Thrombin induces GPIb-IX-mediated fibrin binding to alphaIIbbeta3 in a reconstituted Chinese hamster ovary cell model.
BACKGROUND
The interaction of thrombin with platelet glycoprotein (GP) Ib-IX-V has been recently suggested to induce fibrin-dependent platelet aggregation associated with signaling events. The approaches used to avoid the protease-activated receptor (PAR) thrombin receptors in platelets have provided controversial conclusions regarding the precise mechanism and molecules involved in the response.


OBJECTIVE
In the present study, we developed a cellular model to investigate the functional consequences following the binding of thrombin to GPIb-IX.


METHODS
We used Chinese hamster ovary (CHO) cells stably expressing human alpha(IIb)beta(3) and/or GPIb-IX complexes (CHO-alpha(IIb)beta(3)-IbIX cells) to analyze the effect of thrombin on the binding of polymerizing fibrin by using fluorescein isothiocyanate-fibrinogen as precursor.


RESULTS
Thrombin induces, in a dose-dependent manner, the binding of polymerizing fibrin to CHO-alpha(IIb)beta(3)-IbIX cells. This response is not observed in cells expressing only one of the receptors, and it can be blocked by monoclonal antibodies against alpha(IIb)beta(3) and GPIbalpha. We show that the reaction is not due to simple cell trapping by the fibrin clot, and provide data supporting a role of a signaling pathway in which the 14-3-3zeta adaptor and calcium-calmodulin-dependent events are involved.


CONCLUSIONS
The present data support a significant role of GPIb-IX and alpha(IIb)beta(3) receptors in an alternative fibrin-mediated pathway of platelet activation induced by thrombin.
Pabón
D
D
Department of Molecular and Cellular Pathophysiology, Centro de Investigaciones Biológicas (CSIC), Madrid, Spain.
Jayo
A
A
Xie
J
J
Lastres
P
P
González-Manchón
C
C
D000818:Animals; D001792:Blood Platelets / Q000378:metabolism; D016466:CHO Cells; D002118:Calcium / Q000737:chemistry; D002147:Calmodulin / Q000737:chemistry; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D005337:Fibrin / Q000737:chemistry*; D005340:Fibrinogen; D016650:Fluorescein-5-isothiocyanate / Q000494:pharmacology; D006801:Humans; D020935:MAP Kinase Signaling System; D009154:Mutation; D019000:Phosphotyrosine / Q000737:chemistry; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000737:chemistry* / Q000378:metabolism; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000737:chemistry*; D013917:Thrombin / Q000737:chemistry / Q000502:physiology*
D002147:Calmodulin; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D019038:Platelet Glycoprotein GPIb-IX Complex; D019000:Phosphotyrosine; D005337:Fibrin; D005340:Fibrinogen; D013917:Thrombin; D016650:Fluorescein-5-isothiocyanate; D002118:Calcium