Role of Disabled-2 in Sulfatide-Mediated Platelet Aggregation

Platelets form a clump at the site of vascular injury to stop bleeding. One negative regulator of platelet aggregation is Disabled-2 (Dab2), a protein released to the extracellular surface upon platelet activation. Dab2 inhibits platelet aggregation through its phosphotyrosine-binding (PTB) domain by competing with fibrinogen for alphaIIb-beta3 integrin receptor binding. Sulfatides are sphingolipids found on the platelet surface, which interact with coagulation proteins, playing a major role in haemostasis. One of them is P-selectin, a protein that promotes platelet-platelet and platelet-leukocyte interactions by binding to sulfatides at the target cell surface. Using protein-lipid overlay and liposome-binding assays, we identified that the N-terminal region of Dab2, including its PTB domain (N-PTB), specifically interacts with sulfatides. Moreover, we determined that such interaction is mediated by two conserved basic motifs with a dissociation constant (Kd) of 0.6 microM as estimated by surface plasmon resonance analysis. In addition, liposome-binding assays combined with mass spectroscopy studies revealed that thrombin, a strong platelet agonist, cleaved N-PTB at a site located between the basic motifs, a region that becomes protected from thrombin cleavage when bound to sulfatides. Our results show that sulfatides recruit N-PTB to the platelet surface, sequestering it from integrin receptor binding during platelet activation. This is a transient recruitment that follows N-PTB internalization by an actin-dependent process. We demonstrate that sulfatide binding by N-PTB specifically inhibited P-selectin expression in activated platelets and P-selectin-mediated platelet-leukocyte aggregation. Our experimental data support a model where two pools of Dab2 co-exist at the platelet surface, in both sulfatide- and integrin receptor-bound states, and their balance controls the extent of the clotting response. Furthermore, we propose that Dab2 modulates P-selectin expression by competing off its interaction with sulfatides. Consequently, Dab2 inhibits homotypical and heterotypical aggregation, revealing a novel role of this protein in blood clotting. This project is in collaboration with Drs. Carla Finkielstein, Richey Davis, and David Bevan (Virginia Tech).

 

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Daniel G. S. Capelluto
Associate Professor
Biological Sciences Department
Biocomplexity Institute 263C
1015 Life Science Circle
Blacksburg, VA 24061-0477
E-mail: capellut@vt.edu
Phone: (540) 231-0974

College of Science

 

 

Department of Biological Sciences
2125 Derring Hall
Mail Code 0406
Virginia Tech
Blacksburg, VA 24061-0406
Phone: (540) 231-8930
Fax: (540) 231-9307

Last update 31 May 2017