Heparin Forms Macromolecular Complexes with Protamine and Lysozyme.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1316-1316
Author(s):  
Shalini Chudasama ◽  
Benjamin Espinasse ◽  
Mark R Wiesner ◽  
Gowthami M Arepally
Keyword(s):  
Complex Formation ◽  
Electrostatic Interactions ◽  
Conflicts Of Interest ◽  
Particle Formation ◽  
Biophysical Properties ◽  
Platelet Factor ◽  
Macromolecular Complexes ◽  
Molar Ratios ◽  
Colloidal Interactions ◽  
Positively Charged

Abstract Abstract 1316 Poster Board I-340 The immune response in Heparin-Induced Thrombocytopenia (HIT) is directed to and initiated by large multimolecular complexes of Platelet Factor 4 (PF4) and heparin. We, and others, have previously shown that PF4 and heparin multimolecular assembly occurs through colloidal interactions, wherein heparin, a negatively charged polymeric compound, facilitates macromolecular assembly by binding and neutralizing PF4's positive charge (Suvarna, Blood 2007). In these same studies, we also demonstrated that changes in the molar ratios of the two reactants result in PF4/heparin (PF4:H) complexes with markedly altered biophysical properties and immunogenicity. Because PF4:H electrostatic interactions are non-specific, we hypothesized that other positively charged proteins would exhibit similar colloidal interactions with heparin. To test this hypothesis, we selected two positively charged proteins (protamine and lysozyme) and studied heparin-dependent complex formation by spectrophotometry (A280nm), and zeta potential (Zeta Sizer, Malvern, UK). Protamine sulfate (250, 125, 62.5, 31.2, 15.6 and 7.8 mcg/mL; Mw 5.1kDa) and lysozyme (1000, 500, 250 and 125 mcg/mL; Mw 14.3kDa) were mixed with various heparin concentrations (0-160 U/mL; activity 140U/mg; Mw 12kDa) and biophysical properties characterized by both instruments. Both protamine and lysozyme showed heparin-dependent complex formation, with peak particle formation occurring over a range of heparin concentrations (2-25 U/mL ) for both compounds. For protamine, particle formation was maximal at protamine:heparin (Pr:H) molar ratios of ∼2.5-3:1, whereas lysozyme formed peak particles at lysozyme;heparin (Ly:H) molar ratios of ∼5:1 (See figure). As with PF4:H complexes, size of complexes was dependent on mass amounts of protamine or lysozyme, with particle size increasing or decreasing in proportion to the amounts of protamine or lysozyme available for complex formation. These findings indicate that heparin is capable of forming macromolecular complexes with other proteins through charge dependent interactions. Additional in vitro and in vivo studies are underway to determine if Pr:H or Lys:H complexes exhibit cross-reactivity with PF4/heparin antibodies and if complex formation is associated with immunological consequences. Disclosures No relevant conflicts of interest to declare.

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

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 723-723
Author(s):  
Manali Joglekar ◽  
Pedro Quintana ◽  
Stephen Marcus ◽  
Jian Liu ◽  
Gowthami M. Arepally
Keyword(s):  
Complex Formation ◽  
Electrostatic Interactions ◽  
Conflicts Of Interest ◽  
Anticoagulant Activity ◽  
Neutralization Assay ◽  
Cross Reactivity ◽  
Antibody Binding ◽  
Fixed Amount ◽  
Platelet Factor ◽  
Molar Ratios

Abstract Abstract 723 Recent 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. Disclosures: No relevant conflicts of interest to declare.

Histones, Like Platelet Factor 4 (PF4), Affect Generation of Activated Protein C: Implications for the Pathogenesis of Severe Sepsis

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 530-530
Author(s):  
M. Anna Kowalska ◽  
Guohua Zhao ◽  
George David ◽  
Mortimer Poncz
Keyword(s):  
Severe Sepsis ◽  
Protein C ◽  
Conflicts Of Interest ◽  
Platelet Factor 4 ◽  
Endothelial Protein C Receptor ◽  
Maximal Increase ◽  
Platelet Factor ◽  
Positively Charged ◽  
Formation Of Complexes

Abstract Abstract 530 Platelet factor 4 (PF4) increases aPC generation by the thrombin (IIa)/thrombomodulin (TM) complex and may impact outcome in sepsis. PF4's effect on aPC generation follows a biphasic curve when tested in solution, on human TM expressing HEK293, and on primary endothelial cells (ECs) with a peak concentration at around 25 μg/ml. Formation of complexes at a specific molar ratio between positively-charged tetramers of PF4 and negatively-charged chondroitin sulfate (CS) on the TM glycosaminoglycan (GAG) is crucial for the increase in aPC generation. Other positively-charged molecules like protamine sulfate (PRT) affect aPC generation in a similar manner, and heparin, which is known to bind PF4 and PRT more avidly than CS, lowers effective PF4 or PRT concentrations. Here we examined whether histones, that are also small positively-charged molecules, affect aPC generation. Histones released from cells in sepsis are cytotoxic toward ECs and lethal when injected into mice, and aPC reverses this lethality. May histones affect aPC generation by the same mechanism as other positively-charged molecules, and how does the presence of PF4 or heparin influence this effect? We have addressed these questions both in solution and with TM-expressing cells, in the absence or presence of endothelial protein C receptor. We found that individual, or mixed histones affect aPC formation following a similar biphasic curve seen with PF4 with a peak effect at around 10 μg/ml but to lesser extent (2-fold maximal increase compared to 6-fold for PF4). Histones and PF4 are additive at low concentrations; however, more importantly, histones only decreased aPC generation when tested in the presence of optimal or higher PF4 concentration (>25 μg/ml). Just as with PF4, added heparin decreased effective histone concentration and shifted the curve for aPC generation to the right, both in the absence or presence of PF4. We hypothesize that normally PF4 released from platelets augments aPC generation and low concentration of histones have similar effect. But when histones are released in sepsis in high concentrations, their interaction with CS on TM blocks formation of complexes between PF4 and TM's CS that are optimal for maximal increase of aPC generation. Further we tested the effect of histones on aPC generation in vivo. Injection of histones in mice increased IIa-induced (2U/kg) aPC generation in plasma. This increase was concentration dependent (at 1 to 20 mg/kg increasing aPC generation up to 10-fold), but injection of higher amount of histones (40 mg/kg) became lethal. Mice that were overexpressing human PF4 had an increased lethality when histones at 40 mg/kg were co-injected with thrombin (2U/kg) over the littermate mice deficient in murine PF4 (60% vs. 0% mortality, respectively, n=5 for each group) suggesting that in vivo histones may also act additively with PF4 on aPC generation. We propose that in severe septic patients, especially those with high levels of released PF4, concurrently available histones suppress aPC generation. By binding to the excess of PF4 and/or histones, heparin may be beneficial in severe sepsis by allowing improved aPC generation. Disclosures: No relevant conflicts of interest to declare.

Novel Techniques for Measurement of Variable Sized PF4/H Complexes.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2204-2204
Author(s):  
Benjamin Espinasse ◽  
Manali Joglekar ◽  
Giancarlo Valiente ◽  
Gowthami M. Arepally
Keyword(s):  
Flow Cytometry ◽  
Electrostatic Interactions ◽  
Conflicts Of Interest ◽  
Correlation Spectroscopy ◽  
Size Exclusion ◽  
Size Determination ◽  
Becton Dickinson ◽  
Cationic Protein ◽  
Platelet Factor ◽  
Laser Illumination

Abstract Abstract 2204 Electrostatic interactions between Platelet factor 4 (PF4), a cationic protein, and heparin, an anionic carbohydrate result in the formation of ultra-large complexes (ULCs) that are immunogenic in mice (Suvarna, Blood 2007) and contribute to the immune pathogenesis of Heparin-induced thrombocytopenia (HIT). Previous studies (Rauova, Blood 2005; Greinacher, Arterioscler Thromb Vasc Biol, 2006) have shown that the size of ULCs is determined by the concentration and the molar ratios of PF4:H (PHRs) of each compound. Size determination of PF4/H complexes has been problematic due to technical limitations of two commonly employed methods for sizing complexes, photon correlation spectroscopy (PCS) and size exclusion chromatography (SEC). PCS is a technique for measuring particles in solution using laser illumination is based on principles of Brownian motion. PCS performs optimally with monodisperse populations and is biased by the presence of large aggregates. SEC, a liquid chromatography method, is technically cumbersome, requires sample labeling and not feasible for measuring large numbers of samples. To address these limitations, we examined two novel approaches for measuring a broad range of PF4/H complex size (100–3000 nm) in vitro: Nanosight and flow cytometry (FC). Nanosight (Nanosight Ltd, Wiltshire, United Kingdom),was employed for measuring small-sized complexes using physiologic concentrations of hPF4 (10 ug/mL). Nanosight uses proprietary software to track nanoparticles (range 10–1000nm) in solution by laser illumination with real-time tracking of the motion of individual particles by a camera. Analysis parameters provided by the software include: 1) Particle size distributions displayed as histograms 2) direct visualization of particles 3) particle counting and sizing and 4) particle scatter intensity vs. count and size. For measuring intermediate to large sized particles, formed at high hPF4 concentrations (95 ug/mL), we used flow cytometry calibrated with sizing beads on side scatter channel (SSC). FC was performed using a BD LSRII cell analyzer (Becton Dickinson, Franklin Lakes, NJ), a high throughput flow analyzer with the threshold channel for SSC set to 200 and a flow rate of 1 ul per second. The instrument was calibrated using sizing beads ranging from 0.3–6 μm in size (Figure A). For both techniques, PF4/H ULCs were formed by adding hPF4 (10 or 95 ug/mL)and various UFH concentrations in HBSS to yield the indicated PHRs. Complexes were incubated for 60 minutes and measured by NanoSight or FC. Results of experiments using Nanosight are shown in Table 1 with results showing size and particle counts for each PHR. Results of FC are shown in Figure B and Table 2 (median, 5% and 95% size in nm). Both studies showed reproducibility for measurements for a given concentration and showed changes in complex size as a function of PHR (Figure B). Both methodologies are technically simple and provide complementary approaches to PCS for PF4/H complex size determination. Disclosures: No relevant conflicts of interest to declare.

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

2012 ◽  
Vol 107 (04) ◽  
pp. 717-725 ◽  
Author(s):  
Manali V. Joglekar ◽  
Pedro M. Diez ◽  
Stephen Marcus ◽  
Rui Qi ◽  
Benjamin Espinasse ◽  
...  
Keyword(s):  
Immune Response ◽  
Complex Formation ◽  
Unfractionated Heparin ◽  
Electrostatic Interactions ◽  
Essential Role ◽  
Platelet Factor ◽  
Heparin Induced Thrombocytopenia ◽  
Immune Mediated ◽  
Novel Strategy ◽  
American Society

SummaryRecent 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 52nd American Society of Hematology Annual Meeting and Exposition, December 6th, 2010, Orlando, Florida, USA.

scholarly journals Heparin modifies the immunogenicity of positively charged proteins

Blood ◽  
2010 ◽  
Vol 116 (26) ◽  
pp. 6046-6053 ◽  
Author(s):  
Shalini L. Chudasama ◽  
Benjamin Espinasse ◽  
Fred Hwang ◽  
Rui Qi ◽  
Manali Joglekar ◽  
...  
Keyword(s):  
Electrostatic Interactions ◽  
Cell Activation ◽  
High Titer ◽  
Interleukin 12 ◽  
Dendritic Cell Activation ◽  
Platelet Factor ◽  
Heparin Induced Thrombocytopenia ◽  
Positively Charged

Abstract 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.

Heparin Modifies the Immunogenicity of Positively-Charged Proteins.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1435-1435
Author(s):  
Gowthami M. Arepally ◽  
Shalini Chudasama ◽  
Benjamin Espinasse ◽  
Fred Hwang ◽  
Rui Qi ◽  
...  
Keyword(s):  
Immune Response ◽  
Dendritic Cell ◽  
Immune Responses ◽  
Electrostatic Interactions ◽  
Cell Activation ◽  
Dendritic Cell Activation ◽  
Platelet Factor ◽  
Stoichiometric Ratios ◽  
Positively Charged

Abstract Abstract 1435 Platelet Factor 4 (PF4)/heparin (H) multimolecular complexes initiate an immune response that can ultimately lead to complications of Heparin-Induced Thrombocytopenia (HIT), a life-threatening prothrombotic disorder. We have previously shown that PF4:H multimolecular complexes assemble through non-specific electrostatic interactions and that other unrelated positively-charged proteins such as protamine (PRT) and lysozyme (Lys) exhibit similar biophysical interactions with heparin (ASH 2009; abstract # 1316). In these earlier studies, we showed that PRT/H and Lys/H, like PF4/H, show heparin-dependent binding over a range of heparin concentrations and that formation of multimolecular complexes occurs at distinct stoichiometric ratios (PRT/H at 3:1 and Lys/H at 5:1 molar ratios). We now extend these observations in vivo to show relevance to human disease. Using a murine immunization model, we show that mice injected with PRT/H and Lys/H multimolecular complexes, but not PRT alone, Lys alone or buffer, develop antigen-specific immune responses. In additional studies, we show that the immune response to PRT/H or Lys/H shares important biologic similarities with the humoral response to murine (m) PF4/H multimolecular complexes. Specifically, we demonstrate that antibody formation to PRT/H and Lys/H is heparin-dependent (occurs optimally at certain stoichiometric ratios) dose-dependent (requires threshold amounts of multimolecular complexes) and shows serologic transience. To demonstrate the clinical relevance of our findings, we examined patients undergoing cardiopulmonary bypass (CPB) for development of PRT/H antibodies. For these studies, we assayed the plasma from healthy subjects (n=45) and patients undergoing CPB (n=15) at three time points {baseline (BL), 5 days (5D) and 30 days (30D) after CPB} for the presence of PRT/H antibodies. As shown Figure 1A, plasma from normal subjects and patients undergoing CPB patients at BL and D5 displayed minimal reactivity in the PRT/H ELISA. However, by 30D, we observed that 4/15 patients (27%) developed significantly elevated levels of antibodies to PRT/H as compared to normals, or their respective samples obtained at baseline or 5D after surgery. Seropositive patients (filled symbols, n=4) as compared to seronegative patients (open symbols, n=3) recognized PRT/H and to some extent, PRT alone, but did not cross-react with other antigens including PRT/H, BSA, Lys, Lys/H or human PF4/H, Figure 1B; p<0.001). To identify the mechanism by which protein/heparin multimolecular complexes triggered immune activation, we incubated murine dendritic cells from non-immunized C57Bl/6 mice with heparin or buffer, protein (mPF4, PRT or Lys), or protein/H complexes and measured IL-12, a marker of dendritic cell activation. As shown in Figure 1C, we demonstrated that IL-12 levels were significantly increased in wells containing protein/H complexes as compared to wells containing uncomplexed protein, buffer or heparin. Taken together, these studies indicate that heparin significantly alters the biophysical and biological properties of positively-charged compounds through formation of macromolecular complexes that lead to dendritic cell activation and trigger immune responses in vivo. Disclosures: Arepally: Glaxo Smith Kline: Speakers Bureau; Paringenix: Research Funding; University Of New Mexico: Patents & Royalties; Amgen: Speakers Bureau.

The NET Effect: Platelet Factor 4 and DNA-Histone Interactions in Sepsis

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2197-2197 ◽  
Author(s):  
Kandace Gollomp ◽  
Johnston Ian ◽  
Diarra Fatoumata ◽  
Guohua Zhao ◽  
Sriram Krishnaswamy ◽  
...  
Keyword(s):  
Nuclear Dna ◽  
Conflicts Of Interest ◽  
Calcium Ionophore ◽  
Platelet Factor 4 ◽  
Extracellular Dna ◽  
Microfluidic Channels ◽  
Chromatin Decondensation ◽  
Platelet Factor ◽  
Competitive Binding Assay ◽  
Positively Charged

Abstract In response to infection and inflammation, neutrophils release neutrophil extracellular traps (NETs), web like structures composed of nuclear DNA associated with histones that may have both beneficial and deleterious effects. The formation of NETs alters the course of late-stage sepsis and the associated release of histones has been shown to contribute to many of the observed pathologic complications of sepsis. Histones are octamers comprised of two copies of H2A, H2B, H3 and H4, each of which is highly positively charged. NET formation is dependent on chromatin decondensation mediated by the enzyme peptidylarginine deiminase 4 (PAD4). PAD4 potentiates chromatin decondensation by decreasing the overall positive charge of histones through citrullinating many of their lysines residues, forming Cit-histones, which have a decreased affinity for negatively charged DNA. Platelets also contribute to events in late sepsis by undergoing significant activation and degranulation. We propose that one way platelets may affect outcomes in late sepsis is through the release of large amounts of the highly positively charged chemokine platelet factor 4 (PF4, CXCL4). After its release, we believe that PF4 can displace histones and cit-histones from cell free DNA, altering the composition of NETs. We chose to investigate whether PF4 might liberate cit-histones from NET fibers more effectively than non-citrullinated histones. We initially sought to examine the effect of PF4 on histone attachment to DNA. In a competitive binding assay, we found that PF4 binds to DNA with greater affinity than histones. Of note, cit-histones were approximately 5 times more easily displaced from DNA than non-cit-histones consistent with a model of decreased DNA affinity of cit-histones. Furthermore, using immunofluorescence studies and confocal microscopy, we showed that when NETs are generated in the presence of platelets, endogenous PF4 adheres readily to NET DNA. We have also demonstrated that exogenous PF4 avidly binds to NETs generated from neutrophils isolated with minimal platelet contamination. Based on the results of these experiments, we decided to investigate the interaction between PF4, NETs and histones in a novel microfluidic system that is designed to mimic intravascular flow conditions. We isolated neutrophils from fresh whole blood samples obtained from healthy human donors and stimulated them with TNFα to promote adherence to fibronectin coated microfluidic channels. After the neutrophils had firmly bound to the channels, we exposed them to NET stimuli, including lipopolysaccharide (LPS) and calcium ionophore and visualized NET formation. Extracellular DNA was detected using the cell membrane impermeable dye, SYTOX Green. After NET formation occurred, PF4 was flowed through the channels at 25-100 μg/mL, concentrations similar to those observed in terminal sepsis. Exposure to PF4 at these concentrations lead to the dissolution of NET fibers. Interestingly, although the residual NET fibers continued to stain positive for non-cit-histones, they no longer stained positive for cit-histones. In conclusion, cit-histones are present in NETs and may contribute to the pathobiology of late sepsis. We propose that cit-histones are competitively displaced from NETs by PF4. This may be due to their decreased relative affinity for DNA binding. These studies provide new insights into how histones are released from NET fibers into the circulation during sepsis. This information sheds new light on the interaction of chemokines and NETs and may lead to the identification of new therapeutic strategies in the treatment of sepsis. Disclosures No relevant conflicts of interest to declare.

scholarly journals Bovine Heparin Demonstrates the Same Interaction with HIT Antibodies As Porcine Heparin

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2351-2351
Author(s):  
Jeanine Walenga ◽  
Walter Jeske ◽  
Sabrina Bertini ◽  
Giulia Risi ◽  
Michelle Sung ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Conflicts Of Interest ◽  
Essential Medicine ◽  
Biological Behavior ◽  
Platelet Factor ◽  
Short Supply ◽  
Molar Ratios ◽  
The Us ◽  
Activation Response ◽  
Medical Grade

Heparin, an anticoagulant widely used in numerous medical applications, is considered an essential medicine by the WHO. Due to its high volume use and that it is the parent material for low molecular weight heparins, there is potential for the raw material to be in short supply. The African swine fever epidemic in China, ongoing since August 2018, has added further restraints on heparin source material supply. At present medical grade heparin in the US is only derived from porcine intestinal mucosa; however, there are explorations into using bovine, ovine, and other sources. Bovine heparin, once common place in the US pharmaceutical sector, is again under consideration by the US FDA. This study focused on the primary immunogenic activity associated with heparin, that is heparin-induced thrombocytopenia (HIT), and how the interaction of bovine heparin with functional HIT antibodies compares to that of porcine heparin. Materials and Methods Bovine unfractionated heparin from multiple manufacturers was compared to commercial medical grade porcine heparin obtained from US pharmacies. The US Pharmacopeia porcine heparin standard was used to determine potency equivalence. Antibodies to the complex of platelet factor 4/heparin (PF4/heparin) from banked clinically confirmed HIT patient apheresis fluids were combined with heparin and donor platelet rich plasma (PRP; blood collected in citrate from volunteers after signing a consent document). Heparins were tested at final concentrations of 0.1, 0.4, 0.8, 1, and 100 U/mL. The platelet activation response was determined on the BioData PAP-8 Platelet Aggregometer and quantitated in terms of primary slope (PS), area under the curve (AUC), maximum aggregation (MA), and final aggregation (FA). Characterization of the biophysical interaction between varying molar ratios of human PF4 and heparin was performed using photon correlation spectroscopy (PCS) and zeta potential (Zp) measurements of PF4/heparin complexes using Zetasizer Nano ZS instrumentation and software. Differences between bovine and porcine heparin were assessed by t-test or Mann-Whitney test. Concentration-response curves were analyzed by two-way ANOVA followed by the Holm-Sidak multiple comparison test using SigmaPlot software. Results Platelet activation to PF4/heparin antibodies at bovine and porcine heparin concentrations of 0.1 U/mL (56 ± 9 % vs. 54 ± 11 % MA) and 0.4 U/mL (59 ± 10 % vs. 65 ± 8 % MA) were the same with the expected inhibition (9 ± 4% MA) at the supra-therapeutic concentration of 100 U/mL. Consistent responses were obtained across 21 lots of bovine heparin, 30 lots of porcine heparin, and 38 platelet-HIT antibody combinations. The HIT potential of bovine heparin and porcine heparin was not statistically different (p>0.05). At higher medical use doses, the platelet aggregation response in the presence of HIT antibodies was actually lower for bovine heparin than porcine heparin (0.8 U/mL, 49 ± 10 % vs. 64 ± 9 % MA, p<0.05; and 1.0 U/mL, 45 ± 11 % vs. 62 ± 9 % MA, p<0.05). By PCS, it was observed that the maximal aggregation between PF4 and either porcine or bovine heparin occurred at comparable molar ratios (7.3 ± 1.5 vs. 6.4 ± 0). Although the porcine and bovine heparins exhibited comparable molecular weights (16,333 ± 153 vs. 16,790 ± 230 Da) and polydispersities (1.19 ± 0.02 vs. 1.15 ± 0.01), porcine heparin formed somewhat larger complexes with PF4 (1113 ± 65 nm) than did bovine heparin (863 ± 68 nm). The molar ratios of PF4 to heparin at which the charge of the complex was fully neutralized (Zp = 0) was comparable for porcine and bovine heparin (9.04 ± 0.19 vs. 9.97 ± 0.65). Consistent responses were obtained across 4 lots of bovine heparin and 3 lots of porcine heparin. Conclusions Bovine heparin and porcine heparin had the same in vitro functional platelet activation response in the presence of HIT antibodies, the same potential to form complexes with human PF4, and the same associated features that make PF4 immunogenic. This investigation demonstrates that bovine heparins should have a similar immunogenic response as porcine heparin at equi-unit dosing. Current refinements in the manufacturing process for bovine and porcine heparins have led to well-characterized and purified products which is reflected in their comparable biological behavior. Disclosures No relevant conflicts of interest to declare.

Characteristics of the Interaction between Thrombin Exosite1 and the Sequence 269-297 of Platelet Glycoprotein Ibα

1998 ◽  
Vol 80 (08) ◽  
pp. 310-315 ◽  
Author(s):  
Marie-Christine Bouton ◽  
Christophe Thurieau ◽  
Marie-Claude Guillin ◽  
Martine Jandrot-Perrus
Keyword(s):  
Platelet Activation ◽  
Electrostatic Interactions ◽  
Platelet Glycoprotein ◽  
Thrombin Receptor ◽  
Central Position ◽  
Amidolytic Activity ◽  
N Terminus ◽  
Hydrolysis Of ◽  
Chemical Cross Linking ◽  
Positively Charged

SummaryThe interaction between GPIb and thrombin promotes platelet activation elicited via the hydrolysis of the thrombin receptor and involves structures located on the segment 238-290 within the N-terminal domain of GPIbα and the positively charged exosite 1 on thrombin. We have investigated the ability of peptides derived from the 269-287 sequence of GPIbα to interact with thrombin. Three peptides were synthesized, including Ibα 269-287 and two scrambled peptides R1 and R2 which are comparable to Ibα 269-287 with regards to their content and distribution of anionic residues. However, R2 differs from both Ibα 269-287 and R1 by the shifting of one proline from a central position to the N-terminus. By chemical cross-linking, we observed the formation of a complex between 125I-Ibα 269-287 and α-thrombin that was inhibited by hirudin, the C-terminal peptide of hirudin, sodium pyrophosphate but not by heparin. The complex did not form when γ-thrombin was substituted for α-thrombin. Ibα 269-287 produced only slight changes in thrombin amidolytic activity and inhibited thrombin binding to fibrin. R1 and R2 also formed complexes with α-thrombin, modified slightly its catalytic activity and inhibited its binding to fibrin. Peptides Ibα 269-287 and R1 inhibited platelet aggregation and secretion induced by low thrombin concentrations whereas R2 was without effect. Our results indicate that Ibα 269-287 interacts with thrombin exosite 1 via mainly electrostatic interactions, which explains why the scrambled peptides also interact with exosite 1. Nevertheless, the lack of effect of R2 on thrombin-induced platelet activation suggests that proline 280 is important for thrombin interaction with GPIb.

scholarly journals Cooperation/Competition between Halogen Bonds and Hydrogen Bonds in Complexes of 2,6-Diaminopyridines and X-CY3 (X = Cl, Br; Y = H, F)

Symmetry ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 766
Author(s):  
Barbara Bankiewicz ◽  
Marcin Palusiak
Keyword(s):  
Complex Formation ◽  
Hydrogen Bonds ◽  
Dft Calculations ◽  
Electrostatic Interactions ◽  
Dipole Moments ◽  
Main Role ◽  
Halogen Bonds ◽  
Topological Parameters ◽  
Leading Role ◽  
The Impact

The DFT calculations have been performed on a series of two-element complexes formed by substituted 2,6-diaminopyridine (R−PDA) and pyridine (R−Pyr) with X−CY3 molecules (where X = Cl, Br and Y = H, F). The primary aim of this study was to examine the intermolecular hydrogen and halogen bonds in the condition of their mutual coexistence. Symmetry/antisymmetry of the interrelation between three individual interactions is addressed. It appears that halogen bonds play the main role in the stabilization of the structures of the selected systems. However, the occurrence of one or two hydrogen bonds was associated with the favourable geometry of the complexes. Moreover, the impact of different substituent groups attached in the para position to the aromatic ring of the 2,6-diaminopyridine and pyridine on the character of the intermolecular hydrogen and halogen bonds was examined. The results indicate that the presence of electron-donating substituents strengthens the bonds. In turn, the presence of electron-withdrawing substituents reduces the strength of halogen bonds. Additionally, when hydrogen and halogen bonds lose their leading role in the complex formation, the nonspecific electrostatic interactions between dipole moments take their place. Analysis was based on geometric, energetic, and topological parameters of the studied systems.

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