Abstract
Heparin cofactor II (HC) is a plasma serine proteinase inhibitor (serpin) that inhibits the coagulant proteinase alpha-thrombin. We have recently demonstrated that proteolysis of HC by catalytic amounts of polymorphonuclear leukocyte proteinases (elastase or cathepsin G) generates leukocyte chemotaxins (Hoffman, M., Pratt, C. W., Brown, R. L., and Church, F. C. (1989) Blood 73, 1682-1685). One of four peptides produced when HC is degraded by neutrophil elastase has chemotactic activity for both monocytes and neutrophils with maximal migration comparable to formyl-Met-Leu-Phe, the "gold standard" bacterially derived chemotaxin. The amino-terminal sequence of this HC peptide is Asp-Phe-His-Lys-Glu-Asn-Thr-Val-... and the peptide corresponds to Asp-39 to Ile-66 of HC. A variety of synthetic peptides derived from this sequence were evaluated for leukocyte migration activity, and a dodecapeptide from Asp-49 to Tyr-60 (Asp-Trp-Ile-Pro-Glu-Gly-Glu-Glu-Asp-Asp-Asp-Tyr) was identified as the active site for leukocyte chemotactic action. The 12-mer synthetic peptide possesses significant neutrophil chemotactic action at 1 nM (60% of the maximal activity of formyl-Met-Leu-Phe), while a peptide with the reverse sequence has essentially no chemotactic activity. Cross-desensitization experiments also show that pretreatment of neutrophils with a 19-mer peptide (Asn-48 to Ile-66) greatly reduces subsequent chemotaxis to HC-neutrophil elastase proteolysis reaction products. When injected intraperitoneally in mice, the HC-neutrophil elastase digest elicits neutrophil migration. Our results demonstrate that not only does HC function as a thrombin inhibitor, but that limited proteolysis of HC near the amino terminus yields biologically active peptide(s) which might participate in inflammation and in wound healing and tissue repair processes.
Highlights
Heparin cofactor I1 (HC) is a plasma serine protein- chicken egg white, and endosperm protein Z from barley (1ase inhibitor that inhibits the coagulant pro- 3)
The amino acid sequence of peak 2 (see Figs. 1 and duced whenHC isdegraded by catalytic amountsof neutrophil elastase were purified by reverse-phase HPLC and testedfor chemotactic activity (Fig. 1).Of the four peaks separated by HPLC, only peak 2 was found to have significant chemotactic
Reaction of ATandprothrombin with neutrophil elastaseand cathepsin G was performed using the conditions for HC-neutrophil proteinase mixtures as detailedpreviously [13]
Summary
Isolation of a Unique Chemotactic Peptide followingLimited digest (-100 pg of HC) was chromatographed on a Phenomenex W- NeutrophilElastase Proteolysis of HC-The peptidespro-. Amino acid analysis of this peptide matches the sequence in HC from Asp-39 to Ile-66 (data notincluded) It is not known whether the chemotactic activity generatedfrom HC by cathepsin G is localized to this solving medium, Flow Laboratories) from the blood of healthy vol- same region; cathepsin G hydrolyzes HC at the unteers as described previously [13, 14]. These results suggest that theleukocyte chemotactic action of HC peptides (containing Asp-49 to Tyr-60) is dependent upon a unique structural determinant derived from its amino acid sequence and this activity is not dependent upon interactions based solely oncharge. Neutrophils preincubated with the 19-mer peptide did not migrate to a gradient of the HC-neutrophil elastase digest on subsequent testing (Table I). A reverse sequence peptide based on Asp-49 to Tyr-60 has no chemotactic action
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