Activation of integrin α IIb β 3 in the glycoprotein Ib‐high population of a megakaryocytic cell line, CMK, by inside‐out signaling

Summary.  Affinity/avidity state of integrin α IIb β 3 is regulated by intracellular inside‐out signaling. Although several megakaryocytic cell lines have been established, soluble ligand binding to α IIb β 3 expressed in these cells by cellular agonists has not been demonstrated. We have re‐examined agonist‐induced α IIb β 3 activation on megakaryocytic cell lines with a marker of the late stage of megakaryocytic differentiation, glycoprotein Ib (GPIb). Activation of α IIb β 3 was assessed by PAC1 and soluble fibrinogen binding to the cells. We found that α IIb β 3 expressed in CMK cells with high GPIb expression was activated by a phorbor ester, phorbol myristate acetate (PMA). Although the population of the GPIb high cells was <0.5% of the total cells, incubation with a nucleoside analog, ribavirin, efficiently increased the PMA‐reactive GPIb high cells. Not only PMA but also a calcium ionophore, A23187, induced α IIb β 3 activation, and PMA and A23187 had an additive effect on α IIb β 3 activation. Ligand binding to the activated α IIb β 3 in the GPIb high CMK cells is totally abolished by an α IIb β 3 ‐specific antagonist, and inhibited by wortmannin, cytochalasin‐D and prostaglandin E 1 , and the effects of these inhibitors on α IIb β 3 activation in the GPIb high CMK cells were compatible with those in platelets. We have also demonstrated that the ribavirin‐treated CMK cells express PKC‐α, ‐β, ‐δ and ‐θ, and suggested that PKC‐α and/or ‐β appear to be responsible for PMA‐induced activation of α IIb β 3 in CMK cells.