Dataset Information

Binding of platelet factor 4 to heparin oligosaccharides.

ABSTRACT: Heparin fractions of differing Mr (7800-18 800) prepared from commercial heparin by gel filtration and affinity chromatography on immobilized anti-thrombin III had specific activities when determined by anti-Factor Xa and anti-thrombin assays that ranged from 228 to 448 units/mg. The anti-Factor Xa activity of these fractions could be readily and totally neutralized by increasing concentrations of platelet factor 4 (PF4). That these fractions bound to immobilized PF4 was indicated by the complete binding under near physiological conditions of 3H-labelled unfractionated commercial heparin. An anti-thrombin III-binding oligosaccharide preparation (containing predominantly eight to ten saccharide units), prepared by degradation of heparin with HNO2 had high (800 units/mg) anti-Factor Xa, but negligible anti-thrombin, specific activity. The anti-Factor Xa activity of this material could not be readily neutralized by PF4, and the 3H-labelled oligosaccharides did not completely bind to immobilized PF4. A heterogeneous anti-thrombin III-binding preparation containing upwards of 16 saccharides had anti-thrombin specific activity of just less than one-half the anti-Factor Xa specific activity. This material was completely bound to immobilized PF4 and was eluted with similar concentrations of NaCl to those that were required to elute unfractionated heparins from these columns. Furthermore, increasing concentrations of PF4 neutralized the anti-Factor Xa activity of this material in a manner similar to that of unfractionated heparin. It is concluded that heparin oligosaccharides require saccharide units in addition to the anti-thrombin III-binding sequence in order to fully interact with PF4.

SUBMITTER: Denton J

PROVIDER: S-EPMC1154112 | biostudies-other | 1983 Feb

REPOSITORIES: biostudies-other

ACCESS DATA

Similar Datasets

Project description:Oligosaccharides of well-defined molecular size were prepared from heparin by nitrous acid depolymerization, affinity chromatography on immobilized antithrombin III (see footnote on Nomenclature) and gel chromatography on Sephadex G-50. High affinity (for antithrombin III) octa-, deca-, dodeca-, tetradeca-, hexadeca- and octadeca-saccharides were prepared, as well as oligosaccharides of larger size than octadecasaccharide. The inhibition of Factor Xa by antithrombin III was greatly accelerated by all of these oligosaccharides, the specific anti-Factor Xa activity being invariably greater than 1300 units/mumol. The anti-Factor Xa activity of the decasaccharide was not significantly decreased in the presence of platelet factor 4, even at high platelet factor 4/oligosaccharide ratios. Measurable but incomplete neutralization of the anti-Factor Xa activities of the tetradeca- and hexadeca-saccharides was observed, and complete neutralization of octadeca- and larger oligo-saccharides was achieved with excess platelet factor 4. The octa-, deca-, dodeca-, tetradeca- and hexadeca-saccharides had negligible effect on the inhibition of thrombin by antithrombin III, whereas specific anti-thrombin activity was expressed by the octadeca-saccharide and by the larger oligosaccharides. An octadecasaccharide is therefore the smallest heparin fragment (prepared by nitrous acid depolymerization) that can accelerate thrombin inhibition by antithrombin III. The anti-thrombin activities of the octadecasaccharide and larger oligosaccharides were more readily neutralized by platelet factor 4 than were their anti-Factor Xa activities. These findings are compatible with two alternative mechanisms for the action of platelet factor 4, both involving the binding of the protein molecule adjacent to the antithrombin III-binding site. Such binding results in either steric interference with the formation of antithrombin III-proteinase complexes or in displacement of the antithrombin III molecule from the heparin chain.

Project description:It is well-known that heparin and other glycosaminoglycans (GAGs) inhibit complement activation. It is however not known whether fractionation and/or modification of GAGs might deliver pathway-specific inhibition of the complement system. Therefore, we evaluated a library of GAGs and their derivatives for their functional pathway specific complement inhibition, including the MASP-specific C4 deposition assay. Interaction of human MASP-2 with heparan sulfate/heparin was evaluated by surface plasmon resonance, ELISA and in renal tissue. In vitro pathway-specific complement assays showed that highly sulfated GAGs inhibited all three pathways of complement. Small heparin- and heparan sulfate-derived oligosaccharides were selective inhibitors of the lectin pathway (LP). These small oligosaccharides showed identical inhibition of the ficolin-3 mediated LP activation, failed to inhibit the binding of MBL to mannan, but inhibited C4 cleavage by MASPs. Hexa- and pentasulfated tetrasaccharides represent the smallest MASP inhibitors both in the functional LP assay as well in the MASP-mediated C4 assay. Surface plasmon resonance showed MASP-2 binding with heparin and heparan sulfate, revealing high Kon and Koff rates resulted in a Kd of ~2 ?M and confirmed inhibition by heparin-derived tetrasaccharide. In renal tissue, MASP-2 partially colocalized with agrin and heparan sulfate, but not with activated C3, suggesting docking, storage, and potential inactivation of MASP-2 by heparan sulfate in basement membranes. Our data show that highly sulfated GAGs mediated inhibition of all three complement pathways, whereas short heparin- and heparan sulfate-derived oligosaccharides selectively blocked the lectin pathway via MASP-2 inhibition. Binding of MASP-2 to immobilized heparan sulfate/heparin and partial co-localization of agrin/heparan sulfate with MASP, but not C3b, might suggest that in vivo heparan sulfate proteoglycans act as a docking platform for MASP-2 and possibly prevent the lectin pathway from activation.

Project description:Association of platelet factor 4 (PF4) with heparin is a first step in formation of aggregates implicated in the development of heparin-induced thrombocytopenia (HIT), a potentially fatal immune disorder affecting 1-5% of patients receiving heparin. Despite being a critically important element in HIT etiology, relatively little is known about the specific molecular mechanism of PF4-heparin interactions. This work uses native mass spectrometry to investigate PF4 interactions with relatively short heparin chains (up to decasaccharides). The protein is shown to be remarkably unstable at physiological ionic strength in the absence of polyanions; only monomeric species are observed, and the extent of multiple charging of corresponding ions indicates a partial loss of conformational integrity. The tetramer signal remains at or below the detection threshold in the mass spectra until the solution's ionic strength is elevated well above the physiological level, highlighting the destabilizing role played by electrostatic interactions vis-à-vis quaternary structure of this high-pI protein. The tetramer assembly is dramatically facilitated by relatively short polyanions (synthetic heparin-mimetic pentasaccharide), with the majority of the protein molecules existing in the tetrameric state even at physiological ionic strength. Each tetramer accommodates up to six pentasaccharides, with at least three such ligands required to guarantee the higher-order structure integrity. Similar results are obtained for PF4 association with longer and structurally heterogeneous heparin oligomers (decamers). These longer polyanions can also induce PF4 dimer assembly when bound to the protein in relatively low numbers, lending support to a model of PF4/heparin interaction in which the latter wraps around the protein, making contacts with multiple subunits. Taken together, these results provide a more nuanced picture of PF4-glycosaminoglycan interactions leading to complex formation. This work also advocates for a greater utilization of native mass spectrometry in elucidating molecular mechanisms underlying HIT, as well as other physiological processes driven by electrostatic interactions.

Dataset Information

Binding of platelet factor 4 to heparin oligosaccharides.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure