This study investigates 3 aspects of the adhesive interaction operating between platelet GPIb/lX and integrin o-4βj. These include ( 1 ) examining the sufficiency of GPIb/lX and integrin Otllbβ3 to mediate irreversible cell adhesion on a vWf matrix under flow; (2) the ability of the vWf-GPIb interaction to induce integrin Ollbβ3 activation independent of endogenous platelet stimuli; and (3) the identification of key second messengers linking the vWf-GPIb/IX interaction to integrin <xllbβ3 activation. Using CHO or K562 cells transfected with GPIb/lX and integrin <xnbβ3, we demonstrate that these receptors are both necessary and sufficient to mediate irreversible cell adhesion under flow, wherein GPIb/IX mediates cell tethering and rolling on immobilized vWf and integrin o-4βj mediates cell arrest. Moreover, we demonstrate direct signaling between GPIb/IX and integrin allbβ3. Studies on human platelets pretreated with inhibitors of ADP and TXA2, demonstrate that vWf binding to GPIb/IX is able to induce integrin (x,-4 activation independent of endogenous platelet stimuli under both static and physiological flow conditions (150 s-1, 600 s ' or 1800 s'). Similarly, K562 cells expressing GPIb/IX and integrin O1]bβ3 were able to adhere and spread on vWf and specifically bound the activation specific anti-integrin Ctnbβ3 mAb, PAC-1. Analysis of the key second messengers linking the vWf-GPIb interaction to integrin (XIIbβ3 activation demonstrated a key role for cytoplasmic calcium and PKC in this process. The first step in the activation process involves the mobilization of calcium from internal stores, whereas transmembrane calcium influx is a secondary event, potentiating integrin ot-4βj activation. Taken together, our studies demonstrate the existence of direct signaling pathways operating between GPIb/IX and integrin allbβ3 in platelets and transfected cell lines. Furthermore, they establish a critical role for intracellular calcium mobilization in initiating GPIb/IX-mediated integrin Otllbβ3 activation.
|11 PART I
|Published - 1 Dec 2000