Inhibition of Thrombin by Heparin Cofactor II: Thrombin (IIa), heparin (HEP) and heparin cofactor II (HCII) interact to form a ternary complex via a random order bireactant mechanism. Heparin subsequently dissociates from the complex as thrombin forms a covalent complex with the heparin cofactor II. Heparin cofactor II (HCII) (heparin cofactor A, antithrombin BM, dermatan sulfate cofactor, human leuserpin-2) is a single chain glycoprotein (Mr=65,600, 10% carbohydrate) member of the serine protease inhibitor (serpin) family. Amino acid sequence analysis of heparin cofactor II reveals 3 potential N-glycosylation sites and 2 sulfated tyrosines located in two internal repeats of 7 residues located near the NH2-terminal. The plasma concentration is estimated to be 90ug/ml. In the coagulation cascade, heparin cofactor II inhibits thrombin by formation of a bimolecular complex in the presence (k2=4.5x10e8 M-1min-1) and absence (k2=5.0x10e5 M-1min-1) of heparin. This complex is stable to denaturants and essentially irreversible. In addition to heparin, a wide variety of polyanionic compounds accelerate inhibition of thrombin by HCII. Noteworthy among these compounds is dermatan sulfate, which accelerates inhibition of thrombin by HCII, but not antithrombin III. This feature has been utilized to develop a plasma based assay s. Although HCII shows only ~25% sequence homology to other serpins, the reactive site peptide (COOH-terminal 36 amino acids) released on inhibition of thrombin is 53% homologous to that released from antithrombin III (AT III) by thrombin (4,9). The scissile bond in HCII is Leu-Ser which is unusual for an arginine specific protease inhibitor. In contrast to AT III, the only coagulation protease inhibited by HCII is thrombin, Other proteases inhibited by HCII include a-chymotrypsin, neutrophil cathepsin G and streptomyces griseus protease B. The major physiologic role of heparin cofactor II may remain to be discovered. Extinction Coefficient ( E1%1cm, 280nm): 5.93 Specific Activity: ~730u/mg, One unit will inhibit one NIH unit of thrombin. Storage and Stability: May be stored at 4C for short-term only. Aliquot to avoid repeated freezing and thawing. Store at -20C. Aliquots are stable for 6 months after receipt at -20C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer.
Molekulargewicht:
65600
Reinheit:
Heparin cofactor II is prepared from fresh frozen plasma by a modification of the procedure of Griffith, et al., (9). Purity is 95% by SDS PAGE and activity is determined based on thrombin inhibition in the presence of dermatan sulfate.
Formulierung:
Supplied as a liquid in 50% glycerol/H2O.
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