Heparin Dodecasaccharide Binding to Platelet Factor-4 and Growth-related Protein-α

Abstract

␣-Chemokines are known heparin-binding proteins. Here, a heparin dodecasaccharide (H12) was purified and used in NMR studies to investigate binding to growth-related protein-␣ (Gro-␣) and to platelet factor-4-M2 (PF4-M2), an N-terminal chimera of PF4. Pulsed field gradient NMR was used to derive diffusion coefficients as the protein (monomer):H12 ratio was varied. In the absence of H12, both PF4-M2 and Gro-␣ give diffusion coefficients consistent with the presence of mostly dimers. As the PF4-M2:H12 ratio is increased from 1:6 to 2:1, the diffusion coefficient increases, indicating dissociation to the monomer state. On addition of H12 to either protein, 15 N/ 1 H heteronuclear single quantum coherence NMR data demonstrate loss of 1 H resonance dispersion and intensity, particularly at protein:H12 ratios of 2:1 to 4:1, indicating significant perturbation to native structures. For Gro-␣ in particular, 1 H resonance dispersion appears random coil-like. At these same ratios, circular dichroism (CD) data show general retention of secondary structure elements with a slight shift to additional helix formation. Random coil NMR resonance dispersion suggests a shift to a less compact, partially folded, and/or more flexible state. Further addition of H12 causes resonance intensity and dispersion to return making NMR spectra appear native-like. At low PF4-M2:H12 ratios, loss of resonance intensity for residues proximal to Arg-20 and Arg-22 in three-dimensional NMR HCCH-TOCSY spectra suggests that the Arg-20-Arg-22 loop either interacts most strongly with H12 and/or that binding at this site is heterogeneous. This domain was previously shown to be crucial to heparin binding. Of particular interest to the biology of PF4-heparin complex formation, heparin-induced thrombocytopenia antibody binding occurs at about the same PF4-M2:H12 ratio as does this transition to a partially folded PF4-M2 state, strongly suggesting that heparin-induced thrombocytopenia antibody recognizes a less folded, lower aggregate state of the protein.

Suppression of Insulin Aggregation by Heparin

“…The monosaccharides are sulfated to various extents and linked via 1f 4 glycosidic bonds. [21][22][23] Most common to porcine mucosal heparins (75%) are disaccharide repeats of 2-Osulfated-iduronic acid and 6-O-sulfated, N-sulfated-glucosamine. 23 The influence of heparin on protein aggregation is complicated.…”

“…The purpose of the present study was to investigate this last form of aggregation, the so-called "isoelectric precipitation" of insulin 19 at low I and the effect on it of heparin, a highly charged biopolymer whose biological functions are intimately connected with its ability to bind biofunctionally numerous proteins. 20,21 A member of the glycosaminoglycan family, heparin is composed of variable disaccharide units of uronic acid and glucosamine. The monosaccharides are sulfated to various extents and linked via 1f 4 glycosidic bonds.…”

Platelet Factor 4 and Interleukin-8 CXC Chemokine Heterodimer Formation Modulates Function at the Quaternary Structural Level

“…In vitro, IL8 has been shown to function both as a monomer and dimer (17). In its well known capacity to bind heparin, PF4 functions optimally as a tetramer in vitro (18,19) and presumably as a tetramer in vivo due both to the presence of heparan sulfate normally found on the surface of cells and because it is released from ␣-granules of platelets in relatively large quantities upon tissue injury.Previous studies (20,21) have shown that mixing various chemokines in solution can significantly modify their biological activities, but no thorough explanation as to why this occurs has been given. Here we use NMR spectroscopy and computational modeling to demonstrate that when present together in solution, CXC chemokines PF4 and IL8 interact with each other and exchange subunits to form AB-type heterodimers.…”

“…In vitro, IL8 has been shown to function both as a monomer and dimer (17). In its well known capacity to bind heparin, PF4 functions optimally as a tetramer in vitro (18,19) and presumably as a tetramer in vivo due both to the presence of heparan sulfate normally found on the surface of cells and because it is released from ␣-granules of platelets in relatively large quantities upon tissue injury.…”

Binding of a Large Chondroitin Sulfate/Dermatan Sulfate Proteoglycan, Versican, to L-selectin, P-selectin, and CD44

“…Several of the biological activities of proteoglycans are attributable to the GAGs. For example, heparin binds anti-thrombin III (1), and heparin and HS bind fibroblast growth factor (2) and various chemokines (3)(4)(5). Furthermore, CS binds a subset of chemokines, such as RANTES (regulated on activation normal T cell expressed and secreted) (6) and PF-4 (platelet factor-4) (7).…”