Role of the A+ Helix in Heparin Binding to Protein C Inhibitor

Marc G L M Elisen; Machiel H H Maseland; Frank C Church; Bonno N Bouma; Joost C M Meijers

doi:10.1055/s-0038-1650363

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 1996; 75(05): 760-766
DOI: 10.1055/s-0038-1650363

Original Article

Schattauer GmbH Stuttgart

Role of the A⁺ Helix in Heparin Binding to Protein C Inhibitor

Marc G L M Elisen

¹The Department of Haematology, University Hospital, Utrecht, The Netherlands

,

Machiel H H Maseland

¹The Department of Haematology, University Hospital, Utrecht, The Netherlands

,

Frank C Church

²The Department of Pathology and the Center for Thrombosis and Hemostasis, University of North Carolina School of Medicine, Chapel Hill, North Carolina, USA

,

Bonno N Bouma

¹The Department of Haematology, University Hospital, Utrecht, The Netherlands

,

Joost C M Meijers

¹The Department of Haematology, University Hospital, Utrecht, The Netherlands

› Author Affiliations

Abstract

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Summary

Interactions between proteins and heparin(-like) structures involve electrostatic forces and structural features. Based on charge distributions in the linear sequence of protein C inhibitor (PCI), two positively charged regions of PCI were proposed as possible candidates for this interaction. The first region, the A⁺ helix, is located at the N-terminus (residues 1-11), whereas the second region, the H helix, is positioned between residues 264 and 280 of PCI. Competition experiments with synthetic peptides based on the sequence of these regions demonstrated that the H helix has the highest affinity for heparin. In contrast to previous observations we found that the A⁺ helix peptide competed for the interaction of PCI with heparin, but its affinity was much lower than that of the H helix peptide.

Recombinant PCI was also used to investigate the role of the A⁺ helix in heparin binding. Full-length (wild-type) rPCI as well as an A⁺ helix deletion mutant of PCI (rPCI-Δ2-l 1) were expressed in baby hamster kidney cells and both had normal inhibition activity with activated protein C and thrombin. The interaction of the recombinant PCIs with heparin was investigated and compared to plasma PCI. The A⁺ helix deletion mutant showed a decreased affinity for heparin in inhibition reactions with activated protein C and thrombin, but had similar association constants compared to wild-type rPCI. The synthetic A⁺ helix peptide competed with rPCI-Δ2-11 for binding to heparin. This indicated that the interaction between PCI and heparin is fairly non-specific and that the interaction is primarily based on electrostatic interactions.

In summary, our data suggest that the H helix of PCI is the main heparin binding region of PCI, but the A⁺ helix increases the overall affinity for the PCI-heparin interaction by contributing a second positively charged region to the surface of PCI.

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References

References
1 Suzuki K, Nishioka J, Hashimoto S. Protein C inhibitor. Purification from human plasma and characterization. J Biol Chem 1983; 258: 163-168
2 Meijers JCM, Kanters DHAJ, Vlooswijk RAA, van Erp HE, Hessing M, Bouma BN. Inactivation of human plasma kallikrein and factor XIa by protein C inhibitor. Biochemistry 1988; 27: 4231-4237
3 Shirk RA, Phillips JC, Church FC. The molecular biology of protein C inhibitor. In: Molecular Basis of Thrombosis and Hemostasis High KA, Roberts HR. (eds). New York: Marcel Dekker; 1995: 447-457
4 Heeb MJ, Espana F, Geiger M, Collen D, Stump DC, Griffin JH. Immunological identity of heparin-dependent plasma and urinary protein C inhibitor and plasminogen activator inhibitor-3. J Biol Chem 1987; 263: 15813-15816
5 Geiger M. Protein C inhibitor/plasminogen activator inhibitor 3. Fibrinolysis 1988; 2: 183-188
6 Huber R, Carrell RW. Implications of the three-dimensional structure of a j-antitrypsin for structure and function of serpins. Biochemistry 1989; 28: 8951-8966
7 Travis J, Salvesen GS. Human plasma proteinase inhibitors. Annu Rev Biochem 1983; 52: 655-709
8 Suzuki K, Nishioka J, Kusumoto H, Hashimoto S. Mechanism of inhibition of activated protein C by protein C inhibitor. J Biochem 1984; 95: 187-195
9 Suzuki K, Deyashiki Y, Nishioka J, Toma K. Protein C inhibitor: structure and function. Thromb Haemost 1989; 61: 337-342
10 Pratt CW, Church FC. Heparin binding to protein C inhibitor. J Biol Chem 1992; 267: 8789-8794
11 Griffith MJ. Heparin-catalyzed inhibitor protease reactions: kinetic evidence for a common mechanism of action of heparin. Proc Natl Acad Sci USA 1983; 80: 5460-5465
12 Kazama Y, Niwa M, Yamagishi R, Takahashi K, Sakuragawa N, Koide T. Specificity of sulfated polysaccharides to accelerate the inhibition of activated protein C by protein C inhibotor. Thromb Res 1987; 48: 179-185
13 Cardin AD, Weintraub HJR. Molecular modeling of protein-glycosamino-glycan interactions. Arteriosclerosis 1989; 9: 21-32
14 Margalit H, Fisher N, Ben-Sasson S. Comparative analysis of structurally defined heparin binding sequences reveals a distinct spatial distribution of basic residues. J Biol Chem 1993; 268: 19228-19231
15 Kuhn LA, Griffin JH, Fisher CL, Greengard JS, Bouma BN, Espana F, Tainer JA. Elucidating the structural chemistry of glycosaminoglycan recognition by protein C inhibitor. Proc Natl Acad Sci USA 1990; 87: 8506-8510
16 Pratt CW, Whinna HC, Church FC. A comparison of three heparin binding serine protease inhibitors. J Biol Chem 1992; 267: 8795-8801
17 Shirk RA, Elisen MGLM, Meijers JCM, Church FC. Role of the H helix in heparin binding to protein C inhibitor. J Biol Chem 1994; 269: 28690-28695
18 Meijers JCM, Chung DW. Evidence for a glycine residue at position 316 in human protein C inhibitor. Thromb Res 1990; 59: 389-393
19 Vandeyar MA, Weiner MP, Hutton CJ, Batt CA. A simple and rapid method for the selection of oligodeoxynucleotide-directed mutants. Gene 1988; 65: 129-133
20 Meijers JCM, Davie EW, Chung DW. Expression of human blood coagulation factor XI: characterization of the defect in factor XI type III deficiency. Blood 1992; 79: 1435-1440
21 Laurell M, Carlson T, Stenflo J. Monoclonal antibodies against the heparin dependent protein C inhibitor suitable for purification and assay of inhibitor complexes. Thromb Haemost 1988; 60: 334-339
22 Chase T, Shaw E. Titration of trypsin, plasmin and thrombin with p-nitro-phenylp’-guanidinobenzoate HC1. Meth Enzymol 1970; 19: 20-27
23 Hackeng TM, Hessing M, van’t Veer C, Meijer-Huizinga F, Meijers JCM, de Groot PG, van Mourik JA, Bouma BN. Protein S binding to human endothelial cells is required for expression of cofactor activity for activated protein C. J Biol Chem 1983; 268: 3993-4000
24 Faller B, Cadene M, Bieth JG. Demonstration of a two-step reaction mechanism for the inhibition of heparin-bound neutrophil elastase by a,-proteinase inhibitor. Biochemistry 1993; 32: 9230-9235
25 Hermans JM, Stone SR. Interaction of activated protein C with serpins. Biochem J 1993; 295: 239-245
26 Morrison JF. The slow-binding and slow tight-binding inhibition of enzyme catalysed reactions. TIBS 1982; 7: 102-105
27 Cha S. Tight binding inhibitors -1. Kinetic behaviour. Biochemical Pharmacology 1975; 25: 2695-2702
28 Morrison JF, Walsh CT. The behaviour and significance of slow binding enzyme inhibitors. Adv Enzymol 1988; 61: 201-301
29 Suzuki K, Deyashiki J, Nishioka K, Kurachi M, Akiras S, Yamamoto S, Hashimoto S. Characterization of a cDNA for human protein C inhibitor. A new member of the plasma serine protease superfamily. J Biol Chem 1987; 262: 611-616
30 Laurell M, Stenflo J. Protein C inhibitor from human plasma: characterisation of native and cleaved inhibitor and demonstration of inhibitor complexes with plasma kallikrein. Thromb Haemost 1989; 62: 885-891
31 Toma K, Yamamoto S, Deyashiki Y, Suzuki K. Three dimensional structure of protein C inhibitor predicted from structure of a j-antitrypsin with computer graphics. Protein Engineering 1987; 1: 471-475