Platelet-Derived Chemokine CXCL7 Dimer Preferentially Exists in the Glycosaminoglycan-Bound Form: Implications for Neutrophil-Platelet Crosstalk.

Aaron J. Brown,Kirti V. Sawant,Krishna Mohan Sepuru,Krishna Rajarathnam

doi:10.3389/fimmu.2017.01248

Abstract

Platelet-derived chemokine CXCL7 (also known as NAP-2) plays a crucial role in orchestrating neutrophil recruitment in response to vascular injury. CXCL7 exerts its function by activating the CXC chemokine receptor 2 (CXCR2) receptor and binding sulfated glycosaminoglycans (GAGs) that regulate receptor activity. CXCL7 exists as monomers, dimers, and tetramers, and previous studies have shown that the monomer dominates at lower and the tetramer at higher concentrations. These observations then raise the question: what, if any, is the role of the dimer? In this study, we make a compelling observation that the dimer is actually the favored form in the GAG-bound state. Further, we successfully characterized the structural basis of dimer binding to GAG heparin using solution nuclear magnetic resonance (NMR) spectroscopy. The chemical shift assignments were obtained by exploiting heparin binding-induced NMR spectral changes in the WT monomer and dimer and also using a disulfide-linked obligate dimer. We observe that the receptor interactions of the dimer are similar to the monomer and that heparin-bound dimer is occluded from receptor interactions. Cellular assays also show that the heparin-bound CXCL7 is impaired for CXCR2 activity. We conclude that the dimer–GAG interactions play an important role in neutrophil–platelet crosstalk, and that these interactions regulate gradient formation and the availability of the free monomer for CXCR2 activation and intrathrombus neutrophil migration to the injury site.

Highlights

Akin to a well-equipped first aid kit used during emergencies, platelet granules contain hundreds of proteins that are rapidly released in response to vascular injury [1,2,3,4]
Ex vivo, and biophysical studies for a wide variety of chemokines have shown that the dimer and higher order oligomers, compared to the monomer, bind GAGs such as heparan sulfate with higher affinity [24, 25, 34,35,36,37,38]
Recent nuclear magnetic resonance (NMR) studies of GAG heparin binding to WT CXCL1, CXCL5, and CXCL8 have shown that the dimer is the high-affinity GAG ligand and that the differences in affinities are more pronounced for longer GAGs

Summary

INTRODUCTION

Akin to a well-equipped first aid kit used during emergencies, platelet granules contain hundreds of proteins that are rapidly released in response to vascular injury [1,2,3,4]. CXCL7 was initially identified in platelet-derived releasates more than 25 years ago as a neutrophil-activating chemokine (NAC) [14,15,16]. CXCL7 is a member of a subset of seven chemokines characterized by their N-terminal ELR motif, which function as agonists for the CXC chemokine receptor 2 (CXCR2) receptor These chemokines share the properties of reversibly existing as monomers and dimers and interacting with glycosaminoglycans (GAGs). Considering the local CXCL7 concentration can vary by orders of magnitude during active neutrophil recruitment, GAG-dimer interactions most likely regulate the levels of free monomer available for receptor activation. In the context of platelet-neutrophil crosstalk, that the relative ratios of the monomer and dimer in the free and GAG-bound form could be critical for the repair process and to minimize collateral tissue damage and disease

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION