Manali Joglekar

Heparin modifies the immunogenicity of positively-charged proteins

The immune response in heparin-induced thrombocytopenia is initiated by and directed to large multimolecular complexes of platelet factor 4 (PF4) and heparin (H). We have previously shown that PF4:H multimolecular complexes assemble through electrostatic interactions and, once formed, are highly immunogenic in vivo. Based on these observations, we hypothesized that other positively charged proteins would exhibit similar biologic interactions with H. To test this hypothesis, we selected 2 unrelated positively charged proteins, protamine (PRT) and lysozyme, and studied H-dependent interactions using in vitro and in vivo techniques. Our studies indicate that PRT/H and lysozyme/H, like PF4/H, show H-dependent binding over a range of H concentrations and that formation of complexes occurs at distinct stoichiometric ratios. We show that protein/H complexes are capable of eliciting high-titer antigen-specific antibodies in a murine immunization model and that PRT/H antibodies occur in patients undergoing cardiopulmonary bypass surgery. Finally, our studies indicate that protein/H complexes, but not uncomplexed protein, directly activate dendritic cells in vitro leading to interleukin-12 release. Taken together, these studies indicate that H significantly alters the biophysical and biologic properties of positively charged compounds through formation of multimolecular complexes that lead to dendritic cell activation and trigger immune responses in vivo.

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

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.

Heparin enhances uptake of platelet factor 4/heparin complexes by monocytes and macrophages

Heparin‐induced thrombocytopenia (HIT) is an iatrogenic complication of heparin therapy caused by antibodies to a self‐antigen, platelet factor (4) and heparin. The reasons why antibodies form to PF4/heparin, but not to PF4 bound to other cellular glycosaminoglycans are poorly understood.

Platelets, glycoprotein Ib-IX, and von Willebrand factor are required for FeCl3-induced occlusive thrombus formation in the inferior vena cava of mice

Venous thromboembolism is a leading cause of death from cardiovascular disease. Despite the importance of the glycoprotein (GP) Ib-IX/von Willebrand factor (vWF) axis in arterial thrombosis, its requirement in venous, not venule thrombosis in response to endothelial injury (not stenosis or stasis) is uncharacterized. GPIbα-vWF participation in FeCl3-induced thrombus formation was evaluated in the inferior vena cava (IVC). Stable, occlusive thrombus formation in response to FeCl3-induced injury of the IVC was studied. FeCl3 (20% FeCl3, 10 minutes)-induced occlusive thrombosis required platelets as confirmed by a lack of occlusion in thrombocytopenic mice, and stable occlusion in control animals. No IVC occlusion was observed using GPIbα-deficient animals, a model of the human Bernard-Soulier syndrome (BSS). Transgenic IL-4 R/GPIbα mice (lack murine GPIbα, but express the extracellular domain of the human interleukin (IL-4 receptor fused to the transmembrane and cytoplasmic domains of human GPIbα) were studied to determine if the absence of IVC occlusion in the BSS mouse was caused by GPIbα extracellular domain deficiency rather than platelet BSS phenotype associated abnormalities. As with GPIbα knock-out mice, no occlusion was observed in the IVC of IL-4 R/GPIbα mice. The IVC of vWF-deficient mice also failed to occlude in response to FeCl3 treatment. The chimeric protein GPIbα(2V)-Fc prevented occlusion, demonstrating that GPIbα-vWF A1 domain interaction is required for FeCl3-induced stable thrombus formation in the IVC. Therefore, FeCl3-induced stable, occlusive thrombus formation in the IVC is platelet, and apparently GPIbα-vWF interaction dependent, despite the large diameter and low venous flow rate in the IVC.

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

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 understand the biologic significance of anti-PRT/heparin antibodies, we developed a murine monoclonal antibody (ADA) specific for PRT/heparin complexes and compared it to patient-derived anti-PRT/heparin antibodies, as well as comparing polyclonal and monoclonal antibodies with anti-platelet factor 4 (PF4)/heparin. Using monoclonal antibodies and polyclonal patient-derived antibodies, we show distinctive binding patterns of anti-PRT/heparin antibodies as compared with PF4/heparin antibodies. Whereas heparin-induced thrombocytopenia (HIT) antibody binding to PF4/heparin is inhibited by relatively low doses of heparin (0-1 U/mL), anti-PRT/heparin antibodies, including ADA, retain binding to PRT/heparin over a broad range of heparin concentrations (0-50 U/mL). Unlike PF4/heparin antibodies, which recognize PF4 complexed to purified or cell-associated glycosaminoglycans (GAGs), anti-PRT/heparin antibodies show variable binding to cell-associated GAGs. Further, binding of anti-PRT/heparin antibodies to PRT/dextran complexes correlates closely with the ability of antibodies to bind to cell-surface PRT. These findings suggest that antibody binding to PRT/dextran may identify a subset of clinically relevant anti-PRT/heparin antibodies that can bind to cell-surface GAGs. Together, these findings show important serologic differences between HIT and anti-PRT/heparin antibodies, which may account for the variability in disease expression of the two classes of heparin-dependent antibodies.