Platelet Factor 4 Interactions with Short Heparin Oligomers: Implications for Folding and Assembly

Abstract

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.

Molecular architecture and platelet-activating properties of small immune complexes assembled on intact heparin and their possible involvement in heparin-induced thrombocytopenia

“…While heparinoids as short as decasaccharides 38 ). Our previous work 22 demonstrated that such heparinoids (dp10) are incapable of PF4 bridging, 22 and the results of the present study indicate that even heparin fragments that are twice as long (dp20) are not sufficiently large for PF4 bridging.…”

“…Previously we have shown that short heparin fragments (at least up to a decasaccharide level, dp10) are not sufficient for PF4 bridging (instead, multiple heparinoids associate with a single PF4 tetramer). 22 Similar behavior is exhibited by significantly longer chains, such as dp20 (the length that corresponds to an average size of the low-molecular weight heparin products, LMWH 23 ), as can be seen in Figure 1A. Incubation of dp20 with PF4 at an approximately equimolar ratio gives rise to an abundant ionic signal corresponding to a PF4•(dp20) 2 complex, although the 1:1 protein/heparinoid complexes are also observed (Figure 1A, top).…”

The Utility of Native MS for Understanding the Mechanism of Action of Repurposed Therapeutics in COVID-19: Heparin as a Disruptor of the SARS-CoV-2 Interaction with Its Host Cell Receptor

“…Addition of a structurally homogeneous pentasaccharide heparin mimetic (fondaparinux) to the RBD solution results in a noticeable shift of the ionic signal in native MS ( Figure 1 B). The magnitude of this shift (1521 Da) correlates with the ligand mass (1505 Da); importantly, only 1:1 protein/ligand complexes are observed (in contrast to high-pI proteins, which act as heparin “sponges” by accommodating multiple polyanionic chains 29 ). Similar behavior is observed when a fixed-length heparin oligomer (eicosasaccharide, or dp20) is added to the RBD solution ( Figure 1 C).…”

Solution- and gas-phase behavior of decavanadate: implications for mass spectrometric analysis of redox-active polyoxidometalates

“…Decavanadate is a versatile member of the POM family displaying a variety of unique properties that can be exploited for a range of diverse applications. Our own interest in this molecule stems from its polyanionic nature that lends V10 as a viable candidate for applications in medicine as a disruptor of electrostatically-driven biopolymer interactions such as the platelet factor 4/heparin interactions 42 underlying the etiology of heparin-induced thrombocytopenia. The unique chemical properties of V10 are also enjoying considerable attention in areas ranging from chemical catalysis to electronics.…”