Heparin modulates the conformation and signaling of platelet integrin αIIbß3.

INTRODUCTION: The glycosaminoglycan heparin has been shown to bind to platelet integrin αIIbß3 and induce platelet activation and aggregation, although the relationship between binding and activation is unclear. We analyzed the interaction of heparin and αIIbß3 in detail, to obtain a better understanding of the mechanism by which heparin acts on platelets. METHODS: We assessed conformational changes in αIIbß3 by flow cytometry of platelets exposed to unfractionated heparin. In human platelets and K562 cells engineered to express αIIbß3, we assayed the effect of heparin on key steps in integrin signaling: phosphorylation of the ß3 chain cytoplasmic tail, and activation of src kinase. We measured the heparin binding affinity of purified αIIbß3, and of recombinant fragments of αIIb and ß3, by surface plasmon resonance. RESULTS AND CONCLUSIONS: Heparin binding results in conformational changes in αIIbß3, similar to those observed upon ligand binding. Heparin binding alone is not sufficient to induce tyrosine phosphorylation of the integrin ß3 cytoplasmic domain, but the presence of heparin increased both ß3 phosphorylation and src kinase activation in response to ligand binding. Specific recombinant fragments derived from αIIb bound heparin, while recombinant ß3 did not bind. This pattern of heparin binding, compared to the crystal structure of αIIbß3, suggests that heparin-binding sites are located in clusters of basic amino acids in the headpiece and/or leg domains of αIIb. Binding of heparin to these clusters may stabilize the transition of αIIbß3 to an open conformation with enhanced affinity for ligand, facilitating outside-in signaling and platelet activation.

Heparin modulates integrin-mediated cellular adhesion: specificity of interactions with alpha and beta integrin subunits.

Heparin is known to influence the growth, proliferation, and migration of vascular cells, but the precise mechanisms are unknown. We previously demonstrated that unfractionated heparin (UH) binds to the platelet integrin alpha(IIb)beta(3), and enhances ligand binding. To help define the specificity and site(s) of heparin-integrin interactions, we employed the erythroleukemic K562 cell line, transfected to express specific integrins (alpha(v)beta(3), alpha(v)beta(5), and alpha(IIb)beta(3)). By comparing K562 cells expressing a common alpha subunit (Kalpha(v)beta(3), Kalpha(v)beta(5)) with cells expressing a common beta subunit (Kalpha(v)beta(3), Kalpha(IIb)beta(3)), we observed that heparin differentially modulated integrin-mediated adhesion to vitronectin. UH at 0.5-7.5 microg/ml consistently enhanced the adhesion of beta(3) expressing cells (Kalpha(v)beta(3),Kalpha(IIb)beta(3)). In contrast, UH at 0.5-7.5 microg/ml inhibited Kalpha(v)beta(5) adhesion. Experiments using integrin-blocking antibodies, appropriate control ligands, and nontransfected native K562 cells revealed that heparin's actions were mediated by the specific integrins under study. Preincubation of heparin with Kalpha(v)beta(3) cells enhanced adhesion, while preincubation of heparin with the adhesive substrate (vitronectin) had minimal effect. There was a structural specificity to heparin's effect, in that a low molecular weight heparin and chondroitin sulfate showed significantly less enhancement of adhesion. These findings suggest that heparin's modulation of integrin-ligand interactions occurs through its action on the integrin. The inhibitory or stimulatory effects of heparin depend on the beta subunit type, and the potency is dictated by structural characteristics of the glycosaminoglycan.

Novel vascular endothelial growth factor binding domains of fibronectin enhance vascular endothelial growth factor biological activity.

Interactions between integrins and growth factor receptors play a critical role in the development and healing of the vasculature. This study mapped two binding domains on fibronectin (FN) that modulate the activity of the angiogenic factor, vascular endothelial growth factor (VEGF). Using solid-phase assays and surface plasmon resonance analysis, we identified two novel VEGF binding domains within the N- and C-terminus of the FN molecule. Native FN bound to VEGF enhanced endothelial cell migration and mitogen-activated protein (MAP) kinase activity, but FN that is devoid of the VEGF binding domains failed to do so. Coprecipitation studies confirmed a direct physical association between VEGF receptor-2 (Flk-1) and the FN integrin, alpha5beta1, which required intact FN because FN fragments lacking the VEGF binding domains failed to support receptor association. Thrombin-activated platelets released intact VEGF/FN complexes, which stimulated endothelial cell migration and could be inhibited by soluble high affinity VEGF receptor 1 and antibodies to alpha5beta1 integrin. This study demonstrates that FN is potentially a physiological cofactor for VEGF and provides insights into mechanisms by which growth factor receptors and integrins cooperate to influence cellular behavior.