Proteins, complexes with heparin

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... 

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

Mechanism of Action A protein that complexes with heparin to form a stable salt. Therapeutic Effect Reduces anticoagulant activity of heparin. 

Antagonists Protamine sulfate, a strongly basic protein, forms a complex with heparin to an inactive compound 1 mg protamine for 100 units of heparin Vitamin K, whole blood, fresh plasma... 

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). 

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-... 

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... 

Although the order of affinity of PF-4 for different glycosaminogly-cans, and dissociation of their complexes with salts, are typical of nonspecific, electrostatic interactions, PF-4 is not strictly a cationic protein.452 It is probable that heparin binds to clusters of basic amino acids (two lysine pairs) near the carboxyl terminal of a polypeptide chain that has an overall preponderance of acidic amino acid residues.457 High-molecular-weight heparin species can bind two PF-4 molecules, with formation of complexes 10 to 100 times as strong as those with antithrombin.217... 

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). 

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. 

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. 

The structure of PTPo is consistent with its role in binding ligands on the surface of other cells or in the extracellular matrix. It interacts with heparin sulphate proteoglycan in the basement membrane (Sajnani-Perez et al. 2003). It may also bind to the C-terminal domain of cell surface-exposed nucleolin, a normally nuclear protein that is presented on the surface of developing muscle cells (Alete et al. 2006). However, these complexes seem to mediate structural or long-term regulatory interactions rather than short-term signaling to the neuronal secretory machinery. 

Fondaparinux is a chemically synthesized pentasaccharide that mimics the antithrombin-binding site of heparin and LMWH. Its molecular size (1728Da) is too small to bind to thrombin molecules while it is bound to antithrombin, Therefore, it is a pure anti-Xa inhibitor. It binds very little to platelets, proteins, or endothelium and is excreted in the urine, It does not form a complex with PF4 or other positively charged molecules. It is not neutralizable by protamine sulfate, Recent clinical trials have resulted in FDA approval for prophylaxis of deep vein thrombosis in orthopedic surgery, It has been shown to be effective and safe for the treatment of pulmonary embolism (20,21) and ACS (non-ST-elevation Ml) (OASIS 5—Michelangelo Trial) (17). 

With all the assays based on Clq binding, it is important to remember that the types of complexes detected are restricted by the specificity of Clq for IgM and the IgGl, IgC2, and IgC3 subclasses. The tests may also be affected by the Clq concentration in the test sample (Gl, H16, L21, M10, R8, Z3). More importantly, a number of nonimmunoglobulin substances that are Clq reactive may occur in sera and interfere with the results. Polyanions such as DNA, heparin, and bacterial endotoxins bind Clq (A7, Gl, L2, L21, S28, S39, T5, W26, Z3). C-Reactive protein reacts with Clq through a calcium-dependent bond, a problem usually circum-... 

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. "... 

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