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Available assays for circulating levels of activated protein C (APC) are either time-consuming or difficult to use in a routine laboratory, or have a detection limit above normal levels. We have developed a simplified assay that measures both the in vivo free APC and the in vivo APC complexed to PC inhibitor (PCI). We measured APC levels, with both assays, in 339 plasma samples, 165 from patients with venous thromboembolism (VTE) and 174 from healthy individuals. The mean APC level in the 339 samples was 0.038±0.010 nM, using a previous assay that measures only the in vivo APC level, and 0.041±0.010 nM with the present new assay. The coefficient of correlation between assays was r=0.954 (P

Protein C inhibitor was purified from human plasma by use of a dermatan sulfate or heparin column, followed by hydroxyapatite, gel filtration and ion exchange columns. A dimer of protein C inhibitor was detected by SDS-PAGE under reducing conditions, in addition to two forms of monomer species. One of the monomers, 52-kDa PCI, formed a stable complex with activated protein C, urokinase, plasma and tissue kallikrein, but the dimer species and 48-kDa PCI were inactive. When the monomer and dimer forms of protein C inhibitor were applied to 2D-PAGE, more than 20 spots were observed by Western blot analysis and were confirmed to be protein C inhibitor by MALDI-TOF mass spectrometry. The heterogeneity of the protein C inhibitor species was not due to glycosylation or phosphorylation.

The activation of coagulation factors V and X by Russell's viper venom (RVV) has been implicated in the development of consumptive coagulopathies in severely envenomed patients. However, factor Va is prone to inactivation by activated protein C (APC), an important serine protease that negatively regulates blood coagulation. It is therefore hypothesized that APC may be down-regulated by some of the venom components. In this study, we managed to isolate a potent Kunitz-type APC inhibitor, named DrKIn-I. Using chromogenic substrate, DrKIn-I dose-dependently inhibited the activity of APC. Heparin potentiated the inhibition and reduced the IC(50) of DrKIn-I by 25-fold. DrKIn-I, together with heparin, also protected factor Va from APC-mediated inactivation. Using surface plasmon resonance, DrKIn-I exhibited fast binding kinetics with APC (association rate constant = 1.7 × 10(7) M(-1) s(-1)). Direct binding assays and kinetic studies revealed that this inhibition (K(i) = 53 pM) is due to the tight binding interactions of DrKIn-I with both heparin and APC. DrKIn-I also effectively reversed the anticoagulant activity of APC and completely restored the thrombin generation in APC-containing plasma. Furthermore, although the injection of either DrKIn-I or RVV-X (the venom factor X-activator) into ICR mice did not significantly deplete the plasma fibrinogen concentration, co-administration of DrKIn-I with RVV-X resulted in complete fibrinogen consumption and the deposition of fibrin thrombi in the glomerular capillaries. Our results provide new insights into the pathogenesis of RVV-induced coagulopathies and indicate that DrKIn-I is a novel APC inhibitor that is associated with potentially fatal thrombotic complications in Russell's viper envenomation.

Using a proteomic approach, a study was conducted for determination of the effects of 2,3,4,7,8-pentachlorodibenzofuran (2,3,4,7,8-PCDF) on proteins secreted by HepG2 cells. Briefly, HepG2 cells were exposed to various concentrations of 2,3,4,7,8-PCDF for 24 or 48 h. MTT and comet assays were then conducted for determination of cytotoxicity and genotoxicity, respectively. Results of an MTT assay showed that 1 nM of 2,3,4,7,8-PCDF was the maximum concentration that did not cause cell death. In addition, a dose- and time dependent increase of DNA damage was observed in HepG2 cells exposed to 2,3,4,7,8-PCDF. Therefore, two different concentrations of 2,3,4,7,8-PCDF, 1 and 5 nM, were selected for further analysis of proteomic biomarkers using two different pI ranges (4–7 and 6–9) and large two dimensional gel electrophoresis. Results showed identification of 32 proteins ( 29 up- and 3 down-regulated) by nano-LC-ESI-MS/MS and nano-ESI on a Q-TOF2 MS. Among these, the identities of pyridoxine-5'-phosphate oxidase, UDP-glucose 6-dehydrogenase, plasminogen activator inhibitor I precursor, plasminogen activator inhibitor-3, proteasome activator complex subunit 1, isoform 1 of 14-3-3 protein sigma, peptidyl-prolyl cis-trans isomerase A, 14-3-3 protein gamma, protein DJ-1, and nucleoside diphosphate kinase A were confirmed by western blot analysis. The differential expression of protein DJ-1, proteasome activator complex subunit 1 and plasminogen activator inhibitor-3 was further validated in plasma proteins from rats exposed to 2,3,4,7,8-PCDF. These proteins could be used as potential toxicological biomarkers of 2,3,4,7,8-PCDF.

Introduction Behcet's disease is a vasculitis of unknown cause in which thrombosis occurs in about 25% of patients. Two haplotypes of the endothelial protein C receptor gene, H1 and H3, are associated with the risk of thrombosis. Thus, the objective of this study was to evaluate the influence of these haplotypes on the thrombosis risk in Behcet's disease. Material and Methods We evaluated the H1 and H3 haplotypes in 87 patients with Behcet's disease, 19 with and 68 without a history of thrombosis, and in 260 healthy individuals. We also measured protein C, activated protein C, and soluble endothelial protein C receptor levels in all individuals. Results The presence of the H1 haplotype seemed to protect Behcet's patients against thrombosis (odds ratio 0.21; 95% CI 0.1-0.8; p = 0.023), whereas the frequency of the H3 haplotype was lower in patients than in control individuals (0.19; 0.1-0.5; p = 0.006). Furthermore, the H1 haplotype was associated with increased levels of activated protein C, whereas the H3 haplotype was associated with the highest soluble endothelial protein C levels. Moreover, activated protein C levels were lower in patients with than in patients without posterior uveitis ( p Conclusions These findings indicate that the H1 haplotype protects Behcet's patients from thrombosis, likely via increased levels of activated protein C, whereas individuals carrying the H3 haplotype seem to be protected from the clinical manifestations associated with Behcet's disease, probably via increased soluble endothelial protein C levels.

Summary. Background: Replacement therapy for hemophilic patient treatment is costly, because of the high price of pharmacologic products, and is not affordable for the majority of patients in developing countries. Objective: To generate and evaluate low molecular weight agents that could be useful for hemophilia treatment. Methods: Potential agents were generated by synthesizing specific inhibitors [6-(Lys-Lys-Thr-[homo]Arg)amino-2-(Lys[carbobenzoxy]-Lys[carbobenzoxy]-O-benzyl)naphthalenesulfonamide] (PNASN-1)] for activated protein C (APC) and tested in plasma and fresh blood from hemophilia A patients. Results: In the activated partial thromboplastin time-based APC resistance assay, PNASN-1 partially neutralized the effect of APC. In calibrated automated thrombography, PNASN-1 neutralized the effect of APC on thrombin generation in normal and congenital factor VIII-deficient plasma (FVIII:C

Hemophilia is a family of rare bleeding disorders. The two primary types, hemophilia A and hemophilia B, are caused by recessive X-chromosome linked mutations that result in deficiency of coagulation factor VIII (FVIII) or factor IX (FIX), respectively. Clinically, hemophilia is manifested by spontaneous bleeding, particularly into the joints (haemarthrosis) and soft tissue, and excessive bleeding following trauma or surgery. The total overall number of hemophilia patients worldwide is approximately 400,000, however only about 100,000 of these individuals are treated. The first treatment of hemophilia was initiated when it was determined that the clotting deficiency could be corrected by a plasma fraction taken from normal blood. The discovery of factor VIII enrichment by cryoprecipitation of plasma opened a new era of therapy which eventually led to the production of factor concentrates and the subsequent development of highly purified forms of plasma factors. The most significant improvements have been the availability of recombinant forms of factors VIII and IX. Unfortunately, recombinant factors still retain some of the limitations of plasma concentrates. These limitations include development of antibody responses in patients and the relatively short half-life of the molecules requiring frequent injection to maintain effective concentration. Treatment beyond replacement of native factors has been tried. They include the development of modified factor VIII and IX molecules with improved potency, stability and circulating half-life and enhancement of a prothrombotic responses and/or stabilization of coagulation factors via inhibition of key negative regulatory pathways. These approaches will be reviewed in this commentary.

BACKGROUND: Thrombin activation measured by the levels of the complex between activated protein C (APC) and the protein C inhibitor (PCI) is elevated in several atherosclerotic disorders. The aim of this study was to evaluate whether levels of the APC-PCI complex are related to the prognosis in peripheral arterial disease (PAD). Longitudinal study performed at the Vascular Centre, Malmo University Hospital, Sweden. METHODS: APC-PCI complex levels were analyzed in 268 consecutive patients hospitalized for PAD and in 42 healthy controls (median age, 74 years). Patients (n = 35) with warfarin treatment less than 4 weeks before APC-PCI sampling were excluded from analysis. Data-based medical records of all 233 remaining patients (median age, 72 [64-79] years) were searched for vascular events such as hospitalization because of atherosclerotic disease, operative or endovascular recanalization of peripheral arteries, transtibial or transfemoral amputation because of PAD, acute coronary syndrome, stroke, or death. RESULTS: Median duration of follow-up was 16 months (interquartile range, 12-23 months). APC-PCI complex levels were higher in PAD patients than in controls (0.240 [0.180-0.320] mug/L vs. 0.140 [0.190-0.220] mug/L; p < 0.0001) but not associated with an increased risk for death (p = 0.2054) or events during follow-up (p = 0.2850). Independent predictors of future events were low b-hemoglobin (p = 0.0084), high b-leukocytes (p = 0.0034), and history of a previous vascular event (p = 0.0032). Age (p = 0.0286), high p-creatinine (p = 0.0165), and history of a previous event (p = 0.0311) were independent predictors of death. CONCLUSION: APC-PCI complex levels were higher in PAD patients than in controls, but did not predict the clinical outcome. The effect of a possible prethrombotic state, as reflected in increased APC-PCI levels, on prognosis and severity of atherosclerotic disease has to be further investigated. (Less)

Background Protein C inhibitor (PCI) is a plasma serine protease inhibitor (serpin) that regulates several serine proteases in coagulation including thrombin and activated protein C. However, the physiological role of PCI remains under investigation. The cysteine protease, cathepsin L, has a role in many physiological processes including cardiovascular diseases, blood vessel remodeling, and cancer. Methods and results We found that PCI inhibits cathepsin L with an inhibition rate (k 2 ) of 3.0 × 10 5 M − 1 s − 1 . Whereas, the PCI P1 mutant (R354A) inhibits cathepsin L at rates similar to wild-type PCI, mutating the P2 residue results in a slight decrease in the rate of inhibition. We then assessed the effect of PCI and cathepsin L on the migration of human breast cancer (MDA-MB-231) cells. Cathepsin L was expressed in both the cell lysates and conditioned media of MDA-MB-231 cells. Wound-induced and transwell migration of MDA-MB-231 cells was inhibited by exogenously administered wtPCI and PCI P1 but not PCI P14 mutant. In addition, migration of MDA-MB-231 cells expressing wtPCI was significantly decreased compared to non-expressing MDA-MB-231 cells or MDA-MB-231 cells expressing the PCI P14 mutant. Downregulation of cathepsin L by either a specific cathepsin L inhibitor or siRNA technology also resulted in a decrease in the migration of MDA-MB-231 cells. Conclusions Overall, our data show that PCI regulates tumor cell migration partly by inhibiting cathepsin L. General significance Consequently, inhibiting cathepsin L by serpins like PCI may be a new pathway of regulating hemostasis, cardiovascular and metastatic diseases.

INTRODUCTION: Elevated levels of markers for thrombin activation are associated with plaque echogenicity and degree of stenosis in patients with carotid artery stenosis. The Activated Protein C-Protein C Inhibitor (APC-PCI) complex reflects activation of the Protein C system and is a measure of thrombin generation. The aim of the present study was to examine APC-PCI complex in patients undergoing thrombendartherectomy for carotid artery stenosis, and to relate the findings to clinical characteristics and plaque morphology as determined by ultrasound. MATERIALS AND METHODS: Blood was obtained from 125 patients (39 female, median age 71 years) with carotid artery stenosis admitted from September 2005 to May 2007. The APC-PCI complex was measured using a sandwich immunofluorometric method and compared to an age- and sex-matched healthy control-group. Clinical and demographic characteristics, routine laboratory markers and ultrasound characteristics were analysed using univariate and multivariate analysis. RESULTS: APC-PCI complex concentration was significantly increased in patients with carotid artery stenosis (median 0.21 microg/L; 10th to 90th percentile 0.15-0.36) compared to a healthy control-group (0.19 microg/L; 0.11-0.31; P=.009). There was no significant difference in APC-PCI-values between asymptomatic (n=48) and symptomatic (n=77) patients with carotid artery stenosis (0.22 vs. 0.20 microg/L; p=0.626). Patients with minor stroke (n=31) had a higher median APC-PCI-concentration (0.27 microg/L; 0.15-0.63) than patients with amaurosis fugax (0.19 microg/L; 0.15-0.36) or transient ischemic attack (0.21 microg/L; 0.12-0.36) (p=0.016). No association was found between APC-PCI-values and the degrees of carotid artery stenosis or the time from the latest neurological symptoms to blood sampling. Patients with echolucent plaques had significantly lower APC-PCI concentrations (0.20 microg/L; 0.14-0.35 vs. 0.24 microg/L; 0.15-0.60; p=0.043), according to the Gray-Weale classification. CONCLUSIONS: Patients with carotid artery disease exhibit increased concentrations of APC-PCI compared to a healthy control-group, particularly those patients with echogenic plaques, who have significantly higher APC-PCI levels than patients with echolucent plaques. (Less)

Introduction: The concentration of carboxypeptidase B activation peptide (CAPAP) is proposed to be a predictor of severe acute pancreatitis. The activated protein C (APC)-protein C inhibitor (PCI; APC-PCI) complex in plasma could be useful in detecting the hypercoagulative condition in severe acute pancreatitis. Method: In this prospective study, mild (n = 50) and severe (n = 9) cases of acute pancreatitis were compared with respect to levels of CAPAP and APC-PCI, and sorted in time intervals from onset of symptoms to sampling. The peak values of the C-reactive protein (CRP) within the 1st week were also compared. Results: CRP detected the severe cases with a sensitivity of 0.89 and a specificity of 0.74 (cut-off level 200 mg/l). In the interval 0-72 h, CAPAP could predict the severity of the disease in serum and urine (sensitivity 0.52/0.29, specificity 0.73/0.93, cut-off 2 nM/60 nM). The level of APC-PCI in plasma could predict the severe condition in the interval 0-24 h after the onset of symptoms (sensitivity 0.6, specificity 0.66, cut-off level 0.54 mug/l). Conclusion: Of the parameters explored, CRP is still the best biochemical marker to distinguish between severe and mild acute pancreatitis. CAPAP could be useful in combination with other tests, but the APC-PCI complex's diagnostic time interval is too short to be used in the clinical routine. and IAP. (Less)

Objective: The etiology of abdominal aortic aneurysm (AAA) includes atherosclerotic, inflammatory, immunological and coagulatory mechanisms. The aim of this study was to evaluate associations between markers for some of these mechanisms and AAA-size, in order to identify markers which might later be evaluated in relation to aneurysm growth. Material and methods: Prospectively 360 AAA-patients and an age and sex-matched healthy control group (n=219) were analyzed. AAA-patients were divided in three groups according to AAA-diameter (small 55mm, n=130). Associated diseases, blood pressures and routine laboratory markers were analyzed. Additionally we evaluated endothelin (ET)-1, tumour necrosis factor (TNF)-alpha, interleukin (IL)-6, activated protein C-protein C inhibitor (APC-PCI) complex, and CD40 ligand. Groups were compared with the Kruskall-Wallis test and the Mann-Whitney U test. Results: Of routine markers platelet count was lower (p=0.0006) and creatinine level was higher (p=0.028) in patients with large AAA. Almost all non-routine markers analyzed were highly elevated in AAA-patients compared to the control group. IL-6 (p=0.0002) and thrombin activation measured as APC-PCI (p

SummaryActivated protein C (APC) is a serine protease with anticoagulant, anti-inflammatory, and cytoprotective properties. Using recombinant APC, we identified a class of single-stranded DNA aptamers (HS02) that selectively bind to APC with high affinity. Interaction of HS02 with APC modulates the protease activity in a way such that the anticoagulant functions of APC are inhibited and its reactivity toward the protein C inhibitor is augmented in a glysoaminoglycan-like fashion, whereas APC's antiapoptotic and cytoprotective functions remain unaffected. Based on these data, the binding site of HS02 was localized to the basic exosite of APC. These characteristics render the exosite-specific aptamers a promising tool for the development of APC assays and a potential therapeutic agent applicable for the selective control of APC's anticoagulant activity.

Protein C inhibitor (PCI) is a serine protease inhibitor, displaying broad protease specificity, found in blood and other tissues. In blood, it is capable of inhibiting both procoagulant and anticoagulant proteases. Mechanisms that provide specificity to PCI remain largely unrevealed. In this study we have for the first time provided a full explanation for the marked size heterogeneity of blood-derived PCI and identified functional differences between naturally occurring PCI variants. The heterogeneity was caused by differences in N-glycan structures, N-glycosylation occupancy, and the presence of a Delta6-N-cleaved form. Bi-, tri-, and tetra-antennary complex N-glycans were identified. Fucose residues were identified both on the core GlcNAc and as parts of sialyl-Le(a/x) epitopes. Moreover, a glycan with a composition that implied a di-sialyl antenna was observed. PCI was N-glycosylated at all three potential N-glycosylation sites, Asn-230, Asn-243, and Asn-319, but a small fraction of PCI lacked the N-glycan at Asn-243. The overall removal of N-glycans affected the maximal heparin- and thrombomodulin-enhanced rates of thrombin inhibition differently in different solution conditions. In contrast, the Delta6-N-region increased both the heparin- and the thrombomodulin-enhanced rates of thrombin inhibition at all conditions examined. These results thus demonstrate that the N-linked glycans and the N-terminal region of blood-derived PCI in different ways affect the cofactor-enhanced rates of thrombin inhibition and provide information on the mechanisms by which this may be achieved. The findings are medically important, in view of the documented association of PCI with atherosclerotic plaques and the promising effect of PCI on reducing hypercoagulability states.

Introduction Activated protein C (APC) is well-established as a physiologically important anticoagulant. During development, plasma concentrations of protein C and α 2 macroglobulin, factors involved in APC generation, differ from adult levels. Chemotherapy drugs can perturb endothelial expression of PC-activating receptors. This study examines the effect of chemotherapy treatment of endothelium on APC generation in newborn and adult plasma. Materials and methods APC generations were initiated on endothelial cells treated with vincristine or media by recalcifying defibrinated plasma with buffer containing thromboplastin. APC generation was terminated by mixing timed subsamples into FFRCMK-EDTA or heparin, followed by EDTA. APC-PCI and APC-α 1 AT were assayed by ELISA. APC-α 2 M was measured chromogenically. Since heparin converts free APC to APC-PCI, the difference between APC-PCI detected in heparin subsamples and APC-PCI detected in FFRCMK-EDTA subsamples gave the free APC. Cellular expression of EPCR and TM were measured by flow cytometry and Western blot. Results Vincristine-treated endothelium decreased free APC generation in newborn plasma to a greater degree than in adult plasma. APC-PCI levels in both adult and newborn plasma were unaffected by chemotherapy. Vincristine treatment reduced levels of APC-α 1 AT and APC-α 2 M to a greater degree in newborn plasma versus adult plasma. Expression of EPCR was reduced in cells treated with vincristine. Conversely, TM was reduced on the cell surface, but increased in whole cell lysates. Conclusions The differential response of newborn and adult plasma PC components to chemotherapy-mediated changes in cell surface components may be a factor in the increased risk of thrombosis in children receiving chemotherapy.

Background Activated protein C (APC) reduces mortality in severe sepsis. Protecting APC in the circulatory system from inactivation by serine protease inhibitors (serpins) could improve its therapeutic efficiency. Significantly elevated levels of a serpin plasminogen activator inhibitor 1 (PAI-1) correlate with a lethal outcome in severe sepsis and disseminated intravascular coagulation. Intermolecular mechanisms were employed to redirect the reaction between APC and PAI-1 from the inhibitory to the substrate pathway, which results in the catalytic neutralization of the serpin. Methods The effects of anti-PAI-1 monoclonal antibodies (mAbs) and vitronectin, as well as their fragments, on the kinetics and stoichiometry of the reaction between PAI-1 and APC were studied using SDS PAGE and fluorescence spectroscopy. Results MAbs with epitopes at α-helix F redirected 70–80% of the reaction between PAI-1 and APC, to the substrate pathway. Vitronectin and its SMB domain did not affect the stoichiometry of acyl-enzyme formation, but enhanced the effect of mAbs. While vitronectin induced a more than two-fold increase in the rate of the reaction between PAI-1 and APC, neither mAbs (mAb fragments), nor SMB domain of vitronectin affected it. Conclusions Ligands interacting with α-helix F of PAI-1 demonstrated a potential for the protection of APC from inactivation by PAI-1. Since the mechanism of proteinase/serpin interaction is universal, a similar design and approach could be employed for enhancing the inactivation of other serpins in order to preserve APC activity in the circulation. Rational pharmacological targeting of the inhibitors of APC could have therapeutic utility.

Hemostasis and fibrinolysis, the biological processes that maintain proper blood flow, are the consequence of a complex series of cascading enzymatic reactions. Serine proteases involved in these processes are regulated by feedback loops, local cofactor molecules, and serine protease inhibitors (serpins). The delicate balance between proteolytic and inhibitory reactions in hemostasis and fibrinolysis, described by the coagulation, protein C and fibrinolytic pathways, can be disrupted, resulting in the pathological conditions of thrombosis or abnormal bleeding. Medicine capitalizes on the importance of serpins, using therapeutics to manipulate the serpin-protease reactions for the treatment and prevention of thrombosis and hemorrhage. Therefore, investigation of serpins, their cofactors, and their structure-function relationships is imperative for the development of state-of-the-art pharmaceuticals for the selective fine-tuning of hemostasis and fibrinolysis. This review describes key serpins important in the regulation of these pathways: antithrombin, heparin cofactor II, protein Z-dependent protease inhibitor, alpha(1)-protease inhibitor, protein C inhibitor, alpha(2)-antiplasmin and plasminogen activator inhibitor-1. We focus on the biological function, the important structural elements, their known non-hemostatic roles, the pathologies related to deficiencies or dysfunction, and the therapeutic roles of specific serpins.

Summary. Background: Hepatocyte growth factor (HGF)plays an important role in tissue repair and regeneration. HGFactivator (HGFA), a factor XIIa-like serine protease, activatesHGFprecursortoHGF.TheprecursorofHGFA,proHGFA,is activated by thrombin generated at sites of tissue injury. It isknown that protein C inhibitor (PCI), an inhibitor of activatedprotein C (APC), also inhibits thrombin–thrombomodulin(TM) complex. Objectives: In the present study we evaluatedthe effect of PCI on thrombin-catalyzed proHGFA activationin the presence of TM, and on HGFA activity. Results: PCIdid not inhibit thrombin-TM-mediated proHGFA activation,butitdirectlyinhibitedactivatedHGFAbyforminganenzymeinhibitorcomplex.Thesecond-orderrateconstants( M )1 min )1 )of the reaction between HGFA and PCI in the presence orabsence of heparin (10 U mL )1 )were4.3· 10 6 and 4.0 · 10 6 ,respectively. The inhibition of HGFA by PCI resulted in asignificant decrease of HGFA-catalyzed activation of HGFprecursor. Exogenous HGFA added to normal human plasmaformed a complex with plasma PCI, and this complexformation was competitively inhibited by APC in the presenceof heparin, but very weakly in the absence of heparin. We alsodemonstrated using recombinant R362A-PCI that Arg362residue of PCI is important for HGFA inhibition by PCI asjudged from the three-dimensional structures constructedusingdockingmodelsofPCIandHGFAorAPC. Conclusion:These observations indicate that PCI is a potent inhibitor ofactivated HGFA, suggesting a novel function for PCI in theregulation of tissue repair and regeneration.Keywords: hepatocyte growth factor activator, hepatocytegrowth factor, homology modeling, liver regeneration, proteinC inhibitor.IntroductionBloodclottingisanessentialprocessforproperhemostasisandwound healing. Thrombin is a clotting factor that plays acentral role in the promotion of blood coagulation and tissueregeneration in the wound-healing process [1]. Hepatocytegrowth factor (HGF) plays a critical role in tissue regenerationby stimulating the proliferation and motility of various celltypes, including epithelial and endothelial cells [2]. HGF issynthesized and secreted as an inactive single-chain precursorfrom liver sinusoidal endothelial cells and megakaryocytes(platelets) [3]. Limited proteolytic activation of this precursor isrequiredforthebiologicalactivityofHGF[4].Themostpotentactivator of HGF precursor is the factor XIIa-like serineprotease, HGF activator (HGFA) [5]. HGFA is synthesized inhepatocytes, and its inactive proHGFA circulates in blood as a98-kDa single-chain precursor protein [6]. Thrombin activatesproHGFA by limited proteolysis generating an activated 98-kDa HGFA molecule that contains a disulfide-linked 65-kDaheavy chain and a 31-kDa light chain [7]. The HGFA heavychain is then further cleaved in the systemic circulation bykallikrein releasing the 34-kDa mature form of HGFA [7]. Cellmembrane-bound Kunitz-type HGFA inhibitors (HAI) havebeen recently isolated [8,9], and it has been found that one ofthem, HAI-1, acts as inhibitor and receptor of HGFA on cellsurface [10]. The role of HAI as a physiological inhibitor ofHGFAhasnotbeenasyetreported.Todate,theexistenceofapotent physiological inhibitor of thrombin-catalyzed pro-HGFA activation or activated HGFA has not been as yetidentified.Protein C inhibitor (PCI) (SERPINA5), a member of theplasma serine protease inhibitor (serpin) family, was isolatedfrom human plasma as an inhibitor of activated protein C(APC), the main protease of the anticoagulant protein Cpathway [11]. PCI forms an acyl-bonded complex with APC,and this complex formation is enhanced in the presence of

Introduction Coagulation activation may be related to complications during surgery for abdominal aortic aneurysm. The complex formed between activated protein C (APC) and the serpin, protein C inhibitor (PCI), is a sensitive indicator of the activation of blood coagulation. The purpose of the study was to establish whether the APC–PCI complex can provide information useful for the assessment of outcome after aortic surgery. Materials and methods In 38 patients, the APC–PCI complex was initially determined every 6 h and daily from day three. Protein C, antithrombin, global haemostatic tests, and clinical scores were investigated. Length of stay at the intensive care unit (ICU) and hospital, and vital status up to two years were recorded. Results The median APC–PCI complex concentration in samples drawn 0–6 h after surgery was more than 20-fold higher than the upper limit of the reference interval. The level then declined rapidly, but remained elevated during the first two days. In patients with higher initial APC–PCI complex concentrations, Sequential Organ Failure Assessment (SOFA) scores were higher, the ICU stay was longer, and survival up to two years was lower. Patients who did not survive the ICU care had higher APC–PCI complex levels at 6–12 h and 12–18 h. Conclusions High concentrations of the APC–PCI complex within 6–18 h after the aortic surgery predict a sinister outcome. The results suggest that the APC–PCI complex is indicative of the severity of the disease.

In this issue, Nishii et al. [1] from Mie University in Japan provide interesting evidence that mice over-expressing protein C inhibitor (PCI) are protected from monocrotaline-induced pulmonary hypertension, due in part to the ability of PCI to inhibit thrombin and down-regulate coagulation. The transgenic mouse over-expressing PCI has enhanced lung secretion of PCI, and the clinical, biochemical and pathological parameters show a reduction in pulmonary hypertension in this experimental model. The pathophysiological role of the human plasma serine protease inhibitor (serpin), protein C inhibitor (PCI, also named plasminogen activator inhibitor-3, systematic name of SERPINA5) has remained elusive since its description in the 1980s [2,3]. From its name, PCI is an inhibitor of activated protein C (APC) [4–14]. However, PCI also inhibits thrombin (IIa) [8–10,12], factor Xa and factor XIa [8,11–14], kallikrein [8,10,12,15–17], urokinase-plasminogen activator and tissue-plasminogen activator (t-PA) [8,18–23], acrosin [24–26], prostate specific antigen [27,28], and remarkably, thrombin-thrombomodulin [29–31], which is responsible for generating APC (Fig. 1). The broad protease inhibitory profile of PCI has led many to postulate both specific and generic roles for this serpin. To further complicate matters, the tissue distribution of PCI in humans compared with mice is quite different. Humans show a broad tissue expression pattern for PCI, including the liver, kidney, pancreas, prostate, testes and ovaries [32–37]. Thus, this explains why human PCI (hPCI) is found not only in circulating blood, but also in urine, saliva, amniotic fluid, milk, tears and other body fluids [32]. In contrast, the mouse and rat express PCI only in the reproductive organs and it is not found in the circulating blood [34,38–42]. Through the creation of a PCI knockout mouse by homologous recombination, one non-hemostatic function of PCI was determined [26]. Male PCI−/− mice were infertile due to abnormal spermatogenesis caused by loss of the Sertoli cell barrier. Unopposed proteolytic activity in these mice brought about the degradation of the cell barrier [26]. Two transgenic mouse models expressing hPCI have been developed. The first was described by Wagenaar et al. [43], in which hPCI was expressed in the liver and found in the circulation. The second hPCI transgenic mouse was described by Hayashi et al. [44] and it expressed hPCI not only in the liver, but also in the kidney, heart, brain, lung and reproductive organs. Fig. 1 Role of protein C inhibitor and other serpins (antithrombin, heparin cofactor II, and plasminogen activator inhibitor-1) in the regulation of serine proteases thrombin, activated protein C (APC), APC generation by thrombin-thrombomodulin, and tissue plasminogen ... Concerning human health, the presence of PCI in various lung diseases has been described [45]. Fujimoto et al. [45] reported that bronchoalveolar lavage fluid contained increased amounts of both PCI and thrombin-activatable fibrinolysis inhibitor (TAFI) in patients with interstitial lung disease (ILD), particularly in patients with cryptogenic-organizing pneumonia, collagen vascular disease-associated ILD, and sarcoidosis. One explanation of their findings was that the levels of intra-alveolar PCI inhibited both APC activity and activation, which contributed to the pathogenesis of ILD. Therefore, a key question asked in the current study by Nishii et al. [1] was concerning the contribution of PCI to the pathogenesis of pulmonary hypertension. This study uses the hPCI over-expressing transgenic mouse described by Hayashi et al. [44] to begin to address this question regarding pulmonary hypertension, and also provides data on the physiological function of PCI (Fig. 1). Nishii et al. [1] treat mice with monocrotaline to induce pulmonary hypertension. This murine model is representative of pulmonary hypertension caused by a known etiology and not a secondary consequence of cardiovascular disease. Overall, hPCI reduces the disease state in the mouse lung compared with the wild-type mouse. The increase in pressure associated with pulmonary hypertension is not seen in the hPCI over-expressing transgenic mice. Pulmonary hypertension also results in endothelium dysfunction. The vessels in the lungs are hypercoagulant as a result of a decrease in prostaglandin and nitric oxide production. Platelets become activated and will adhere to the vessel wall. Hypercoagulability can be assessed by measuring the formation of thrombin:AT (TAT) complex. Although there is an increase of TAT complex in the hPCI over-expressing transgenic mice when treated with monocrotaline, this increase is significantly less than that in wild-type animals. These results suggest that either there is a decrease in thrombin production that would reduce TAT levels, or the increased presence of PCI is competing with AT to inhibit thrombin, which would also reduce the TAT levels. Either way, the hPCI over-expressing transgenic mouse does not exhibit an increase in activation of coagulation upon treatment with monocrotaline. Although PCI can be procoagulant through its inhibition of the protein C system of proteases, when hPCI is over-expressed in the mouse, its anticoagulant function is more prominent. Fibrinolysis is increased in mice upon treatment with monocrotaline, as indicated by an increase in t-PA activity. As PAI-1 levels are similar between the wild-type and hPCI over-expressing transgenic mice when treated with monocrotaline, the only explanation for a decrease in free t-PA and lowered fibrinolysis in the transgenic mice is the increase in hPCI. Pulmonary vascular endothelium dysfunction also results in the release of various cytokines, such as tumor necrosis factor-alpha (TNF-α) and monocyte chemoattractant protein-1 (MCP-1), and growth factors, such as platelet-derived growth factor (PDGF) and interleukin-13 (IL-13), that will promote inflammation and vascular wall thickening. The monocrotaline-treated wild-type mice show an increase in all of these protein levels. The hPCI over-expressing transgenic mice treated with monocrotaline exhibit little to no change in these same proteins. Therefore, hPCI reduces the endothelial dysfunction and inflammation associated with pulmonary hypertension. Furthermore, measurements of the pulmonary arteries in the hPCI over-expressing transgenic mouse show a smaller change in vessel wall and lumen area upon monocrotaline treatment. Overall, their results suggest that hPCI inhibits thrombin, a pro-inflammatory and pro-migratory factor, thus, reducing the effects of monocrotaline-induced pulmonary hypertension. The data presented in this paper raise more questions than answers about the in vivo protease specificity of PCI. Their results suggest that PCI exerts both anti-inflammatory and anticoagulant action by inhibiting thrombin, a known participant in coagulation, inflammation and tissue remodeling. This function of PCI is more prominent than its role as an inhibitor of APC. Inhibition of APC alone would result in an increase in coagulation and inflammation, and a reduction of tissue remodeling, which was not seen in the mouse model described. PCI is also antifibrinolytic through its inhibition of t-PA. Whether or not PCI can be used therapeutically for treating pulmonary hypertension remains to be studied. With the aid of new ELISAs reported for PCI-protease complexes [46–49], these tools can provide answers regarding the role of PCI in coagulation and inflammation.

Summary. Background: Protein C inhibitor (PCI) plays a role in multiple biological processes including fertilization, coagulation, fibrinolysis and kinin systems. Objectives: We hypothesized that PCI participates in the pathogenesis of pulmonary hypertension. To demonstrate this, we compared the development of pulmonary hypertension in mice overexpressing PCI in the lung with wild-type (WT) mice. Pulmonary hypertension was induced by s.c. injection of 600 mg kg−1 of monocrotaline weekly for 8 weeks. Results: Right ventricular arterial pressure was significantly increased in monocrotaline-treated WT mice compared with that in monocrotaline-treated transgenic mice. Bronchoalveolar lavage fluid (BALF) levels of thrombin–antithrombin complex, monocyte chemoattractant protein-1 and platelet-derived growth factor, and the plasma level of tumor necrosis factor-α were significantly increased in monocrotaline-treated WT mice as compared with monocrotaline-treated PCI transgenic mice. Increased level of PCI-thrombin complex was detected in BALF from monocrotaline-treated PCI transgenic mice as compared with saline-treated PCI transgenic mice. Conclusions: This study showed that increased expression of PCI in the lung is protective against monocrotaline-induced pulmonary hypertension, suggesting a potential beneficial effect of PCI for the therapy of this disease.

The Protein C pathway plays a crucial role in the regulation of thrombosis and inflammation. One of the tools that researchers presently use to elucidate mechanisms of action of activated protein C (APC) is the use of transgenic or gene deletion murine models. To correlate observations in these murine models with the APC levels, there is a need for a sensitive and specific assay for circulating murine APC in plasma. We developed an immunological assay to measure the physiological and pharmacologic levels of circulating murine APC. The sandwich ELISA uses an anti-murine anti-protein C antibody capture antibody and human protein C inhibitor (PCI) as a detection reagent, taking advantage of the facts that the mouse lacks plasma PCI and that human PCI forms a 1/1 stable complex with mouse APC. The amount of complex APC:PCI is detected with an anti-human PCI monoclonal antibody. The assay shows improved sensitivity versus enzyme immunocapture assays commonly used to detect human APC and considerably reduces the processing time.

Background α 1 -Antitrypsin (AAT)-Z deficiency is a risk factor for the development of COPD. Compared to wild-type M, AAT-Z has an increased tendency to polymerize, rendering it inactive as a serine proteinase inhibitor. It has been demonstrated that wild-type M- and Z-deficiency AAT polymers are chemotactic for human neutrophils. However, our own studies dispute a proinflammatory role for polymerized AAT-M and AAT-Z, suggesting rather that they are predominantly antiinflammatory, exhibiting inhibitory effects on lipopolysaccharide-stimulated human monocyte activation. The discrepancies between these observations prompted us to re-examine the effects of AAT. Methods and results The effects of native and polymerized AAT-M and AAT-Z with varying levels of endotoxin contamination (0.08 to 2.55 endotoxin units [EU]/mg protein) on human neutrophil chemotaxis and interleukin (IL)-8 release, in vitro , were evaluated. Neither native nor polymerized (M- or Z-deficient) AAT contaminated with low levels of endotoxin (≤ 0.08 EU/mg protein) stimulated neutrophil chemotaxis, whereas N-formyl methionyl leucyl phenylalanine (fMLP), a positive control, increased chemotaxis fourfold. A small but nonsignificant increase in neutrophil chemotaxis, however, was observed with AAT preparations containing higher levels of endotoxin (≥ 0.88 EU/mg protein), and significant chemotaxis occurred when AAT was spiked with either endotoxin or zymosan. In support, native and polymeric AAT-M with low endotoxin contamination completely inhibited neutrophil IL-8 release triggered by the zymosan, while AATs with high endotoxin contamination strongly induced IL-8 release and did not inhibit zymosan-stimulated IL-8 release. Conclusions The proinflammatory effects of native and polymeric AAT may be critically dependent on the presence of other cell activators, bacterial or otherwise, while pure preparations of AAT appear to exert predominantly antiinflammatory activity.

The concentration of the complex between activated protein C and the protein C inhibitor reflects the degree of activation of blood coagulation. A sandwich method has been devised that measures the complex concentration in blood plasma. A key feature of the method is that the catching monoclonal antibody recognizes a complex-dependent neoepitope in PCI, which is a prerequisite, since the concentration of uncomplexed PCI is approximately 10(4)-fold higher than that of the complex. In patients with atherosclerotic disease, those with aortic aneurysms exhibit a three-fold increase in complex concentration compared to that of normal subjects.

The serine protease inhibitor (serpin) protein C inhibitor (PCI; also named plasminogen activator inhibitor-3) regulates serine proteases in hemostasis, fibrinolysis, and reproduction. The biochemical activity of PCI is not fully defined partly due to the lack of a convenient expression system for active rPCI. Using pET-15b plasmid, Ni(2+)-chelate and heparin-Sepharose affinity chromatography steps, we describe here the expression, purification and characterization of wild-type recombinant (wt-rPCI) and two inactive mutants, R354A (P1 residue) and T341R (P14 residue), expressed in Escherichia coli. Wild-type rPCI, but not the two mutants, formed a stable bimolecular complex with thrombin, activated protein C and urokinase. In the absence of heparin, wt-rPCI-thrombin, -activated protein C, and -urokinase inhibition rates were 56.7, 3.4, and 2.3 x 10(4) M(-1) min(-1), respectively, and the inhibition rates were accelerated 25-, 71-, and 265-fold in the presence of 10 mug/mL heparin for each respective inhibition reaction. The stoichiometry of inhibition (SI) for wt-rPCI-thrombin was 2.0, which is comparable to plasma-derived PCI. The present report describes for the first time the expression and characterization of recombinant PCI in a bacterial expression system and demonstrates the feasibility of using this system to obtain adequate amounts of biologically active rPCI for future structure-function studies.

Summary. In humans, protein C inhibitor (PCI) is expressed in various tissues and present in many body fluids including plasma and seminal fluid. In rodents, PCI is expressed in reproductive organs only and is absent in plasma. In this study, we characterized the tissue expression and physiological role of PCI in novel human PCI gene transgenic (TG) mice. Northern blot and immunohistochemical analyses demonstrated that human PCI is expressed in liver hepatocytes, renal epithelial cells as well as heart, brain and reproductive organs of the TG mice. This PCI tissue distribution is similar to that found in humans. PCI in plasma of TG mice showed the same immunological and functional properties as human plasma PCI. Next, we evaluated the effect of PCI on coagulation, inflammation and tissue damage in lipopolysaccharide-treated TG mice. The results suggested that PCI efficiently inhibits not only the anticoagulant and anti-inflammatory activities of exogenously injected human activated protein C (APC) but also that of endogenously produced APC in mice with endotoxemia. These findings suggest that PCI exerts a procoagulant and proinflammatory effect by inhibiting APC. We believe our results also show how useful these TG mice may be for assessing the therapeutic effect of human APC in vivo and for evaluating the role of PCI in human physiological and pathological conditions.

Serpins are the predominant protease inhibitors in the higher organisms and are responsible, in humans, for the control of many highly regulated processes including blood coagulation and fibrinolysis. The serpin inhibitory mechanism has recently been revealed by the solution of a crystallographic structure of the final serpin-protease complex. The serpin mechanism, in contrast to the classical lock-and-key mechanism, involves dramatic conformational change in both the inhibitor and the inhibited protein. The final result is a stable covalent complex in which the properties of each component are altered so as to allow clearance from the circulation. Several serpins are involved in hemostasis: antithrombin (AT) inhibits many coagulation proteases, most importantly factor Xa and thrombin; heparin cofactor II (HCII) inhibits thrombin; protein C inhibitor (PCI) inhibits activated protein C and thrombin bound to thrombomodulin; plasminogen activator inhibitor 1 inhibits tissue plasminogen activator; and alpha2-antiplasmin inhibits plasmin. Nearly all of these reactions are accelerated through interactions with glycosaminoglycans (GAGs) such as heparin or heparan sulfate. Recent structures of AT, HCII and PCI have revealed how in each case the serpin mechanism has been fine-tuned by evolution to bring about high levels of regulatory control, and how seemingly disparate mechanisms of GAG binding and activation can share critical elements. By considering the serpins involved in hemostasis together it is possible to develop a deeper understanding of their complex individual roles.

The role of lysines 37-39 (chymotrypsin numbering) in the 37-loop of the serine protease activated protein C (APC) was studied by expressing acidic and neutral recombinant APC (rAPC) mutants. Activity of the APC mutants was assessed using human plasma and plasma-purified and recombinant derivatives of protein C inhibitor (PCI; also known as plasminogen activator inhibitor-3) and alpha(1)-antitrypsin, with and without heparin. The catalytic properties of the mutants to small peptidyl substrates were essentially the same as wild-type rAPC (wt-rAPC), yet their plasma anticoagulant activities were diminished. Analysis of the rAPC-protease inhibitor complexes formed after addition of wt-rAPC and mutants to plasma revealed no change in the inhibition pattern by alpha(1)-antitrypsin but a reduction in mutant complex formation by PCI in the presence of heparin. Using purified serpins, we found that inhibition rates of the mutants were the same as wt-rAPC with alpha(1)-antitrypsin; however, PCI (plasma-derived and recombinant forms) inhibition rates of the acidic mutants were slightly faster than that of wt-rAPC without heparin. By contrast, PCI-heparin inhibition rates of the mutants were not substantially accelerated compared to wt-rAPC. The mutants had reduced heparin-binding properties compared to wt-rAPC. Molecular modeling of the PCI-APC complex with heparin suggests that heparin may function not only to bridge PCI to APC, but also to alleviate putative non-optimal intermolecular interactions. Our results suggest that the basic residues of the 37-loop of APC are involved in macromolecular substrate interactions and in heparin binding, and they influence inhibition by PCI (with or without heparin) but not by alpha(1)-antitrypsin, two important blood plasma serpins.

Protein C inhibitor (PCI) is a member of the serpin family that has many biological functions. In blood it acts as a procoagulant, and, in the seminal vesicles, it is required for spermatogenesis. The activity of PCI is affected by heparin binding in a manner unique among the heparin binding serpins, and, in addition, PCI binds hydrophobic hormones with apparent specificity for retinoids. Here we present the 2.4 Å crystallographic structure of reactive center loop (RCL) cleaved PCI. A striking feature of the structure is a two-turn N-terminal shortening of helix A, which creates a large hydrophobic pocket that docking studies indicate to be the retinoid binding site. On the basis of surface electrostatic properties, a novel mechanism for heparin activation is proposed.

Activated protein C (APC) is a vitamin K-dependent anticoagulant serine protease in plasma that downregulates the coagulation cascade by degrading cofactors Va and VIIIa by limited proteolysis. In addition to its anticoagulant function, APC also exhibits potent profibrinolytic and anti-inflammatory properties. The proteolytic activity of APC in plasma is slowly inhibited by three serpins: protein C inhibitor, plasminogen activator inhibitor-1, and alpha(1)-antitrypsin. Recent structural and mutagenesis data have indicated that basic residues of three exposed surface loops known as the 39-loop (Lys(37)-Lys(39)), 60-loop (Lys(62), Lys(63)), and 70-80-loop (Arg(74), Arg(75), and Lys(78)) (chymotrypsin numbering) constitute an anion-binding exosite in APC that interacts with these macromolecular substrates and inhibitors. Moreover, this exosite plays a critical role in the thrombomodulin-dependent activation of the zymogen protein C by thrombin. This article briefly reviews how the binding of physiological protein and polysaccharide cofactors on this exosite modulates the protein C anticoagulant pathway in plasma.

Recently, a highly purified human menopausal gonadotrophin preparation (HMG) was launched. The composition and purity of this HMG (Menopur® Ferring Pharmaceuticals) with a claimed 1:1 ratio of FSH and LH was determined. Three gonadotrophins were observed: FSH, LH and human chorionic gonadotrophin (HCG). The immunoactivity for HCG was three-fold higher than the immunoactivity for LH. Because of the longer half-life of HCG as compared with LH, about 95% of the in-vivo LH-receptor-mediated bioactivity is attributable to the presence of HCG. This is substantiated by biochemical analyses. To the best of the authors' knowledge, this relatively high amount of HCG can only be explained by assuming the addition of HCG from external sources, which is a well established practice for standardization purposes. In addition to gonadotrophins, a number of other proteins were detected. The amount of these impurities, as determined by reversed-phase high-performance liquid chromatography on a peak-area basis, is at least 30%. Therefore, it is concluded that this HMG preparation contains at most 70% gonadotrophins. Via a proteomics approach three major impurities were identified: leukocyte elastase inhibitor, protein C inhibitor, and zinc-α 2 -glycoprotein. On the basis of the results obtained in this study, a comparison is made with recombinant FSH.

Protein S (PS) is a vitamin K-dependent plasma protein and serves as a cofactor for the anticoagulant activities of activated protein C (APC). We investigated the effects of different PS concentrations on prothrombin activation and thrombin generation in cord and adult plasma containing APC and different amounts of alpha 2-macroglobulin (a2-M). Prothrombin activation was assessed by monitoring the time-course of prothrombin fragment 1+2 (F1+2) generation. Thrombin generation curves were determined by means of a subsampling technique using the chromogenic substrate S-2238. We demonstrate a dose-dependent inhibition of the anticoagulant action of PS by a2-M: suppression of F1+2 and thrombin generation due to addition of PS was stronger in plasma containing low amounts of a2-M than in plasma with elevated a2-M levels. Since no complex formation between a2-M and PS was observed by means of SDS-PAGE, we attribute decreased anticoagulant action of PS at high a2-M levels to enhanced complex formation between APC and a2-M. Thereby, APC is subtracted from its cofactor PS, resulting in suppressed formation of the anticoagulant APC/PS complex. Thus, our data suggest that a2-M, besides its well-known anticoagulant effects, also acts as a procoagulant by suppressing the formation of the anticoagulant APC/PS complex. Our findings have implications particularly on thrombin generation and inhibition in cord plasma, since a2-M levels in newborns are elevated over adult values and the antithrombotic APC/PS pathway is up-regulated at birth. Therefore, elevated levels of a2-M might restrict the up-regulation of the APC/PS pathway.

The endothelial cell protein C receptor (EPCR) is an endothelial cell-specific transmembrane protein that binds both protein C and activated protein C (APC). EPCR regulates the protein C anticoagulant pathway by binding protein C and augmenting protein C activation by the thrombin-thrombomodulin complex. EPCR is homologous to the MHC class 1/CD1 family, members of which contain two α-helices that sit upon an 8-stranded β-sheet platform. In this study, we identified 10 residues that, when mutated to alanine, result in the loss of protein C/APC binding (Arg-81, Leu-82, Val-83, Glu-86, Arg-87, Phe-146, Tyr-154, Thr-157, Arg-158, and Glu-160). Glutamine substitutions at the four N-linked carbohydrate attachment sites of EPCR have little affect on APC binding, suggesting that the carbohydrate moieties of EPCR are not critical for ligand recognition. We then mapped the epitopes for four anti-human EPCR monoclonal antibodies (mAbs), two of which block EPCR/Fl-APC (APC labeled at the active site with fluorescein) interactions, whereas two do not. These epitopes were localized by generating human-mouse EPCR chimeric proteins, since the mAbs under investigation do not recognize mouse EPCR. We found that 5 of the 10 candidate residues for protein C/APC binding (Arg-81, Leu-82, Val-83, Glu-86, Arg-87) colocalize with the epitope for one of the blocking mAbs. Three-dimensional molecular modeling of EPCR indicates that the 10 protein C/APC binding candidate residues are clustered at the distal end of the two α-helical segments. Protein C activation studies on 293 cells that coexpress EPCR variants and thrombomodulin demonstrate that protein C binding to EPCR is necessary for the EPCR-dependent enhancement in protein activation by the thrombin-thrombomodulin complex. These studies indicate that EPCR has exploited the MHC class 1 fold for an alternative and possibly novel mode of ligand recognition. These studies are also the first to identify the protein C/APC binding region of EPCR and may provide useful information about molecular defects in EPCR that could contribute to cardiovascular disease susceptibility.

The aim of this study was to determine whether total fertilization failure in human IVF can be partially explained by alterations in the gene that codes for protein C inhibitor. Forty-six men had IVF total fertilization failure and 51 controls with normal fertilization were screened for mutations in the protein C inhibitor gene by direct sequencing. The main finding was that in men involved in total fertilization failure, a heterozygous adenosine/guanine (A/G) base combination in position 1389 (rs2069990) (exon 6) in the protein C inhibitor gene was significantly more common compared with controls (10.9% vs. 0).

Protein C inhibitor (PCI) is a member of the serine protease inhibitor (serpin) family, which was initially found to be an inhibitor of activated protein C (APC) and later a potent inhibitor of the thrombin-thrombomodulin complex, suggesting that PCI plays a pivotal role in the regulation of the anticoagulant protein C pathway in human plasma. PCI is also known as a plasminogen activator inhibitor-3 (PAI-3), since this serpin was found in urine forming a complex with urokinase type-plasminogen activator (uPA). Human PCI also inhibits several other serine proteases involved in blood coagulation and fibrinolysis. Precursor proteins of PCI deduced from human, rhesus monkey, bovine, rabbit, rats and mouse cDNAs have sequence homology from 62 to 93%. Human PCI gene is located in a region involving genes of related serpins in chromosome 14q32.1. As regulatory mechanism of PCI gene expression, Sp1- and AP2-binding sites in the 5′-flanking region are the promoter and the enhancer, respectively. PCI mRNA is expressed in many organs, such as liver, kidney, spleen, pancreas, and reproductive organs (including testis, prostate, seminal vesicles and ovary in humans) and also in the liver and reproductive organs in bovines and rabbits; though in rats and mice only in the reproductive organs. PCI appears to play a role in the regulation of fertilization, presumably by inhibiting prostate specific antigen and acrosin in the male reproductive organs or by inhibiting protease(s) in the ovaries. In addition to the roles of PCI in coagulation, fibrinolysis and fertilization, human PCI is also suggested to regulate wound healing and renal tumour metastasis by inhibiting hepatocyte growth factor activator and uPA, respectively. Thus, PCI is a unique multi-functional serpin member playing several roles depending on species and organ tissues.

Human glandular kallikrein (hK2) is a possible new marker for prostate cancer that is homologous to prostate specific antigen. Purified hK2 added to serum or plasma reacted with endogenous protease inhibitors to form complexes of >350, 135, and 80 kDa, and some hK2 remained free, as judged by immunoblotting. The former two complexes could be removed by specific antibodies to alpha2-macroglobulin and to C1- inactivator, respectively, and they comigrated on SDS-PAGE with complexes formed between hK2 and purified alpha2-macroglobulin or C1-inactivator. hK2 complexes of 80 kDa could not be completely removed with any anti-serpin antibody used. Thus, these may consist of more than one type of hK2 complex. In contrast, essentially all hK2 complexes were removed from seminal plasma by antibody to protein C inhibitor, demonstrating that protein C inhibitor is the only significant inhibitor of hK2 in semen. hK2 reacted more rapidly with alpha2-macroglobulin than with any other inhibitor in plasma or serum. Divalent metal ions and heparin did not appreciably affect the rate of formation of any of the hK2 complexes in serum or plasma or with purified alpha2-macroglobulin or C1-inactivator. Measurement of one or more of the hK2 forms identified here may have diagnostic or prognostic potential for prostate cancer.

Background . The protein C system is important in the regulation of hemostasis. We studied its behavior during coronary artery bypass grafting procedures with and without aprotinin treatment using assays sensitive for activation of the protein C system. Methods . In a prospective, double-blind, randomized study of 48 patients we investigated the levels of antigen to proteins C and S and of the complexes between activated protein C with its two major plasma inhibitors, protein C inhibitor and α 1 -antitrypsin in patients treated with placebo (n = 17), low-dose (n = 15), and high-dose (n = 16) aprotinin during elective coronary artery bypass grafting. Results . The levels of proteins C and S showed a rapid decrease after heparinization, decreased greatly after start of cardiopulmonary bypass, and remained stable during cardiopulmonary bypass. Activated protein C inhibitor complexes were markedly elevated at the start of the procedure. Activated protein C–α 1 -antitrypsin decreased greatly after the start of cardiopulmonary bypass and remained stable during cardiopulmonary bypass. A significant peak was observed at the intensive care unit. Activated protein C–protein C inhibitor levels showed a peak after heparinization in accordance with the accelerating effect of heparin on the complex formation but decreased thereafter. Treatment with aprotinin did not notably alter any of the measured patterns. Conclusions . In this study no evidence was found for increased activation of the protein C system during coronary artery bypass grafting. Administration of aprotinin did not result in different patterns of activation of the protein C system.

A soluble form of the endothelial cell protein C receptor (EPCR) was analyzed for the ability to modulate the functional properties of protein C and activated protein C (APC). In a plasma clotting system initiated with factor Xa, EPCR blocked the anticoagulant activity of APC in a dose-dependent fashion. EPCR had no influence on clotting in the absence of APC. Consistent with the plasma results, EPCR slowed the proteolytic inactivation of factor Va by slowing both of the key proteolytic cleavages in the heavy chain of factor Va. EPCR did not prevent protein C activation by the soluble thrombin-thrombomodulin complex, did not alter the inactivation of APC by alpha1-antitrypsin or protein C inhibitor, and did not influence the kinetics of peptide paranitroanilide substrate cleavage significantly. We conclude that EPCR binds to an exosite on APC that selectively modulates the enzyme specificity in a manner reminiscent of the influence of thrombomodulin on thrombin.

Heat shock protein 47 (HSP47) is a molecular chaperone which assists procollagen triple helix assembly and secretion. As a molecular chaperone it is unique in that it binds specifically to a very narrow range of protein "substrates" (i.e., procollagen and collagen only), and it is also a member of the well-characterized serine protease inhibitor (serpin) superfamily. In the absence; of any X-ray crystallographic data, a novel tandem-modeling procedure is used to obtain three-dimensional structural information on mature recombinant mouse HSP47 (mrmHSP47). MrmHSP47 is shown to have 30% amino acid sequence identity and 70% sequence similarity with human protein C inhibitor (hPCI). Therefore, molecular models of inhibitory and latent states of hPCI are generated, using the X-ray crystal structure coordinates of proteolytically cleaved hPCI, and used as templates for the homology modeling of inhibitory and latent states of mrmHSP47. The validity of the models is discussed and the latent state model of mrmHSP47 is shown to have a suitable candidate binding groove for procollagen/collagen peptides, which appears to account for experimental observations made with aminoacid deletion experiments.

Protein C inhibitor (PCI), a plasma serine protease inhibitor, inhibits several proteases including the anticoagulant enzyme, activated protein C (APC), and the coagulation enzymes, thrombin and factor Xa. Previous studies have shown that thrombin and APC are inhibited at similar rates by PCI and that heparin accelerates PCI inhibition of both enzymes more than 20-fold. We now demonstrate that the thrombin-binding proteoglycan, rabbit thrombomodulin, accelerates inhibition of thrombin by PCI approximately equal to 140-fold (k2 = 2.4 x 10(6) in the presence of TM compared to 1.7 x 10(4) M-1 S-1 in the absence of TM). Most of this effect is mediated by protein-protein interactions since the active fragment of TM composed of epidermal growth factor-like domains 4-6 (TM 4-6) accelerates inhibition by PCI approximately equal to 59-fold (k2 = 1.0 x 10(6) M-1 S-1). The mechanism by which TM alters reactivity with PCI appears to reside in part in an alteration of the S2 specificity pocket. Replacing Phe353 with Pro at the P2 position in the reactive loop of PCI yields a mutant that inhibits thrombin better in the absence of TM (k2 = 6.3 x 10(5) M-1 S-1), but TM 4-6 enhances inhibition by this mutant approximately equal to 9-fold (k2 = 5.8 x 10(6) M-1 S-1) indicating that TM alleviates the inhibitory effect of the less favored Phe residue. These results indicate that PCI is a potent inhibitor of the protein C anticoagulant pathway at the levels of both zymogen activation and enzyme inhibition.

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, α1-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.

Acrosin is a trypsin-like serine protease present in its zymogen form in the acrosome of spermatozoa. The activated enzyme is thought to digest a pathway for the sperm through the zona pellucida of the ovum during the process of fertilisation. We have shown that in a purified system boar acrosin was inhibited by human protein C inhibitor with an apparent second order rate constant (k app ) of 3.7×10 4 M −1 s −1 1. Protein C inhibitor is present in high concentrations in seminal plasma and endogenous protein C inhibitor was found in the immediate vicinity of disrupted acrosomal membranes of washed human spermatozoa. This serpin could therefore function as a scavenger of prematurely activated acrosin in the male reproductive tract. Since little is known about the interaction of acrosin with other serpin type inhibitors, we analysed in this study the interaction of boar acrosin with other purified human serpins. Antithrombin III, plasminogen activator inhibitor-1, plasminogen activator inhibitor-2, and α 1 -antitrypsin inhibited acrosin activity. The following apparent k app s were calculated: Antithrombin III: 19.5×10 4 M −1 s −1 , plasminogen activator inhibitor-1: 21.5×10 4 M −1 s −1 , plasminogen activator inhibitor-2: 3.3×10 4 M −1 s 1 , α 1 -antitrypsin: 0.09×10 4 M −1 s 1 . α 2 -antiplasmin and heparin cofactor II did not inhibit acrosin. SDS-stable acrosin/serpin complexes were only seen with antithrombin III; all other acrosin inhibitors were cleaved by the enzyme. As determined by enzyme-linked immunosorbent assays, the concentrations of protein C inhibitor, plasminogen activator inhibitor-1, and plasminogen activator inhibitor-2 in individual seminal plasma samples from healthy donors were 5.33±0.47 μM, 88±24 pM and 163±17 pM (means±SD), respectively. The concentrations of antithrombin III in seminal plasma were ⩽50 nM (semiquantitative immunoblotting). Therefore considering both, the k app calculated for the inhibition of boar acrosin in a purified system and the concentration of each serpin in seminal plasma, protein C inhibitor seems to be the best candidate to function as a physiological acrosin inhibitor in the male reproductive tract.

Urokinase-type plasminogen activator (u-PA) occurs either in a single-chain form or in a two chain form (scu-PA or tcu-PA, respectively). The occurrence of the different forms of u-PA and the formation of u-PA-inhibitor complexes was studied in urine samples from 10 healthy donors. Measurement by means of a bio-immunoassay showed that the urine samples contained 307±189ng/ml u-PA (mean±SD), of which 72±15% occurred in the active two chain form and 28±15% in the plasmin-activatable single chain form. SDS-polyacrylamide gel electrophoresis followed by fibrin zymography revealed that fresh urine contained no, or only traces of, u-PA-inhibitor complexes. In contrast to what has previously been published, the bulk of u-PA appeared to be in an uncomplexed 55 kD form. Incubation of urine at 37°C for 8 h did not lead to the formation of u-PA-inhibitor complexes. However, after rapid removal of the low molecular weight fraction of urine on a gel filtration column a 100kD complex, consisting of u-PA and plasminogen activator inhibitor-3/protein C inhibitor, was formed during incubation (t1/2≈45 min). Complex formation was dependent on stimulation by urinary glycosaminoglycans and only occurred at a low ionic strength, which explains why no complexes were formed in the untreated urine samples of healthy donors.

Protein C inhibitor (PCI) is a heparin-dependent serine protease inhibitor which, in vitro, inhibits activated protein C (APC, urokinase (u-PA) and tissue plasminogen activator (t-PA), plasma kallikrein (KK), thrombin and factors Xa and Ma. We have previously shown in vivo complexes of PCI with APC, u-PA and KK. Here we study the contribution of PCI to the inhibition of t-PA both in vitro and in vivo. PCI complexed to t-PA (t-PA:PCI) was measured by a sandwich ELISA using antibodies directed against each protein in the complex. The formation of t-PA:PCI complexes paralleled the inhibition of t-PA amidolytic activity by PCI in a purified system. In the plasma milieu, after incubation of 8 μg/mL t-PA (final concentration) with human plasma for 2 h at 37°C, in the presence of heparin, the residual t-PA activity was about 38% and plasma contained 0.56 μg/mL t-PA:PCI complexes. To study complex formation in vivo, human plasma samples from patients (n=5) with myocardial infarction were analyzed following infusion of 100 mg recombinant t-PA for 2.5h (10% initial bolus; 40% by a 20min infusion, and 50% by a 2-h infusion). Maximum peak values of t-PA:PCI complexes were measured immediately after the second and third t-PA doses, ranging from 16 to 135 ng/mL. In contrast, during t-PA infusion the level of complexes of t-PA with plasminogen activator inhibitor-1 (t-PA:PAI-1) increased only slightly, from 2.4±1.5 to 7.2±4.6 ng/mL, and reached a maximum mean value of 17.2±20.6 ng/mL 2h post-infusion. PCI antigen decreased during t-PA infusion from 80%±16% to 71%±7%. Protein C antigen levels decreased during t-PA infusion, with the subsequent appearance of complexes between APC and PCI (APC:PCI), and free protein S gradually declined during infusion, reaching levels of 69% of the basal value 2h post-infusion. These data show that in vivo interaction of PCI and t-PA exists, which in turn support the view that PCI may play a role in the regulation of the fibrinolysis pathway. They also show that thrombolytic therapy with t-PA produces activation and a subsequent decrease in protein C antigen levels as well as in free protein S levels.

Two functional assays for protein C inhibitor (PCI) were evaluated in parallel for the same plasma samples. One assay is specific for active PCI and measures its ability to form complexes with activated protein C (APC) in the presence of heparin (Espana et al, Thromb Res 64, 309, 1991) (Method A). After incubation of samples with heparin and an excess of APC, the amount of APC:PCI formed is measured by an ELISA. The second functional PCI assay is based on the determination of the residual amidolytic activity of the APC added to the sample. In all cases a good correlation was obtained with both methods. For 20 healthy subjects and 20 patients before surgery, there were no significant between-assay differences in the levels of functional PCI activity. On the other hand, there were significant between-assays differences in the levels of functional PCI of the 20 patients measured 1 and 3 days after surgery. In this case, method B gave higher values than method A (38% ± 13% versus 29% ± 11% at day 1 and 59% ± 16% versus 43% ± 12% at day 3) (mean ± SD). The level of α1-antitrypsin (α1AT), the second major plasma APC inhibitor, was significantly increased in patients at day 1 and day 3 (133% ± 30% and 190% ± 57%, respectively). Furthermore, there was a positive correlation between the level of α1AT and the normalized difference between the level of functional PCI measured by method B and that measured by method A. Additionally, when normal plasma was supplemented with increasing amounts of purified α1AT, the level of PCI activity measured by method B increased parallel to the increase in α1AT added, but that measured by method A did not. Determination of APC:inhibitor complexes in aliquots of the final mixtures utilized in the PCI assays revealed that the level of APC:α1AT complex increased parallel to the increase in the amount of α1AT added, whereas the APC:PCI complex level remained unchanged. We conclude that current functional PCI assays based on the determination of APC residual activity in plasma are not specific for PCI, are influenced by the level of plasma α1AT and are not reliable for the determination of functional PCI levels in patients with altered levels of α1AT. In contrast, the method based on the determination of the APC:PCI complexes formed following incubation of plasma samples with APC is specific for active PCI and is not influenced for the presence of other APC inhibitors.

Protein C and protein S are two plasma proteins that participate in an anticoagulant pathway. Protein C circulates as an inactive precursor that is converted to an active serine protease by a complex between thrombin and the endothelial cell-surface protein thrombomodulin. Activated protein C and protein S form an anticoagulant complex on cell surfaces that inactivates two of the regulatory proteins of coagulation, factors Va and VIIIa. Activated protein C is then cleared from the circulation by a relatively slow inactivation by α 1 antitrypsin and the protein C inhibitor. Deficiencies in protein C and protein S are associated with thrombotic complications. With protein S, this can arise as the result of a deficiency in protein S synthesis, proteolytic cleavage, and/or due to an increase in binding to the complement regulatory protein C4bBP, which behaves like an acute phase reactant. Inflammatory mediators not only elevate C4bBP levels, but also lead to downregulation of thrombomodulin expression. Animal experiments suggest that activated protein C and protein S may be effective and safe antithrombotic agents.

The purpose of the study was to test whether APC: α 1 AT complex is a useful clinical marker of the activation of coagulation. The rationale for this is that activated protein C may appear in circulation at an early stage of blood coagulation, when subcoagulant amounts of thrombin are formed. Given the relatively higher half-life of APC: α 1 AT as compared to that of thrombin:AT-III (TAT) complexes, we hypothesized that APC: α 1 AT could represent an amplification of the thrombin generated in the first events of coagulation. Using sandwich ELISA's we measured APC: α 1 AT and TAT complexes as well as complexes of AT-III with its target proteases in normal subjects and in several clinical groups of patients prone to thrombotic episodes, including pregnancy, preeclampsia, hemodialysis, gynecological tumors, diabetes and oral contraceptives. APC: α 1 AT complex was significantly increased in all clinical groups as compared to normal subjects and showed relatively higher increases than did TAT and ATM complexes in the majority of the groups studied. There was a significant and positive correlation between APC: α 1 AT and TAT complex levels in the majority of the groups, as well as between TAT and ATM and between APC: α 1 AT and ATM complex levels. We conclude that APC: α 1 AT complex can be used as a sensitive marker of prethrombotic states.

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.