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

 

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Summary

Recent studies have shown that ultra-large complexes (ULCs) of platelet factor 4 (PF4) and heparin (H) play an essential role in the pathogenesis of heparin-induced thrombocytopenia (HIT), an immune-mediated disorder caused by PF4/H antibodies. Because antigenic PF4/H ULCs assemble through non-specific electrostatic interactions, we reasoned that disruption of charge-based interactions can modulate the immune response to antigen. We tested a minimally anticoagulant compound (2-O, 3-O desulfated heparin, ODSH) with preserved charge to disrupt PF4/H complex formation and immunogenicity. We show that ODSH disrupts complexes when added to pre-formed PF4/H ULCs and prevents ULC formation when incubated simultaneously with PF4 and UFH. In other studies, we show that excess ODSH reduces HIT antibody (Ab) binding in immunoassays and that PF4/ODSH complexes do not cross-react with HIT Abs. When ODSH and unfractionated heparin (UFH) are mixed at equimolar concentrations, we show that there is a negligible effect on amount of protamine required for heparin neutralisation and reduced immunogenicity of PF4/UFH in the presence of ODSH. Taken together, these studies suggest that ODSH can be used concurrently with UFH to disrupt PF4/H charge interactions and provides a novel strategy to reduce antibody mediated complications in HIT.

Presented in part at the 52^nd American Society of Hematology Annual Meeting and Exposition, December 6^th, 2010, Orlando, Florida, USA.

• References

• 1 Frederiksen JW. Cardiopulmonary bypass in humans: bypassing unfractionated heparin. Ann Thorac Surg 2000; 70: 1434-1443.
  CrossrefPubMedGoogle Scholar
• 2 Greinacher A. The use of direct thrombin inhibitors in cardiovascular surgery in patients with heparin-induced thrombocytopenia. Semin Thromb Hemost 2004; 30: 315-327.
  Article in Thieme ConnectPubMedGoogle Scholar
• 3 Kurup V. et al. Cardiac surgery in a patient with heparin-induced thrombocytopenia--cautions with use of the direct thrombin inhibitor, argatroban. Conn Med 2006; 70: 245-250.
  PubMedGoogle Scholar
• 4 Nimjee SM. et al. The potential of aptamers as anticoagulants. Trends Cardiovasc Med 2005; 15: 41-45.
  CrossrefPubMedGoogle Scholar
• 5 Nimjee SM. et al. A novel antidote-controlled anticoagulant reduces thrombin generation and inflammation and improves cardiac function in cardiopulmonary bypass surgery. Mol Ther 2006; 14: 408-415.
  CrossrefPubMedGoogle Scholar
• 6 Arepally GM, Ortel TL. Heparin-Induced Thrombocytopenia. N Engl J Med 2006; 355: 809-817.
  CrossrefPubMedGoogle Scholar
• 7 Warkentin TE. et al. Anti-platelet factor 4/heparin antibodies in orthopedic surgery patients receiving antithrombotic prophylaxis with fondaparinux or enoxaparin. Blood 2005; 106: 3791-3796.
  CrossrefPubMedGoogle Scholar
• 8 Cuker A. Heparin-induced thrombocytopenia (HIT) in 2011: An epidemic of overdiagnosis. Thromb Haemost 2011; 106: 993-994.
  Article in Thieme ConnectPubMedGoogle Scholar
• 9 Lo GK. et al. What is the potential for overdiagnosis of heparin-induced throm-bocytopenia?. Am J Hematol 2007; 82: 1037-1043.
  CrossrefPubMedGoogle Scholar
• 10 Krauel K. et al. Heparin-induced thrombocytopenia--therapeutic concentrations of danaparoid, unlike fondaparinux and direct thrombin inhibitors, inhibit formation of platelet factor 4-heparin complexes. J Thromb Haemost 2008; 06: 2160-2167.
  CrossrefPubMedGoogle Scholar
• 11 Meuleman DG. Orgaran (Org 10172): its pharmacological profile in experimental models. Haemostasis 1992; 22: 58-65.
  PubMedGoogle Scholar
• 12 Danhof M. et al. Pharmacokinetic considerations on Orgaran (Org 10172) therapy. Haemostasis 1992; 22: 73-84.
  PubMedGoogle Scholar
• 13 Rao N V, Argyle B, Xu XY. et al. Low anticoagulant heparin targets multiple sites of inflammation, suppresses heparin-induced thrombocytopenia, and inhibits interaction of RAGE with its ligands. Am J Physiol Cell Physiol 2010; 299: C97-C110.
  CrossrefPubMedGoogle Scholar
• 14 Fryer A. et al. Selective O-desulfation produces nonanticoagulant heparin that retains pharmacological activity in the lung. J Pharmacol Exp Ther 1997; 282: 208-219.
  PubMedGoogle Scholar
• 15 Atha DH. et al. Contribution of monosaccharide residues in heparin binding to antithrombin III. Biochemistry 1985; 24: 6723-6729.
  CrossrefPubMedGoogle Scholar
• 16 Arepally GM. et al. Characterization of a murine monoclonal antibody that mimics heparin-induced thrombocytopenia antibodies. Blood 2000; 95: 1533-1540.
  PubMedGoogle Scholar
• 17 Suvarna S. et al. Determinants of PF4/heparin immunogeneicity. Blood 2007; 110: 4253-4260.
  CrossrefPubMedGoogle Scholar
• 18 Suvarna S. et al. PF4/heparin complexes are T cell-dependent antigens. Blood 2005; 106: 929-931.
  CrossrefPubMedGoogle Scholar
• 19 Rauova L. et al. Ultralarge complexes of PF4 and heparin are central to the pathogenesis of heparin-induced thrombocytopenia. Blood 2005; 105: 131-138.
  CrossrefPubMedGoogle Scholar
• 20 Saggin L. et al. Neutralization of the antiheparin activity of platelet factor 4 by a monoclonal antibody. Thromb Haemost 1992; 06: 137-143.
  Article in Thieme ConnectPubMedGoogle Scholar
• 21 Stringer SE, Gallagher JT. Specific binding of the chemokin platelet factor 4 to heparan sulfate. J Biol Chem 1997; 272: 20508-20514.
  CrossrefPubMedGoogle Scholar
• 22 Chudasama SL. et al. Heparin modifies the immunogenicity of positively-charged proteins. Blood 2010; 116: 6046-6053.
  CrossrefPubMedGoogle Scholar
• 23 Wilson WW. Light scattering as a diagnostic for protein crystal growth--a practical approach. J Struct Biol 2003; 142: 56-65.
  CrossrefPubMedGoogle Scholar
• 24 Suvarna S. et al. Optimization of a murine immunization model for study of PF4/heparin antibodies. J Thromb Haemost 2009; 07: 857-864.
  CrossrefPubMedGoogle Scholar
• 25 Hopfner M. et al. Selectin-blocking semisynthetic sulfated polysaccharides as promising anti-inflammatory agents. J Pharm Pharmacol 2003; 55: 697-706.
  CrossrefPubMedGoogle Scholar
• 26 Ferrer-Lopez P. et al. Heparin inhibits neutrophil-induced platelet activation via cathepsin G. J Lab Clin Med 1992; 119: 231-239.
  PubMedGoogle Scholar
• 27 Krauel K. et al. Heparin-induced thrombocytopenia: in vitro studies on the interaction of dabigatran, rivaroxaban, and low-sulfated heparin, with platelet factor 4 and anti-PF4/heparin antibodies. Blood. 2011 epub ahead of print.
  PubMedGoogle Scholar
• 28 Grocott H P. et al. Neurological injury during cardiopulmonary bypass in the rat. Perfusion 2001; 16: 75-81.
  PubMedGoogle Scholar
• 29 Mackensen GB. et al. Cardiopulmonary bypass induces neurologic and neurocognitive dysfunction in the rat. Anesthesiology 2001; 95: 1485-1491.
  CrossrefPubMedGoogle Scholar

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