Biomedical subjects

Philip A Patston

Publications and source records attributed to Philip A Patston.

5 recordsLinked to original sources

PubMed2004

Serpin-ligand interactions.

One of the more common features of serpins is the ability to bind various ligands. Ligand binding can occur so that the inhibitory properties of the serpin are regulated, so that the serpin can be localized, or to produce or modulate some other biological function of the serpin. Ligands known to affect serpin biologic activity include glycosaminoglycans such as heparin, heparan sulfate and dermatan sulfate, DNA, extracellular matrix proteins such as vitronectin and collagen, and small organic molecule hormones. Many different biochemical and biophysical techniques in conjunction with molecular biology and cell biology approaches have been used to study the binding of various ligands to serpins and to assess the influence of this binding on activity and structure. We summarize here the different approaches that have been used to identify serpin ligands and the many methods that have been used to characterize the interactions of these ligands with their cognate serpins.

Binding Sites↗

PubMed2004

Inhibition of plasma kallikrein by C1-inhibitor: role of endothelial cells and the amino-terminal domain of C1-inhibitor.

Activation of plasma prekallikein and generation of bradykinin are responsible for the angioedema attacks observed with C1-inhibitor deficiency. Heterozygous individuals with <50% levels of active C1-inhibitor are susceptible to angioedema attacks indicating a critical need for C1-inhibitor to be present at maximum levels to prevent unwanted prekallikrein activation. Studies with purified proteins do not adequately explain this observation. Therefore to investigate why reduction of C1-inhibitor to levels seen in angioedema patients results in excessive kallikrein generation we examined the effect of endothelial cells on the inhibition of kallikrein by C1-inhibitor. Surprisingly, it was found that a C1-inhibitor concentration of greater than 1 microM was needed to inhibit 3 nM kallikrein. We propose that this apparent protection from inhibition was mediated by kallikrein binding to the cells via the heavy chain in a high molecular weight kininogen and zinc independent manner. Protection of kallikrein from inhibition was not observed when C1-inhibitor truncated in the amino-terminal domain by the StcE metalloproteinase was used, which suggests a novel function for this unique domain. The requirement for high concentrations of C1-inhibitor to fully inhibit kallikrein is consistent with the fact that reduced levels of C1-inhibitor result in the kallikrein activation seen in angioedema.

Angioedema↗

PubMed2003

Characterization of different high molecular weight angiotensinogen forms.

BACKGROUND: Angiotensinogen is the substrate for renin in the system that releases angiotensin II. This renin-angiotensin system is an important regulator of blood pressure (BP), and defects in the system are linked to the development of hypertension. Native angiotensinogen is a 62,000-dalton monomer, but various high molecular weight forms also exist, which have not been well characterized. High molecular weight angiotensinogen has been reported to be 5% of the total angiotensinogen, and increases to 60% of the total during pregnancy and hypertension. The purpose of this investigation was to study high molecular weight angiotensinogen in normal plasma. METHODS: Normal human plasma was run on a gel filtration column, and high molecular weight angiotensinogen detected by Western blotting. Further purification was by ion-exchange chromatography. In vitro polymerization of angiotensinogen was analyzed by sodium dodecyl sulfate (SDS) and native gels. RESULTS: Two forms of high molecular weight angiotensinogen were found with molecular weights of 500,000 and 250,000 daltons on gel filtration, and 140,000 and 110,000 daltons, respectively, on nonreduced SDS-polyacrylamide gel electrophoresis, and at 62,000 daltons on reduced gels. Our estimation of the amount of high molecular weight angiotensinogen present is close to that reported previously. We also describe some of the in vitro polymerization characteristics of angiotensinogen, which can be explained by angiotensinogen being a member of the serpin family of proteins. CONCLUSIONS: The angiotensinogen polymers produced in vitro might provide a model system for some of the high molecular weight forms produced in vivo, and help in understanding their function.

Angiotensinogen↗

PubMed2002

Release and degradation of angiotensin I and angiotensin II from angiotensinogen by neutrophil serine proteinases.

Cathepsin G, elastase, and proteinase 3 are serine proteinases released by activated neutrophils. Cathepsin G can cleave angiotensinogen to release angiotensin II, but this activity has not been previously reported for elastase or proteinase 3. In this study we show that elastase and proteinase 3 can release angiotensin I from angiotensinogen and release angiotensin II from angiotensin I and angiotensinogen. The relative order of potency in releasing angiotensin II by the three proteinases at equivalent concentrations is cathepsin G > elastase > proteinase 3. When all three proteinases are used together, the release of angiotensin II is greater than the sum of the release when each proteinase is used individually. Cathepsin G and elastase can also degrade angiotensin II, reactions which might be important in regulating the activity of angiotensin II. The release and degradation of angiotensin II by the neutrophil proteinases are reactions which could play a role in the local inflammatory response and wound healing.

Angiotensin I↗

PubMed2002

alpha(1)-Proteinase inhibitor mutants with specificity for plasma kallikrein and C1s but not C1.

Coagulation and complement proteinases are activated in sepsis, and one approach to therapy is to develop proteinase inhibitors that will specifically inhibit these proteinases without inhibiting activated protein C, a proteinase that is beneficial to survival. In this study, we made mutants of the serpin alpha(1)-PI, designed to mimic the specificity of C1-inhibitor. The P3-P2-P1 residues of alpha1-PI were changed from IPM to LGR and PFR, sequences preferred by C1s and kallikrein, respectively. Inhibition of C1s, kallikrein, factor XIIa, and activated protein C was assessed by SDS-PAGE, and by determination of the k(app) and SI. alpha(1)-PI-LGR inhibited C1s with a rate of 7790 M(-1)s(-1), but only minimal inhibition of C1 in a hemolytic assay was observed. Kallikrein, factor XIIa, and activated protein C were inhibited with rates of 382,180 M(-1)s(-1), 10,400 M(-1)s(-1), and 3500 M(-1)s(-1), respectively. alpha(1)-PI-PFR was a poor inhibitor of C1s, factor XIIa, and activated protein C, but had enhanced reactivity with kallikrein. Changing the P4' residue of alpha(1)-PI-LGR Pro to Glu reduced the activity with C1s, consistent with the idea that C1s requires hydrophobic residues in this region of the serpin for optimal interaction. The data provide insight into the requirements for kallikrein and C1s inhibition necessary for designing inhibitors with appropriate properties for further investigation as therapeutic agents.

Animals↗