Interaction Of Heparin Research Articles

Kinetic and Structural Studies on the Interactions of Heparin and Proteins of Human Seminal Plasma using Surface Plasmon Resonance

Heparin is naturally occurring polysaccharides which interacts with seminal plasma proteins and regulate multiple steps in fertilization process. Qualitative and quantitative information regarding the affinity for heparin-seminal plasma proteins interactions is not generally well documented and there are no reports of a comprehensive analysis of these interactions in human seminal plasma. Such information should improve our understanding of how GAGs especially heparin present in the reproductive tract regulate fertilization. In this study, we use SPR to study interactions of heparin with various seminal plasma heparin-binding proteins (HBPs). HBPs like lactoferrin (LF), fibronectin fragment (FNIII), semenogelinI (SGI) and prostate specific antigen (PSA) all bind heparin with different binding kinetics and affinities. Kinetic data suggests that FNIII binds heparin with a high affinity (KD=3.2 nM), while PSA binds heparin with a micromolar affinity (KD=11.1 μM). Preincubation of SGI with heparin inhibits the binding of SGI to immobilized PSA in a dose-dependent manner, while FNIII incubated with heparin binds with an increased affinity to PSA. Solution-competition studies show that the minimum size of a heparin oligosaccharide capable of binding with PSA is greater than a tetrasaccharide, with LF and SGI is larger than a hexasaccharide and for FNIII is larger than an octasaccharide.

Abstract PR2: A systems biology approach to elucidating the consequences of complex ternary interactions of heparin, FGF ligands, and FGF receptor on downstream signaling in NSCLC cells

Abstract Autocrine/paracrine FGF signaling has been implicated in non-small cell lung cancer (NSCLC) development and progression. In multiple NSCLC cell lines, a switch from FGFR2-IIIb expressed in lung epithelial cells to FGFR1-IIIc and FGFR2-IIIc, results in autocrine response to FGF2 and FGF9 ligands also expressed by these cell lines. FGF2 and FGFR1 expression have also been detected in patient samples and shown to correlate with short survival times. The FGF signaling complex involves a ternary interaction of FGF ligand, receptor, and heparin sulfate glycosaminoglycans (HSGAGs). HSGAGs bind directly to both ligand and receptor, and can serve both to stabilize the receptor-ligand complex and to sequester autocrine/paracrine ligands, increasing local concentrations. A systems approach combining experiment and modeling was used to understand the complex interplay of these components and their implications on cell signaling and behavior. We focused on the signaling behavior of NSCLC cell line NCI-H1703, which predominantly expressed FGFR1. We interrogated pathway dynamics with both FGF2 and FGF9 ligands and found varying Erk1/2 activation kinetics. Using a model encompassing the ternary complex formation in the cellular membrane, ligand-mediated receptor dimerization and phosphorylation, and subsequent kinase cascade to activate Erk1/2, we were able to identify potential mechanisms driving these differences. Additional perturbations of extracellular interactions and intracellular signaling were able to support these hypotheses. These results highlight the role of the extracellular interactions between ligand, receptor, and HSGAGs in controlling downstream behavior. This proffered talk is also presented as Poster A5. Citation Format: Diana H. Chai, Jaeyeon Kim, Jannik Vollmer, Andrijana Radivojevic, Jitendra Kanodia, Marco Muda, Birgit Schoeberl. A systems biology approach to elucidating the consequences of complex ternary interactions of heparin, FGF ligands, and FGF receptor on downstream signaling in NSCLC cells [abstract]. In: Proceedings of the AACR Special Conference on Chemical Systems Biology: Assembling and Interrogating Computational Models of the Cancer Cell by Chemical Perturbations; 2012 Jun 27-30; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2012;72(13 Suppl):Abstract nr PR2.

Heparin‐integrin interaction in endothelial cells: Downstream signaling and heparan sulfate expression

Endothelial cells (ECs) are a source of physiologically important molecules that are synthesized and released to the blood and/or to the subendothelial extracellular matrix such as a heparan sulfate proteoglycan (HSPG) with antithrombotic properties. Previously, we have shown that heparin stimulates the synthesis and modifies the sulfation pattern of this HSPG. Here the molecular mechanisms involved in the up-regulation of HSPG synthesis by heparin in endothelial cells were decoded. The cells were stimulated with heparin and the expression of HSPG and intracellular pathways were evaluated by a combination of methods involving confocal microscopy, flow cytometry, Western blotting analyses, and [(35) S]-sulfate metabolically labeling of the cells. We observed that the up-regulation of HSPG synthesis evoked by heparin is dependent on the interaction of heparin with integrin since RGD peptide abolishes the effect. The activation of integrin leads to tyrosine-phosphorylation of focal adhesion-associated proteins such as FAK, Src, and paxillin. In addition, heparin induces ERK1/2 phosphorylation and inhibitors of Ras and MEK decreased heparin-dependent HSPG synthesis. Moreover, heparin also induced intracellular Ca(2+) release, PLCγ1 (phospholipase Cγ1) and CaMKII (calcium calmodulin kinase II) activation, as well as an increase in nitric oxide (NO) production. Finally, an intracellular Ca(2+) chelator, Ca(2+) signaling inhibitors, and an endothelial NO synthase inhibitor were all able to abolish the effect in heparan sulfate synthesis. In conclusion, the heparin-induced up-regulation of HSPG expression is associated with the phosphorylation of focal adhesion proteins and Ras/Raf/MEK/ERK MAP and Ca(2+) /NO pathways.

Heparin‐modified dendrimer crosslinked collagen matrices for the delivery of heparin‐binding epidermal growth factor

A tissue engineered corneal equivalent (TECEs) requires host integration to achieve adequate anchorage and long-term device stability. Corneal integration through epithelialization and stromal integration can be manipulated by growth factors. We investigated the potential of heparin-binding epidermal growth factor (HB-EGF) for mediating interactions with human corneal epithelial cells (HCEC) and compared its efficacy to epidermal growth factor (EGF) in vitro. Furthermore, we utilized heparinized dendrimer crosslinked collagen gels, intended for use as TECE, for delivery of HB-EGF in a sustained manner. HCEC were exposed to HB-EGF at varying concentrations between 0.1 and 1000 ng/mL. Cell proliferation increased with growth factor concentration up to a concentration of 50 ng/mL, suggesting growth factor receptor down-regulation at higher HB-EGF concentrations. Response to HB-EGF was comparable to EGF at low concentrations of 0.1 and 1 ng/mL but at a concentration of 10 ng/mL, HB-EGF induced significantly better proliferation than EGF. Proliferation was found to be dependent on the initial seeding density. Heparinized dendrimer crosslinked collagen (CHG) gels were capable of HB-EGF uptake, which was influenced by heparin concentration within the gel, growth factor concentration and exposure time to the growth factor. HB-EGF release followed first order kinetics, with ∼90% of the growth factor released after 2 weeks. Growth factor stability was verified with in vitro HCEC culture studies. Bioavailability was maintained in the gels through heparin interaction. Overall, HB-EGF induced proliferation of HCEC in vitro and can be released from heparinized collagen gels making it potentially suitable for promoting epithelialization of TECEs.

The Thymidine Phosphorylase Inhibitor 5′-O-Tritylinosine (KIN59) Is an Antiangiogenic Multitarget Fibroblast Growth Factor-2 Antagonist

5'-O-Tritylinosine (KIN59) is an allosteric inhibitor of the angiogenic enzyme thymidine phosphorylase. Previous observations showed the capacity of KIN59 to abrogate thymidine phosphorylase-induced as well as developmental angiogenesis in the chicken chorioallantoic membrane (CAM) assay. Here, we show that KIN59 also inhibits the angiogenic response triggered by fibroblast growth factor-2 (FGF2) but not by VEGF in the CAM assay. Immunohistochemical and reverse transcriptase PCR analyses revealed that the expression of laminin, the major proteoglycan of the basement membrane of blood vessels, is downregulated by KIN59 administration in control as well as in thymidine phosphorylase- or FGF2-treated CAMs, but not in CAMs treated with VEGF. Also, KIN59 abrogated FGF2-induced endothelial cell proliferation, FGF receptor activation, and Akt signaling in vitro with no effect on VEGF-stimulated biologic responses. Accordingly, KIN59 inhibited the binding of FGF2 to FGF receptor-1 (FGFR1), thus preventing the formation of productive heparan sulphate proteoglycan/FGF2/FGFR1 ternary complexes, without affecting heparin interaction. In keeping with these observations, systemic administration of KIN59 inhibited the growth and neovascularization of subcutaneous tumors induced by FGF2-transformed endothelial cells injected in immunodeficient nude mice. Taken together, the data indicate that the thymidine phosphorylase inhibitor KIN59 is endowed with a significant FGF2 antagonist activity, thus representing a promising lead compound for the design of multitargeted antiangiogenic cancer drugs.

Heparin Interacting Protein Mediated Assembly of Nano-Fibrous Hydrogel Scaffolds for Guided Stem Cell Differentiation

A new methodology is developed to conjugate hyaluronic acid (HA) hydrogel with novel nano-fibrous architectures via non-covalent assembly that specifically allows for targeted adipose-derived stem cells (ASCs) differentiation and soft tissue engineering. The assembly of non-covalently associated hydrogel network produced via the interaction of a low molecular weight heparin (LMWH) modified HA derivative and heparin interacting protein (HIP). The multifunctional star poly(ethylene glycol) (PEG) and HIP copolymer has the capability to mediate the non-covalent assembly of nano-fibrous HA hydrogel networks via affinity interactions with LMWH. The effect of the HIP mediation on in vitro gelation, rheological characteristics, degradation, equilibrium swelling, adipose-derived stem cells (ASCs) proliferation and differentiation of nano-fibrous hydrogel is examined. The results suggest the potential utility of this unique design of the bioactive nano-fibrous HA hydrogel in directing the differentiation of ASCs and adipogenesis in ECM-mimetic scaffolds in vitro. These studies demonstrate that this nano-fibrous HA hydrogel can render the formulation of a therapeutically effective platform for in vitro adipogenesis applications.

Anticoagulation in large-volume leukapheresis: comparison between citrate- versus heparin-based anticoagulation on safety and CD34 + cell collection efficiency

Little is known of the effect of anticoagulation on peripheral blood progenitor cell (PBPC) harvest during large-volume leukapheresis (LVL). Because of the interaction of heparin with stromal cell-derived factor (SDF)-1α, it has been proposed that a heparin-based anticoagulation may result in an increased PBPC collection efficiency compared with standard citrate-based anticoagulation. We conducted a prospective randomized trial to address the effect of both anticoagulation regimes on safety, subjective comfort and CD34 (+) collection efficiency in 90 adult patients undergoing standardized LVL. Anticoagulation consisted of either citrate (group C) or a combination of heparin and low-dose citrate (group H). The overall incidence of adverse reactions (AR) during LVL was 17%. AR consisted only of citrate-related AR; no bleeding complications were observed. Determination of parameters of the acid-base balance revealed a higher frequency of metabolic alkalosis in group C. Analysis of serum SDF-1α revealed no differences in SDF-1α plasma levels. There were no differences in the CD34 (+) cell collection efficiency, resulting in the harvest of equal CD34 (+) cell yields independent of the anticoagulation used. Our data show no clinical relevant effect of a heparin containing anticoagulation in terms of an increased overall CD34 (+) cell collection during LVL, although this regime shows some benefits in terms of the incidence and subjective tolerance towards AR. Based on our results the decision between a citrate- and heparin-substituted anticoagulation for LVL should be driven by patient-related factors, and should concern potential contraindications of both methods.

Capillary electrochromatography and quartz crystal microbalance, valuable techniques in the study of heparin–lipoprotein interactions

Atherosclerosis is initiated when lipoproteins bind to proteoglycans (PGs) in arterial walls. The binding is mediated by apolipoprotein apoB-100 and/or apoE, both of which have binding affinity toward heparin. We developed covalently bound heparin coatings for APTES-modified silica capillaries and SiO2 chips and carried out capillary electrochromatography (CEC) and quartz crystal microbalance (QCM) studies on the interactions of heparin with selected peptide fragments of apoB-100 and apoE and, for CEC, also with low- and high-density lipoproteins (LDL and HDL), the latter with and without apoE. The peptides are known to mediate interactions of HDL and LDL with arterial PGs. Interactions and affinities were expressed in CEC as retention factors and reduced mobilities and in continuous flow QCM techniques as affinity constants. Both techniques showed heparin interactions to be stronger with apoB-100 peptide than with apoE peptide fragment, and they confirmed that the sulfate groups in heparin play an especially important role in interactions with apoB-100 peptide fragments. In addition, CEC confirmed the importance of sulfate groups of heparin in interactions between heparin and LDL and between heparin and apoE-containing HDL. CEC and QCM acted as excellent platforms to mimic these biologically important interactions, with small sample and reagent consumption.

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

AbstractHeparin is a widely used anticoagulant. Because of its negative charge, it forms complexes with positively charged platelet factor 4 (PF4). This can induce anti-PF4/heparin IgG Abs. Resulting immune complexes activate platelets, leading to the prothrombotic adverse drug reaction heparin-induced thrombocytopenia (HIT). HIT requires treatment with alternative anticoagulants. Approved for HIT are 2 direct thrombin inhibitors (DTI; lepirudin, argatroban) and danaparoid. They are niche products with limitations. We assessed the effects of the DTI dabigatran, the direct factor Xa-inhibitor rivaroxaban, and of 2-O, 3-O desulfated heparin (ODSH; a partially desulfated heparin with minimal anticoagulant effects) on PF4/heparin complexes and the interaction of anti-PF4/heparin Abs with platelets. Neither dabigatran nor rivaroxaban had any effect on the interaction of PF4 or anti-PF4/heparin Abs with platelets. In contrast, ODSH inhibited PF4 binding to gel-filtered platelets, displaced PF4 from a PF4-transfected cell line, displaced PF4/heparin complexes from platelet surfaces, and inhibited anti-PF4/heparin Ab binding to PF4/heparin complexes and subsequent platelet activation. Dabigatran and rivaroxaban seem to be options for alternative anticoagulation in patients with a history of HIT. ODSH prevents formation of immunogenic PF4/heparin complexes, and, when given together with heparin, may have the potential to reduce the risk for HIT during treatment with heparin.

Heparin-induced Leukocytosis Requires 6-O-Sulfation and Is Caused by Blockade of Selectin- and CXCL12 Protein-mediated Leukocyte Trafficking in Mice

Leukocytosis refers to an increase in leukocyte count above the normal range in the blood and is a common laboratory finding in patients. In many cases, the mechanisms underlying leukocytosis are not known. In this study, we examined the effects, the structural determinants, and the underlying mechanisms of heparin-induced leukocytosis, a side effect occurring in 0.44% of patients receiving heparin. We observed that heparin induced both lymphocytosis and neutrophilia, and the effects required heparin to be 6-O-sulfated but did not require its anticoagulant activity. Cell mobilization studies revealed that the lymphocytosis was attributable to a combination of blockage of lymphocyte homing and the release of thymocytes from the thymus, whereas the neutrophilia was caused primarily by neutrophil release from the bone marrow and demargination in the vasculature. Mechanistic studies revealed that heparin inhibits L- and P-selectin, as well as the chemokine CXCL12, leading to leukocytosis. Heparin is known to require 6-O-sulfate to inhibit L- and P-selectin function, and in this study we observed that 6-O-sulfate is required for its interaction with CXCL12. We conclude that heparin-induced leukocytosis requires glucosamine 6-O-sulfation and is caused by blockade of L-selectin-, P-selectin-, and CXCL12-mediated leukocyte trafficking.

Mimicking the receptor-aided binding of HIV-1 TAT protein transduction domains to phospholipid monolayers at the air–water interface

We have designed heparin-incorporated model lipid monolayers and monitored the adsorption behaviours of cell penetrating peptides (CPPs) on a molecular scale at the air–water interface. We found initially that heparin could incorporate homogeneously into 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoserine (DPPS), and DPPC/DPPS mixed monolayers, allowing improved adsorption of transcription-activating factor (TAT) derived peptide (TAT-TDP) molecules. X-ray reflectivity measurements, as well as the surface pressure changes from surface pressure–area isotherms, suggest that a preferred interaction of heparin with TAT-TDP occurs, and is responsible for the effective penetration. This behaviour resembles the ubiquitous activities of glycosaminoglycan (GAG) molecules as cellular receptors that promote intracellular transport of cell-penetrating peptide domains in biological systems. We suggest that heparin–TAT-TDP complex formation can be exploited in a primary step of CPP translocation.

Heparin modulates the conformation and signaling of platelet integrin αIIbβ3

IntroductionThe glycosaminoglycan heparin has been shown to bind to platelet integrin αIIbβ3 and induce platelet activation and aggregation, although the relationship between binding and activation is unclear. We analyzed the interaction of heparin and αIIbβ3 in detail, to obtain a better understanding of the mechanism by which heparin acts on platelets. MethodsWe assessed conformational changes in αIIbβ3 by flow cytometry of platelets exposed to unfractionated heparin. In human platelets and K562 cells engineered to express αIIbβ3, we assayed the effect of heparin on key steps in integrin signaling: phosphorylation of the β3 chain cytoplasmic tail, and activation of src kinase. We measured the heparin binding affinity of purified αIIbβ3, and of recombinant fragments of αIIb and β3, by surface plasmon resonance. Results and conclusionsHeparin binding results in conformational changes in αIIbβ3, similar to those observed upon ligand binding. Heparin binding alone is not sufficient to induce tyrosine phosphorylation of the integrin β3 cytoplasmic domain, but the presence of heparin increased both β3 phosphorylation and src kinase activation in response to ligand binding. Specific recombinant fragments derived from αIIb bound heparin, while recombinant β3 did not bind. This pattern of heparin binding, compared to the crystal structure of αIIbβ3, suggests that heparin-binding sites are located in clusters of basic amino acids in the headpiece and/or leg domains of αIIb. Binding of heparin to these clusters may stabilize the transition of αIIbβ3 to an open conformation with enhanced affinity for ligand, facilitating outside-in signaling and platelet activation.

Biophysical Investigations on the Interaction of the Major Bovine Seminal Plasma Protein, PDC-109, with Heparin

PDC-109, the major bovine seminal plasma protein, binds to sperm plasma membrane and modulates capacitation in the presence of heparin. In view of this, the PDC-109/heparin interaction has been investigated employing various biophysical approaches. Isothermal titration calorimetric studies yielded the association constant and changes in enthalpy and entropy for the interaction at 25 °C (pH 7.4) as 1.92 (±0.2) × 10(5) M(-1), 18.6 (±1.6) kcal M(-1), and 86.5 (±5.1) cal M(-1) K(-1), respectively, whereas differential scanning calorimetric studies indicated that heparin binding results in a significant increase in the thermal stability of PDC-109. The affinity decreases with increase in pH and ionic strength, consistent with the involvement of electrostatic forces in this interaction. Circular dichroism spectroscopic studies indicated that PDC-109 retains its conformational features even up to 70-75 °C in the presence of heparin, whereas the native protein unfolds at about 55 °C. Atomic force microscopic studies demonstrated that large oligomeric structures are formed upon binding of PDC-109 to heparin, indicating an increase in the local density of the protein, which may be relevant to the ability of heparin to potentiate PDC-109 induced sperm capacitation.

Heparin Induces Harmless Fibril Formation in Amyloidogenic W7FW14F Apomyoglobin and Amyloid Aggregation in Wild-Type Protein In Vitro

Glycosaminoglycans (GAGs) are frequently associated with amyloid deposits in most amyloid diseases, and there is evidence to support their active role in amyloid fibril formation. The purpose of this study was to obtain structural insight into GAG-protein interactions and to better elucidate the molecular mechanism underlying the effect of GAGs on the amyloid aggregation process and on the related cytotoxicity. To this aim, using Fourier transform infrared and circular diochroism spectroscopy, electron microscopy and thioflavin fluorescence dye we examined the effect of heparin and other GAGs on the fibrillogenesis and cytotoxicity of aggregates formed by the amyloidogenic W7FW14 apomyoglobin mutant. Although this protein is unrelated to human disease, it is a suitable model for in vitro studies because it forms amyloid-like fibrils under physiological conditions of pH and temperature. Heparin strongly stimulated aggregation into amyloid fibrils, thereby abolishing the lag-phase normally detected following the kinetics of the process, and increasing the yield of fibrils. Moreover, the protein aggregates were harmless when assayed for cytotoxicity in vitro. Neutral or positive compounds did not affect the aggregation rate, and the early aggregates were highly cytotoxic. The surprising result that heparin induced amyloid fibril formation in wild-type apomyoglobin and in the partially folded intermediate state of the mutant, i.e., proteins that normally do not show any tendency to aggregate, suggested that the interaction of heparin with apomyoglobin is highly specific because of the presence, in protein turn regions, of consensus sequences consisting of alternating basic and non-basic residues that are capable of binding heparin molecules. Our data suggest that GAGs play a dual role in amyloidosis, namely, they promote beneficial fibril formation, but they also function as pathological chaperones by inducing amyloid aggregation.

Dual Roles of the Cardin-Weintraub Motif in Multimeric Sonic Hedgehog

The fly morphogen Hedgehog (Hh) and its mammalian orthologs, Sonic, Indian, and Desert hedgehog, are secreted signaling molecules that mediate tissue patterning during embryogenesis and function in tissue homeostasis and regeneration in the adult. The function of all Hh family members is regulated at the levels of morphogen multimerization on the surface of producing cells, multimer release, multimer diffusion to target cells, and signal reception. These mechanisms are all known to depend on interactions of positively charged Hh amino acids (the Cardin-Weintraub (CW) motif) with negatively charged heparan sulfate (HS) glycosaminoglycan chains. However, a precise mechanistic understanding of these interactions is still lacking. In this work, we characterized ionic HS interactions of multimeric Sonic hedgehog (called ShhNp) as well as mutant forms lacking one or more CW residues. We found that deletion of all five CW residues as well as site-directed mutagenesis of CW residues Lys(33), Arg(35), and Lys(39) (mouse nomenclature) abolished HS binding. In contrast, CW residues Arg(34) and Lys(38) did not contribute to HS binding. Analysis and validation of Shh crystal lattice contacts provided an explanation for this finding. We demonstrate that CW residues Arg(34) and Lys(38) make contact with an acidic groove on the adjacent molecule in the multimer, suggesting a new function of these residues in ShhNp multimerization rather than HS binding. Therefore, the recombinant monomeric morphogen (called ShhN) differs in CW-dependent HS binding and biological activity from physiologically relevant ShhNp multimers, providing new explanations for functional differences observed between ShhN and ShhNp.

Regulatory role of heparin compounds with low molecular ligands of blood in plasma and thromocyte hemostasis

Aanalysis of the literature and our own research on the physiological effects of complex compounds of heparin with low molecular ligands (amino acids, regulatory peptides) is presented. It is proved that anticoagulative effects in blood flow were conditioned by the interaction of heparin with glioproline, immunopeptides, and other low molecular substances with formation of complex compounds. The presence of structural regions of binding of heparin and other components is established. It is indicated that in the blood of animals heparin complexes with low molecular ligands possess protective anticoagulative and antithrombotic effects. We made an attempt to reveal the possible mechanism of anticoagulative-fibrinolytic and antithrombotic action of complex compounds of heparin in the organism.

Investigation of the Neisseria Gonorrhoeae Opacity Associated Membrane Protein a Structure, Dynamics, and Host-Receptor Interactions

Opacity associated (Opa) outer membrane proteins from the bacteria Neisseria meningitides and Neisseria gonorrhoeae bind to human host cell receptors, which triggers phagocytosis of the bacteria. Opa proteins are classified into two groups (OpaCEA and OpaHS) based on host receptor specificity. OpaCEA interacts with carcinoembryonic antigen-related cell adhesion molecules (CEACAM) and OpaHS binds heparansulfate proteoglycan (HSPG) receptors. Two hypervariable extracellular loops are proposed to determine receptor specificity; however, the molecular determinants of the interaction and host receptor specificity are unknown. The aim of this study is to determine the structure of OpaA (an OpaHS from N. gonorrhoeae MS11) using NMR, and to map the specific loop interactions of OpaA with heparin, a competitive inhibitor of the OpaHS and HSPG interaction. The expression, purification, and folding of OpaA will be presented as well as the progress towards the NMR backbone assignment. In addition, the feasibility of using chemical shift perturbation to map the OpaA - heparin interaction will be explored. These invesitgations of the binding specificity of OpaA to heparin will further the understanding of the pathogenesis of Neisseria, which will in turn increase our understanding of an attractive function for receptor-targeted therapeutics.

Heparin-induced thrombocytopenia in the ICU: an overview

Heparin, a negatively charged glycosaminoglycan (3000–30,000 Da), is an anticoagulant released by mast cells and basophils during the normal clotting process [1]. Heparin is widely used for the treatment and prophylaxis of thromboembolic diseases in medical and surgical patients [1]. Heparin-induced thrombocytopenia (HIT) is one of the most serious adverse events associated with this drug. HIT is an immune-mediated, prothrombotic complication that occurs with unfractionated heparin (UFH) and to a lesser extent with low-molecular-weight heparin (LMWH) [2]. The fundamental paradox of HIT results from a platelet-activating immune response triggered by the interaction of heparin with a specific platelet protein, platelet factor 4 (PF4) [3].

Disruption of PF4/Hep Multimolecular Complex Assembly Using a Minimally Anticoagulant Heparin (ODSH)

AbstractAbstract 723Recent studies indicate that multimolecular complexes of platelet factor 4 (PF4) and heparin (H) are central to the pathogenesis of Heparin-Induced Thrombocytopenia (HIT). PF4/H multimolecular complexes are recognized preferentially by HIT antibodies (Rauova, Blood 2005) and are potently immunizing in a murine immunization model (Suvarna, Blood 2005). Because PF4/H multimolecular complexes assemble through non-specific electrostatic interactions, we hypothesized that disruption of PF4/H charge-dependent interactions could reduce immune mediated complications. To test this hypothesis, we employed a minimally anticoagulant compound (2-O, 3-O desulfated heparin, or ODSH, ParinGenix, Inc.) and characterized the charge-dependent interactions of murine PF4 (mPF4), ODSH and unfractionated heparin (UFH). In chromogenic assays of thrombin (IIa) generation, UFH was >80-fold more potent than ODSH in inactivating heparin (IC50 of residual IIa generation for UFH=3.1 nM v. ODSH= 259 nM, (Figure 1A). However, when equimolar amounts of UFH or ODSH (1.7 mM) were tested in a PF4 neutralization assay (Saggin, Thrombosis and Haemostasis 1992), the amount of mPF4 required to neutralize 50% of the anticoagulant activity of ODSH (IC50) was 25μg/mL, as compared to 73μg/mL for UFH (~3-fold difference), indicating that charge-dependent interactions, but not anticoagulant activity, were preserved between PF4 and ODSH (Figure 1B). When ODSH was added at 2.5, 5 or 10 fold molar excess to a fixed amount of UFH (6nM) in the PF4 neutralization assay, a proportionate increase in the amount of PF4 was needed to neutralize UFH, indicating that ODSH promotes the anticoagulant effect of UFH through preferential binding of PF4. To further characterize the biophysical interactions of PF4, ODSH and UFH, we used spectrophotometry and zeta potential to study the multimolecular complex formation (Suvarna, Blood 2007). We noted that mPF4 and ODSH formed multimolecular complexes at molar ratios of 2:1, whereas mPF4 and UFH complexes occurred at molar ratios of 1:1. When increasing concentrations of ODSH were added to pre-formed PF4/H multimolecular complexes, we noted a decrease in absorbance with increasing amounts of ODSH, indicating disruption of PF4/H multimolecular complexes (Figure 1C). However, when increasing amounts of UFH was added to preformed PF4/ODSH multimolecular complexes, a plateau in signal was noted, suggesting a higher affinity of ODSH for PF4. In PF4/H immunoassays, incubation of ODSH (1μg/mL) with HIT antibodies was effective in reducing antibody binding by >50% as compared to wells without ODSH. HIT antibodies did not recognize hPF4 (10mg/mL) in complex with ODSH (0.4-3.2 mg/mL), indicating minimal cross-reactivity of HIT antibodies with PF4/ODSH complexes (Figure 1D). In summary, we show that ODSH, a minimally anticoagulant heparin, can disrupt PF4/H multimolecular complex formation through charge dependent interactions and interfere with HIT antibody binding. These studies suggest that manipulation of PF4:H charge interactions can be a potential therapeutic strategy in the management of HIT. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Elucidation of Inositol Hexaphosphate and Heparin Interaction Sites and Conformational Changes in Arrestin-1 by Solution Nuclear Magnetic Resonance

Arrestins specifically bind activated and phosphorylated G protein-coupled receptors and orchestrate both receptor trafficking and channel signaling through G protein-independent pathways via direct interactions with numerous nonreceptor partners. Here we report the first successful use of solution NMR in mapping the binding sites in arrestin-1 (visual arrestin) for two polyanionic compounds that mimic phosphorylated light-activated rhodopsin: inositol hexaphosphate (IP6) and heparin. This yielded an identification of residues involved in the binding with these ligands that was more complete than what has previously been feasible. IP6 and heparin appear to bind to the same site on arrestin-1, centered on a positively charged region in the N-domain. We present the first direct evidence that both IP6 and heparin induced a complete release of the arrestin C-tail. These observations provide novel insight into the nature of the transition of arrestin from the basal to active state and demonstrate the potential of NMR-based methods in the study of protein-protein interactions involving members of the arrestin family.