIa to thrombin-activated platelets was in- hibited by plasmin (7). In a later study, however, the GPIIb/IIIa receptor was shown to mediate the binding of FXIIIa to throm- bin-stimulated platelets, and indirect binding of FXIIIa through fibrinogen associated with its receptor was also demonstrated (8). In contrast to previous studies, here we investigated the bind- ing of intact non-activated plasma factor XIII to human platelets stimulated with thrombin-receptor activating peptide (TRAP). Our working hypothesis was that FXIII can bind to platelets in non-activated form, then on the surface of activated platelets it becomes easily activated by locally formed thrombin and exerts its cross-linking action. We confirmed that intracellular platelet FXIII-A 2 is not exposed on the cell surface during platelet acti- For personal or educational use only. No other uses without permission. All rights reserved. Downloaded from www.thrombosis-online.com on 2018-04-21 | ID: 1001066444 | IP: 54.70.40.11