Disruption of PF4/H multimolecular complex formation with a minimally anticoagulant heparin (ODSH)

Joglekar, Manali; Diez, Pedro M. Quintana; Marcus, S.; Rui, Qi; Espinasse, Benjamin; Wiesner, Mark R.; Pempe, Elizabeth H.; Liu, Jian; Monroe, Dougald M.; Arepally, Gowthami M.

doi:10.1160/th11-11-0795

Cited by 30 publications

(28 citation statements)

References 27 publications

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Section: Discussionmentioning

confidence: 99%

“…In support of this mechanistic concept, other groups have recently reported that anticoagulant heparin 2-O,3-O-desulfated heparin, a heparinoid molecule, can disrupt PF4:heparin complexes and decrease platelet activation. 34,35 This article describes the initial steps in the identification and characterization of small molecules that bind to specific sites on PF4 and thereby (1) inhibit PF4 tetramerization, (2) inhibit formation of PF4:heparin ULCs, (3) disrupt preformed ULCs, and (4) prevent HIT antibody and Fc␥RIIA-mediated platelet activation.We also report the development of a robust and reproducible new assay to measure HIT antibodies capable of inducing cellular activation via Fc␥RIIA by transfecting the receptor and an NFATLuc reporter into DT40 cells. We have shown that these cells are activated by the HIT-like monoclonal antibody KKO, as well as by antibodies from several patients with HIT, when PF4 and heparin are present and that activation is via Fc␥RIIA.…”

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confidence: 99%

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Rational design and characterization of platelet factor 4 antagonists for the study of heparin-induced thrombocytopenia

Sachais

Rux

Cines

et al. 2012

Blood

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Patients with heparin-induced thrombocytopenia (HIT) remain at risk for recurrent thromboembolic complications despite improvements in management. HIT is caused by antibodies that preferentially recognize ultralarge complexes (ULCs) of heparin and platelet factor 4 (PF4) tetramers. We demonstrated previously that a variant PF4 K50E forms dimers but does not tetramerize or form ULCs. Here, we identified small molecules predicted to bind PF4 near the dimer-dimer interface and that interfere with PF4 tetramerization. Screening a library of small molecules in silico for binding at this site, we identified 4 compounds that inhibited tetramerization at micromolar concentrations, designated PF4 antagonists (PF4As). PF4As also inhibited formation of pathogenic ULCs, and 3 of these PF4As promoted the breakdown of preformed ULCs. To characterize the ability of PF4As to inhibit cellular activation, we developed a robust and reproducible assay IntroductionHeparin-induced thrombocytopenia (HIT) is a serious complication of heparin therapy. HIT develops in ϳ 1% to 3% of patients treated with unfractionated heparin for 5 to 10 days but also in patients treated with low-molecular-weight heparin and other polysaccharide anticoagulants. [1][2][3] It is generally accepted that the clinical manifestations of HIT are caused by antibodies that recognize a complex composed of heparin and platelet factor 4 (PF4) tetramers. 4 PF4 is a 70-amino acid, lysine-rich 7.8-kDa platelet-specific CXC chemokine that is secreted in high concentrations when platelets are activated. PF4 tetramers bind avidly to heparin and to cellular glycosaminoglycans. [5][6][7] This interaction is central to the pathogenesis of HIT. 4,[8][9][10][11] HIT antibodies bind preferentially to PF4 over a narrow molar ratio of reactants, that is, ϳ 1-2 tetramers: 1 molecule of heparin, average molecular mass 15 kDa. 4,10,[12][13][14] We and others have shown that at these ratios, PF4 tetramers and heparin form stable but reversible ultralarge complexes (ULCs) 10 that preferentially bind HIT antibodies and activate platelets through FcR␥IIA. 4,10,15 Therefore, the biochemical basis for ULC formation is of importance in understanding antigen formation and potentially as a means to disrupt antigen formation and prevent or manage HIT.The crystal structure of PF4 reveals that the ␤-sheets of each monomer face the tetramer interface, with the N and C termini lying on the exterior surface of the molecule. 16 The lysine-rich C-terminal ␣-helices contribute to a circumferential band of positively charged amino acid residues 17 that have been implicated in the binding of heparin. 18 Importantly, the energy contributed by salt bridges, specifically involving glutamic acid 28 and lysine 50, drive PF4 tetramer formation. 16 In solution, PF4 exists in a dynamic equilibrium between monomeric, dimeric, and tetrameric forms. 19 We have reported previously that when Lys50 is mutated to glutamic acid (K50E), PF4 readily forms dimers but not tetramers. 10 Importantly, we also have demonstra...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Rational design and characterization of platelet factor 4 antagonists for the study of heparin-induced thrombocytopenia

Sachais

Rux

Cines

et al. 2012

Blood

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“…The amount of UFH needed to reverse these effects and again enhance aPC generation off of TM CS may markedly exceed typical heparinization doses, resulting in a marked anticoagulant effect. However, partially desulfated 2-O, 3-O desulfated heparin (ODSH) has been noted to bind well to PF4, 31 yet it has ≈60-fold less antithrombin activity. 32 We reasoned that ODSH at an optimal level may bind and neutralize much of the free histones and PF4, allowing maximum aPC generation without causing undue anticoagulant effects.…”

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confidence: 99%

Modulation of Protein C Activation by Histones, Platelet Factor 4, and Heparinoids

Kowalska

Zhao

Zhai

et al. 2014

ATVB

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Objective-Histones are detrimental in late sepsis. Both activated protein C (aPC) and heparin can reverse their effect.Here, we investigated whether histones can modulate aPC generation in a manner similar to another positively charged molecule, platelet factor 4, and how heparinoids (unfractionated heparin or oxygen-desulfated unfractionated heparin with marked decrease anticoagulant activity) may modulate this effect. Approach and Results-We measured in vitro and in vivo effects of histones, platelet factor 4, and heparinoids on aPC formation, activated partial thromboplastin time, and murine survival. In vitro, histones and platelet factor 4 both affect thrombin/thrombomodulin aPC generation following a bell-shaped curve, with a peak of >5-fold enhancement. Heparinoids shift these curves rightward. Murine aPC generation studies after infusions of histones, platelet factor 4, and heparinoids supported the in vitro data. Importantly, although unfractionated heparin and 2-O, 3-O desulfated heparin both reversed the lethality of high-dose histone infusions, only mice treated with 2-O, 3-O desulfated heparin demonstrated corrected activated partial thromboplastin times and had significant levels of aPC. Conclusions-Our data provide a new contextual model of how histones affect aPC generation, and how heparinoid therapy may be beneficial in sepsis. These studies provide new insights into the complex interactions controlling aPC formation and suggest a novel therapeutic interventional strategy. Kowalska et al Histones, PF4, and Heparinoids and aPC Formation 121range that can be achieved locally at sites of platelet activation. 26 Moreover, other small positive proteins, such as protamine sulfate (PRT), can modulate aPC generation with a similar bell-shaped profile. 24 We have also shown that unfractionated heparin (UFH), which binds PF4 with greater affinity than does CS, effectively reduces available PF4, shifting the aPC generation bell-shaped curve to the right. Thus, added UFH enhances aPC formation at high PF4 concentrations while inhibiting at low PF4 concentrations.24 A similar effect of UFH was seen for the modulation of PRT on aPC.Chromatin has been shown to be released from activated neutrophils by a process called NETosis and can become a source for free histones 27 that, in turn, can worsen the outcome in sepsis in humans. 28 In murine models, infused histones alone can simulate many of the outcomes seen in endotoxemic lethal shock.2 Infused aPC 2 or UFH 29 reverse histoneinduced lethality in mice. Relevant to our studies, histones were shown to inhibit aPC generation in vitro in the presence of TM and IIa. 30 However, because histones are positively charged small proteins, such as PF4 and PRT, we hypothesized that they would modulate aPC generation by IIa complexed to TM CS along a bell-shaped curve, enhancing aPC generation at low concentrations and only inhibiting aPC generation at high concentrations. We reasoned that in severe sepsis, large amounts of both histones and PF4 are released and inhibit ...

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“…Binding of antibodies was then measured by ELISA as previously described. 8 GAGs were purchased from Sigma Aldrich (chondroitin sulfates A and C, dermatan sulfate, dextran sulfate [molecular weight 6500-10 000], and heparan sulfate). Binding of anti-PRT/heparin or anti-PF4/heparin antibodies to PRT/GAGs or PF4/GAGs was performed as previously described.…”

Section: Elisa Studiesmentioning

confidence: 99%

Serologic characterization of anti-protamine/heparin and anti-PF4/heparin antibodies

Lee¹,

Joglekar²,

Kuchibhatla

et al. 2017

Blood Advances

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Key Points• Monoclonal and polyclonal anti-PRT/heparin antibodies are serologically distinct from anti-PF4/heparin antibodies.• Binding of anti-PRT/ heparin antibodies to PRT/dextran complexes correlates with PRT/GAG reactivity.Anti-protamine (PRT)/heparin antibodies are a newly described class of heparin-dependent antibodies occurring in patients exposed to PRT and heparin during cardiac surgery. To

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Disruption of PF4/H multimolecular complex formation with a minimally anticoagulant heparin (ODSH)

Cited by 30 publications

References 27 publications

Rational design and characterization of platelet factor 4 antagonists for the study of heparin-induced thrombocytopenia

Rational design and characterization of platelet factor 4 antagonists for the study of heparin-induced thrombocytopenia

Modulation of Protein C Activation by Histones, Platelet Factor 4, and Heparinoids

Serologic characterization of anti-protamine/heparin and anti-PF4/heparin antibodies

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