Overexpression of the partially activated αIIb β3D723H integrin salt bridge mutant downregulates RhoA activity and induces microtubule-dependent proplatelet-like extensions in Chinese hamster ovary cells

Abstract

Background: We have recently reported a novel mutation in the β3 subunit of the platelet fibrinogen receptor (αIIbβ3 D723H) identified in a patient with dominantly inherited macrothrombocytopenia, and we have shown that this mutation promotes a new phenotype in Chinese hamster ovary (CHO) cells, characterized by fibrinogen-dependent, microtubule-driven proplatelet-like cell extensions. Results: Here we demonstrate that the partially activated αIIbβ3 D723H or αIIb β3 D723A salt bridge mutants, but not fully activated αIIbβ3 mutants, cause this phenotype. Time-lapse videomicroscopy clearly differentiated these stable microtubule-driven and nocodazole-sensitive extensions from common dynamic actin-driven pseudopodia. In addition, overexpression of a mitochondrial marker confirmed their functional role in organelle transport. Comparative immunofluorescence analysis of the subcellular localization of αIIbβ3, the focal adhesion proteins talin or vinculin and actin revealed a similar membrane labeling of CHO cell extensions and CD34+-derived megakaryocyte proplatelets. Mutant αIIb β3 D723H signaling was independent of Src, protein kinase C or phosphoinositide 3-kinase, but correlated with decreased RhoA activity as compared with wild-type αIIb β3 signaling, reminiscent of integrin signaling during neurite outgrowth. Accordingly, overexpression of constitutively active RhoA in CHO αIIbβ3 D723H cells prevented protrusion formation on fibrinogen. Most interestingly, RhoA/ ROCK inhibition was necessary, but not sufficient, and integrin activity was additionally required to induce CHO cell extension formation. Conclusions: CHO αIIbβ3 D723H cell protrusions and megakaryocyte proplatelets, like neuronal cell neurites, result from a common integrin-dependent signaling pathway, promoting strongly decreased RhoA activity and leading to microtubule-driven formation of cytoplasmic extensions. © 2009 International Society on Thrombosis and Haemostasis.