Disabled-2 is required for efficient hemostasis and platelet activation by thrombin in mice.

OBJECTIVE: The essential role of platelet activation in hemostasis and thrombotic diseases focuses attention on unveiling the underlying intracellular signals of platelet activation. Disabled-2 (Dab2) has been implicated in platelet aggregation and in the control of clotting responses. However, there is not yet any in vivo study to provide direct evidence for the role of Dab2 in hemostasis and platelet activation. APPROACH AND RESULTS: Megakaryocyte lineage-restricted Dab2 knockout (Dab2(-/-)) mice were generated to delineate in vivo functions of Dab2 in platelets. Dab2(-/-) mice appeared normal in size with prolonged bleeding time and impaired thrombus formation. Although normal in platelet production and granule biogenesis, Dab2(-/-) platelets elicited a selective defect in platelet aggregation and spreading on fibrinogen in response to low concentrations of thrombin, but not other soluble agonists. Investigation of the role of Dab2 in thrombin signaling revealed that Dab2 has no effect on the expression of thrombin receptors and the outside-in signaling. Dab2(-/-) platelets stimulated by low concentrations of thrombin were normal in Gαq-mediated calcium mobilization and protein kinase C activation, but were defective in Gα12/13-mediated RhoA-ROCKII activation. The attenuated Gα12/13 signaling led to impaired ADP release, Akt-mammalian target of rapamycin and integrin αIIbß3 activation, fibrinogen binding, and clot retraction. The defective responses of Dab2(-/-) platelets to low concentrations of thrombin stimulation may contribute to the impaired hemostasis and thrombosis of Dab2(-/-) mice. CONCLUSIONS: This study sheds new insight in platelet biology and represents the first report demonstrating that Dab2 is a key regulator of hemostasis and thrombosis by functional interplay with Gα12/13-mediated thrombin signaling.

The endocytic adaptor protein Disabled-2 is required for cellular uptake of fibrinogen.

Endocytosis is pivotal for uptake of fibrinogen from plasma into megakaryocytes and platelet α-granules. Due to the complex adaptor and cargo contents in endocytic vehicles, the underlying mechanism of fibrinogen uptake is not yet completely elucidated. In this study, we investigated whether the endocytic adaptor protein Disabled-2 (DAB2) mediates fibrinogen uptake in an adaptor-specific manner. By employing primary megakaryocytes and megakaryocytic differentiating human leukemic K562 cells as the study models, we found that fibrinogen uptake is associated with the expression of integrin αIIbß3 and DAB2 and is mediated through clathrin-dependent manner. Accordingly, constitutive and inducible knockdown of DAB2 by small interfering RNA reduced fibrinogen uptake for 53.2 ± 9.8% and 59.0 ± 10.7%, respectively. Culturing the cells in hypertonic solution or in the presence of clathrin inhibitor chlorpromazine abrogated clathrin-dependent endocytosis and diminished the uptake of fibrinogen. Consistent with these findings, 72.2 ± 0.2% of cellular DAB2 was colocalized with clathrin, whereas 56.4±4.1% and 54.6 ± 2.0% of the internalized fibrinogen were colocalized with clathrin and DAB2, respectively. To delineate whether DAB2 mediates fibrinogen uptake in an adaptor-specific manner, K562 stable cell lines with knockdown of the adaptor protein-2 (AP-2) or double knockdown of AP-2/DAB2 were established. The AP-2 knockdown cells elicited normal fibrinogen uptake activity but the uptake of collagen was diminished. In addition, collagen uptake was further reduced in DAB2/AP-2 knockdown cells. These findings thereby define an adaptor-specific mechanism in the control of fibrinogen uptake and implicate that DAB2 is the key adaptor in the clathrin-associated endocytic complexes to mediate fibrinogen internalization.