Ferric chloride-induced thrombus formation in murine inferior vena cava requires platelets, platelet receptor GPIb-IX and von Willebrand factor

Venous thromboembolism (VTE) is the 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 has not been tested. We evaluated FeCl3-induced thrombus formation in inferior vena cava (IVC) of mice. We first established FeCl3 injury conditions (20% FeCl3, 10 minutes) requiring platelets for IVC occlusion as confirmed by a lack of occlusion in severely thrombocytopenic ( less than 23% circulating platelet count) mice (n=7) and stable occlusion in control animals. Using similar injury conditions, no IVC occlusion was observed using platelet GPIbα-deficient animals (n=6), a model of human Bernard-Soulier syndrome (BSS). Transgenic IL-4R/GPIbα mice were studied to determine if the absence of IVC occlusion in the mouse model of BSS was caused by extracellular GPIbα deficiency or by other platelet abnormalities associated with BSS phenotype. IL-4R/GPIbα mice lack murine GPIbα, but express a transgenic fusion protein compound of an extracellular domains of human IL-4 receptor fused to the transmembrane and cytoplasmic domains of human GPIbα ; an ameliorated form of mouse BSS lacking macrothrombocytopenia. No occlusion was observed in the IVC of IL-4R/GPIbα mice (n=8), just as observed with the murine model of BSS. VWF-deficient mice were also studied and failed to occlude in response to FeCl3 treatment (n=7). These data establish experimental conditions for FeCl3-induced thrombus formation in the IVC that is platelet, platelet receptor GPIbα and VWF-dependent despite the low venous flow rate in the IVC. Also unlike venules, thrombus formation in IVC’s is GPIb-IX dependent, and fibronectin cannot substitute for VWF in IVC’s as it can in arterioles

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 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