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The structures and proteolytic specificities of autolysed human thrombin.

ABSTRACT: Three Arg/Lys-Xaa bonds in the B-chain of human alpha-thrombin were found to be the major autolytic sites. Under the conditions of 1 mg of alpha-thrombin/ml in 50 mM-ammonium bicarbonate solution at 25 degrees C, the 50% cleavage times of Lys-Gly (residues 154-155), Arg-Tyr (residues 70-71) and Arg-Asn (residues 73-74) were 32 h, 72 h and 96 h respectively. Fragments generated from these three major autolytic sites were purified and analysed. In addition, minor and random autolytic cleavages occurred simultaneously that eventually led to the complete breakdown of the enzyme. These results reveal several novel aspects about the process of autolysis and the structure of autolysed human thrombin. It identifies a major autolytic site at Arg-Tyr (residues 70-71) that has not been previously reported. It demonstrates that beta-thrombin is not an obligatory intermediate during the process of conversion of alpha-thrombin into gamma-thrombin. There exists a new form of autolysed thrombin, designated as beta'-thrombin (with cleavage at Lys-Gly only), which also serves as the intermediate in the conversion of alpha-thrombin into gamma-thrombin. It shows that autolysis of human alpha-thrombin does not proceed in an absolutely clear-cut manner. Numerous minor cleavages, which amount to approx. 20% of the three major autolytic sites, occur simultaneously. It is the first time that several autolytic sites of human alpha-thrombin have been quantitatively analysed, and that it has been shown that formation of beta-, beta'- and gamma-thrombins can be quantitatively followed by the h.p.l.c. method. Furthermore, the data demonstrate that alpha-thrombin and the autolysed thrombin (mixture of beta-, beta'- and gamma-thrombins) have comparable proteolytic activity and specificity towards various sizes of non-fibrinogen polypeptide substrates with relative molecular masses ranging from 3000 to 25,000.

SUBMITTER: Chang JY

PROVIDER: S-EPMC1147489 | biostudies-other | 1986 Dec

REPOSITORIES: biostudies-other

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Project description:Binding of 125I-thrombin to human umbilical vein endothelial cells (HUVECs) was specifically displaced by the synthetic tetradecapeptide SFLLRNPNDKYEPF, named thrombin receptor agonist peptide (TRAP), which has recently been described as a peptide mimicking the new N-terminus created by cleavage of the thrombin receptor, and F-14, a tetradecapeptide representing residues 365-378 of the human alpha-thrombin B chain. Binding of 125I-TRAP to HUVECs was time-dependent, reversible and saturable, showing high affinity (KD = 1.5 +/- 0.4 microM) and high binding capacity (Bmax. = 7.1 +/- 0.6 x 10(6) sites/cell) (n = 3). Unlabelled thrombin and TRAP competitively and selectively inhibited the specific binding of 125I-TRAP with IC50 values of 5.8 +/- 0.7 nM and 2.8 +/- 0.4 microM respectively, whereas F-14 remained ineffective at displacing 125I-TRAP from its binding sites, suggesting the presence of at least two different types of thrombin-binding sites on HUVECs. TRAP was a potent mitogen for HUVECs in culture. Both TRAP and alpha-thrombin stimulated the proliferation of HUVECs with half-maximum mitogenic responses between 1 and 10 nM. F-14 also promoted HUVEC growth. The mitogenic effects of F-14 and TRAP were additive. N alpha-(2-Naphthylsulphonylglycyl)-DL-p-amidinophenylalanylpiper idine (NAPAP) and hirudin (two specific inhibitors of the enzyme activity of thrombin) specifically inhibited thrombin-induced HUVEC growth (IC50 values 400 +/- 60 and 52 +/- 8 nM respectively) but remained without effect on the mitogenic effect of TRAP or F-14. This demonstrated that the mitogenic effect of alpha-thrombin for HUVECs was intimately linked to its esterolytic activity but also showed that thrombin can stimulate HUVEC growth via another non-enzymic pathway. This hypothesis was further reinforced by the fact that F-14-induced proliferation of HUVECs remained unaltered by two antibodies directed against TRAP or the cleavage site on the extracellular portion of the thrombin receptor, which both strongly reduced thrombin-induced proliferation of HUVECs. Thrombin-, TRAP- or F-14-induced HUVEC proliferation was strongly inhibited by a neutralizing monoclonal antibody directed against basic fibroblast growth factor (bFGF), suggesting that thrombin regulates the autocrine release of bFGF in HUVECs.

Project description:Through their hemagglutinin-neuraminidase glycoprotein, parainfluenza viruses bind to sialic acid-containing glycoconjugates to initiate infection. Although the virus-receptor interaction is a key factor of infection, the exact nature of the receptors that human parainfluenza viruses recognize has not been determined. We evaluated the abilities of human parainfluenza virus types 1 (hPIV-1) and 3 (hPIV-3) to bind to different types of gangliosides. Both hPIV-1 and hPIV-3 preferentially bound to neolacto-series gangliosides containing a terminal N-acetylneuraminic acid (NeuAc) linked to N-acetyllactosamine (Galbeta1-4GlcNAc) by the alpha2-3 linkage (NeuAcalpha2-3Galbeta1-4GlcNAc). Unlike hPIV-1, hPIV-3 bound to gangliosides with a terminal NeuAc linked to Galbeta1-4GlcNAc through an alpha2-6 linkage (NeuAcalpha2-6Galbeta1-4GlcNAc) or to gangliosides with a different sialic acid, N-glycolylneuraminic acid (NeuGc), linked to Galbeta1-4GlcNAc (NeuGcalpha2-3Galbeta1-4GlcNAc). These results indicate that the molecular species of glycoconjugate that hPIV-1 recognizes are more limited than those recognized by hPIV-3. Further analysis using purified gangliosides revealed that the oligosaccharide core structure is also an important element for binding. Gangliosides that contain branched N-acetyllactosaminoglycans in their core structure showed higher avidity than those without them. Agglutination of human, cow, and guinea pig erythrocytes but not equine erythrocytes by hPIV-1 and hPIV-3 correlated well with the presence or the absence of sialic acid-linked branched N-acetyllactosaminoglycans on the cell surface. Finally, NeuAcalpha2-3I, which bound to both viruses, inhibited virus infection of Lewis lung carcinoma-monkey kidney cells in a dose-dependent manner. We conclude that hPIV-1 and hPIV-3 preferentially recognize oligosaccharides containing branched N-acetyllactosaminoglycans with terminal NeuAcalpha2-3Gal as receptors and that hPIV-3 also recognizes NeuAcalpha2-6Gal- or NeuGcalpha2-3Gal-containing receptors. These findings provide important information that can be used to develop inhibitors that prevent human parainfluenza virus infection.

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The structures and proteolytic specificities of autolysed human thrombin.

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