Plasma Serine Protease Inhibitor Research Articles

Interactions of α1–proteinase inhibitor with small ligands of therapeutic potential: binding with retinoic acid

Human alpha(1)-proteinase inhibitor (alpha(1)-PI), also known as alpha(1)-antitrypsin, is the most abundant plasma serine protease inhibitor (serpin). It is best recognized for inhibition of neutrophil elastase. The alpha(1)-PI interactions with non-protease ligands were investigated mainly in regards to those molecules that may block the aggregation of alpha(1)-PI Z mutant. The objective of this study was to evaluate the potential of alpha(1)-PI to bind small non-peptide ligands of pharmaceutical interest that may attain additional properties to currently available alpha(1)-PI therapeutic preparations. Among putative ligands of bio-medical interest examined in this study, all-trans retinoic acid (RA) was selected due to its recently proposed roles in the lungs, and as an efficient optical probe. The results of this study, including absorption spectroscopy data, fluorescence quenching and the protein-induced chirality of the visible circular dichroism strongly suggest that alpha(1)-PI does bind RA in vitro to non-covalent complexes of up to two moles of RA per one mole of the protein. To our knowledge, this is the first report that provides experimental evidence of the alpha(1)-PI potential towards bi-functional drugs via a combination with RA, or potentially other molecules of pharmaceutical interest, that ultimately, may enhance currently available alpha(1)-PI therapies.

Effects of emersion and re-immersion on physiological and immunological variables in creel-caught and trawled Norway lobsterNephrops norvegicus

Immune defence in creel-caught and trawled Nephrops norvegicus was investigated to assess a possible relationship between phenoloxidase (PO) activation and the total haemocyte count (THC). Capture, capture method and emersion evoked physiological and immunological responses that may have implications for the ability of N. norvegicus to survive the effects of such stressors. Haemolymph THC was always negatively related to PO activity in the trawled samples, suggesting a decreased level of the plasma serine proteinase inhibitors which reportedly regulate the ProPO system (Le Moullac et al. 1998; Fish shellfish Immunol 8:621-629). In contrast, creel-caught samples showed increased levels of both PO and THC (cf. control N. norvegicus), after a 12 h emersion period. Trawling and emersion evoked progressive and significant increases (p < 0.05) in the mean levels of haemolymph L-lactate, glucose and total ammonia. The evidence of overt activity and measured haemolymph parameters suggest that creel fishing yields N. norvegicus that are more likely to survive post-harvest treatments than those that are trawled.

Alpha-1 Antitrypsin Genotypes in Breast Cancer Patients

Proteolytic enzymes play a significant role in malignancy including, loss of growth regulation, invasiveness and metastases formation. Alpha-1 antitrypsin (A1AT) is a secretory glycoprotein produced mainly in liver and monocytes. It is the most abundant serine protease inhibitor in human plasma. Deficiency of A1AT is an inherited disorder characterized by reduced serum level of A1AT. Protease inhibitor Z (PiZ) and protease inhibitor S (PiS) are the most common deficient genotypes of A1AT. The association of deficient A1AT subtypes with several tumors such as primary liver carcinoma, lung cancer, bladder cancer and malignant hepatoma was reported. This study was aimed to test the relationship between A1AT genotypes Z and S and breast cancer in Jordanian female patients. Blood samples were collected from 111 patients. DNA was isolated and polymerase chain reaction (PCR) was performed to amplify the regions contain the Z and S mutations in exon V, and III, respectively. Genotyping of Z and S alleles was performed by restriction fragment length polymorphism technique using Taq1 restriction enzyme. Our results demonstrated that 100% of the breast cancer patients were homozygous for the normal allele Protease inhibitor MM (PiMM) and no PiZ and PiS genotypes were found. In conclusion, there is no relationship between A1AT deficient genotypes Z and S and breast cancer in Jordanian female patients.

New therapies for hereditary angioedema: Disease outlook changes dramatically

Hereditary angioedema (HAE) is an autosomal dominant disease associated with episodic attacks of nonpitting edema that may affect any external or mucosal body surface. Attacks most often affect the extremities, causing local swelling, the GI tract, leading to severe abdominal pain, and the mouth and throat, at times causing asphyxiation. Most patients with HAE have low levels of the plasma serine protease inhibitor C1 inhibitor. The edema in these patients is caused by unregulated generation of bradykinin. Effective chronic therapy of patients with impeded androgens or plasmin inhibitors has been available for decades, but in the United States, we do not have therapy for acute attacks. Five companies have completed or are in the process of conducting phase 3 clinical trials, double-blind, placebo-controlled studies of products designed to terminate acute attacks or to be used in prophylaxis. Two companies, Lev Pharmaceuticals and CSL Behring, have preparations of C1 inhibitor purified from plasma that have been used in Europe for decades (trade names Cinryze and Berinert P, respectively). One company, Pharming, has developed a recombinant C1 inhibitor preparation. One company, Dyax, is testing a kallikrein inhibitor (ecallantide), and one company, Jerini, is completing testing of a bradykinin type 2 receptor antagonist (Icatibant). Although little has been published thus far, all of these products may prove effective. It is likely that HAE treatment will change dramatically within the next few years.

Inhibition of Factor VIIa-Tissue Factor by Protein C Inhibitor.

Protein C inhibitor (PCI) is a plasma serine protease inhibitor (serpin) that inhibits several proteases in blood coagulation including thrombin, thrombin-thromobomodulin and activated protein C. Although PCI has been shown to inhibit these key blood coagulation proteases, a clearly defined hemostatic role for PCI remains to be elucidated. Preliminary results from our laboratory show that exogenously added PCI prolongs venous thrombus formation upon blood vessel injury in a murine model of vascular injury. These results suggest that PCI may be responsible for inhibiting other proteases in blood coagulation, possibly including factor VIIa. Blood coagulation is initiated when factor VIIa binds to TF on cell surfaces. We investigated the ability of PCI to inhibit factor VIIa, both in the presence and absence of TF in vitro, and in a cellular environment. It has been shown previously and we have confirmed that the serpin antithrombin (AT) inhibits both factor VIIa and factor VIIa/TF; however, the ability of PCI to inhibit this protease has not been fully investigated. PCI and AT inhibit factor VIIa in the presence of TF and heparin (AT: k2=1.1 × 104 M−1 s−1; PCI: k2=7.2 × 103 M−1s−1). In the absence of heparin the inhibition of factor VIIa/TF by PCI is reduced 10-fold. Interestingly, PCI is unable to inhibit VIIa in the absence of TF with or without heparin. Furthermore, both PCI and AT form SDS-stable complexes with factor VIIa (in the presence of TF). We also investigated whether PCI is able to inhibit VIIa-mediated cell migration of the breast cancer cell line MDA-MB-231 that constitutively expresses TF. AT and PCI are able to inhibit cell surface derived factor VIIa/TF. Interestingly, the rate of factor VIIa inhibition on MDA-MB-231 cells is 1.2 and 0.4 × 103 M−1s−1 for PCI and AT, respectively. The factor VIIa-mediated cell migration of MD-MB-231 cells is attenuated in the presence of PCI, but not in the presence of the PCI inactive mutant (T341R). Given that both AT and PCI are able to inhibit factor VIIa/TF both in vitro and in a cellular environment, suggests that more potent derivatives (prepared by site-directed mutagenesis) of these serpins may be useful alternatives as novel therapeutics anticoagulants that target the initial step in the coagulation pathway.

Longitudinal studies of inter-alpha inhibitor proteins in severely septic patients: A potential clinical marker and mediator of severe sepsis*

To determine the clinical relevance and prognostic significance of serial measurement of inter-alpha inhibitor proteins (IalphaIp) in severely septic patients. A laboratory-based study of serial plasma samples over the first 5 days of severe sepsis from a prospective clinical trial. Small business and academic medical center research laboratories. Two hundred sixty-six patients with severe sepsis from a multiple-center phase III clinical trial. None. Inter-alpha inhibitor proteins serve as endogenous serine protease inhibitors in human plasma. The levels of IalphaIp were markedly reduced to a mean value of 290+/-15 microg/mL at the onset of severe sepsis compared with normal plasma levels (617+/-197 microg/mL). Failure of IalphaIp levels to recover over the first 5 days of sepsis was associated with an unfavorable outcome (p<.001). IalphaIp levels were inversely correlated with interleukin-6 levels at study entry and over the first 5 days of management of severe sepsis. IalphaIp levels were significantly lower in women, with increased age, in the presence of multiple organ failure and in patients with intra-abdominal sources of sepsis. Inter-alpha inhibitor proteins are markedly reduced in severe sepsis, and failure of recovery of IalphaIp levels over the course of sepsis is associated with an unfavorable outcome.

Development and evaluation of an ELISA for quantification of human alpha-1-proteinase inhibitor in complex biological mixtures

Human alpha-1-proteinase inhibitor 1 Also known as antitrypsin. 1 (α 1-PI) is the most abundant serine protease inhibitor in plasma. Its major function is inhibition of neutrophil elastase in lungs. α 1-PI deficiency may result in severe, ultimately fatal emphysema. Three plasma-derived (pd-) α 1-PI products are licensed in the US for replacement therapy of deficient patients. The recombinant versions (r-α 1-PI), proposed as alternatives to pd-α 1-PI products, have been under intensive investigation. For accurate determination of α 1-PI from different sources and in various forms, there is an obvious need for reliable standardized assays for α 1-PI quantification and potency measurements. As a part of our multi-step research focused on α 1-PI structure-function investigation, we have established a simple and reproducible double-sandwich ELISA based on commercially available polyclonal antibodies. The developed ELISA allows the quantification of both pd-α 1-PI and r-α 1-PI in various complex matrices. A validation of the ELISA was performed with the working range of the assay (3.1–50 ng/ml) established on the bases of the following parameters: linearity (3–100 ng/ml, r 2 = 0.995); accuracy (87.3–114.6% recovery); intra-assay precision (%CV, 2.8%); inter-assay plate-to-plate precision (3.9% per day and 4.1% day-to-day); detection limit (1.10 ng/ml); and quantification limit (3.34 ng/ml). The analytical performance of the α 1-PI ELISA indicates that this assay can be used for monitoring concentration levels of α 1-PI in multi-component biological matrices, based on the following: (a) quantification of r -α 1-PI in various fermentation mixtures ( E. coli and A. niger); (b) investigation of α 1-PI enzymatically digested in the conditions of harsh fungal proteolysis; (c) evaluation of thermally polymerized α 1-PI; (d) quantification of α 1-PI in human serum; and (e) comparative quantification of α 1-PI in commercially available products.

Expression of human α1-proteinase inhibitor in Aspergillus niger

BackgroundHuman α1-proteinase inhibitor (α1-PI), also known as antitrypsin, is the most abundant serine protease inhibitor (serpin) in plasma. Its deficiency is associated with development of progressive, ultimately fatal emphysema. Currently in the United States, α1-PI is available for replacement therapy as an FDA licensed plasma-derived (pd) product. However, the plasma source itself is limited; moreover, even with efficient viral inactivation steps used in manufacture of plasma products, the risk of contamination from emerging viruses may still exist. Therefore, recombinant α1-PI (r-α1-PI) could provide an attractive alternative. Although r-α1-PI has been produced in several hosts, protein stability in vitro and rapid clearance from the circulation have been major issues, primarily due to absent or altered glycosylation.ResultsWe have explored the possibility of expressing the gene for human α1-PI in the filamentous fungus Aspergillus niger (A. niger), a system reported to be capable of providing more "mammalian-like" glycosylation patterns to secretable proteins than commonly used yeast hosts. Our expression strategy was based on fusion of α1-PI with a strongly expressed, secreted leader protein (glucoamylase G2), separated by dibasic processing site (N-V-I-S-K-R) that provides in vivo cleavage. SDS-PAGE, Western blot, ELISA, and α1-PI activity assays enabled us to select the transformant(s) secreting a biologically active glycosylated r-α1-PI with yields of up to 12 mg/L. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis further confirmed that molecular mass of the r-α1-PI was similar to that of the pd-α1-PI. In vitro stability of the r-α1-PI from A. niger was tested in comparison with pd-α1-PI reference and non-glycosylated human r-α1-PI from E. coli.ConclusionWe examined the suitability of the filamentous fungus A. niger for the expression of the human gene for α1-PI, a medium size glycoprotein of high therapeutic value. The heterologous expression of the human gene for α1-PI in A. niger was successfully achieved to produce the secreted mature human r-α1-PI in A. niger as a biologically active glycosylated protein with improved stability and with yields of up to 12 mg/L in shake-flask growth.

Regulation of Cathpsin L by the Serpin Protein C Inhibitor.

Protein C inhibitor (PCI) is a plasma serine protease inhibitor (serpin) that regulates several serine proteases in coagulation and fibrinolysis including thrombin and activated protein C (APC). The physiological role of PCI, however, remains under investigation since PCI both inhibits and promotes thrombin generation. The cysteine protease, cathepsin L, has been shown to play a role in many physiological processes including cardiovascular disease, atherosclerosis, blood vessel remodeling, and tumor cell invasion. Recently, several serpins have been described to inhibit both serine and cysteine proteases and they are termed “cross-class” inhibitors. The goal of this project was to determine if PCI inhibits cathepsin L activity and if so, does this inhibition process mimic the mechanism of serine proteases. Previous studies have shown that the prototypical serpin, antithrombin (AT), inhibits the cysteine proteases papain and cathepsin L. We found that PCI is a more efficient inhibitor of cathepsin L than AT with an inhibition rate (k2) of 1.5 × 106 M−1min−1 compared to 5.2 × 104 M−1min−1 for AT. Also, PCI is a more efficient inhibitor of cathepsin L than either thrombin or APC whose inhibition rates are 5.7 × 105 M−1min−1 and 3.4 × 104 M−1min−1, respectively. In contrast to AT, PCI does not inhibit papain. Thrombin inhibition by AT and PCI is accelerated in the presence of glycosaminoglycans such as heparin and heparan sulfate. The inhibition of cathepsin L by PCI is not accelerated in the presence of heparin suggesting either that cathepsin L does not bind heparin or that heparin is not required for accelerated inhibition of cysteine proteases. Interestingly, a reactive site P1 mutant (R354A) of PCI does not inhibit thrombin but does inhibit cathepsin L at rates comparable to wild-type PCI. This implies that the P1 residue of PCI does not determine specificity for inhibition of cathepsin L unlike for thrombin and APC. We believe that the specificity is primarily determined by the hydrophobic Phe residue located at the P2 position since other serpins that inhibit cathepsin L contain either a Phe or Val at the P2 position. Mutating the P14 residue (T341R; mutation in the hinge region) of PCI results in the conversion of PCI from an inhibitor to a substrate. As expected, the PCI-P14 mutant does not inhibit either thrombin or cathepsin L. Another characteristic of the serpin inhibition mechanism is formation of a bi-molecular SDS stable complex. We found that wild-type PCI and PCI-P1 mutant both form a stable complex with cathepsin L under non-reducing conditions. Lastly, the wild-type PCI-cathepsin L interaction has a stoichiometry of inhibition (SI) value of 1.6. This indicates that PCI is an effective and possibly a physiologically relevant inhibitor of cathepsin L. Regulating cathepsin L by serpins like PCI may be a novel and new pathway of regulation of hemostasis-thrombosis, cardiovascular and metastatic diseases.

Association of Moderate Alpha-1 Antitrypsin Deficiency with Lung Cancer in the Serbian Population

Alpha-1 antitrypsin (AAT) is an important serine protease inhibitor in human plasma. Its major physiological role is to inhibit neutrophil elastase (NE) in the lower respiratory tract and protect lung tissue from destruction. Recent studies indicated an etiological role of NE in lung cancer development. The aim of this study was to investigate the association of alpha-1 antitrypsin deficiency (AATD) with lung cancer in patients with four different histological types of cancer: squamous cell carcinoma, adenocarcinomas, large cell carcinoma and small cell carcinoma. Phenotyping was carried out by isoelectric focusing (pH 4.2-4.9). We compared the frequency of AATD phenotypes in 186 lung cancer patients with the value obtained in our previous study in a healthy Serbian population (3.7%) using the Fisher exact test. Allele frequencies in patients were Pi *M 0.9677, Pi *Z 0.0215, Pi *S 0.0081 and Pi *other rare 0.0027. Eleven of the 186 lung cancer patients (5.9%) were AATD heterozygotes with moderate deficiencies (PiMZ and PiMS). When this value was compared with AATD heterozygote frequency obtained in the healthy individuals (3.7%), the difference was close to the level of significance (p = 0.055). However, individuals with AATD phenotypes had a higher risk of developing squamous cell lung cancer then those with non-deficient AAT variants (OR = 4.51, 95% CI = 1.66-12.29). Our findings provide evidence of an association between AAT phenotypes with moderate deficiency and squamous cell lung cancer.

Protein C Inhibitor Expression by Adult Rat Sertoli Cells: Effects of Testosterone Withdrawal and Replacement

Protein C inhibitor (PCI), a member of the plasma serine protease inhibitor family, has been reported to be abundantly expressed in the seminal vesicles and testes. In this study, we examine the localization and regulation of the PCI gene and protein expression in testes and freshly isolated Sertoli cells from control rats, rats treated with luteinizing hormone-suppressive testosterone/estradiol (TE)-containing Silastic capsules for 7, 14, 28, and 56 days, and rats treated with TE for 56 days, followed by high levels of testosterone for 7 or 14 days. The administration of the TE capsules for 56 days resulted in reduced testicular testosterone, from approximately 100 ng/mL in the controls to approximately 10 ng/mL, accompanied by a 73% reduction in testicular weight. PCI mRNA levels in freshly isolated Sertoli cells were reduced by 30% and 54% following TE treatment for 28 and 56 days, respectively. When rats that had received TE capsules for 56 days were provided replacement testosterone, there was a 40% increase in PCI mRNA levels within 7 days in the absence of any change in testicular weight, and PCI mRNA levels returned to control values by 14 days. The decrease in PCI mRNA levels in TE-treated rats was paralleled by a decrease in PCI protein levels in whole testis lysates and in seminiferous tubule fluid (STF). Protease activity was significantly increased in STF following 56 days of TE treatment. Taken together, these results indicate that 1) PCI in the testis is expressed by Sertoli cells; 2) the testicular expression of PCI is responsive to intratesticular testosterone levels; and 3) protease activity within the seminiferous epithelium is elevated when intratesticular concentration is decreased, perhaps as a consequence of decreased PCI.

Inter-Alpha-Inhibitor Proteins Are Endogenous Furin Inhibitors and Provide Protection against Experimental Anthrax Intoxication

Inter-alpha-inhibitor protein (IalphaIp) functions as an endogenous serine protease inhibitor in human plasma, and IalphaIp levels diminish rapidly during acute inflammatory states. One potential target for IalphaIp is furin, a cell-associated serine endopeptidase essential for the activation of protective antigen and the formation of anthrax lethal toxin (LT). IalphaIp blocks furin activity in vitro and provides significant protection against cytotoxicity for murine peritoneal macrophages exposed to up to 500 ng/ml LT. A monoclonal antibody (MAb), 69.31, that specifically blocks the enzymatic activity of IalphaIp eliminates its protective effect against LT-induced cytotoxicity. IalphaIp (30 mg/kg of body weight) administered to BALB/c mice 1 hour prior to an intravenous LT challenge resulted in 71% survival after 7 days compared with no survivors among the control animals (P < 0.001). We conclude that human IalphaIp may be an effective preventative or therapeutic agent against anthrax intoxication.

Aggrecanase-1 (ADAMTS-4) interacts with α1-antitrypsin

In degradative articular diseases such as rheumatoid arthritis and osteoarthritis, loss of the extracellular matrix occurs, resulting in the destruction of joint cartilage. Proteolysis of aggrecan is one of the early events that leads to breakdown of the extracellular matrix. Aggrecanase-1 (ADAMTS--4) is considered to play a pivotal role in the abrasion of cartilage aggrecan in rheumatoid arthritis and osteoarthritis. To identify an endogenous inhibitor of aggrecanase-1, we performed a yeast two-hybrid screen using the catalytic domain of human aggrecanase-1 as a bait and transformed an EGY 48 yeast strain carrying the bait plasmid with a human liver cDNA library plasmid. This screen identified alpha1-antitrypsin, a member of the family of plasma serine proteinase inhibitors, as a prey. Recombinant aggrecanase-1 and alpha1-antitrypsin were expressed in mammalian cells and used in co-immunoprecipitation experiments, which showed that full-length aggrecanase-1 and alpha1-antitrypsin are also associated in vivo. However, aggrecanase-1 did not interfere with the inhibitory activity of alpha1-antitrypsin against elastase, and alpha1-antitrypsin had no effect on the proteolytic activity of aggrecanase-1. Taken together, these data suggest that aggrecanase-1 and alpha1-antitrypsin bind in vivo, although the physiological significance of the interaction between aggrecanase-1 and alpha1-antitrypsin remains unclear.

Alpha-1 antitrypsin genotypes in breast cancer patients

Alpha-1 antitrypsin (1-AT) is a secretory glycoprotein mainly produced in the liver and monocytes. It is the most abundant serine protease inhibitor in human plasma. Proteolytic enzymes play a significant role in the expression of the malignant phenotype, including the loss of growth regulation, invasiveness and formation of metastases. Deficiency of 1-AT is an inherited disorder characterized by reduced serum level of 1-AT. Protease inhibitors Z (PiZ) and protease inhibitors S (PiS) are the most common deficient genotypes of 1-AT. The association of deficient 1-AT subtypes with several tumors such as primary liver carcinoma, lung cancer, bladder cancer and malignant hepatoma was reported. This study aimed to determine the incidence of 1-AT genotypes (PiZ and PiS) in breast cancer female patients. Blood samples were collected from 111 patients. DNA was isolated and the PCR technique was performed to amplify the regions contain the Z and S mutations in exon V and exon III, respectively. Genotyping of the Z and S alleles was performed by restriction fragment length polymorphism analysis using the Taq1 restriction enzyme. Our results demonstrated that 100% of the breast cancer patients were homozygous for the normal allele (PiMM) and no PiZ and PiS genotypes were found.

Oxalate-inducible AMBP gene and its regulatory mechanism in renal tubular epithelial cells

The AMBP [A1M (alpha1-microglobulin)/bikunin precursor] gene encodes two plasma glycoproteins: A1M, an immunosuppressive lipocalin, and bikunin, a member of plasma serine proteinase inhibitor family with prototypical Kunitz-type domain. Although previously believed to be constitutively expressed exclusively in liver, the present study demonstrates the induction of this gene by oxalate in porcine proximal tubular LLC-PK1 cells and rat kidney. In liver, the precursor protein is cleaved in the Golgi network by a furin-like enzyme to release constituent proteins, which undergo glycosylation before their export from the cell. In the renal tubular cells, A1M and bikunin co-precipitate, indicating lack of cleavage of the precursor protein. As the expression of the AMBP gene is regulated by A1M-specific cis elements and transcription factors, A1M protein was studied as a representative of AMBP gene expression in renal cells. Oxalate treatment (500 microM) resulted in a time- and dose-dependent induction of A1M protein in LLC-PK1 cells. Of the four transcription factors, HNF-4 (hepatocyte nuclear factor-4) has been reported previously to be a major regulator of AMBP gene expression in liver. Electrophoretic mobility-shift assay, supershift assay, immunoreactivity assay and transfection-based studies showed the presence of an HNF-4 or an HNF-4-like protein in the kidney, which can affect the expression of the AMBP gene. In situ hybridization and immunocytochemical studies showed that the expression of this gene in kidney was mainly restricted to cells lining the renal tubular system.

Haplotypes of the alpha-1 antitrypsin gene in healthy controls and Z deficiency patients

Alpha-1 antitrypsin (AAT; HUGO symbol, SERPINA1) is one of the major serine protease inhibitors (serpins) in human plasma. Deficiency of AAT is a recognised risk factor for chronic obstructive pulmonary disease (COPD), attributed to uninhibited neutrophil elastase released into the lung tissue during inflammatory states. In this study we used sequencing to screen the exonic regions, 5' and 3' flanking sequence of the AAT gene in order to generate a high density map of single nucleotide polymorphisms (SNPs). 16 SNPs were identified throughout AAT. Haplotypes based on SNPs with a minor allele frequency of > or =5% were estimated using genotypic information from 225 healthy control individuals and 41 AAT deficient Pi-ZZ individuals. AAT shows a large amount of variation in the control population, with 17 haplotypes accounting for 88% of the observed variation. The haplotype distribution of the common deficiency Pi-Z variant of AAT was significantly different when compared to the normal variants. In addition to the haplotype information, we present evidence for a functional effect of a SNP in intron 1.

Genetic diversity of the alpha-1-antitrypsin gene in Africans identified using a novel genotyping assay.

The highly polymorphic human alpha-1-antitrypsin (AAT) gene, more recently named SERPINA1, codes for the most abundant circulating plasma serine protease inhibitor, protease inhibitor 1 (PI). Most studies determining AAT haplotype frequencies have been restricted first by the limited accuracy of the phenotypic method used and secondly by the analysis of predominantly Caucasian populations. Limited studies have been performed on African-based populations. Here a new comprehensive assay for genotyping the entire coding region, including splice junctions, of the AAT gene was designed. This assay, based on denaturing gradient gel electrophoresis (DGGE), allows for the complete analysis of a single individual in two lanes on a gel. Application of the assay resulted in the identification of nine known AAT variants as well as 13 novel sequence variants, five of which are single nucleotide polymorphisms (SNPs), occurring exclusively in the African-based populations. This is the first comprehensive analysis of the genetic diversity of the AAT gene in a cohort from sub-Saharan Africa.

Proteases in blood clotting.

The serine proteases, cofactors and cell-receptor molecules that comprise the haemostatic mechanism are highly conserved modular proteins that have evolved to participate in biochemical reactions in blood coagulation, anticoagulation and fibrinolysis. Blood coagulation is initiated by exposure of tissue factor, which forms a complex with factor VIIa and factor X, which results in the generation of small quantities of thrombin and is rapidly shutdown by the tissue factor pathway inhibitor. The generation of these small quantities of thrombin then activates factor XI, resulting in a sequence of events that lead to the activation of factor IX, factor X and prothrombin. Sufficient thrombin is generated to effect normal haemostasis by converting fibrinogen into fibrin. The anticoagulant pathways that regulate blood coagulation include the protein C anticoagulant mechanism, the serine protease inhibitors in plasma, and the Kunitz-like inhibitors, tissue factor pathway inhibitor and protease nexin 2. Finally, the fibrinolytic mechanism that comprises the activation of plasminogen into plasmin prevents excessive fibrin accumulation by promoting local dissolution of thrombi and promoting wound healing by reestablishment of blood flow.

Activation of Heparin Cofactor II by Calcium Spirulan

Heparin cofactor II (HCII) is a plasma serine protease inhibitor whose ability to inhibit alpha-thrombin is accelerated by a variety of sulfated polysaccharides in addition to heparin and dermatan sulfate. Previous investigations have indicated that calcium spirulan (Ca-SP), a novel sulfated polysaccharide, enhanced the rate of inhibition of alpha-thrombin by HCII. In this study, we investigated the mechanism of the activation of HCII by Ca-SP. Interestingly, in the presence of Ca-SP, an N-terminal deletion mutant of HCII (rHCII-Delta74) inhibited alpha-thrombin, as native recombinant HCII (native rHCII) did. The second-order rate constant for the inhibition of alpha-thrombin by rHCII-Delta74 was 2.0 x 10(8) M(-1) min(-1) in the presence of 50 microgram/ml Ca-SP and 10, 000-fold higher than in the absence of Ca-SP. The rates of native rHCII and rHCII-Delta74 for the inhibition of gamma-thrombin were increased only 80- and 120-fold, respectively. Our results suggested that the anion-binding exosite I of alpha-thrombin was essential for the rapid inhibition reaction by HCII in the presence of Ca-SP and that the N-terminal acidic domain of HCII was not required. Therefore, we proposed a mechanism by which HCII was activated allosterically by Ca-SP and could interact with the anion-binding exosite I of thrombin not through the N-terminal acidic domain of HCII. The Arg(103) --> Leu mutant bound to Ca-SP-Toyopearl with normal affinity and inhibited alpha-thrombin in a manner similar to native rHCII. These results indicate that Arg(103) in HCII molecule is not critical for the interaction with Ca-SP.

Measurement of protein C inhibitor in seminal plasma is useful for detecting agenesis of seminal vesicles or the vas deferens.

Protein C inhibitor (PCI), a plasma serine protease inhibitor of activated protein C, is present at high concentrations in the seminal plasma of normal subjects and is decreased in some infertile patients. We measured the concentrations of PCI, prostate-specific antigen, and fructose in the seminal plasma of infertile patients (n = 125) and of normal subjects (n = 13). We also measured time-dependent changes in the concentrations of PCI and fructose in seminal plasma after ejaculation. A weak correlation was found between the levels of PCI and fructose (r = 0.268, P = 0.016). The PCI level in seminal plasma of patients with seminal vesicle and/or vasal agenesis was significantly lower (P < .01) than in normal subjects. The level of fructose in seminal plasma decreased in vitro in a time-dependent manner after ejaculation, whereas the concentration of PCI was stable at 48 hours after ejaculation. These data suggest that PCI in seminal plasma, as well as fructose, may become one of the markers for agenesis of seminal vesicles and/or the vas deferens.