אילמ"ר - האיגוד הישראלי למדעי המעבדה הרפואית

אילמ"ר - האיגוד הישראלי למדעי המעבדה הרפואית

כינוס שנתי ה-46 | 2012

פוסטרים להצגה בכינוס

ACTIVATION OF αIIbβ3 AND ITS MEDIATED CLOT RETRACTION ARE DEPENDENT ON THE PRESENCE OF FREE THIOLS

Ronit Mor-Cohen1, Nurit Rosenberg1, Yulia Averbukh1, Uri Seligsohn1, Judith Lahav2
1. The Amalia Biron Research Institute of Thrombosis and Hemostasis, Sheba Medical Center, Tel-Hashomer and the
2. Hemostasis Laboratory, Rabin Medical Center, Beilinson Campus, Petah Tiqva, and Sackler Faculty of Medicine, Tel Aviv University, Israel.     
Sheba Medical Center, Tel-Hashomer 52621

 

Introduction: The platelet integrin αIIbβ3 plays a major role in the process of platelet aggregation and formation of a hemostatic plug at the site of vascular injury. αIIbβ3 is inactive in resting platelets, but following its activation by inside-out signaling, it undergoes conformational changes resulting in fibrinogen binding which gives rise to platelet aggregation. Fibrinogen binding also initiates outside-in signaling leading to spreading of the platelets on extracellular matrix and clot retraction. We recently showed that disruptions of integrin-unique disulfide bonds in the EGF-like domains of the β3 subunit constitutively activate αIIbβ3 which is variably dependent on the presence of free thiols (JBC, 2012 Feb 3 Epub)

Aim: We investigated the role of free thiols in the processes of αIIbβ3 activation and αIIbβ3 mediated clot retraction.
Methods: We co-expressed in baby hamster kidney cells WT-aIIb together with WT-β3 or β3 containing cysteine substitutions that disrupt the unique C523-C544 bond (C523S and C544S) and the unique C560-C583 bond (C560S). We measured the binding of the fibrinogen-mimetic antibody PAC-1 to the WT or mutant integrins by flow cytometry and conducted a time-course of fibrin clot retraction in the presence or absence of the thiol blocker DTNB.

Results: All mutants displayed a pronounced increase in PAC-1 binding compared to WT αIIbβ3 which was not reduced by DTNB. WT αIIbβ3 bound PAC-1 only when it was pre incubated with the activating antibody anti-LIBS6 and this binding was inhibited by 70% in the presence of DTNB. Both WT αIIbβ3 and the three constitutively active mutants retracted the fibrin clot, but the mutants retracted faster. DTNB significantly inhibited clot retraction by WT αIIbβ3, but had a much smaller effect on the mutants. LIBS6 significantly increased the rate of WT αIIbβ3 clot retraction, and this increase was partially inhibited by DTNB only when it was added before but not after LIBS6.

Conclusions: Integrin αIIbβ3 has to be in an active conformation for ligand binding but not for clot retraction. The presence of free thiols is necessary for αIIbβ3 activation and for clot retraction only when the integrin is in its resting conformation but not when it is in its active conformation. Taken together our results suggest a role for free thiols in both αIIbβ3 inside-out and outside-in signaling.

 

 

לתכנית הכנס     חזרה לריכוז הפוסטרים 2012
דף הבית | מי אנחנו | רישום לאילמ"ר ולאתר | תקנון | חברי הועד | מערכת האתר | צור קשר | מקצוע המעבדנות הרפואית | מישיבות ועד אילמ"ר | המועצה למעבדות במשה"ב |
חיפוש פרטי חבר | עדכון פרטים | ועדות מקצועיות | מפגשים וועדות המטולוגיה וקרישה | פורום אנדוקריני | פורום האיכות | כנסים שנתיים | ימי עיון | קפה מדע | כנסים של אגודות אחרות | פעילות בינלאומית | אבטחת איכות סקירת ספרות | מכשור ושיטות | דיווחי המעבדות | פרסים | לזכר חברנו | קישורים | דרושים | מידע מקצועי מחברות תומכות    
מומלץ לצפייה ברזולוציה של 768X1024