Antithrombin heparin binding

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... 

The fluorescent labelling of heparin with F-D by this technique did not observably alter the biologic activity of the heparin as regards to its binding to antithrombin and catalysis of antithrombin s neutralization of activated coagulation factors. F-D labelled heparins also bound to other known heparin-binding proteins in a saturable and reversible manner, as demonstrated by the dot-blot assay technique (Figure 6). 

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. 

Based on the mixed-phase method, ACE is introduced for studying the interaction of heparin with the serine protease inhibitors, antithrombin III (ATIII) and secretory leukocyte proteinase inhibitor (SLPI) (85). An etched capillary, to which heparin has been covalently immobilized, was used in this study. This modified capillary both afforded an improvement in the separation of heparin-binding proteins and required a lower quantity of loaded protein. 

Heparin binds to antithrombin III and induces a conformational change that accelerates the interaction of antithrombin III with the coagulation factors. Heparin also catalyzes the inhibition of thrombin by heparin cofactor II, a circulating inhibitor. Smaller amounts of heparin are needed to prevent the formation of free thrombin than are needed to inhibit the protease activity of clot-bound thrombin. Inhibition of free thrombin is the basis of low-dose prophylactic therapy. 

Interactions of heparinoids with the most diverse proteins such as enzymes and enzyme inhibitors, cytokines, and adhesion molecules have been described. To date, many more than a hundred heparin binding proteins are known. A number of heparin binding proteins are members of the serpin family of serine protease inhibitors. The best described example is antithrombin [4]. Antithrombin III (AT III) is able to inhibit various serine proteases involved in the blood coagulation process by formation of stable, equimolar complexes. Binding of heparin to AT III accelerates the kinetics of this complex formation by several orders of magnitude. This has been the basis for the successful clinical use of heparin as an anticoagulant for nearly sixty years. 

Cesaretti, M., Luppi, E., Maccari, M., and Volpi, N. (2004). Isolation and characterization of a heparin with high anticoagulant activity from the clam Tapes philippinarum. Evidence for the presence of a high content of antithrombin Ill-binding site. Glycobiology 14,1275-1284. 

Deficiency of antithrombin predisposes the patient to thrombotic complications. Antithrombin deficiencies can be the result of low protein levels or due to functionally abnormal molecules. Low protein levels can be brought about by reduced synthesis or an increased turnover of the molecule, Functional deficiencies can be brought about by mutations in either the reactive site or heparin binding sites, A number of such mutations have been documented (81,86,87),... 

Like antithrombin, heparin cofactor II inhibits proteases by forming a I I stoichiometric complex with the enzyme. The protease attacks the reactive site of heparin cofactor II located on the C-terminus, resulting in the formation of a covalent bond. Heparin cofactor II has higher protease specificity than antithrombin. Of the coagulation enzymes, heparin cofactor II is known only to inhibit thrombin (92). Additionally heparin cofactor II has been shown to inhibit chymotrypsin (93) and leukocyte cathepsin G (94), This protease specificity appears to be due to the active site bond present in heparin cofactor II. Whereas antithrombin contains an Arg-Ser bond as its active site, heparin cofactor II is unique in containing a Leu-Ser bond. This suggests than another portion of the heparin cofactor II molecular may be required for protease binding,... 

Heparin is a glycosaminoglycan extracted from animal tissues (porcine mucosa, beef lung, etc.). It is a mixture of molecules having a mean molecular weight of 15,000 Da. A pentasaccharide sequence found in approximately one third of the molecules binds to antithrombin in mammalian blood, enhancing its inhibitory effects on the enzymes thrombin, factor Xa, factor Vila, and factor IXa. The reaction is reversible, heparin being released after the antithrombin molecule binds to the procoagulant enzymes. Heparin binds to platelets, platelet factor-4 (which neutralizes it), histidine-rich GP vWp and a number of other proteins. Its half-life is about one hour in the circulation (18). Antibodies to heparin... 

In order to make equation 6 more specific it will be assumed that heparin binds to antithrombin III... 

Heparin binds to antithrombin (a protease inhibitor that inactivates factors Ha, IXa, Xa and XIa) and markedly accelerates its inhibitory effect on coagulation. In addition, heparin inhibits platelet function, The newer low-molecular-weight heparins augment antithrombin activity preferentially against factor Xa and do not prolong the APTT like standard (unfractionated) heparin does. 

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