Interaction Of Heparin Research Articles

Addressing bioreactor hiPSC aggregate stability, maintenance and scaleup challenges using a design of experiment approach

BackgroundStem cell-derived therapies hold the potential for treatment of regenerative clinical indications. Static culture has a limited ability to scale up thus restricting its use. Suspension culturing can be used to produce target cells in large quantities, but also presents challenges related to stress and aggregation stability.MethodsUtilizing a design of experiments (DoE) approach in vertical wheel bioreactors, we evaluated media additives that have versatile properties. The additives evaluated are Heparin sodium salt (HS), polyethylene glycol (PEG), poly (vinyl alcohol) (PVA), Pluronic F68 and dextran sulfate (DS). Multiple response variables were chosen to assess cell growth, pluripotency maintenance and aggregate stability in response to the additive inputs, and mathematical models were generated and tuned for maximal predictive power.ResultsExpansion of iPSCs using 100 ml vertical wheel bioreactor assay for 4 days on 19 different media combinations resulted in models that can optimize pluripotency, stability, and expansion. The expansion optimization resulted in the combination of PA, PVA and PEG with E8. This mixture resulted in an expansion doubling time that was 40% shorter than that of E8 alone. Pluripotency optimizer highlighted the importance of adding 1% PEG to the E8 medium. Aggregate stability optimization that minimizes aggregate fusion in 3D culture indicated that the interaction of both Heparin and PEG can limit aggregation as well as increase the maintenance capacity and expansion of hiPSCs, suggesting that controlling fusion is a critical parameter for expansion and maintenance. Validation of optimized solution on two cell lines in bioreactors with decreased speed of 40 RPM, showed consistency and prolonged control over aggregates that have high frequency of pluripotency markers of OCT4 and SOX2 (> 90%). A doubling time of around 1–1.4 days was maintained after passaging as clumps in the optimized medium. Controlling aggregate fusion allowed for a decrease in bioreactor speed and therefore shear stress exerted on the cells in a large-scale expansion.ConclusionThis study resulted in a control of aggregate size within suspension cultures, while informing about concomitant state control of the iPSC state. Wider application of this approach can address media optimization complexity and bioreactor scale-up challenges.

Protamine and Heparin Interactions: A Narrative Review.

Protamine, first isolated from salmon fish sperm and now produced through recombinant biotechnology, is an antidote that neutralizes the anticoagulant properties of heparin. Protamine function is based on the capacity to dissociate the heparin-antithrombin III (AT III) complex (an important link that promotes blood fluidification by inhibiting coagulation), forming the inactive heparin-protamine complex. Protamine has itself dose-dependent anticoagulant properties: It interferes with coagulation factors and platelet function; it stimulates fibrinolysis; it can lead to thrombocytopenia and reduction in thrombin-related platelet aggregation; it decreases platelet response to thrombin receptor agonist in a dose-dependent manner. In this review, we will focus on protamine and its interaction with heparin. Notably, protamine is able to antagonize not only unfractionated heparin (UFH) but also low molecular weight heparins to various degrees. Protamine-allergic and anaphylactoid systemic reactions may affect up to 1 in 10 people and should be prevented and treated early.

Marine-Derived Sulfated Glycans Inhibit the Interaction of Heparin with Adhesion Proteins of Mycoplasma pneumoniae.

Mycoplasma pneumoniae, a notable pathogen behind respiratory infections, employs specialized proteins to adhere to the respiratory epithelium, an essential process for initiating infection. The role of glycosaminoglycans, especially heparan sulfate, is critical in facilitating pathogen-host interactions, presenting a strategic target for therapeutic intervention. In this study, we assembled a glycan library comprising heparin, its oligosaccharide derivatives, and a variety of marine-derived sulfated glycans to screen the potential inhibitors for the pathogen-host interactions. By using Surface Plasmon Resonance spectroscopy, we evaluated the library's efficacy in inhibiting the interaction between M. pneumoniae adhesion proteins and heparin. Our findings offer a promising avenue for developing novel therapeutic strategies against M. pneumoniae infections.

Interaction of heparin with human cardiac troponin complex and its influence on the immunodetection of troponins in human blood samples.

Heparin is a highly charged polysaccharide used as an anticoagulant to prevent blood coagulation in patients with presumed myocardial infarction and to prepare heparin plasma samples for laboratory tests. There are conflicting data regarding the effects of heparin on the measurement of cardiac isoforms of troponin I (cTnI) and troponin T (cTnT), which are used for the immunodiagnosis of acute myocardial infarction. In this study, we investigated the influence of heparin on the immunodetection of human cardiac troponins. Gel filtration (GF) techniques and sandwich fluoroimmunoassay were performed. The regions of сTnI and cTnT that are affected by heparin were investigated with a panel of anti-cTnI and anti-cTnT monoclonal antibodies, specific to different epitopes. Heparin was shown to bind to the human cardiac full-size ternary troponin complex (ITC-complex) and free cTnT, which increased their apparent molecular weights in GF studies. Heparin did not bind to the low molecular weight ITC-complex and to binary cTnI-troponin С complex. We did not detect any sites on cTnI in the ITC-complex that were specifically affected by heparin. In contrast, cTnT regions limited to approximately 69-99, 119-138 and 145-164 amino acid residues (aar) in the ITC-complex and a region that lies approximately between 236 and 255 aar of free cTnT were prone to heparin influence. Heparin binds to the ITC-complex via cTnT, interacting with several sites on the N-terminal and/or central parts of the cTnT molecule, which might influence the immunodetection of analytes in human blood.

Identification of heparin interaction sites on thrombin-activatable fibrinolysis inhibitor that modulate plasmin-mediated activation, thermal stability, and antifibrinolytic potential

BackgroundThrombin-activatable fibrinolysis inhibitor (TAFI) is a plasma zymogen that provides a molecular link between coagulation and fibrinolysis. Studies have shown that the presence of glycosaminoglycans accelerates TAFI activation by plasmin and stabilizes activated TAFI (TAFIa). ObjectivesWe aimed to define the elements of TAFI structure that allow these effects. MethodsBased on crystallographic studies and homology to heparin-binding proteins, we performed mutagenesis of surface-exposed charged residues on TAFI that putatively constitute heparin-binding sites. We determined heparin binding, kinetics of activation by plasmin in the presence or absence of heparin, thermal stability, and antifibrinolytic potential of each variant. ResultsMutagenesis of Lys211 and Lys212 did not impair heparin binding but affected the ability of TAFI to be activated by plasmin. Mutagenesis of Lys306 and His308 did not impair heparin binding, but mutation of His308 had a severe negative effect on TAFI/TAFIa function. Mutation of Arg320 and Lys324 in combination markedly decreased heparin binding but had no effect on heparin-mediated acceleration of TAFI activation by plasmin while somewhat decreasing TAFIa stabilization by heparin. Mutagenesis of Lys327 and Arg330 decreased (but did not eliminate) heparin binding while decreasing the ability of heparin to accelerate plasmin-mediated TAFI activation, stabilize TAFIa, and increase the antifibrinolytic ability of TAFIa. A quadruple mutant of Arg320, Lys324, Lys327, and Arg330 completely lost heparin-binding ability and stabilization of the enzyme by heparin. ConclusionBasic residues in the dynamic flap of TAFIa define a functionally relevant heparin-binding site, but additional heparin-binding sites may be present on TAFI.

Differential Solvent DEEP-STD NMR and MD Simulations Enable the Determinants of the Molecular Recognition of Heparin Oligosaccharides by Antithrombin to Be Disentangled.

The interaction of heparin with antithrombin (AT) involves a specific sequence corresponding to the pentasaccharide GlcNAc/NS6S-GlcA-GlcNS3S6S-IdoA2S-GlcNS6S (AGA*IA). Recent studies have revealed that two AGA*IA-containing hexasaccharides, which differ in the sulfation degree of the iduronic acid unit, exhibit similar binding to AT, albeit with different affinities. However, the lack of experimental data concerning the molecular contacts between these ligands and the amino acids within the protein-binding site prevents a detailed description of the complexes. Differential epitope mapping (DEEP)-STD NMR, in combination with MD simulations, enables the experimental observation and comparison of two heparin pentasaccharides interacting with AT, revealing slightly different bound orientations and distinct affinities of both glycans for AT. We demonstrate the effectiveness of the differential solvent DEEP-STD NMR approach in determining the presence of polar residues in the recognition sites of glycosaminoglycan-binding proteins.

Marine sulfated glycans inhibit the interaction of heparin with S-protein of SARS-CoV-2 Omicron XBB variant.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a worldwide COVID-19 pandemic, leading to 6.8million deaths. Numerous variants have emerged since its outbreak, resulting in its significantly enhanced ability to spread among humans. As with many other viruses, SARS‑CoV‑2 utilizes heparan sulfate (HS) glycosaminoglycan (GAG) on the surface of host cells to facilitate viral attachment and initiate cellular entry through the ACE2 receptor. Therefore, interfering with virion-HS interactions represents a promising target to develop broad-spectrum antiviral therapeutics. Sulfated glycans derived from marine organisms have been proven to be exceptional reservoirs of naturally existing HS mimetics, which exhibit remarkable therapeutic properties encompassing antiviral/microbial, antitumor, anticoagulant, and anti-inflammatory activities. In the current study, the interactions between the receptor-binding domain (RBD) of S-protein of SARS-CoV-2 (both WT and XBB.1.5 variants) and heparin were applied to assess the inhibitory activity of 10 marine-sourced glycans including three sulfated fucans, three fucosylated chondroitin sulfates and two fucoidans derived from sea cucumbers, sea urchin and seaweed Saccharina japonica, respectively. The inhibitory activity of these marine derived sulfated glycans on the interactions between RBD of S-protein and heparin was evaluated using Surface Plasmon Resonance (SPR). The RBDs of S-proteins from both Omicrion XBB.1.5 and wild-type (WT) were found to bind to heparin, which is a highly sulfated form of HS. All the tested marine-sourced sulfated glycans exhibited strong inhibition of WT and XBB.1.5S-protein binding to heparin. We believe the study on the molecular interactions between S-proteins and host cell glycosaminoglycans provides valuable insight for the development of marine-sourced, glycan-based inhibitors as potential anti-SARS-CoV-2 agents.

Investigating the single-molecule interactions of heparin and FGF-1 proteins using solid-state nanopore

Investigating the single-molecule interactions of heparin and FGF-1 proteins using solid-state nanopore

Molecular interaction of heparin and proteins–A relationship with SARS-CoV-2

Interactions between proteins and drugs can lead to the formation of stable complexes and have important implications for several processes related to human health. These interactions can affect, for example, the distribution, free concentration, biological activity and metabolism of drugs in the bloodstream. This work presents an investigation based on spectrophotometry (UV–Vis) on the interaction of albumin with heparin (HEP) in aqueous solution and in the form of films, under physiological conditions. Results from analytical models, molecular dynamics and calculations of binding constants indicate that increasing temperature leads to a decrease in the molecular interaction of HEP with albumin, with dynamic interaction, and secondary bonds. Morphological analyzes with fractality assessment corroborate the results. Simulation results suggest that the interaction of HEP with the SARS-CoV-2 spike protein should be stronger, with little variation over time. The weak interaction between HEP and albumin contributes to greater availability of the drug, which inhibits viral invasion in addition to disrupting microthrombi that prevent oxygenation in patients infected with SARS-CoV-2. The investigation of biological systems in solutions or in the form of films has allowed the analysis of complex formation using unconventional pharmacokinetic techniques.

SPR Sensor-Based Analysis of the Inhibition of Marine Sulfated Glycans on Interactions between Monkeypox Virus Proteins and Glycosaminoglycans.

Sulfated glycans from marine organisms are excellent sources of naturally occurring glycosaminoglycan (GAG) mimetics that demonstrate therapeutic activities, such as antiviral/microbial infection, anticoagulant, anticancer, and anti-inflammation activities. Many viruses use the heparan sulfate (HS) GAG on the surface of host cells as co-receptors for attachment and initiating cell entry. Therefore, virion-HS interactions have been targeted to develop broad-spectrum antiviral therapeutics. Here we report the potential anti-monkeypox virus (MPXV) activities of eight defined marine sulfated glycans, three fucosylated chondroitin sulfates, and three sulfated fucans extracted from the sea cucumber species Isostichopus badionotus, Holothuria floridana, and Pentacta pygmaea, and the sea urchin Lytechinus variegatus, as well as two chemically desulfated derivatives. The inhibitions of these marine sulfated glycans on MPXV A29 and A35 protein-heparin interactions were evaluated using surface plasmon resonance (SPR). These results demonstrated that the viral surface proteins of MPXV A29 and A35 bound to heparin, which is a highly sulfated HS, and sulfated glycans from sea cucumbers showed strong inhibition of MPXV A29 and A35 interactions. The study of molecular interactions between viral proteins and host cell GAGs is important in developing therapeutics for the prevention and treatment of MPXV.

Interactions of heparin with key glycoproteins of human respiratory syncytial virus.

Introduction: The unexpected surge of respiratory syncytial virus (RSV) cases following pandemic phase of COVID-19 has drawn much public attention. Drawing on the latest antiviral research, revisiting this heightened annual outbreak of respiratory disease could lead to new treatments. The ability of sulfated polysaccharides to compete for a variety of viruses binding to cell surface heparan sulfate, suggests several drugs that might have therapeutic potential for targeting RSV-glycosaminoglycan interactions. Methods: In the current study, the binding affinity and kinetics of two RSV glycoproteins (RSV-G protein and RSV-F protein) to heparin were investigated by surface plasmon resonance. Furthermore, solution competition studies using heparin oligosaccharides of different lengths indicated that the binding of RSV-G protein to heparin is size-dependent, whereas RSV-F protein did not show any chain length preference. Results and discussion: The two RSV glycoproteins have slightly different preferences for heparin sulfation patterns, but the N-sulfo group in heparin was most critical for the binding of heparin to both RSV-G protein and RSV-F protein. Finally, pentosan polysulfate and mucopolysaccharide polysulfate were evaluated for their inhibition of the RSV-G protein and RSV-F protein-heparin interaction, and both highly negative compounds showed strong inhibition.

Heparin and calreticulin interact on the surface of early G0/G1 dividing rat mesangial cells to regulate hyperglycemic glucose-induced responses

Heparin can block pathological responses associated with diabetic nephropathy in animal models and human patients. Our previous studies showed that the interaction of heparin on the surface of rat mesangial cells (RMCs) entering G1 of cell division in hyperglycemic glucose: 1) blocked glucose uptake by glucose transporter 4; 2) inhibited cytosolic uridine diphosphate-glucose elevation that would occur within 6h from G0/G1; and 3) prevented subsequent activation of hyaluronan synthesis in intracellular compartments and subsequent inflammatory responses. However, specific proteins that interact with heparin are unresolved. Here, we showed by live cell imaging that fluorescent heparin was rapidly internalized into the cytoplasm and then into the endoplasmic reticulum, Golgi, and nuclei compartments. Biotinylated-heparin was applied onto the surface of growth arrested G0/G1 RMCs in order to extract heparin-binding protein(s). SDS-PAGE gels showed two bands at ∼70kDa in the extract that were absent when unlabeled heparin was used to compete. Trypsin digests of the bands were analyzed by MS and identified as calreticulin and prelamin A/C. Immunostaining with their antibodies identified the presence of calreticulin on the G0/G1 RMC cell surface. Previous studies have shown that calreticulin can be on the cell surface and can interact with the LDL receptor-related protein, which has been implicated in glucose transport by interaction with glucose transporter 4. Thus, cell surface calreticulin can act as a heparin receptor through a mechanism involving LRP1, which prevents the intracellular responses in high glucose and reprograms the cells to synthesize an extracellular hyaluronan matrix after division.

Designing ECM-inspired supramolecular scaffolds by utilizing the interactions between a minimalistic neuroactive peptide and heparin.

Short bioactive peptide-based supramolecular hydrogels are emerging as interesting candidates for developing scaffolds for tissue engineering applications. However, proteins and peptides represent only a single class of molecules present in the native ECM, thus, recapitulating the complete ECM microenvironment via only peptide-based biomaterials is extremely challenging. In this direction, complex multicomponent-based biomaterials have started gaining importance for achieving the biofunctional complexity and structural hierarchy of the native ECM. Sugar-peptide complexes can be explored in this direction as they provide essential biological signaling required for cellular growth and survival in vivo. In this direction, we explored the fabrication of an advanced scaffold by employing heparin and short bioactive peptide interactions at the molecular level. Interestingly, the addition of heparin into the peptide has significantly modulated the supramolecular organization, nanofibrous morphology and the mechanical properties of the scaffold. Additionally, the combined hydrogels demonstrated superior biocompatibility as compared to the peptide counterpart at certain ratios. These newly developed scaffolds were also observed to be stable under 3-D cell culture conditions and supported cellular adhesion and proliferation. Most importantly, the inflammatory response was also minimized in the case of combined hydrogels as compared to heparin. We expect that this approach of using simple non-covalent interactions between the ECM-inspired small molecules to fabricate biomaterials with improved mechanical and biological properties could advance the current knowledge on designing ECM mimetic biomaterials. Such an attempt would create a novel, adaptable and simplistic bottom-up strategy for the invention of new and more complex biomaterials of ECM origin with advanced functions.

Structural Characteristics of Heparin Binding to SARS-CoV-2 Spike Protein RBD of Omicron Sub-Lineages BA.2.12.1, BA.4 and BA.5.

The now prevalent Omicron variant and its subvariants/sub-lineages have led to a significant increase in COVID-19 cases and raised serious concerns about increased risk of infectivity, immune evasion, and reinfection. Heparan sulfate (HS), located on the surface of host cells, plays an important role as a co-receptor for virus-host cell interaction. The ability of heparin and HS to compete for binding of the SARS-CoV-2 spike (S) protein to cell surface HS illustrates the therapeutic potential of agents targeting protein-glycan interactions. In the current study, phylogenetic tree of variants and mutations in S protein receptor-binding domain (RBD) of Omicron BA.2.12.1, BA.4 and BA.5 were described. The binding affinity of Omicron S protein RBD to heparin was further investigated by surface plasmon resonance (SPR). Solution competition studies on the inhibitory activity of heparin oligosaccharides and desulfated heparins at different sites on S protein RBD-heparin interactions revealed that different sub-lineages tend to bind heparin with different chain lengths and sulfation patterns. Furthermore, blind docking experiments showed the contribution of basic amino acid residues in RBD and sulfo groups and carboxyl groups on heparin to the interaction. Finally, pentosan polysulfate and mucopolysaccharide polysulfate were evaluated for inhibition on the interaction of heparin and S protein RBD of Omicron BA.2.12.1, BA.4/BA.5, and both showed much stronger inhibition than heparin.

Proline-Rich Region II (PRR2) Plays an Important Role in Tau-Glycan Interaction: An NMR Study.

(1) Background: Prion-like transcellular spreading of tau pathology in Alzheimer's disease (AD) is mediated by tau binding to the cell-surface glycan heparan sulfate (HS). However, the structural determinants for tau-HS interaction are not well understood. (2) Methods and Results: Binding-site mapping using NMR showed two major binding regions in full-length tau responsible for heparin interaction. Thus, two tau constructs, tau PRR2* and tau R2*, were designed to investigate the molecular details at the tau-heparin binding interface. The 2D 1H-15N HSQC of tau PRR2* and tau R2* lacked dispersion, which is characteristic for intrinsically disordered proteins. NMR titration of Arixtra into 15N-labeled tau R2* induced large chemical shift perturbations (CSPs) in 275VQIINK280 and downstream residues K281-D283, in which L282 and I278 displayed the largest shifts. NMR titration of Arixtra into 15N-labeled tau PRR2* induced the largest CSPs for residue R209 followed by residues S210 and R211. Residue-based CSP fitting showed that tau PRR2*-Arixtra interaction had a much stronger binding affinity (0.37-0.67 mM) than that of tau R2*-Arixtra (1.90-5.12 mM) interaction. (3) Conclusions: Our results suggested that PRR2 is a crucial domain for tau-heparin and tau-HS interaction.

Kinetic and Structural Aspects of Glycosaminoglycan-Monkeypox Virus Protein A29 Interactions Using Surface Plasmon Resonance.

Monkeypox virus (MPXV), a member of the Orthopoxvirus genus, has begun to spread into many countries worldwide. While the prevalence of monkeypox in Central and Western Africa is well-known, the recent rise in the number of cases spread through intimate personal contact, particularly in the United States, poses a grave international threat. Previous studies have shown that cell-surface heparan sulfate (HS) is important for vaccinia virus (VACV) infection, particularly the binding of VACV A27, which appears to mediate the binding of virus to cellular HS. Some other glycosaminoglycans (GAGs) also bind to proteins on Orthopoxviruses. In this study, by using surface plasmon resonance, we demonstrated that MPXV A29 protein (a homolog of VACV A27) binds to GAGs including heparin and chondroitin sulfate/dermatan sulfate. The negative charges on GAGs are important for GAG–MPXV A29 interaction. GAG analogs, pentosan polysulfate and mucopolysaccharide polysulfate, show strong inhibition of MPXV A29–heparin interaction. A detailed understanding on the molecular interactions involved in this disease should accelerate the development of therapeutics and drugs for the treatment of MPXV.

Interaction of Heparin with Proteins: Hydration Effects.

We present a thermodynamic investigation of the interaction of heparin with lysozyme in the presence of potassium glutamate (KGlu). The binding constant Kb is measured by isothermal titration calorimetry (ITC) in a temperature range from 288 to 310 K for concentrations of KGlu between 25 and 175 mM. The free energy of binding ΔGb derived from Kb is strongly decreasing with increasing concentration of KGlu, whereas the dependence of ΔGb on temperature T is found to be small. The decrease of ΔGb can be explained in terms of counterion release: Binding of lysozyme to the strong polyelectrolyte heparin liberates approximately three of the condensed counterions of heparin, thus increasing the entropy of the system. The dependence of ΔGb on T, on the other hand, is traced back to a change of hydration of the protein and the polyelectrolyte upon complex formation. This dependence is quantitatively described by the parameter Δw that depends on T and vanishes at a characteristic temperature T0. A comparison of the complex formation in the presence of KGlu with the one in the presence of NaCl demonstrates that the parameters related to hydration are changed considerably. The characteristic temperature T0 in the presence of KGlu solutions is considerably smaller than that in the presence of NaCl solutions. The change of specific heat Δcp is found to become more negative with increasing salt concentration: This finding agrees with the model-free analysis by the generalized van't Hoff equation. The entire analysis reveals a small but important change of the free energy of binding by hydration. It shows that these ion-specific Hofmeister effects can be modeled quantitatively in terms of a characteristic temperature T0 and a parameter describing the dependence of Δcp on salt concentration.

Heparin, klotho, and FGF23: the 3-beat waltz of the discordant heart

Excess fibroblast growth factor (FGF) 23 signaling in patients with chronic kidney disease induces left ventricular hypertrophy. In this issue, Yanucil etal. investigated the interaction of soluble klotho and heparin with FGF23 and FGF receptor isoforms. They concluded that heparin promotes the FGF23-FGF receptor isoform 4 interaction and FGF23 pathogenic effects, supporting an important role of heparin in the pathogenesis of FGF23-mediated left ventricular hypertrophy in chronic kidney disease.

Heparin activation of protein Z-dependent protease inhibitor (ZPI) allosterically blocks protein Z activation through an extended heparin-binding site

Protein Z (PZ)-dependent protease inhibitor (ZPI) is a plasma anticoagulant protein of the serpin superfamily, which is activated by its cofactor, PZ, to rapidly inhibit activated factor X (FXa) on a procoagulant membrane surface. ZPI is also activated by heparin to inhibit free FXa at a physiologically significant rate. Here, we show that heparin binding to ZPI antagonizes PZ binding to and activation of ZPI. Virtual docking of heparin to ZPI showed that a heparin-binding site near helix H close to the PZ-binding site as well as a previously mapped site in helix C was both favored. Alanine scanning mutagenesis of the helix H and helix C sites demonstrated that both sites were critical for heparin activation. The binding of heparin chains 72 to 5-saccharides in length to ZPI was similarly effective in antagonizing PZ binding and in inducing tryptophan fluorescence changes in ZPI. Heparin binding to variant ZPIs with either the helix C sites or the helix H sites mutated showed that heparin interaction with the higher affinity helix C site most distant from the PZ-binding site was sufficient to induce these tryptophan fluorescence changes. Together, these findings suggest that heparin binding to a site on ZPI extending from helix C to helix H promotes ZPI inhibition of FXa and allosterically antagonizes PZ binding to ZPI through long-range conformational changes. Heparin antagonism of PZ binding to ZPI may serve to spare limiting PZ and allow PZ and heparin cofactors to target FXa at different sites of action.

Comparative Studies on the Interaction of Heparins from Various Origins with Heparin Cofactor II and Antithrombin

IntroductionUnfractionated heparins (UFH) of mucosal origin are currently obtained from bovine, ovine, and porcine sources. In the USP potency assays, bovine mucosal heparin (BMH) exhibit much lower activities (140 U/mg) in comparison to porcine mucosal heparin (PMH; 195 U/mg) and ovine mucosal heparin (OMH; 200 U/mg). Besides molecular and structural differences, these heparins have differential interactions with serpins such as antithrombin (AT) and heparin cofactor II (HCII). The purpose of this study is to compare the inhibitory profiles of various heparins in systems enriched with AT and HCII.Materials andMethodsThe active pharmaceutical ingredients (API) of BMH, OMH, and PMH were obtained from Kin Master (Brazil), Ronssi (China), and Medefil (USA). Bovine (B.LMWH), ovine (O.LMWH), and porcine (P.LMWH) derived low‐molecular‐weight heparins (LMWH, enoxaparin) were obtained from Ronssi (China). Purified human AT, HCII, and thrombin (IIa) were obtained from Enzyme Research (USA). All heparins (UFH’s and LMWH’s) were supplemented to AT and HCII enriched buffer systems at various gravimetric and potency equated concentrations. The relative inhibition of IIa was measured in terms of percent inhibition using an amidolytic method employing IIa specific substrates. Percent inhibition was calculated for each concentration in both systems for each of the three heparins. Inhibitory concentrations required to inhibit 50% of thrombin (IC‐50) was calculated for each UFH and LMWH in terms of µg/mL and U/mL.ResultsIn the AT supplemented system using gravimetric concentrations of various UFH’s, PMH and OMH showed comparable values, whereas BMH exhibited much higher IC‐50’s. However, the LMWH’s of various origins showed comparable IC‐50 values. In this same system, the USP potency adjusted heparins exhibited comparable results for all UFH’s (Figure 1). In the HCII supplemented system, UFH’s of various origins exhibited similar IC‐50 values in both gravimetric and USP potency adjusted studies. Similarly, the LMWH’s when compared in gravimetric amounts demonstrated similar IC‐50 values (Figure 2). The relative IC‐50 values of the LMWH’s in both the AT and HCII enriched systems were higher than the UFH’s.ConclusionThese results show that potency adjusted UFH’s exhibit comparable inhibition of IIa in AT and HCII enriched systems. The differing BMH results observed in these two systems at gravimetric concentrations may partly be due to differences in charge density and AT‐binding consensus sequences. The depolymerized enoxaparin of different origins did not reveal any significant differences in the IC‐50 values. This may be due to the chemical modifications that occur during the manufacturing of these agents. Future studies may be warranted to further investigate these cofactors and their impacts on the function of various heparins.