Heparin-protein complex

Most of the common methods of isolation of heparin (described in sufficient detail in monographs128-30) are based on a procedure, developed by Charles and Scott,31 involving autolysis of the tissue (originally beef liver and beef lung), extraction with alkali, coagulation of proteins by heating, and precipitation of a heparin - protein complex by acidification. Heparin is recovered from the complex by reprecipitation with ethanol, or acetone, or both. Fats are removed by extraction with ethanol, and proteins by treatment with trypsin. Modifications of this proce-... 

Mast Cells and Basophils. The chief sites of histamine storage are mast cells in the tissues and basophils in blood. These cells synthesize histamine and store it in secretory granules along with a heparin-protein complex. In response to specific antigens, mast cells or basophils are sensitized. Histamine is then secreted from the storage granules. Besides the histamine stores in mast cells and basophils, there is evidence of non-mast cell histamine in some tissues, particularly gastric and intestinal mucosa (60). 

The kinetics of the lytic effect displayed by the complexes of immobilized heparin with thrombin and fibrinogen, in distinction from those with plasmin, are described by their saturation curves. The observed slowing down of the dissolution of unstabilized fibrin is probably due to the inhibiton of the lytic activity of the complexes by the soluble products of the reaction. In fact, as it was shown in Ref. 106, further addition of immobilized heparin-protein complex to partially hydrolyzed fibrin results in a complete recovery of the dissolution rate. 

Beef liver (or lung) was minced and then autolyzed for twenty-four hours before extraction with an alkaline solution saturated with ammonium sulfate. Protein was precipitated by warming the extract, and the heparin-protein complex was precipitated from the supernatant liquor on acidification. Extraction of the complex with ethanol removed fatty material, and tryptic digestion removed most of the protein. The heparin was precipitated with ethanol, redissolved in warm alkaline solution to destroy trypsin, and reprecipitated with acetone. This material, crude heparin, was isolated in a yield of 15-50 g. per 100 lb. of animal tissue. In a later paper," the purification of crude heparin by fractionation successively with Lloyd s reagent, cadmium chloride, and acetone, was described. The purified heparin w-as 100 times as active as the crude material. Scott and Charles" reported the presence of nitrogen... 

For the separation of heparin from heparin-protein complexes, Homan and Lens have developed a method which avoids the use of acid media. The heparin-protein complex is dissolved in aqueous solution at pH 7.5 and is extracted with phenol (which removes most of the protein). The method also facilitates the removal of colored impurities wLich are normally difficult to eliminate. Extraction of the heparin-protein-octylamine complex with phenol has been studied. "... 

In view of the uncertainty as to the existence of a heparin-protein complex, Idndahl et al. (1965) analyzed a number of heparin samples for the presence of amino acids. It was found that heparin which had been prepared by use of mild methods, essentially involving only treatment with proteolytic enzymes, contained residual amino acids with serine as the main component. In some preparations serine was the only amino acid found in significant amounts. Samples prepared under more drastic conditions, including alkali extraction or bleaching, contained only traces of amino acids. These findings thus suggest that heparin does occur in the tissues as a covalently bound complex with protein. 

The isolation of a heparin-protein complex containing 13% protein was... 

From animal tissue, especially bovine lung and liver (e. g. autolysis of comminuted tissue parts, heating with ammonium sulfate in alkaline solution, filtration and acidification yield heparin as complex with protein, removal of fat with alcohol and treatment with trypsine for the purpose of decomposition of proteins, precipitation with alcohol and various purification methods). 

Stuckey JA, St Charles R, Edwards BF. A model of the platelet factor 4 complex with heparin. Proteins 1992 14 277-87. 

Heparin has been reported to complex with a variety of basic species, including biogenic amines and drugs for reviews, see Refs. 10 and 391. For its possible relevance to the pharmacological properties of heparin and complexed species, mention is made here of complexes with histamine392,393 and anthracycline antibiotics.394 C.d. studies on the interaction of basic homopolypeptides with heparin and other glycosaminogly-cans have shown that heparin is able to induce an ordered, helical conformation in the polypeptide.395 397 Similar, and even more dramatic, effects were observed with mixed basic polypeptides, presumed to represent better models for the biologically relevant interactions with plasma proteins.368... 

Heparin is a carbohydrate-based (glycosaminoglycan) anticoagulant associated with many tissues, but mainly found stored intracellularly as granules in mast cells that line the endothelium of blood vessels. Upon release into the bloodstream, heparin binds to and thereby activates an additional plasma protein, namely antithrombin. The heparin-antithrombin complex then binds a number of activated clotting factors (including Ha, IXa, Xa, XIa and Xlla), thereby inactivating them. The heparin now disassociates from the complex and combines with another antithrombin molecule, thereby initiating another turn of this inhibitory cycle. 

One of the prospective pathways for the synthesis of HCP involves copolymerization of neutral monomers with a basic ionogenic one. This procedure was used for synthesis of copolymers of acrylonitrile and dimethylaminoethyl methacrylate used for production of dialysis membranes67). Prior to use, the membranes were quater-nized with HC1 and subsequently treated with heparin. The reported high stability of the heparin-polymer complex (the loss of heparin on washing the polymer with distilled water for 70 hours was less than 1 %) does not obviously ensure that the HCP will perform properly when contacted with solutions of proteins and blood. 

Subcutaneous administration leads to a peak heparin level 2 h after injection and an onset of anticoagulant effect within 1-2 h. Intravenous administration leads to an immediate peak heparin level with anticoagulant activity within 20-30 min. Heparin binds extensively to a number of plasma proteins. Its volume of distribution is 0.07lkg in adults. The pharmacokinetics of heparin is complex and incompletely understood. Heparin metabolism occurs primarily in the reticuloendothelial system by desulfation. The LMWH agents have longer half-lives than standard heparin. Heparin s elimination half-life increases disproportionately with increasing dose, indicating saturable kinetics. 

There are now many X-ray crystal structures of proteins complexed with heparin fragments. Despite the possibility of distortion from the free solution conformation by interaction of the protein, in fact the points for the cx-d-GlcNSp(6S)-(l->4)-cx-L-IdoAp(2S) glycosidic link on the pseudo-Ramachandran... 

A property shared by all these substances is the ability to form complexes with many other substances [2]. Thus A. Fischer demonstrated complex formation between heparin and casein visually by observing the precipitation of the protein at the isoelectric point. With casein, this occurred at pH 5.0. When heparin is added to casein, no precipitation occurs at 5.0, but a new isoelectric point is observed at 3.0, i.e. the complex has different properties as a protein. Complexing occurs for heparin with small molecules, such as inorganic salts and simple organic bases. Benzidine, cetyl-pyridinium chloride (C.P.C.), cetyl trimethyl ammonium bromide (Cetavlon) and other long chain amines form insoluble complexes with heparinoids and mucopolysaccharides and are universally used as precipitating agents for these substances. 

This is evidently due to the ability of heparin to complex amines and proteins. Presumably, this will be true of heparinoids when they are used for specific purposes and, therefore, administered in trace amounts to produce the specific biological effect characteristic of the heparinoid used. 

Our laboratory has been involved in the identification and characterization of the transcription factors for RNA polymerase I (Pol I)-directed transcription of ribosomal RNA genes (rDNA). To this end, we initially isolated and partially purified a transcriptionally active protein complex which contains RNA polymerase I and the essential Pol I transcription factors (1). Such a fraction was obtained from whole ceU extracts (1) or from nuclear extracts (2). Subsequently, we demonstrated that a fraction obtained during chromatography of the cell extract on a heparin sepharose coliunn could prevent nonrandom transcription of cloned rat rDNA in an in vitro system (3). The major protein in this fraction exhibited characteristics of purified poly(ADP-ribose) polymerase. The present report summarizes the properties of this protein, and describes experiments showing the dramatic appearance of accurately initiated transcript in an unfiractionated whole ceU extract or nuclear extract from a tissue foUowing addition of the protein factor. 

Chitosan-heparin polyelectrolyte complex nanoparticles Protein delivery [236]... 

Du YJ, Brash JL, McClung G, Berry LR, Klement P, Chan AKC. Protein adsorption on polyurethane catheters modified with a novel antithrombin-heparin covalent complex. J Biomed Mater Res A 2007 80A 216-25. 

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