Antithrombin III

Heparin is a powerful anticoagulant medication because it accelerates the pace at which antithrombin inhibits serine proteases in the blood coagulation pathway. Heparin and heparan sulfate, which are structurally similar, are complex linear polymers made up of a mix of chains of varying lengths and sequences. Heparin binds most strongly with peptides that include a complementary binding site with a high positive charge density. Heparin and heparan sulfate have primarily linear helical secondary structures, with sulfo and carboxyl groups arranged at specific intervals and orientations along the polysaccharide backbone. Heparin and DNA are both highly charged linear polymers that act as polyelectrolytes, hence they have similarities. Heparin is thought to work as an anticoagulant chiefly through its interaction with AT III, which enhances AT III-mediated inhibition of blood coagulation factors such as thrombin and factor Xa. Heparin forms a ternary complex with AT III and thrombin, boosting the bimolecular rate constant for thrombin inhibition by a factor of 2,000. Heparin is found mostly in the granules of tissue mast cells, which are directly involved with the immunological response. Heparin forms multiple interactions with FGF-2 and FGFR-1, stabilizing FGF-FGFR binding. Heparin also interacts with the FGFR-1 of the neighboring FGF-FGFR complex, which appears to enhance FGFR dimerization [1].

Heparin is an anticoagulant agent known to have diverse effects on angiogenesis with some reports suggesting that it can induce angiogenesis while a few have indicated of its inhibitory property. Cancer patients treated for venous thromboembolism with low molecular heparin had a better survival than the unfractionated heparin (UFH). Heparin is known to interact with various angiogenic growth factors based on its sulfation modifications within the glycosaminoglycan chains. Therefore it is important to study the mechanism of action of heparin of different molecular weight to understand its angiogenic property. In this concern, we examined the angiogenic response of higher molecular weight Heparin (15 kDa) of different concentrations using late CAM assay. Growth of blood vessels in terms of their length and size was measured and thickness of the CAM was calculated morphometrically. The observed increase in the thickness of the CAM is suggestive of the formation of capillary like structures at the treated region. Analysis of the diffusion pattern showed internalized action of heparin that could affect gene expression leading to proliferation of endothelial cells. Angiogenesis refers to formation of new blood vessels from the existing ones and occurrence of new blood vessels at the treated area strongly confirms that heparin of 15 kDa molecular weight has the ability to induce angiogenesis on CAM vascular bed in a dose dependent manner. The results demonstrate the affinity of heparin to induce angiogenesis and provide a novel mechanism by which heparin could be used in therapeutics such as in wound healing process [3].

Heparin, a sulfated polysaccharide belonging to the family of glycosaminoglycans, has numerous important biological activities, associated with its interaction with diverse proteins. Heparin is widely used as an anticoagulant drug based on its ability to accelerate the rate at which antithrombin inhibits serine proteases in the blood coagulation cascade. Heparin and the structurally related heparan sulfate are complex linear polymers comprised of a mixture of chains of different length, having variable sequences. Heparan sulfate is ubiquitously distributed on the surfaces of animal cells and in the extracellular matrix. It also mediates various physiologic and pathophysiologic processes. Difficulties in evaluating the role of heparin and heparan sulfate in vivo may be partly ascribed to ignorance of the detailed structure and sequence of these polysaccharides. In addition, the understanding of carbohydrate-protein interactions has lagged behind that of the more thoroughly studied protein-protein and protein-nucleic acid interactions. The recent extensive studies on the structural, kinetic, and thermodynamic aspects of the protein binding of heparin and heparan sulfate have led to an improved understanding of heparin-protein interactions. A high degree of specificity could be identified in many of these interactions. An understanding of these interactions at the molecular level is of fundamental importance in the design of new highly specific therapeutic agents. This review focuses on aspects of heparin structure and conformation, which are important for its interactions with proteins. It also describes the interaction of heparin and heparan sulfate with selected families of heparin-binding proteins [1].

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Human Phosphokinase Array [2]
A membrane-based antibody array that determines the relative levels of 43 different human phosphorylated protein kinases was utilized according to the manufacturer's instructions. Briefly, equal amounts of cell lysates of human RPMI-8226 cells treated without or with 100 μg of exosomes were incubated overnight with the phosphokinase array membrane. The array was washed to remove unbound proteins followed by incubation with a mixture of biotinylated detection antibodies. Streptavidin-HRP and chemiluminescent detection reagents were applied to visualize the signal produced at each capture spot corresponding to the amount of phosphorylated protein bound.

Flow Cytometry Analysis of Exosomes Bound to Beads [2]
Flow cytometry analysis to identify molecules on the surface of exosomes was performed after attaching exosomes to either anti-CD63-bound beads or Heparin-agarose beads. 100 μg of purified exosomes were mixed with the anti-CD63 beads or Heparin agarose beads and incubated on a rotating rack at 4 °C overnight. Exosomes bound to beads were suspended in 200 μl of 1% BSA in PBS and stained with antibodies against fibronectin or syndecan-1 prior to analysis with a Becton Dickinson FACSCalibur flow cytometer located in the UAB Comprehensive Flow Cytometry Core. Fibronectin was stained using a mouse monoclonal anti-human fibronectin-PE-conjugated antibody. Mouse isotype matched (IgG1) PE was used as the control. For detection of syndecan-1, exosomes bound to anti-CD63 beads were treated with bacterial heparitinase for 2 h at 37 °C followed by extensive washing. This enzyme treatment, by releasing heparan sulfate and any bound ligands (e.g. fibronectin), exposes the core protein epitope to the antibody. Syndecan-1 was detected using an affinity-purified polyclonal goat anti-syndecan-1 IgG and PE-conjugated secondary antibody. Normal goat IgG was used for the control.

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Exosome Protein Analysis by MS/MS [2]
Exosomes excluded by an iodixanol cushion were solubilized in 1× LDS sample buffer followed by membrane disruption for 10 min in an ultrasonic bath and heat denaturation as per manufacturer's instructions for the LDS buffer. Protein extracts were then quantified using the BCA protein assay kit. An aliquot containing 20 μg of protein was reduced, denatured, and loaded onto a 10% Bis-Tris gel and separated as a short stack run (∼1 cm). The gel was stained with a colloidal blue staining kit, destained, and visualized. The upper gel section containing protein for each sample was cut out and digested using Trypsin Gold, followed by peptide extraction as per the manufacturer's instructions, and the volumes were reduced using a Savant SpinVac Concentrator. One microgram of peptide extract (diluted to ∼1 μg/10 μl in 0.1% formic acid) was loaded onto a 100 μm × 13-cm capillary column, packed in-house with C18 Monitor 100 A-spherical silica beads, and eluted over a 90-min gradient (0–30% acetonitrile in 0.1% TFA).

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Analysis of Exosome-Cell Interactions [2]
Subconfluent RPMI-8226 myeloma cells or HS-5 bone marrow stromal cells were incubated with CD63-RFP or PKH-labeled myeloma-derived exosomes (100 μg/ml) for 2 h. The cells were washed in PBS and fixed in 3% paraformaldehyde for 15 min on ice. Myeloma cells grow in suspension and therefore were cytospun on to slides for analysis. All samples were analyzed using a Nikon A1 confocal microscope. Fluorescent exosomes bound to cells were quantified by flow cytometry using a FACSCalibur instrument. For experiments designed to block the Hep-II domain of fibronectin on exosomes, the exosomes were preincubated with monoclonal antibody (A32) (25 μg of antibody/100 μg of exosome) that binds specifically to the Hep-II Heparin-binding domain of fibronectin. For experiments designed to block heparan sulfate on the surface of target cells, the cells were incubated with a 40-kDa fragment of human fibronectin (purified from chymotryptic digest of human plasma fibronectin) that contains the Hep-II binding domain (50 μg/ml). After washing to remove unbound fibronectin fragments, exosome-target cell interaction was analyzed by confocal microscopy or flow cytometry. For experiments in which heparan sulfate was removed from cell surface, cells were treated with 5 millunits/ml of bacterial heparitinase for 2 h at 37 °C, followed by extensive washing. For removing heparan sulfate from exosomes, 100 μg of exosomes were treated with 1.5 millunits/ml heparitinase for 3 h at 37 °C; new enzyme was added, and exosomes were incubated overnight. Exosomes were then pelleted by ultracentrifugation to remove enzyme. In some blocking experiments, exogenous heparin (10 μg/ml), Roneparstat (10 μg/ml), or a synthetic heparin dodecasaccahride (100 μg/ml) was added simultaneously with 100 μg of exosomes/ml. Roneparstat (previously known as SST0001) is a proprietary drug of Sigma-Tau Research Switzerland that inhibits growth of myeloma tumors in animal models and is currently in phase I trials in advanced multiple myeloma patients (ClinicalTrials.gov identifier NCT01764880). The heparin dodecasaccharide is a fully sulfated molecule prepared by chemoenzymatic synthesis.

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Fibronectin ELISA [2]
Fibronectin levels were quantified using a commercially available human fibronectin ELISA kit, according to the manufacturer's protocol. The assay is in the format of competitive inhibition ELISA. For some experiments, exosomes were treated without or with 1.5 millunits/ml heparitinase for 3 h at 37 °C followed by the addition of fresh enzyme and incubation overnight. Exosomes were then washed and pelleted by ultracentrifugation or ExoQuick exosome precipitation solution. Exosome protein content of the various samples was determined by BCA protein assay, and an equal amount of exosome protein (8 μg) from each sample was used for the ELISA.

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Western Blot [2]
100 μg of exosomes, based on BCA protein assay, were lysed in RIPA buffer containing protease inhibitors for 10 min on ice, and proteins were separated by electrophoresis in a 4–12% SDS-PAGE. The proteins were electroblotted to nitrocellulose membrane and probed with primary antibodies that recognize fibronectin, clathrin, or flotillin. In some experiments, the exosomes were treated with bacterial heparitinase and probed for syndecan-1. Primary antibodies were detected using HRP-conjugated secondary antibodies, and bands were visualized by chemiluminescence. In some experiments, RPMI-8226 cells were treated with or without heparitinase for 2 h at 37 °C and then exposed to 100 μg/ml of either control or aggressive exosomes for 20 min. Cells were lysed and probed for phosphorylated p38 or total p38.

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Endothelial Cell Invasion [2]
The effect of exosomes on the invasion of human umbilical vein endothelial cells was assessed using Biocoat Matrigel invasion chambers, as described. To study the relevance of fibronectin-heparan sulfate interactions for the functional activity of exosomes, exosomes were preincubated without or with the anti-fibronectin antibody A32 (25 μg of antibody/100 μg of exosome protein) before adding to the endothelial cells.

The in ovo CAM model [3]
Fertilized chicken eggs weighing 50 ± 2gm were incubated at 37°C in a humidified atmosphere (>60% relative humidity) based on protocol for the Hen's Egg Test-Chorioallantoic Membrane (HET-CAM) method. At day 3 of post incubation, 2 to3 ml of albumin was withdrawn, using a 21-gauge needle, through the large blunt edge of the egg in order to minimize adhesion of the shell membrane with CAM. A square window of 1 cm² was opened in the egg shell and sealed with paraffin film to prevent dehydration and the eggs were returned for incubation. On day 9th, gelatin sponges of size of 1 mm³ were placed on the top of growing CAM under sterile condition. The sponges were soaked with 10 μl of 50, 100 and 150 μM concentrations of Heparin. Control CAM was treated with 10 μl of 1X PBS. The window was closed with a transparent adhesive tape and the eggs were returned for further incubation till day 12 (72hours) at which vascularization potential of the CAM reaches its maximum. The experimental groups were divided into 4 with each containing 40 numbers. Group1 represents 1X PBS (control), group 2, 3 and 4 corresponds to 10 μl of 50 μM, 100 μM and 150 μM of Heparin. Treated CAM was photographed at 0, 24, 48 and 72hours using Cannon digital camera and the images were analyzed with Angioquant Toolbox, MATLAB 6.5 software to measure total length and size of blood vessels in micrometer at the area of treatment.

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Histology [3]
CAM treated with Heparin was flooded with Bouin's fixative solution and the treated area was removed, dehydrated and embedded in paraffin wax. Vertical cross sections of 7 μm in thickness were taken using Rotary Microtome. The histological sections were observed under Light Microscope at 40X magnification for qualitative assessment and images were taken using Nikon Camera attached with light microscope at 10X magnification.

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C57BL/6J mice
100, 500, or 2500 units/kg
i.p.

[1]. Heparin-protein interactions. Angew Chem Int Ed Engl. 2002 Feb 1;41(3):391-412.

[2]. Fibronectin on the Surface of Myeloma Cell-derived Exosomes Mediates Exosome-Cell Interactions. J Biol Chem. 2016 Jan 22;291(4):1652-63.

[3]. Angiogenic efficacy of Heparin on chick chorioallantoic membrane. Vascular Cell, [S.l.] v. 4, n. 1, p. 8, apr. 2012. ISSN 2045-824X.

Heparin, a sulfated polysaccharide belonging to the family of glycosaminoglycans, has numerous important biological activities, associated with its interaction with diverse proteins. Heparin is widely used as an anticoagulant drug based on its ability to accelerate the rate at which antithrombin inhibits serine proteases in the blood coagulation cascade. Heparin and the structurally related heparan sulfate are complex linear polymers comprised of a mixture of chains of different length, having variable sequences. Heparan sulfate is ubiquitously distributed on the surfaces of animal cells and in the extracellular matrix. It also mediates various physiologic and pathophysiologic processes. Difficulties in evaluating the role of heparin and heparan sulfate in vivo may be partly ascribed to ignorance of the detailed structure and sequence of these polysaccharides. In addition, the understanding of carbohydrate–protein interactions has lagged behind that of the more thoroughly studied protein–protein and protein–nucleic acid interactions. The recent extensive studies on the structural, kinetic, and thermodynamic aspects of the protein binding of heparin and heparan sulfate have led to an improved understanding of heparin–protein interactions. A high degree of specificity could be identified in many of these interactions. An understanding of these interactions at the molecular level is of fundamental importance in the design of new highly specific therapeutic agents. This review focuses on aspects of heparin structure and conformation, which are important for its interactions with proteins. It also describes the interaction of heparin and heparan sulfate with selected families of heparin-binding proteins. [1]

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The present study evaluates the effects of HMWH on formation of capillary-like tubular structures using CAM assay. UFH of 15 kDa enhances its formation in contrast to LMWH which is known to decrease the formation of tubular endothelial structures. Thus the present in ovo vivo study has demonstrated that heparin possesses the angiogenesis inducing affinity and is significant at 100 μM concentration. These data provide a novel mechanism by which higher molecular weight heparins can promote formation of capillary-like tubular structures and might be of therapeutic significance in inducing angiogenisis during wound healing process while the low molecular weight constituents can be employed to inhibit angiogenesis as in tumor progression.[3]

(C14H25NO20S3)N.XNA

6000-20000

9041-08-1

Heparin sodium salt;9041-08-1;Heparin Lithium salt;9045-22-1;Heparin;9005-49-6

118989588

White to off-white solid

>181°C (dec.)

2

6

6

16

162

0

O[C@H]1[C@H](O[C@H]2[C@H](OS(O)(=O)=O)[C@@H](O)[C@H](OC)[C@H](C(O)=O)O2)[C@@H](COS(O)(=O)=O)O[C@H](OC)[C@@H]1NS(O)(=O)=O.[n].[.xNa]

YOMTXLQWRQUKAK-UHFFFAOYSA-N

InChI=1S/C9H20N3O3.Na/c13-4-1-10-7-11(2-5-14)9-12(8-10)3-6-15;/h13-14H,1-9H2;/q-1;+1

sodium;2-[3,5-bis(2-hydroxyethyl)-1,3,5-triazinan-1-yl]ethanolate

Heparin, sodium salt; 9041-08-1; Nadroparin Sodium; Heparin sodium salt (MW 15kDa); sodium;2-[3,5-bis(2-hydroxyethyl)-1,3,5-triazinan-1-yl]ethanolate;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O: ~20 mg/mL
DMSO: < 1 mg/mL

Solubility in Formulation 1: 25 mg/mL (Infinity mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)