One wonders whether heparin would be approved as an anticoagulant drug using modern criteria. Although this drug has been in clinical use since 1935 (1Barrowcliffe T.W. History of heparin.Handb. Exp. Pharmacol. 2012; 207: 3-22Crossref Scopus (37) Google Scholar), up to 3% of patients still can experience clinical complications of thrombocytopenia (2Arepally G.M. Heparin-induced thrombocytopenia.Blood. 2017; 129: 2864-2872Crossref PubMed Scopus (155) Google Scholar), a severe disorder with a mortality rate around 20% (3Rauova L. Poncz M. Sachais B.S. et al.Ultralarge complexes of PF4 and heparin are central to the pathogenesis of heparin-induced thrombocytopenia.Blood. 2005; 105: 131-138Crossref PubMed Scopus (208) Google Scholar). Heparin-induced thrombocytopenia is low platelet count caused by anti-platelet antibodies against a complex between heparin and endogenous protein platelet factor 4 (PF4), a small cytokine that is released by platelets during coagulation. PF4 exists as a tetramer with a large positively charged “belt” and negatively charged “poles”; it has high affinity for heparin and other negatively charged cellular glycosaminoglycans (4Kowalska M.A. Rauova L. Poncz M. Role of the platelet chemokine platelet factor 4 (PF4) in hemostasis and thrombosis.Thromb. Res. 2010; 125: 292-296Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). What mediates this heparin-PF4 binding and how might it be mitigated? The electrostatic interactions between the basic PF4 protein and the acidic heparin were implicated very early (5Zhang X. Chen L. Maione T.E. et al.Crystal structure of recombinant human platelet factor 4.Biochemistry. 1994; 33: 8361-8366Crossref PubMed Scopus (151) Google Scholar); however, the exact mechanism of complex formation is still not clearly understood. It was demonstrated that large complexes between tetramers of PF4 and heparin, responsible for the immune response, can form only in a narrow range of molar ratios of reactants: 1–2 mol PF4 tetramer/1 mol heparin (3Rauova L. Poncz M. Sachais B.S. et al.Ultralarge complexes of PF4 and heparin are central to the pathogenesis of heparin-induced thrombocytopenia.Blood. 2005; 105: 131-138Crossref PubMed Scopus (208) Google Scholar). Recently, the solved structure of PF4-heparin complex suggested that heparin does not simply wrap around PF4 but serves as an intermolecular binder between several tetramers, initiating formation of the large, disease-triggering complexes (6Cai Z. Yarovoi S.V. Greene M.I. et al.Atomic description of the immune complex involved in heparin-induced thrombocytopenia.Nat. Commun. 2015; 6: 8277Crossref PubMed Scopus (59) Google Scholar). Still, the extreme heterogeneity of the heparin-PF4 complexes and their sensitivity to molar ratios of the ingredients presented a major conundrum. To tease out this problem, the work of Kaltashov and co-workers, published in this issue of Biophysical Journal, employs native mass spectrometry (MS), a technique that allows qualitative evaluation of distributions of different complexes directly from a solution under physiological conditions (7Niu C. Yang Y. Kaltashov I.A. et al.Platelet factor 4 interactions with short heparin oligomers: implications for folding and assembly.Biophys. J. 2020; 119: 1371-1379Abstract Full Text Full Text PDF Scopus (3) Google Scholar). The authors tackle the complexity of the native MS profiles, using the technique of limited charge reduction in the gas phase to assign charge states. Surprisingly, it was discovered that even formation of the PF4 tetramers depends strongly on the presence of polyanions: heparin or endogenous glycosaminoglycans under native conditions. Moreover, even monomeric PF4 molecules revealed a high level of instability in the absence of polyanions at physiological ionic strength. At least three pentasaccharides were required for stability of a single PF4 molecule. Heparin decamers were also found to be effective binders of PF4, although they did not cause formation of the large complexes implicated in heparin-induced thrombocytopenia. This is consistent with the earlier data indicating that heparin has to have at least 12 saccharide residues to cause the immune response (8Visentin G.P. Moghaddam M. Aster R.H. et al.Heparin is not required for detection of antibodies associated with heparin-induced thrombocytopenia/thrombosis.J. Lab. Clin. Med. 2001; 138: 22-31Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). This work demonstrates the power of native MS for the analysis of heparin-PF4 complexes and provides an extremely detailed picture of a very convoluted process of binding between several sets of highly charged proteins and oligomers. The implication is that native MS will help define the structures of the tightest-binding saccharides, a capability that may be useful for screening libraries for compounds that compete with heparin binding to PF4. Platelet Factor 4 Interactions with Short Heparin Oligomers: Implications for Folding and AssemblyNiu et al.Biophysical JournalApril 21, 2020In BriefAssociation 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). Full-Text PDF Open Archive