Biomedical subjects

F C Church

Publications and source records attributed to F C Church.

At least 37 records · Page 2Linked to original sources

PubMed1995

Characterization of the glycosaminoglycan-binding region of lactoferrin.

Lactoferrin is a prominent component of neutrophil secondary granules and its blood concentration is increased in certain inflammatory diseases. Although the biochemical characterization of lactoferrin as an iron-binding protein has been well described, its physiological role in inflammation remains undefined. We examined the ability of lactoferrin to regulate glycosaminoglycan-accelerated thrombin-serine protease inhibitor (serpin) reactions. Lactoferrin effectively reduced the rate of thrombin-serpin (antithrombin and heparin cofactor II) reactions by three physiological glycosamino-glycans including heparin, heparan sulfate, and dermatan sulfate. An enzyme kinetics analysis showed that lactoferrin did not alter the apparent heparin-thrombin or the heparin-antithrombin dissociation constant values for the heparin-catalyzed thrombin-antithrombin reaction. However, the maximum reaction velocity at saturation with respect to either protein was markedly decreased by lactoferrin. The glycosaminoglycan-binding region of lactoferrin was analyzed following limited proteolysis using Staphylococcus aureus V8 protease. Two lactoferrin fragments with Mr's of approximately 8 and approximately 11 kDa were purified based on their affinity to heparin-Sepharose. Amino acid sequence analysis demonstrated that both peptides were from the N-terminus. Although slightly less capable compared to intact lactoferrin, the lactoferrin peptides effectively neutralized heparin, heparan sulfate, and dermatan sulfate-catalyzed serpin-thrombin inhibition reactions. In addition, lactoferrin N-terminal peptides have approximately the same binding affinity to heparin-Sepharose as that of intact lactoferrin. Inspection of both the N-terminal amino acid sequence and the crystal structure of lactoferrin further supports the conclusion that lactoferrin is a novel glycosaminoglycan binding protein and that the putative glycosaminoglycan-binding site is localized to the N-terminus.

Amino Acid Sequence↗

PubMed1995

Neutralization of heparin activity by neutrophil lactoferrin.

Lactoferrin is a prominent component of neutrophil secondary granules, and its blood concentration is increased in certain inflammatory diseases. In contrast to the well-described biochemical characterization of lactoferrin as an iron-binding protein, its physiologic role in the regulation of inflammation and other host defense mechanisms is unclear. In this report, we provide evidence that lactoferrin has a potent heparin-neutralizing activity during thrombin inhibition by the serine proteinase inhibitors (serpins) antithrombin and heparin co-factor II. Activated neutrophil supernatant, which contains lactoferrin and other heparin-binding proteins, could neutralize the heparin-dependent antithrombin-thrombin inhibition reaction. The addition of lactoferrin to plasma corrected the heparin-induced prolongation of blood plasma coagulation as measured by the activated partial thromboplastin time (aPTT). Treatment of whole blood with specific inflammatory mediators, fMLP, lipopolysaccharide (LPS), and tumor necrosis factor-alpha (TNF-alpha) increased the concentration of both plasma lactoferrin and platelet factor 4 while inhibiting the blood anticoagulant activity of heparin as measured by the aPTT. These results suggest that the prothrombotic sequelae of some inflammatory processes may be partly due to various agonists that release neutrophil lactoferrin, which can then neutralize glycosaminoglycan-dependent serpin-thrombin inhibition reactions.

Amino Acid Sequence↗

PubMed1995

Reactive site mutants of recombinant protein C inhibitor.

Protein C inhibitor (PCI) is a heparin-binding serine proteinase inhibitor (serpin) which is thought to be a physiological regulator of activated protein C (APC). The residues F353-R354-S355 (P2-P1-P1') constitute part of the reactive site loop of PCI with the R-S peptide bond being cleaved by the proteinase. Changing the reactive site P1 and P2 residues to those of either proteinase nexin-1, alpha 1-proteinase inhibitor or heparin cofactor II resulted in a decrease in inhibitory activity towards thrombin and APC. Changing the P2 residue F353-->P generated a rPCI which was a better thrombin inhibitor, but was 10-fold less active with APC. While these results support the concept that the P1 and P2 residues are important in the specificity of PCI, they suggest that the reactive site residues are not the only determinant of serpin specificity. Kinetic analysis of the rPCI variants was consistent with PCI operating by a mechanism similar to that proposed for other serpins. In this model an intermediary complex forms between inhibitor and proteinase that can proceed to either cleavage of the inhibitor as substrate or formation of an inactive complex.

Amino Acid Sequence↗

PubMed1995

Monocyte chemoattractant activity of Ser195-->Ala active site mutant recombinant alpha-thrombin.

alpha-Thrombin is chemotactic for human monocytes with optimal activity between 10-100 nM. The mechanism by which this response is mediated remains a point of controversy. The purpose of this study was to compare the chemotactic activity of proteolytically inactive thrombin (active site Ser195-->Ala mutant or Phe-Pro-Arg-chloromethyl ketone-inactivated thrombin) to thrombin and the "tethered ligand" thrombin receptor agonist peptide SFLLRN (single-letter amino acid code). Monocyte chemotaxis was compared to an optimal concentration (10 nM, considered to be 100%) of formyl-Met-Leu-Phe (fMLP). Proteolytically inactive thrombin (38% of fMLP) had similar chemotactic activity to active thrombin (46% of fMLP) at a concentration of 100 nM. Chemotaxis to SFLLRN was comparable to that of a control hexapeptide (FSLNLR) which is not an agonist for the tethered ligand thrombin receptor. Cross-desensitization experiments showed that pretreatment of monocytes with either mutant or active thrombin reduced subsequent chemotaxis to both thrombin chemotaxins. Pretreatment with SFLLRN did not decrease subsequent chemotaxis to either form of thrombin. Calcium flux measurements showed that both active thrombin and SFLLRN induced a rapid increase in monocyte and platelet intracellular calcium concentration. However, there was no intracellular calcium change in response to mutant thrombin or FSLNLR. Likewise, active thrombin and SFLLRN induced a rapid net increase in polymerized actin, but mutant thrombin and FSLNLR did not. By contrast, both active and mutant thrombin induced a polarization of monocyte morphology and actin distribution. This polarization has been associated with directed migration in many cell types. SFLLRN, however, induced a symmetrical increase in polymerized actin. These results suggest that measurements of intracellular calcium and polymerized actin are not perfect surrogate tests for true chemotactic activity. These results show that thrombin proteolysis is not required for monocyte chemotaxis and may be mediated by interaction with a binding site other than the tethered ligand thrombin receptor.

Actins↗

PubMed1995

Characterization of recombinant heparin cofactor II expressed in insect cells.

Recombinant human heparin cofactor II (rHCII) was expressed as a fully active protein in the High-Five insect cell line. A maximal protein concentration of 6 micrograms/10(6) cells was achieved 2 days postinfection. Approximately 40 micrograms of partially purified rHCII was routinely recovered from 50 ml of media after sequential heparin and Q-Sepharose affinity adsorption. rHCII had a slightly lower apparent molecular weight than blood plasma HCII (pHCII) due to differences in N-glycosylation. Like pHCII, rHCII formed a stable bimolecular complex with thrombin when assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The thrombin and chymotrypsin inhibitory properties of rHCII and pHCII were quite similar. In the absence of glycosaminoglycan, the thrombin inhibition rate (k2 x 10(-4) M-1 min-1) was 2.29 +/- 0.36 for rHCII and 3.38 +/- 0.34 for pHCII. Chymotrypsin inhibition rates (k2 x 10(-5) M-1 min-1) were 6.2 +/- 2.0 for rHCII and 8.0 +/- 2.6 for pHCII. In the presence of glycosaminoglycans, the maximal thrombin inhibition rate (k2 x 10(-3) M-1 min-1) for rHCII was 10.4 +/- 2.5 at 100 micrograms/ml heparin and 16.0 +/- 4.3 at 1000 micrograms/ml dermatan sulfate compared to 9.0 +/- 0.7 at 200 micrograms/ml heparin and 18.5 +/- 5.3 at 1000 micrograms/ml dermatan sulfate for pHCII. HCII inhibition of thrombin was blocked by a synthetic sulfated hirudin peptide in both the presence and the absence of glycosaminoglycan. The present report describes for the first time the expression and characterization of HCII in a baculovirus system and demonstrates the feasibility of using this system to obtain adequate amounts of biologically active rHCII for future structure-function studies.

Animals↗

PubMed1995

Engineering of five 88-residue receptor-adhesive modular proteins containing a parallel alpha-helical coiled coil and two RGD ligand sites.

Several 88-residue proteins were designed, synthesized and examined as receptor-adhesive modular proteins (RAMPs). Three covalent and two noncovalent dimers were made from two 44-residue peptide chains containing three structural modules: RGD-A23a (ligand-spacer-coil) and A9a-RGD (coil-spacer-ligand). The ligand module contained the tripeptide Arg-Gly-Asp (RGD). The coil modules A9a and A23a were five-heptad alpha-helices engineered by Hodges and co-workers [Int. J. Peptide Protein Res. (1992) 40, 171-179]. By circular dichroic spectroscopy, each of these five RAMPs contained an alpha-helical coiled coil. The disulfide-bridged dimer RGD-A23a/RGD-A23a and its reduced form (RGD-A23a)2 had two N-terminal RGD sites. The disulfide-bridged dimer A9a-RGD/A9a-RGD and its reduced form (A9a-RGD)2 had two C-terminal RGD sites. However, the disulfide-bridged heterodimer RGD-A23a/A9a-RGD had one RGD site at each terminus with a 50 Angstrum coiled coil between them. The temperature at the midpoint of unfolding for each of the covalent homodimers RGD-A23a/RGD-A23a (67 degrees C) and A9a-RGD/A9a-RGD (69 degrees C) was slightly higher than that of the corresponding noncovalent homodimer (RGD-A23a)2 (62 degrees C) or (A9a-RGD)2 (68 degrees C) but much lower than that of the covalent heterodimer RGD-A23a/A9a-RGD (79 degrees C). The enthalpy and entropy of thermal unfolding were also significantly greater for the heterodimer than for the four homodimers, consistent with the heterodimer having the most stable coiled coil. Although the distance between its RGD sites was at least 50 Angstrum greater than that for the homodimers, this heterodimeric RAMP was only as active as the homodimers A9a-RGD/A9a-RGD and (A9a-RGD)2 in inhibiting the adhesion of A2058 melanoma cells to extracellular matrix proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1995

Designed polyanionic coiled-coil proteins: acceleration of heparin cofactor II inhibition of thrombin.

Novel polyanionic proteins were designed to increase the rate of heparin cofactor II (HC) inhibition of alpha-thrombin, an essential protease in the coagulation cascade. Two alpha-helical coiled-coil proteins, a 62-residue dimer containing 8 Glu residues (E8C) and a 104-residue dimer containing 14 Glu residues (E14C), plus two 31-residue control peptides containing 8 Glu residues each (E8A and E8B), were chemically synthesized, structurally characterized and enzymatically assayed. Circular dichroic spectrophotometry indicated that both E8C and E14C formed stable two-chain alpha-helical coiled coils at pH 7 and 25 degrees C. The control peptides were only partially alpha-helical. E14C remained folded at 90 degrees C but E8C was half unfolded at 49 degrees C. Coiled-coil proteins E8C and E14C maximally accelerated by 35- and 33-fold, respectively, the rate of HC inhibition of alpha-thrombin. None of these compounds accelerated antithrombin inhibition of alpha-thrombin, and neither control peptide accelerated HC inhibition of alpha-thrombin. Acceleration of the HC inhibition of alpha-thrombin showed bimodal dependence on the concentration of the polyanionic protein, which is consistent with formation of a HC-coiled-coil-thrombin ternary complex. The results suggest that antithrombotic polyanionic alpha-helical coiled-coil proteins can be designed and synthesized and that the occurrence of secondary structure can be correlated with biological activity.

Amino Acid Sequence↗

PubMed1994

Role of the H helix in heparin binding to protein C inhibitor.

Protein C inhibitor (PCI) is a plasma serine proteinase inhibitor (serpin) that is a major physiological regulator of activated protein C. Inhibition of its target proteinase is accelerated by heparin in a reaction that involves the binding of both inhibitor and proteinase to heparin to form a ternary complex. This study was undertaken to understand the role of the H helix region (residues 264-278) of PCI in heparin binding and used (i) a recombinant truncated PCI fusion protein of the first 294 residues, (ii) H helix synthetic peptides containing single Arg/Lys-->Glu substitutions, and (iii) site-directed Ala mutagenesis of 4 basic residues (Arg-269, Lys-270, Lys-276, and Lys-277) in the H helix region of full-length recombinant PCI (rPCI) expressed in Baculovirus. The PCI fusion protein interfered in heparin-accelerated PCI-proteinase inhibition reactions, and it bound to heparin-Sepharose. Compared to the wild-type PCI fusion protein, deletion of the H helix from the fusion protein resulted in a reduction of both heparin-Sepharose binding and the ability to compete for heparin during PCI-proteinase inhibition reactions. Competition assays with H helix synthetic peptides revealed that the R269E altered peptide was the least effective at blocking heparin-catalyzed PCI-proteinase inhibition reactions. Compared with full-length active wild-type rPCI, R269A: K270A and K276A:K277A rPCI both had reduced heparin-Sepharose binding, but only R269A:K270A rPCI showed a loss of heparin-accelerated proteinase inhibition for both activated protein C and thrombin. We conclude that a major heparin-binding site of PCI is the H helix, unlike its heparin-binding serpin homologues antithrombin and heparin cofactor II, which bind heparin primarily through the D helix.

Amino Acid Sequence↗

PubMed1994

Mutagenesis of recombinant protein C inhibitor reactive site residues alters target proteinase specificity.

Protein C inhibitor (PCI) is a heparin-binding plasma serine proteinase inhibitor (serpin) which is thought to be a physiological regulator of activated protein C. We are using recombinant PCI (rPCI) to study structural determinants of target proteinase specificity. A cDNA encoding full-length PCI has been expressed as a fully active proteinase inhibitor using Autographa californica nuclear polyhedrosis virus (baculovirus). rPCI was expressed maximally 4 days after infection and could be expressed either in Sf9 or High-Five cells. rPCI bound heparin and was conveniently purified with heparin-Sepharose (eluting > 0.5 M NaCl). The rPCI formed sodium dodecyl sulfate-polyacrylamide gel electrophoresis-stable complexes with thrombin and activated protein C (APC). The inhibitory properties of wild-type rPCI and plasma-derived PCI are essentially the same either in the absence or presence of heparin with thrombin, APC, trypsin, and urokinase. The residues Phe353-Arg354-Ser355 (P2-P1-P1') constitute part of the reactive site loop of PCI with the Arg-Ser peptide bond being cleaved by the proteinase. Using site-directed mutagenesis we studied the contribution of the reactive site FRS for proteinase inhibition in rPCI. Changing the P1 residue Arg354-->Met generated a reactive site similar to alpha 1-proteinase inhibitor which was a much poorer inhibitor of thrombin, APC, trypsin, and urokinase. Changing the P2 residue Phe353-->Gly generated a mutant with a reactive site like antithrombin which was better at inhibiting thrombin or urokinase, but was much less active with APC or trypsin. Changing the P1' residue Ser355-->Met generated a reactive site like plasminogen activator inhibitor-1 and this protein inhibits all the proteinases essentially like wild-type rPCI. These results show the importance of PCI's Phe353 (P2) and Arg354 (P1) in target proteinase specificity, and they further support the concept of reactive site sequences determining serpin function.

Amino Acid Sequence↗

PubMed1994

Modulation of protein C inhibitor activity.

Protein C inhibitor (PCI), antithrombin, and heparin cofactor II are members of the serine proteinase inhibitor (serpin) superfamily that inhibit proteinases at rates which increase in the presence of the glycosaminoglycan heparin. These studies were undertaken to understand how PCI activity is modulated by various substances that are found in or interact with the vascular endothelium/basement membrane. The effects of antithrombin-heparin, thrombomodulin, vitronectin and leukocyte elastase on PCI-thrombin and PCI-activated protein C (APC) interactions were investigated. Antithrombin, which does not inhibit APC but which does bind to heparin/heparan sulphate with higher affinity than PCI, caused only a small decrease in the inhibition rate of PCI-APC in the presence of unfractionated heparin. Thrombomodulin, a chondroitin sulphate-containing proteoglycan, accelerated PCI inhibition of thrombin and APC. PCI-thrombin in the presence or absence of heparin bound plastic absorbed vitronectin, but neither PCI alone nor PCI-APC bound. Vitronectin also decreased the inhibition rate of PCI-thrombin and PCI-APC in the presence of low concentrations of heparin. Leukocyte elastase proteolytically inactivated PCI in a reaction that was accelerated by heparin. Overall, these results indicate that PCI activity is modulated by these endothelial cell/basement membrane-based substances in similar ways as other heparin-binding serpins, especially antithrombin.

Animals↗

PubMed1993

Interaction of heparin cofactor II with biglycan and decorin.

Two small interstitial dermatan sulfate-containing proteoglycans, biglycan and decorin, are present in extracellular matrices of skin, tendon, ligament, and cartilage. We investigated the effects of biglycan and decorin on the inhibition of alpha-thrombin by the serine proteinase inhibitor heparin cofactor II. In solution, heparin cofactor II inhibition of thrombin is accelerated by intact biglycan or decorin and by the dermatan sulfate-containing glycosaminoglycan (GAG) chains prepared from the proteoglycans, while core protein from cartilage biglycan had no effect. L-Iduronic acid-rich skin decorin and GAG chains had a greater accelerating effect than proteoglycan and GAG chains from cartilage that had lower L-iduronic acid content. Treatment of skin decorin and GAG chains with chondroitinase ABC totally eliminated the ability of these compounds to accelerate thrombin inhibition by heparin cofactor II suggesting that dermatan sulfate was responsible for this action. Both biglycan and decorin bound to type V collagen in a saturable and specific manner. Biglycan, decorin, and core protein from biglycan competed for decorin binding to the type V collagen, while only the intact proteoglycans competed for biglycan binding. When bound to type V collagen, both biglycan and decorin accelerated the heparin cofactor II/thrombin inhibition reaction as efficiently as the proteoglycans in solution. Our results demonstrate that heparin cofactor II in the presence of biglycan or decorin bound to type V collagen provides a "thromboresistant surface," further suggesting a physiological function for these proteins in regulating the extravascular activities of thrombin.

Animals↗

PubMed1993

Inhibition of dysthrombins Quick I and II by heparin cofactor II and antithrombin.

Heparin cofactor II and antithrombin are plasma serine proteinase inhibitors whose ability to inhibit alpha-thrombin is accelerated by glycosaminoglycans. Dysfunctional thrombin mutants Quick I (Arg67-->Cys) and Quick II (Gly226-->Val) were used to further compare heparin cofactor II and antithrombin interactions. Quick I, Quick II, and alpha-thrombin were eluted at the same salt concentration from heparin-Sepharose suggesting that the putative heparin-binding site (also termed anion binding exosite-II) is functional. Antithrombin yielded similar inhibition rates for Quick I and alpha-thrombin in the absence or presence of various amounts of heparin. Also, Quick I was inhibited similarly to alpha-thrombin by heparin cofactor II in the absence of glycosaminoglycan. In contrast, glycosaminoglycan-accelerated Quick I inhibition by heparin cofactor II was greatly reduced indicating that anion binding exosite-I (where the mutation occurs in Quick I) is critical for increased inhibition by heparin cofactor II. We also found that heparin cofactor II formed a SDS-resistant bimolecular complex with Quick II and alpha-thrombin at similar rates and the rate of complex formation was accelerated in the presence of glycosaminoglycans. A three-dimensional molecular model of the Quick II active site compared to alpha-thrombin suggested that the heparin cofactor II Leu-Ser-reactive site sequence (P1-P1') is a compatible "pseudosubstrate" in contrast to the Arg-Ser sequence found in antithrombin. The importance of heparin cofactor II as a thrombin regulator will depend upon its ability to interact with glycosaminoglycans and the functional availability of thrombin exosites.

Amino Acid Sequence↗

PubMed1993

Response of blood leukocytes to thrombin receptor peptides.

Thrombin has receptor-mediated effects on a variety of cell types. A recently cloned platelet thrombin receptor exerts its effects by a tethered-ligand mechanism. A similar receptor was shown in at least two nonplatelet cell types, fibroblasts and endothelial cells. Thrombin has biologically important effects on leukocytes, but the type of receptor mediating the effects is not known. Therefore, we examined the responses of monocytes, neutrophils, and lymphocytes to thrombin and to an agonist specific for the platelet-type thrombin receptor. We compared the effects of a peptide (SFLLRNPNDKYEPF) corresponding to residues 42-55 of the cloned platelet thrombin receptor on calcium flux in platelets and leukocytes. The thrombin receptor peptide induced increases in intracellular calcium in platelets and monocytes that reached a maximum at 5 microM peptide. The maximal increase was similar in magnitude to the response to thrombin. Lymphocytes showed a small and variable increase in intracellular calcium in response to thrombin or the thrombin receptor agonist. The thrombin receptor peptide had no effect on neutrophil calcium concentrations. When the amino acid corresponding to Arg 46 was replaced with Ala in the synthetic peptide, the ability to increase intracellular calcium was abolished for both platelets and monocytes. The peptide instead had thrombin antagonist activity. Thus, monocytes respond to thrombin receptor peptides similarly to platelets. We conclude that human monocytes possess a thrombin receptor similar to that present on platelets. Furthermore, the residue corresponding to Arg 46 of the thrombin receptor is critical for receptor agonist activity.

Actins↗

PubMed1993

Interaction of thrombin with antithrombin, heparin cofactor II, and protein C inhibitor.

alpha-Thrombin is a trypsin-like serine proteinase involved in blood coagulation and wound repair processes. Thrombin interacts with many macromolecular substrates, cofactors, cell-surface receptors, and blood plasma inhibitors. The three-dimensional structure of human alpha-thrombin shows multiple surface "exosites" for interactions with these macromolecules. We used these coordinates to probe the interaction of thrombin's active site and two exosites, anion-binding exosite-I and -II, with the blood plasma serine proteinase inhibitors (serpins) antithrombin (AT), heparin cofactor II (HC), and protein C inhibitor (PCI). Heparin, a widely used anticoagulant drug, accelerates the rate of thrombin inhibition by AT, PCI, and HC. Thrombin Quick II is a dysfunctional thrombin mutant with a Gly 226-->Val substitution in the substrate specificity pocket. We found that thrombin Quick II was inhibited by HC, but not by AT or PCI. Molecular modeling studies suggest that the larger Val side chain protrudes into the specificity pocket, allowing room for the smaller P1 side chain of HC (Leu) but not the larger P1 side chain of AT and PCI (both with Arg). gamma T-Thrombin and thrombin Quick I (Arg 67-->Cys) are both altered in anion-binding exosite-I, yet bind to heparin-Sepharose and can be inhibited by AT, HC, and PCI in an essentially normal manner in the absence of heparin. In the presence of heparin, inhibition of these altered thrombins by HC is greatly reduced compared to both AT and PCI. alpha-Thrombin with chemically modified lysines in both anion-binding exosite-I and -II has no heparin accelerated thrombin inhibition by either AT or HC. Thrombin lysine-modified in the presence of heparin has protected residues in anion-binding exosite-II and the loss of heparin-accelerated inhibition by HC is greater than that by AT. Collectively, these results suggest differences in serpin reactive site recognition by thrombin and a more complicated mechanism for heparin-accelerated inhibition by HC compared to either AT or PCI.

Allosteric Site↗

PubMed1993

General features of the heparin-binding serpins antithrombin, heparin cofactor II and protein C inhibitor.

The blood plasma serine proteinase inhibitors (serpins) are glycoproteins whose activities are involved in many important homeostatic reactions. The heparin-dependent plasma serpins, antithrombin, heparin cofactor II and protein C inhibitor, regulate the proteinases of blood coagulation. Heparin and some other glycosaminoglycans increase the rate of proteinase inhibition by these three plasma serpins. Proteinases recognize a specific peptide, termed the reactive site, near the carboxyl-terminus of serpins (for antithrombin and protein C inhibitor this is Arg-Ser and for heparin cofactor II this is Leu-Ser). Additionally, these three serpins contain unique structural elements that confer glycosaminoglycan binding activities. The therapeutic anticoagulant action of the glycosaminoglycan heparin is believed to depend partially on heparin-accelerated inhibition of proteinases by antithrombin. The physiological importance of specific proteoglycans has been attributed to their recognition of these serpins and their biological 'activation' of these proteinase inhibitors.

Amino Acid Sequence↗

PubMed1992

Heparin binding to protein C inhibitor.

Protein C inhibitor is a plasma protein whose ability to inhibit activated protein C, thrombin, and other enzymes is stimulated by heparin. These studies were undertaken to further understand how heparin binds to protein C inhibitor and how it accelerates proteinase inhibition. The region of protein C inhibitor from residues 264-283 was identified as the heparin-binding site. This differs from the putative heparin-binding site in the related proteins antithrombin and heparin cofactor. The glycosaminoglycan specificity of protein C inhibitor was relatively broad, including heparin and heparan sulfate, but not dermatan sulfate. Non-sulfated and non-carboxylated polyanions also enhanced proteinase inhibition by protein C inhibitor. Heparin accelerated inhibition of alpha-thrombin, gamma T-thrombin, activated protein C, factor Xa, urokinase, and chymotrypsin, but not plasma kallikrein. The ability of glycosaminoglycans to accelerate proteinase inhibition appeared to depend on the formation of a ternary complex of inhibitor, proteinase, and glycosaminoglycan. The optimum heparin concentration for maximal rate stimulation varied from 10 to 100 micrograms/ml and was related to the apparent affinity of the proteinase for heparin. There was no obvious relationship between heparin affinity and maximum inhibition rate or degree of rate enhancement. The affinity of the resultant protein C inhibitor-proteinase complex was also not related to inhibition rate enhancement, and the results showed that decreased heparin affinity of the complex is not an important part of the catalytic mechanism of heparin. The importance of protein C inhibitor as a regulator of the protein C system may depend on the relatively large increase in heparin-enhanced inhibition rate for activated protein C compared to other proteinases.

Amino Acid Sequence↗

PubMed1992

A comparison of three heparin-binding serine proteinase inhibitors.

The purpose of this study was to compare three heparin-binding plasma proteinase inhibitors in order to identify common and unique features of heparin binding and heparin-enhanced proteinase inhibition. Experiments with antithrombin, heparin cofactor, and protein C inhibitor were performed under identical conditions in order to facilitate comparisons. Synthetic peptides corresponding to the putative heparin binding regions of antithrombin, heparin cofactor, and protein C inhibitor bound to heparin directly and interfered in heparin-enhanced proteinase inhibition assays. All three inhibitors obeyed a ternary complex mechanism for heparin-enhanced thrombin inhibition, and the optimum heparin concentration was related to the apparent heparin affinity of the inhibitor. The maximum inhibition rate and rate enhancement due to heparin appeared to be unique properties of each inhibitor. In assays with heparin oligosaccharides of known size, only the antithrombin-thrombin reaction exhibited a sharp threshold for rate enhancement at 14-16 saccharide units. Acceleration of antithrombin inhibition of factor Xa, heparin cofactor inhibition of thrombin, and protein C inhibitor inhibition of thrombin, activated protein C, and factor Xa did not require a minimum saccharide size. The differences in heparin size dependence and rate enhancement of proteinase inhibition by these inhibitors might reflect differences in the importance of the ternary complex mechanism and other mechanisms, alterations in inhibitor reactivity, and orientation effects in heparin-enhanced proteinase inhibition.

Amino Acid Sequence↗

PubMed1992

Role of thrombin exosites in inhibition by heparin cofactor II.

We determined the role of specific thrombin "exosites" in the mechanism of inhibition by the plasma serine proteinase inhibitors heparin cofactor II (HC) and antithrombin (AT) in the absence and presence of a glycosaminoglycan by comparing the inhibition of alpha-thrombin to epsilon- and gamma T-thrombin (produced by partial proteolysis of alpha-thrombin by elastase and trypsin, respectively). All of the thrombin derivatives were inhibited in a similar manner by AT, either in the absence or presence of heparin, which confirmed the integrity of both heparin binding abilities and serpin reactivities of epsilon- and gamma T-thrombin compared to alpha-thrombin. Antithrombin activities of HC in the absence of a glycosaminoglycan with alpha-, epsilon, and gamma T-thrombin were similar with rate constants of 3.5, 2.4, and 1.2 x 10(4) M-1 min-1, respectively. Interestingly, in the presence of glycosaminoglycans the maximal inhibition rate constants by HC with heparin and dermatan sulfate, respectively, were as follows: 30.0 x 10(7) and 60.5 x 10(7) for alpha-thrombin, 14.6 x 10(7) and 24.3 x 10(7) for epsilon-thrombin, and 0.017 x 10(7) and 0.034 x 10(7) M-1 min-1 for gamma T-thrombin. A hirudin carboxyl-terminal peptide, which binds to anion-binding exosite-I of alpha-thrombin, dramatically reduced alpha-thrombin inhibition by HC in the presence of heparin but not in its absence. We analyzed our results in relation to the recently determined x-ray structure of D-Phe-Pro-Arg-chloromethyl ketone-alpha-thrombin (Bode, W., Mayr, I., Baumann, U., Huber, R., Stone, S. R., and Hofsteenge, J. (1989) EMBO J. 8, 3467-3475). Our results suggest that the beta-loop region of anion-binding exosite-I in alpha-thrombin, which is not present in gamma T-thrombin, is essential for the rapid inhibition reaction by HC in the presence of a glycosaminoglycan. Therefore, alpha-thrombin and its derivatives would be recognized and inhibited differently by HC and AT in the presence of a glycosaminoglycan.

Amino Acid Chloromethyl Ketones↗