Sulfatides partition disabled-2 in response to platelet activation.
BACKGROUND
Platelets contact each other at the site of vascular injury to stop bleeding. One negative regulator of platelet aggregation is Disabled-2 (Dab2), which is released to the extracellular surface upon platelet activation. Dab2 inhibits platelet aggregation through its phosphotyrosine-binding (PTB) domain by competing with fibrinogen for alphaIIbbeta3 integrin receptor binding by an unknown mechanism.


RESULTS
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 (K(d)) of 0.6 microM as estimated by surface plasmon resonance (SPR) 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. Sulfatides on the platelet surface interact with coagulation proteins, playing a major role in haemostasis. 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.


CONCLUSIONS
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.
Drahos
Karen E
KE
Protein Signaling Domains Laboratory, Virginia Polytechnic Institute and State University, Blacksburg, Virginia, United States of America.
Welsh
John D
JD
Finkielstein
Carla V
CV
Capelluto
Daniel G S
DG
D000199:Actins / Q000737:chemistry; D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D000595:Amino Acid Sequence; D051017:Apoptosis Regulatory Proteins; D005340:Fibrinogen / Q000737:chemistry; D006487:Hemostasis; D006801:Humans; D007700:Kinetics; D008055:Lipids / Q000737:chemistry; D008081:Liposomes / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D019000:Phosphotyrosine / Q000737:chemistry; D015539:Platelet Activation; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000737:chemistry; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013433:Sulfoglycosphingolipids / Q000378:metabolism*; D020349:Surface Plasmon Resonance; D025521:Tumor Suppressor Proteins
D000199:Actins; D048868:Adaptor Proteins, Signal Transducing; D051017:Apoptosis Regulatory Proteins; C102885:DAB2 protein, human; D008055:Lipids; D008081:Liposomes; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D013433:Sulfoglycosphingolipids; D025521:Tumor Suppressor Proteins; D019000:Phosphotyrosine; D005340:Fibrinogen