Essential thrombin residues for inhibition by protein C inhibitor with the cofactors heparin and thrombomodulin.

BACKGROUND: Protein C inhibitor (PCI) and antithrombin (AT) are serine protease inhibitors (serpins) that inhibit a wide array of blood coagulation serine proteases including thrombin. OBJECTIVE: Fifty-five Ala-scanned recombinant thrombin mutants were used to determine thrombin residues important for inhibition by PCI with and without the cofactors heparin and thrombomodulin (TM) and compared with the prototypical serpin, AT. RESULTS: Residues around the active site (Tyr50 and Glu202) and the sodium-binding site (Glu229 and Arg233) were required for thrombin inhibition by PCI with and without cofactors. Exosite-2 residues (Arg89, Arg93, Glu94, Arg98, Arg245, Arg248, and Gln251) were critical for heparin-accelerated inhibition of thrombin by PCI. Exosite-1 residues (especially Lys65 and Tyr71) were required for enhanced PCI inhibition of thrombin-TM. Interestingly, we also found that the TM chondroitin sulfate moiety is not required for the approximately 150-fold enhanced rate of thrombin inhibition by PCI. Using the aforementioned thrombin exosite-2 mutants that were essential for heparin-catalyzed PCI-thrombin inhibition reactions we found no change in PCI inhibition rates for thrombin-TM. CONCLUSIONS: Collectively, these results show that (i) similar thrombin exosite-2 residues are critical for the heparin-catalyzed inhibition by PCI and AT, (ii) PCI and AT are different in their thrombin-TM inhibition properties, and (iii) PCI has a distinct advantage over AT in the regulation of the activity of thrombin-TM.

Tissue distribution and processing of proSAAS by proprotein convertases.

The conversion of inactive precursor proteins into bioactive neuropeptides and peptide hormones involves regulated secretory proteins such as prohormone convertases PC1 and PC2. The neuroendocrine protein 7B2 represents a specific binding protein for PC2, and the protein proSAAS, which interacts with PC1, exhibits certain structural and functional homologies with 7B2. With the intention of better understanding the physiological role of proSAAS and its derived peptides, we investigated its tissue localization using a new radioimmunoassay (RIA) to a C-terminal proSAAS-derived peptide. Immunoreactivity corresponding to this SAAS-derived peptide is mostly localized to the brain and gut. Analysis of the brain distribution of the proSAAS-derived peptides indicates that the hypothalamus and pituitary are the two richest areas, consistent with the previously described high expression of PC1 in these two areas. In order to investigate the cleavage of proSAAS by prohormone convertases, we incubated recombinant His-tagged proSAAS with recombinant mouse proPC2 or furin, separated the cleavage products using high-pressure gel permeation chromatography and analyzed the products by RIA. Our results indicate that either PC2 or furin can accomplish in vitro rapid removal and efficient internal processing of the C-terminal peptide, exposing the inhibitory hexapeptide to possible further digestion by carboxypeptidases. Finally, we also studied proSAAS processing in the brains of wild-type and PC2 null mice and found that proSAAS is efficiently processed in vivo. Whereas the C-terminal peptide is mostly internally cleaved in wild-type mouse brain, it is not processed as efficiently in the brain of PC2 null mice, suggesting that PC2 is partially responsible for this cleavage in vivo.

Processing and sorting of the prohormone convertase 2 propeptide.

The prohormone convertases (PCs) are synthesized as zymogens whose propeptides contain several multibasic sites. In this study, we investigated the processing of the PC2 propeptide and its function in the regulation of PC2 activity. By using purified pro-PC2 and directed mutagenesis, we found that the propeptide is first cleaved at the multibasic site separating it from the catalytic domain (primary cleavage site); the intact propeptide thus generated is then sequentially processed at two internal sites. Unlike the mechanism described for furin, our mutagenesis studies show that internal cleavage of the propeptide is not required for activation of pro-PC2. In addition, we identified a point mutation in the primary cleavage site that does not prevent the folding nor the processing of the zymogen but nevertheless results in the generation of an inactive PC2 species. These data suggest that the propeptide cleavage site is directly involved in the folding of the catalytic site. By using synthetic peptides, we found that a PC2 propeptide fragment inhibits PC2 activity, and we identified the inhibitory site as the peptide sequence containing basic residues at the extreme carboxyl terminus of the primary cleavage site. Finally, our study supplies information concerning the intracellular fate of a convertase propeptide by providing evidence that the PC2 propeptide is generated and is internally processed within the secretory granules. In agreement with this localization, an internally cleaved propeptide fragment could be released by stimulated secretion.

The SAAS granin exhibits structural and functional homology to 7B2 and contains a highly potent hexapeptide inhibitor of PC1.

Prohormone convertases (PCs) 1 and 2 are thought to mediate the proteolytic cleavage of many peptide precursors. Endogenous inhibitors of both PC1 and PC2 have now been identified; the 7B2 protein is a nanomolar inhibitor of PC2, while the novel protein proSAAS was recently reported to be a micromolar inhibitor of PC1 [Fricker et al. (2000) J. Neurosci. 20, 639-648]. We here report evidence that 7B2 and proSAAS exhibit several elements of structural and functional homology. Firstly, 26 kDa human, mouse and rat proSAAS, like all vertebrate 7B2s, contain a proline-rich sequence within the first half of the molecule and also contain a C-terminal 40 residue peptide (SAAS CT peptide) separated from the remainder of the protein by a furin consensus sequence. The SAAS CT peptide contains the precise sequence of a hexapeptide previously identified by combinatorial peptide library screening as a potent inhibitor of PC1, and the vast majority of the inhibitory potency of proSAAS can be attributed to this hexapeptide. Further, like the 7B2 CT peptide, SAAS CT-derived peptides represent tight-binding competitive convertase inhibitors with nanomolar potencies. Lastly, recombinant PC1 is able to cleave the proSAAS CT peptide to a product with a mass consistent with cleavage following the inhibitory hexapeptide. Taken together, our results indicate that proSAAS and 7B2 may comprise two members of a functionally homologous family of convertase inhibitor proteins.

The role of the 7B2 CT peptide in the inhibition of prohormone convertase 2 in endocrine cell lines.

Prohormone convertase (PC) 2 plays an important role in the processing of neuropeptide precursors via the regulated secretory pathway in neuronal and endocrine tissues. PC2 interacts with 7B2, a neuroendocrine protein that is cleaved to a 21-kDa domain involved in proPC2 maturation and a carboxyl-terminal peptide (CT peptide) that represents a potent inhibitor of PC2 in vitro. A role for the CT peptide as an inhibitor in vivo has not yet been established. To study the involvement of the CT peptide in PC2-mediated cleavages in neuroendocrine cells, we constructed a mutant proenkephalin (PE) expression vector containing PE with its carboxyl-terminal peptide (peptide B) replaced with the 7B2 inhibitory CT peptide. This PECT chimera was stably transfected into two PC2-expressing cell lines, AtT-20/PC2 and Rin cells. Although recombinant PECT proved to be a potent (nM) inhibitor of PC2 in vitro, cellular PC2-mediated cleavages of PE were not inhibited by the PECT chimera, nor was proopiomelanocortin cleavage (as assessed by adrenocorticotropin cleavage to alpha-melanocyte-stimulating hormone) inhibited further than in control cells expressing only the competitive substrate PE. Tests of stimulated secretion showed that both the CT peptide and the PE portion of the chimera were stored in regulated secretory granules of transfected clones. In both AtT-20/PC2 and Rin cells expressing the chimera, the CT peptide was substantially internally hydrolyzed, potentially accounting for the observed lack of inhibition. Taken together, our data suggest that overexpressed CT peptide derived from PECT is unable to inhibit PC2 in mature secretory granules, most likely due to its inactivation by PC2 or by other enzyme(s).

An adrenomedullin (ADM) fragment retains the systemic vasodilator activity of human ADM.

The present study was undertaken to investigate the effects of human ADM, a newly discovered peptide present in normal human plasma, as well as a fragment of human ADM, human ADM13-52, on systemic hemodynamics in the anesthetized cat. Intravenous (i.v.) bolus injections of human ADM and human ADM13-52 decreased systemic arterial pressure (SAP) in a dose-dependent manner. Since neither peptide altered cardiac output, the decreases in SAP reflect reductions in systemic vascular resistance. The systemic vasodilator responses to the same doses of human ADM and human ADM13-52 in the cat were similar. The present study demonstrates the systemic vasodilator activity of ADM is conserved across species. The present data suggest that human ADM13-52 or a peptide structurally similar to it may mediate the hemodynamic properties of ADM in vivo in man. Since cardiac output and heart rate were not altered during the marked systemic vasodepressor response to ADM, activation of the ADM vasodilator mechanism may represent a therapeutic alternative in the clinical management of hypertensive diseases.