Enoxaparin Increases D6 Receptor Expression and Restores Cytoskeleton Organization in Trophoblast Cells from Preeclampsia.

D6 is a scavenger receptor for CC chemokines expressed in the human placenta. It prevents excessive leukocyte tissue infiltration by internalizing chemokines through cytoskeleton-dependent intracellular transport. In preeclampsia (PE), the D6 receptor is overexpressed in trophoblast cells, but functionally impaired, due to cytoskeleton destructuring. Low molecular weight heparin (LMWH) represents a potential treatment for PE based on its anti-thrombotic and anti-inflammatory properties. Here, we investigated the effect of enoxaparin on D6 expression, and cytoskeleton organization primary cytotrophoblast cell cultures were obtained from the placentae of women with PE (n = 9) or uncomplicated pregnancy (n = 9). We demonstrated that enoxaparin is able to (i) increase D6 expression, and (ii) improve cytoskeletal fiber alignment in trophoblast cells from PE patients.

Identification of Kukoamine A, Zeaxanthin, and Clexane as New Furin Inhibitors.

The endogenous protease furin is a key protein in many different diseases, such as cancer and infections. For this reason, a wide range of studies has focused on targeting furin from a therapeutic point of view. Our main objective consisted of identifying new compounds that could enlarge the furin inhibitor arsenal; secondarily, we assayed their adjuvant effect in combination with a known furin inhibitor, CMK, which avoids the SARS-CoV-2 S protein cleavage by means of that inhibition. Virtual screening was carried out to identify potential furin inhibitors. The inhibition of physiological and purified recombinant furin by screening selected compounds, Clexane, and these drugs in combination with CMK was assayed in fluorogenic tests by using a specific furin substrate. The effects of the selected inhibitors from virtual screening on cell viability (293T HEK cell line) were assayed by means of flow cytometry. Through virtual screening, Zeaxanthin and Kukoamine A were selected as the main potential furin inhibitors. In fluorogenic assays, these two compounds and Clexane inhibited both physiological and recombinant furin in a dose-dependent way. In addition, these compounds increased physiological furin inhibition by CMK, showing an adjuvant effect. In conclusion, we identified Kukoamine A, Zeaxanthin, and Clexane as new furin inhibitors. In addition, these drugs were able to increase furin inhibition by CMK, so they could also increase its efficiency when avoiding S protein proteolysis, which is essential for SARS-CoV-2 cell infection.

Saturated tetrasaccharide profile of enoxaparin. An additional piece to the heparin biosynthesis puzzle.

Enoxaparin, widely used antithrombotic drug, is a polydisperse glycosaminoglycan with highly microheterogeneous structure dictated by both parent heparin heterogeneity and depolymerization conditions. While the process-related modifications of internal and terminal sequences of enoxaparin have been extensively studied, very little is known about the authentic non-reducing ends (NRE). In the present study a multi-step isolation and thorough structural elucidation by NMR and LC/MS allowed to identify 16 saturated tetramers along with 23 unsaturated ones in the complex enoxaparin tetrasaccharide fraction. Altogether the elucidated structures represent a unique enoxaparin signature, whereas the composition of saturated tetramers provides a structural readout strictly related to the biosynthesis of parent heparin NRE. In particular, both glucuronic and iduronic acids were detected at the NRE of macromolecular heparin. The tetrasaccharides bearing glucosamine at the NRE are most likely associated with the heparanase hydrolytic action. High sulfation degree and 3-O-sulfation are characteristic for both types of NRE.

Enoxaparin prevents CXCL16/ADAM10-mediated cisplatin renal toxicity: Role of the coagulation system and the transcriptional factor NF-κB.

BACKGROUND AND AIMS: C-X-C ligand 16 (CXCL16) is an exceptional chemokine that is expressed as transmembrane and soluble forms. Our aim is to shed lights on the role of CXCL16/ADAM10 (a disintegrin and metalloproteinase) in cisplatin (CP)-induced renal toxicity as well as possible protective effect of enoxaparin. MAIN METHODS: Male albino mice were injected with CP (30 mg/kg, i.p.) in the presence or absence of enoxaparin (ENOX) (5 mg/kg, i.p.). Renal toxicity markers, serum level of cystatin-c, complete blood count (CBC), prothrombin time (Pt) and tissue expression of CXCL16, ADAM10, cluster of differentiation 3 (CD3), fibrinogen, tissue factor (TF), nuclear factor-κB (NF-κB) and tumour necrosis factor α (TNF-α) were measured. Besides, serum CXCL16 and histopathology were also analyzed. KEY FINDINGS: CP increased renal toxicity markers, renal expression of CXCL16/ADAM10, fibrinogen, TF and CD3 tissue expression in a time-dependent manner, and elevated serum cystatin-c, CXCL16 and tissue TNF-α, NF-κB. Alternatively, ENOX restored the deteriorated parameters and reduced tissue level of NF-κB. SIGNIFICANCE: This report, for the first time, showed that soluble CXCL16 resulting from ADAM10 cleavage may recruit T-cells to the renal glomeruli and tubules in CP toxicity. Furthermore, TF and fibrin, have similar expression and location pattern like CXCL16 and ADAM10 suggesting their possible interrelation. ENOX successfully restored the deteriorated parameters suggesting it may be an effective nephroprotective adjuvant therapy.

Preparation of Low Molecular Weight Heparin from a Remodeled Bovine Intestinal Heparin.

Bovine intestinal heparins are structurally distinct from porcine intestinal heparins and exhibit lower specific anticoagulant activity (units/mg). The reduced content of N-sulfo, 3-O-sulfo glucosamine, the central and critical residue in heparin's antithrombin III binding site, is responsible for bovine intestinal heparin's reduced activity. Previous studies demonstrate that treatment of bovine intestinal heparin with 3-O-sulfotransferase in the presence of 3'-phosphoadenosine-5'-phosphosulfate afforded remodeled bovine heparin with an enhanced activity reaching the United States Pharmacopeia's requirements. Starting from this remodeled bovine intestinal heparin, we report the preparation of a bovine intestinal low molecular weight heparin having the same structural properties and anti-factor IIa and anti-factor Xa activities of Enoxaparin. Moreover, this bovine intestinal heparin-derived "Enoxaparin" showed comparable platelet factor-4 binding affinity, suggesting that it should exhibit similarly low levels of heparin induced thrombocytopeneia, HIT.

NETosis Markers in Pregnancy: Effects Differ According to Histone Subtypes.

NETosis is an innate immune response occurring after infection or inflammation: activated neutrophils expel decondensed DNA in complex with histones into the extracellular environment in a controlled manner. It activates coagulation and fuels the risk of thrombosis. Human pregnancy is associated with a mild proinflammatory state characterized by circulatory neutrophil activation which is further increased in complicated pregnancies, placenta-mediated complications being associated with an increased thrombotic risk. This aberrant activation leads to an increased release of nucleosomes in the blood flow. The aim of our study was to initially quantify nucleosome-bound histones in normal pregnancy and in placenta-mediated complication counterpart. We analyzed the role of histones on extravillous trophoblast function. Circulating nucleosome-bound histones H3 (Nu.QH3.1, Nu.QH3PanCit, Nu.QH3K27me3) and H4 (Nu.QH4K16Ac) were increased in complicated pregnancies. In vitro using the extravillous cell line HTR-8/SVNeo, we observed that free recombinant H2B, H3, and H4 inhibited migration in wound healing assay, but only H3 also blocked invasion in Matrigel-coated Transwell experiments. H3 and H4 also induced apoptosis, whereas H2B did not. Finally, the negative effects of H3 on invasion and apoptosis could be restored with enoxaparin, a low-molecular-weight heparin (LMWH), but not with aspirin. Different circulating nucleosome-bound histones are increased in complicated pregnancy and this would affect migration, invasion, and induce apoptosis of extravillous trophoblasts. Histones might be part of the link between the risk of thrombosis and pregnancy complications, with an effect of LMWH on both.

Effective Inhibition of SARS-CoV-2 Entry by Heparin and Enoxaparin Derivatives.

Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) has caused a pandemic of historic proportions and continues to spread globally, with enormous consequences to human health. Currently there is no vaccine, effective therapeutic, or prophylactic. As with other betacoronaviruses, attachment and entry of SARS-CoV-2 are mediated by the spike glycoprotein (SGP). In addition to its well-documented interaction with its receptor, human angiotensin-converting enzyme 2 (hACE2), SGP has been found to bind to glycosaminoglycans like heparan sulfate, which is found on the surface of virtually all mammalian cells. Here, we pseudotyped SARS-CoV-2 SGP on a third-generation lentiviral (pLV) vector and tested the impact of various sulfated polysaccharides on transduction efficiency in mammalian cells. The pLV vector pseudotyped SGP efficiently and produced high titers on HEK293T cells. Various sulfated polysaccharides potently neutralized pLV-S pseudotyped virus with clear structure-based differences in antiviral activity and affinity to SGP. Concentration-response curves showed that pLV-S particles were efficiently neutralized by a range of concentrations of unfractionated heparin (UFH), enoxaparin, 6-O-desulfated UFH, and 6-O-desulfated enoxaparin with 50% inhibitory concentrations (IC50s) of 5.99 µg/liter, 1.08 mg/liter, 1.77 µg/liter, and 5.86 mg/liter, respectively. In summary, several sulfated polysaccharides show potent anti-SARS-CoV-2 activity and can be developed for prophylactic as well as therapeutic purposes.IMPORTANCE The emergence of severe acute respiratory syndrome coronavirus (SARS-CoV-2) in Wuhan, China, in late 2019 and its subsequent spread to the rest of the world has created a pandemic situation unprecedented in modern history. While ACE2 has been identified as the viral receptor, cellular polysaccharides have also been implicated in virus entry. The SARS-CoV-2 spike glycoprotein (SGP) binds to glycosaminoglycans like heparan sulfate, which is found on the surface of virtually all mammalian cells. Here, we report structure-based differences in antiviral activity and affinity to SGP for several sulfated polysaccharides, including both well-characterized FDA-approved drugs and novel marine sulfated polysaccharides, which can be developed for prophylactic as well as therapeutic purposes.

Peri-operative Medication Dosing in Adult Obese Elective Surgical Patients: A Systematic Review of Clinical Studies.

BACKGROUND: Despite the increasing numbers of obese patients undergoing elective surgery, there is a lack of evidence-based dosing guidelines for peri-operative medications in obesity. OBJECTIVE: The objective was to systematically review the dosing and outcomes of peri-operative medications used in obese elective surgical patients. METHODS: Medical subject headings and general keywords were used to systematically search multiple databases (PubMed, EMBASE, Cochrane Library and CINAHL). Studies of medications in obese surgical patients were included if they had a non-obese control or comparative dosing scalar group. The National Health and Medical Research Council GRADE tool was used to assess quality of evidence for each drug. RESULTS: Thirty-three studies of six drug classes were identified: anaesthetics (n = 6), muscle relaxants (n = 10), neuromuscular reversal agents (n = 3), analgesics (n = 2), antibiotics (n = 5) and anticoagulants (n = 7). A variety of dose scalars and/or recommendations was observed for various medications. Lean body weight was proposed as a suitable weight scalar for induction of anaesthesia with propofol whereas total body weight for maintenance of anaesthesia with propofol and depolarizing muscle relaxants. Ideal body weight was reported as an appropriate dosing scalar for non-depolarizing muscle relaxants and neuromuscular reversal agents. Both corrected body weight 40% and ideal body weight were reported as suitable weight scalars for post-operative analgesia with morphine. The standard 2-g dose of cefazolin appeared effective in the prevention of surgical site infection. Body mass index stratified dosing of enoxaparin was effective for venous thromboembolism prevention. CONCLUSION: No drug recommendation achieved an "Excellent" quality of evidence. Limited data suggest that clinicians should consider each individual class of medication when selecting a dose for obese surgical patients. Routine use of fixed-dosing regimens is likely to under- or overdose obese patients thus predisposing them to adverse drug events or treatment failure leading to patient harm.

Quantitative analysis of antithrombin III binding site in low molecular weight heparins by exhausetive heparinases digestion and capillary electrophoresis.

The antithrombin III (ATIII)-binding site, which contains a special 3-O-sulfated, N-sulfated glucosamine residue with or without 6-O-sulfation, is mainly responsible for the anticoagulant activity of heparin. Undergoing the chemical depolymerization process, the preservation of the ATIII-binding site in low molecular weight heparins (LMWHs) are varied leading to the fluctuation of the anticoagulant activity. Herein we report a capillary electrophoresis (CE) method in combination with heparinase digestion and affinity chromatography for the measurement of molar percentage of ATIII-binding site of LMWHs. After exhaustively digesting LMWHs with the mixture of heparinase I, II and III, almost all the resulting oligosaccharide building blocks, including the three 3-O-sulfated tetrasaccharides derived from the ATIII-binding site, were resolved by CE separation. The peak area of each building block permits quantification of the molar percentage of the ATIII-binding site. The peaks corresponding to the 3-O-sulfated tetrasaccharides were assigned based on the linear relationship between the electrophoretic mobilities of the oligosaccharides and their charge to mass ratios. The peak assignment was further confirmed by analysis of the high ATIII affinity fractions, which contains much high 3-O-sulfated tetrasaccharides. With the method, the molar percentage of the ATIII-binding site of enoxaparin from different batches and different manufactures were measured and compared. It was demonstrated that the CE method provides more precise data for assessing the anti-FXa activity than that of the biochemical assay method.

Enhancement of trophoblast differentiation and survival by low molecular weight heparin requires heparin-binding EGF-like growth factor.

STUDY QUESTION: Does low molecular weight heparin (LMWH) require heparin-binding epidermal growth factor (EGF)-like growth factor (HBEGF) signaling to induce extravillous trophoblast differentiation and decrease apoptosis during oxidative stress? SUMMARY ANSWER: LMWH increased HBEGF expression and secretion, and HBEGF signaling was required to stimulate trophoblast extravillous differentiation, increase invasion in vitro and reduce trophoblast apoptosis during oxidative stress. WHAT IS KNOWN ALREADY: Abnormal trophoblast differentiation and survival contribute to placental insufficiency syndromes, including preeclampsia and intrauterine growth restriction. Preeclampsia often manifests as a pro-thrombotic state, with unsuccessful transformation of the spiral arteries that reduces oxygen supply and can produce placental infarction. LMWH improves placental function by increasing blood flow. Recent data suggest that the actions of LMWH transcend its anti-coagulative properties, but the molecular mechanism is unknown. There is evidence that LMWH alters the expression of human HBEGF in trophoblast cells, which regulates human trophoblast pathophysiology. HBEGF, itself, is capable of increasing trophoblast survival and invasiveness. STUDY DESIGN, SIZE, DURATION: First-trimester placental explants and the HTR-8/SVneo cell line, established using extravillous trophoblast outgrowths from first-trimester villous explants, were treated in vitro with LMWH to examine the effects on HBEGF signaling and trophoblast function under normal physiological and pathological conditions. A highly specific antagonist of HBEGF and other inhibitors of HBEGF downstream signaling were used to determine the relationship between LMWH treatment and HBEGF. PARTICIPANTS/MATERIALS, SETTING, METHODS: Placental tissues (n = 5) were obtained with IRB approval and patient consent from first-trimester terminations. Placental explants and HTR-8/SVneo cells were cultured on plastic or Matrigel™ and treated with a therapeutic dose of LMWH (Enoxaparin; 10 IU/ml), with or without CRM197, pan Erb-B2 Receptor Tyrosine Kinase (ERBB) inhibitor, anti-ERBB1 or ERBB4 blocking antibodies, or pretreatment of cells with heparitinase I. Extravillous differentiation was assessed by immunocytochemistry to determine the relative levels of integrins α6ß4 and α1ß1. Trophoblast invasiveness was assessed in villous explants by measuring outgrowth from villous tips cultured on Matrigel, and by invasion assays with HTR-8/SVneo cells cultured on Matrigel-coated transwell insert. Placental explants and HTR-8/SVneo cells were exposed to oxidative stress in a hypoxia-reoxygenation (H-R) model, measuring cell death by TUNEL assay, caspase 3 cleavage, and BCL-2α expression. MAIN RESULTS AND THE ROLE OF CHANCE: LMWH induced extravillous differentiation, according to trophoblast invasion assays and integrin (α6ß4-α1ß1) switching. Treatment with LMWH rescued cytotrophoblasts and HTR-8/SVneo cells from apoptosis during exposure to reoxygenation injury, based on TUNEL, caspase 3 cleavage and BCL-2α expression. Experiments using CRM197, ERBB1 and ERBB4 blocking antibodies, pan-ERBB inhibitor and removal of cell surface heparin demonstrated that the effects of LMWH on trophoblast invasion and survival were dependent upon HBEGF signaling. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: The primary limitation of this study was the use of only in vitro experiments. Patient demographics from elective terminations were not available. WIDER IMPLICATIONS OF THE FINDINGS: These data provide new insights into the non-coagulation-related aspects of perinatal LMWH treatment in the management of placental insufficiency disorders. STUDY FUNDING/COMPETING INTEREST(S): This research was supported by grants from the National Institutes of Health (HD071408 and HL128628), the March of Dimes, and the W. K. Kellogg Foundation. There were no conflicts or competing interests.

Binding of heparin-dependent antibodies to PF4 modified by enoxaparin oligosaccharides: evaluation by surface plasmon resonance and serotonin release assay.

BACKGROUND: The minimal structural requirements of low-molecular-weight heparins that determine the risk of developing heparin-induced thrombocytopenia (HIT) are not fully defined. OBJECTIVES: The ability of enoxaparin-derived oligosaccharides (OS) to induce platelet activation and exposure of platelet-factor 4 (PF4) epitopes recognized by antibodies developed in HIT was studied by surface plasmon resonance (SPR) and serotonin release assay. RESULTS: Decasaccharides with ≥ 11 sulfate groups induced platelet activation in the presence of plasma from patients with confirmed HIT. Serotonin release of > 80% without full inhibition at 100 µg mL(-1) was achieved with decasaccharides containing 14 or 15 sulfate groups, 2 dodecasaccharides and 2 tetradecasaccharides. An SPR method was developed using purified PF4 immobilized on carboxymethylated dextran. Antibodies from all HIT samples bound to PF4/heparin in SPR assays with resonance units (RU) ratio of 109-173 with HIT plasma vs. 88-93 with control plasma. RU ratios > 100 were measured when PF4 was pre-incubated with OS with ≥ 10 saccharide units and one octasaccharide containing 10 sulfate groups. RU ratios > 140, similar to those measured when PF4 was pre-incubated with unfractionated heparin or enoxaparin, were obtained with purified dodeca- and tetradecasaccharides. RU values strongly correlated with the number of sulfate groups in the decasaccharides tested (r = 0.93, P = 0.02). CONCLUSIONS: LMWHs with fragments > 10 saccharides and a large number of sulfate groups are more likely to be associated with a higher risk of HIT. These structure-activity relationships were independent of the ability of the OS to bind antithrombin.

Structural features of low-molecular-weight heparins affecting their affinity to antithrombin.

As part of a more extensive investigation on structural features of different low-molecular-weight heparins (LMWHs) that can affect their biological activities, Enoxaparin, Tinzaparin and Dalteparin were characterised with regards to the distribution of different chain length oligosaccharides as determined by size-exclusion (SE) chromatography, as well as their structure as defined by 2D-NMR spectra (HSQC). The three LMWHs were also fractionated into high affinity (HA) and no affinity (NA) pools with regards to their ability to bind antithrombin (AT). The HA fractions were further subfractionated and characterised. For the parent LMWHs and selected fractions, molecular weight parameters were measured using a SE chromatographic system with a triple detector (TDA) to obtain absolute molecular weights. The SE chromatograms clearly indicate that Enoxaparin is consistently richer in shorter oligosaccharides than Tinzaparin and Dalteparin. Besides providing the content of terminal groups and individual glucosamine and uronic acid residues with different sulfate substituents, the HSQC-NMR spectra permitted us to evaluate and correlate the content of the pentasaccharide, AT-binding sequence A-G-A*-I-A (AT-bs) through quantification of signals of the disaccharide sequence G-A*. Whereas the percent content of HA species is approximately the same for the three LMWHs, substantial differences were observed for the chain distribution of AT-bs as a function of length, with the AT-bs being preferentially contained in the longest chains of each LMWH. The above information will be useful in establishing structure-activity relationships currently under way. This study is therefore critical for establishing correlations between structural features of LMWHs and their AT-mediated anticoagulant activity.

Comparative anticoagulant and platelet modulatory effects of enoxaparin and sulodexide.

Sulodexide represents a novel antithrombotic agent with multiple sites of action on blood coagulation and vascular processes. The purpose of this study was to compare sulodexide and enoxaparin on anticoagulant effects, tissue factor (TF)-induced activation of platelets, inhibition of microparticle generation and to investigate their effect on heparin-induced platelet aggregation (HIPA). Sulodexide was compared with enoxaparin at equigravimetric concentrations. When compared to enoxaparin, sulodexide produced a stronger anticoagulant effect in the prothrombin time (PT), activated partial thromboplastin time (APTT), Heptest, and thrombin time (TT) assays. In addition, sulodexide had a stronger inhibitory effect on TF-mediated microparticle generation (IC(50) = 2.8 microg/ mL), P-selectin expression (IC(50) = 4.8 microg/ml), and platelet aggregate formation (IC(50) = 8.5 microg/mL) compared to higher IC(50) values with enoxaparin. Sulodexide and enoxaparin exhibited a similar effect on heparin-induced thrombocytopenia (HIT) antibody-mediated platelet activation HIPA assays. These results suggest that sulodexide is a relatively stronger anticoagulant agent than enoxaparin. Sulodexide is subcutaneously absorbed. Its ability to inhibit TF-mediated platelet activation may contribute to the observed therapeutic effects of sulodexide in microvascular vasculopathy such as diabetic nephropathy. These results also suggest that inhibition of TF activation of platelets by sulodexide may be independent of its anticoagulant effects. These results warrant further investigation of sulodexide in additional preclinical and clinical studies.

Rat and human HARE/stabilin-2 are clearance receptors for high- and low-molecular-weight heparins.

The human hyaluronic acid (HA) receptor for endocytosis (HARE/stabilin-2) is the primary clearance receptor for systemic HA, chondroitin sulfates, and heparin, but not for heparan sulfate or keratan sulfate (Harris EN, Weigel JA, Weigel PH. J Biol Chem 283: 17341-17350, 2008). HARE is expressed in the sinusoidal endothelial cells (SECs) of liver and lymph nodes where it acts as a scavenger for uptake and degradation of glycosaminoglycans, both as free chains and proteoglycan fragments. Unfractionated heparin (UFH; approximately 14 kDa) and low-molecular-weight heparin (LMWH; approximately 4 kDa) are commonly used in treatments for thrombosis and cancer and in surgical and dialysis procedures. The reported half-lives of UFH and LMWH in the blood are approximately 1 h and 2-6 h, respectively. In this study, we demonstrate that anti-HARE antibodies specifically block the uptake of LMWH and UFH by isolated rat liver SECs and by human 293 cells expressing recombinant human HARE (hHARE). hHARE has a significant affinity (K(d) = 10 microM) for LMWH, and higher affinity (K(d) = 0.06 microM) for the larger UFH. Rat liver SECs or cells expressing the recombinant 190-kDa HARE isoform internalized both UFH and LMWH, and both heparins cross-compete with each other, suggesting that they share the same binding sites. These cellular results were confirmed in ELISA-like assays using purified soluble 190-hHARE ectodomain. We conclude that both UFH and LMWH are cleared by HARE/Stab2 and that the differences in the affinities of HARE binding to LMWH and UFH likely explain the longer in vivo circulating half-life of LMWH compared with UFH.

Investigation of freezing- and thawing-induced biological, chemical, and physical changes to enoxaparin solution.

This study investigated the effect of freezing and thawing on the biological, physical, and chemical properties of enoxaparin solution. Solutions were frozen and thawed under different conditions, in the presence or absence of dimethyl sulfoxide (DMSO) or 1,2-propanediol (1,2-PD), and the antifactor Xa (AFXa) activity was determined. Enoxaparin solution lost more than 60% of its AFXa activity when thawed rapidly after freezing at -196 degrees C. The loss of AFXa activity was less with higher freezing temperatures and increased with the number of freeze/thaw cycles, but was independent of the duration of freezing. Slow freezing to -196 degrees C with rapid thawing, or rapid freezing with slow thawing, resulted in negligible loss of AFXa activity. The loss of AFXa activity did not involve the loss of N-sulfate groups, the breakdown of glycosidic bonds or the glassy state transition. Controlling the freezing or thawing conditions, dilution with water or addition of a small percentage of DMSO ameliorated the loss of enoxaparin AFXa activity. The loss in AFXa activity was found by size exclusion chromatography to be primarily due to aggregation and was reversed by sonication in the presence of DMSO. These results may provide insight into solutions for the long-term storage of concentrated or diluted enoxaparin.

Phosphatidylserine-positive erythrocytes bind to immobilized and soluble thrombospondin-1 via its heparin-binding domain.

Phosphatidylserine (PS)-dependent erythrocyte adhesion to endothelium and subendothelial matrix components is mediated in part via thrombospondin (TSP). Although TSP exhibits multiple cell-binding domains, the PS-binding site on TSP is unknown. Because a cell-binding domain for anionic heparin is located at the amino-terminus, we hypothesized that PS-positive red blood cells (PS(+ve)-RBCs) bind to this domain. We demonstrate that both heparin and its low-molecular-weight derivative enoxaparin (0.5-50 u/mL) inhibited PS(+ve)-RBC adhesion to immobilized TSP in a concentration-dependent manner (21% to 77% inhibition, P < 0.05). Preincubation of immobilized TSP with an antibody against the heparin-binding domain blocked PS(+ve)-RBC adhesion to TSP. Antibodies that recognize the collagen- and the carboxy-terminal CD47-binding domain on TSP had no effect on this process. Although preincubation of PS(+ve)-RBCs with TSP peptides from the heparin-binding domain that contained the specific heparin-binding motif KKTRG inhibited PS(+ve)-erythrocyte adhesion to matrix TSP (P < 0.001), these peptides in the immobilized form supported PS-mediated erythrocyte adhesion. A TSP-peptide that lacks the binding motif neither inhibited nor supported PS(+ve)-RBC adhesion. Additional experiments show that soluble TSP also interacted with PS(+ve)-RBCs via its heparin-binding domain. Our results demonstrate that PS-positive erythrocytes bind to both immobilized and soluble TSP via its heparin-binding domain and that both heparin and enoxaparin, at clinically relevant concentrations, block this interaction. Other studies have shown that heparin inhibited P-selectin- and soluble-TSP-mediated sickle erythrocyte adhesion to endothelial cells. Our results, taken together with the previously documented findings, provide a rational basis for clinical use of heparin or its low-molecular-weight derivatives as therapeutic agents in treating vaso-occlusive pain in patients with sickle cell disease.

Tetradecylmaltoside (TDM) enhances in vitro and in vivo intestinal absorption of enoxaparin, a low molecular weight heparin.

Tetradecylmaltoside (TDM) was evaluated as a potential gastrointestinal absorption enhancer for low molecular weight heparin (LMWH), enoxaparin. The in vitro efficacy of TDM (0.0625, 0.125 and 0.25% w/v) in enhancing transport of 3H-enoxaparin or 14C-mannitol was investigated in human colonic epithelial cells (C2BBel). Metabolic stability of the drug was determined in C2BBel cell extracts. Transepithelial electrical resistance (TEER) was measured before and after exposure of the cells to TDM. Enoxaparin was further administered to anesthetized Sprague-Dawley rats in oral formulations in the absence or presence of increasing concentrations of TDM and drug absorption was monitored by measuring anti-factor Xa activity in rat blood. In vitro permeability study shows that apparent permeability (Papp) of 3H-enoxaparin across C2BBe1 cells was increased by 8-fold in the presence of 0.0625% TDM compared to untreated cells. The movement of 14C-mannitol across the cell monolayer followed a similar pattern in the presence of increasing concentrations of TDM. No degradation or depolymerization of enoxaparin was observed when the drug was incubated in C2BBel cell extract. TEER was reversible after 60 min exposure of the cells to 0.0625% (w/v) TDM. Oral formulations of enoxaparin containing TDM administered to anesthetized rats significantly and rapidly increased gastrointestinal absorption as compared to those animals which received enoxaparin plus saline (p < 0.05). In the presence of 0.125% TDM in the formulation, enoxaparin oral bioavailability was increased by 2.5-fold compared to the saline control group. Overall, the data on the effect of TDM on the in vitro and in vivo intestinal permeation of enoxaparin suggest that TDM may represent a promising excipient for use in oral LMWH formulations.

Few bicyclic acetals at reducing end of low-molecular-weight heparins: might they restrict specification of pharmacopoeia?

The alkaline hydrolysis of heparin benzyl ester originates enoxaparin. The depolymerization by beta-elimination is the primary effect of reaction; but side reactions can happen and the bicyclic acetal at the reducing end of glucosamine N,6-disulphate, called 1,6-anhydro ring, is a product of a side reaction. The amount of this predictable moiety of enoxaparin can be controlled to a lowest extent (6%) and to extent higher than 40% by modulating the alkalinity and duration of the reaction of hydrolysis. With the exclusion of the beta-elimination effects and of these non significative side reactions, the chemical structure of the parent heparin is entirely maintained in enoxaparin as it results by the same profiles of constituent disaccharides. The content of 1,6-anhydro rings is assessed by a not yet validated NMR method. The chains of enoxaparin bearing, at their reducing end, 1,6-anhydro rings could be regarded as Related Substances of enoxaparin. If present, even in an amount less than, or equal to, 30% of chains, these "related substances" affect neither activities nor safety of enoxaparin.

Novel concatameric heparin-binding peptides reverse heparin and low-molecular-weight heparin anticoagulant activities in patient plasma in vitro and in rats in vivo.

Patients given unfractionated heparin (UFH) or low-molecular-weight heparin (LMWH) for prophylaxis or treatment of thrombosis sometimes suffer serious bleeding. We showed previously that peptides containing 3 or more tandem repeats of heparin-binding consensus sequences have high affinity for LMWH and neutralize LMWH (enoxaparin) in vivo in rats and in vitro in citrate. We have now modified the (ARKKAAKA)(n) tandem repeat peptides by cyclization or by inclusion of hydrophobic tails or cysteines to promote multimerization. These peptides exhibit high-affinity binding to LMWH (dissociation constant [K(d)], approximately 50 nM), similar potencies in neutralizing anti-Factor Xa activity of UFH and enoxaparin added to normal plasma in vitro, and efficacy equivalent to or greater than protamine. Peptide (ARKKAAKA)(3)VLVLVLVL was most effective in all plasmas from enoxaparin-treated patients, and was 4- to 20-fold more effective than protamine. Several other peptide structures were effective in some patients' plasmas. All high-affinity peptides reversed inhibition of thrombin-induced clot formation by UFH. These peptides (1 mg/300 g rat) neutralized 1 U/mL anti-Factor Xa activity of enoxaparin in rats within 1 to 2 minutes. Direct blood pressure and heart rate measurements showed little or no hemodynamic effect. These heparin-binding peptides, singly or in combination, are potential candidates for clinical reversal of UFH and LMWH in humans.