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Protein C inhibitor (PCI) maintains hemostasis by inhibiting both procoagulant and anticoagulant serine proteases, and plays important roles in coagulation, fibrinolysis, reproduction, and anti-angiogenesis. The reactive site loop of PCI traps and irreversibly inhibits the proteases like APC (activating protein C), thrombin (FIIa) and factor Xa (FXa). Previous studies on antithrombin (ATIII) had identified Tyr253 and Glu255 as functional exosites that interact and aid in the inhibition of factor IXa and FXa. Presence of exosite in PCI is not known, however a sequence comparison with the PCI from different vertebrate species and ATIII identified Glu239 to be absolutely conserved. PCI residues analogous to ATIII exosite residues were mutated to R238A and E239A. Purified variant PCI in the presence of heparin (10 μg/ml) showed a 2-4 fold decrease in the rate of inhibition of the proteases. However, the stoichiometry of inhibition of FIIa, APC, and FXa by native PCI, R238A and E239A variants were found to be close to 1.0, which also indicated the formation of stable complexes based on SDS-PAGE and western blot analysis with thrombin and APC. Our findings revealed the possible presence of an exosite in PCI that influences the protease inhibition rates., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Aim: To study the profile of biochemical markers of the hemostasis system, to clarify their role and relationships in the pathogenesis of the development of thrombotic complications (TC) of ischemic stroke (IS) and the associated assessment of the possibilities of their diagnostic application., Materials and Methods: The study group included 302 patients (164 men, 138 women) who were admitted to the hospital with a diagnosis of IS within 24 hours of the onset of the disease. The diagnosis was confirmed by computed tomography. The average age of patients was 69 (5088) years. Blood was taken from all patients on the 1st day of the disease to determine the profile of analytes presumably associated with the pathogenesis of TC. Levels of homocysteine, protein C inhibitor, thrombomodulin, plasminogen, tissue plasminogen activator, urokinase, plasminogen activator type 1 inhibitor, t-PA/PAI-1 complex, vitronectin, plasmin-2-antiplasmin complex, D-dimer, fibronectin were determined in blood serum by ELISA., Results: TC in the acute period of IS (up to 21 days) were recorded in 32 (10.6%, 95% CI 7.3714.3) patients, of which pulmonary embolism was observed in 27 (8.94%, 95% CI 5.9812.4) patients, deep vein thrombosis in 5 (1.66%, 95% CI 0.473.47) patients. The results of the study of a panel of specific proteins involved in pathogenetic processes accompanying necrosis of brain tissue in IS demonstrated that of the entire list of markers of the hemostasis system activation selected for the study, the most significant are: the concentration of fibronectin in the prognosis of the absence of TC with a threshold value of more than 61 mkg/ml and OR 4.4 (95% CI 1.512.9, p=0.011), and the concentration of the t-PA/PAI-1 complex in the prognosis of the development of TC with a threshold value of more than 14 ng/ml and OR 11.3 (95% CI 1.18109.3, p=0.03)., Conclusion: The significance of the t-PA/PAI-1 complex and fibronectin as markers of TC in IS may be due to a violation of the activation processes of the fibrinolytic link of hemostasis and the accumulation of non-deposited compounds that damage the vascular wall.

We aimed to identify proteins that were differentially regulated in spermatozoal samples collected from fertile healthy men (FHM) and infertile patients with varicocele (IFPV) before and after varicocelectomy. Seminal samples were collected from 20 IFPV before and after varicocelectomy and from 14 FHM as controls. Samples underwent seminal examination and proteomic analysis. Extracted spermatozoal proteins were analysed using two-dimensional gel electrophoresis, and differentially regulated spermatozoal proteins (DRSPs) were identified. In particular, attention was placed on those DRSPs in which the concentration changed after varicocelectomy and corrected to approximate levels observed in FHM. Varicocelectomy significantly improved the sperm count and concentration in IFPV (p < 0.05). Proteomic analysis showed that 11 DRSPs were identified when comparisons were made among the three groups. Among these 11 proteins, change in the SERPIN A5 concentrations was notable because it was 100-fold downregulated in pre-operative IFPV samples and nearly resembled to control concentrations following varicocelectomy. Western blot analysis using an anti-SERPIN antibody validated the changes observed in SERPIN A5 levels before and after varicocelectomy operation. Increase in SERPIN A5 after varicocelectomy may be due to improvement in semen quality, suggesting that SERPIN A5 is a potential seminal biomarker for assessment of semen quality in varicocele-related infertility., (© 2021 Wiley-VCH GmbH.)

Activated protein C (APC) acts as an "on demand" anticoagulant, reducing thrombin formation. Reduced plasma levels of APC or protein C (PC) are associated with an increased risk of venous thromboembolism. APC also displays cytoprotective functions and its therapeutic use has been evaluated in severe sepsis and is under evaluation in several diseases with an important inflammatory component. In addition, different studies have revealed a potential role of PC/APC in disorders such as obesity, pneumonia, disseminated intravascular coagulation, Alzheimer, stroke, etc. Accordingly, the therapeutic value of different recombinant APC molecules that lack anticoagulant activity but retain the cytoprotective function is being tested in clinical trials for some of these diseases. Therefore, an available method to measure circulating APC in plasma is of great interests. About 16 different methods for the quantification of APC have been reported. Here, we will review the available assays, highlighting their advantages and disadvantages as well as their different stages of implementation and the most appropriate use for each method, including their potential clinical usefulness., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Binding of serine protease inhibitors (serpins) to nonprotein ligands such as glycosaminoglycans or phospholipids has been shown to modify their inhibitory activity and-at least in the case of SERPINA5-to mediate serpin internalization into cells. Also phospholipid functions may be altered when bound to serpins or other proteins.By interacting with phospholipids, serpins might influence a variety of cellular functions. Binding of proteins to phospholipids can be studied by several methods. Here we describe solid-phase assays, in which pure phospholipids are immobilized on nitrocellulose membranes, PVDF membranes, or microtiter plates. Bound proteins are detected with specific antibodies and labeled secondary antibodies. We also describe a method visualizing binding of phospholipids in suspension by non-denaturing polyacrylamide gel electrophoresis (PAGE) followed by Western blotting.

Background/aim: To date there has been no investigation into the epigenetic regulation of the serine protease inhibitor SERPINA5 in prostate cancer, where lack of this gene was considered to facilitate invasive growth patterns., Materials and Methods: Methylation degrees of eight CpG sites of SERPINA5 were analyzed in normal and malignant prostate cells using nucleotide sequencing, methylation-specific high resolution melting and digital droplet PCR techniques., Results: The methylation degree of five CpG sites significantly correlated with lower SERPINA5 expression levels. In contrast, two CpG sites (at -19 bp and -14 bp from the transcription start site) were hypermethylated in normal epithelial prostate cells, benign hyperplasic cells and low-invasive malignant LNCaP cells, whereas in aggressive DU-145 and PC-3 cell lines, these sites were essentially unmethylated., Conclusion: Novel methylation patterns of two distinct CpG sites of the SERPINA5 promoter may be useful for differentiating benign from malignant prostate disease., (Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

SERPINA5 (proteinC inhibitor, plasminogen activator inhibitor-3) is a secreted, extracellular clade A serpin. Its main characteristics are broad protease reactivity and wide tissue distribution (in man). SERPINA5 has originally been described as an inhibitor of activated protein C and independently as an inhibitor of the plasminogen activator urokinase. SERPINA5 binds glycosaminoglycans, phospholipids, and retinoic acid. Glycosaminoglycans and certain phospholipids can modulate its inhibitory activity and specificity. Studies suggest that SERPINA5 may play a role in hemostasis, in male reproduction, in host defense, and as a tumor suppressor. However, its biological role has not yet been defined. So far SERPINA5 deficiency has not been described in man. Mouse models are of limited value, since in mice serpinA5 is almost exclusively expressed in the reproductive tract. Consistently the only obvious phenotype of serpinA5-knockout mice is infertility of homozygous males. SERPINA5 can be internalized by cells and translocated to the nucleus. The internalization is dependent on the phospholipid phosphatidylethanolamine and on the intact N-terminus of SERPINA5, which functions as a cell penetrating peptide. Further functional analysis of intracellular SERPINA5 will contribute to our understanding of the biological role of this molecule., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

It is generally accepted that the phospholipid bilayer of the cell membrane is impermeable for proteins and peptides and that these molecules require special mechanisms for their transport from the extra- to the intracellular space. Recently there is increasing evidence that certain proteins/peptides can also directly cross the phospholipid membrane. SERPINA5 (protein C inhibitor) is a secreted protease inhibitor with broad protease reactivity and wide tissue distribution. It binds glycosaminoglycans and certain phospoholipids, which can modulate its inhibitory activity. SERPINA5 has been shown to be internalized by platelets, granulocytes, HL-60 promyelocytic leukemia cells, and by Jurkat lymphoma cells. Once inside the cell it can translocate to the nucleus. There are several indications that SERPINA5 can directly cross the phospholipid bilayer of the cell membrane. In this review we will describe what is known so far about the conditions, as well as the cellular and molecular requirements for SERPINA5 translocation through the cell membrane and for its penetration of pure phospholipid vesicles.

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<0.001). The mean APC level in VTE patients was 0.034±0.009 nM (previous assay) and 0.037±0.009 nM (new assay), significantly lower than those in controls (P<0.001). In both groups there was a significant correlation between the levels obtained by the two assays (P<0.001). These results show that both assays are equivalent, and confirm that the APC level is lower in VTE patients than in healthy individuals. Therefore, the new simplified assay, which measures the sum of circulating free APC and APC complexed to PCI, may be used to estimate the level of circulating APC, and will allow its use in routine laboratories., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Introduction: Protein C inhibitor (PCI), a member of the serine protease inhibitor family, is expressed in various human tissues, including liver and kidneys. In the plasma, PCI physiologically inhibits an anticoagulant serine protease, activated protein C (APC). PCI expressed by cancer cells suppresses tumor invasion by inhibiting urokinase-type plasminogen activator, and inhibits tumor growth and metastasis, which are independent of its protease-inhibitory activity. In the present study, we clarified the effects of host PCI on growth and metastasis of B16 melanoma (B16) cells by comparing between wild-type mice and mice transgenic for human PCI gene (hPCI-TG), which have a tissue distribution of PCI similar to that observed in humans., Materials and Methods: Growth of intracutaneously-injected B16 cells was evaluated by measuring the tumor volume, and metastatic behavior of intravenously-injected B16 cells by counting the number of metastatic lung nodules., Results: Growth of intracutaneously injected B16 cells was significantly faster in wild-type mice than in hPCI-TG mice; however, hPCI-TG mice developed more metastatic nodules of B16 cells in the lungs. Immunohistochemical analysis using anti-mouse fibrinogen antibody revealed more fibrin deposition in the lung in hPCI-TG mice than in wild-type mice. Furthermore, the more invasive behavior observed in hPCI-TG mice was reduced by rabbit anti-human PCI IgG, APC, or soluble TM administration for 3 consecutive days including the day that B16 cells were injected., Conclusions: Our results suggest that like PCI expressed in tumor cells, host PCI also inhibits tumor growth, but host PCI promotes tumor metastasis via its procoagulant properties., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Protein C inhibitor (PCI) is a serpin with broad protease reactivity. It binds glycosaminoglycans and certain phospholipids that can modulate its inhibitory activity. PCI can penetrate through cellular membranes via binding to phosphatidylethanolamine. The exact mechanism of PCI internalization and the intracellular role of the serpin are not well understood. Here we showed that testisin, a glycosylphosphatidylinositol-anchored serine protease, cleaved human PCI and mouse PCI (mPCI) at their reactive sites as well as at sites close to their N terminus. This cleavage was observed not only with testisin in solution but also with cell membrane-anchored testisin on U937 cells. The cleavage close to the N terminus released peptides rich in basic amino acids. Synthetic peptides corresponding to the released peptides of human PCI (His(1)-Arg(11)) and mPCI (Arg(1)-Ala(18)) functioned as cell-penetrating peptides. Because intact mPCI but not testisin-cleaved mPCI was internalized by Jurkat T cells, a truncated mPCI mimicking testisin-cleaved mPCI was created. The truncated mPCI lacking 18 amino acids at the N terminus was not taken up by Jurkat T cells. Therefore our model suggests that testisin or other proteases could regulate the internalization of PCI by removing its N terminus. This may represent one of the mechanisms regulating the intracellular functions of PCI., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

The occurrence of various serine proteinases and serine proteinases inhibitors (SERPINs) was investigated by RT-PCR in whole testes of 1-, 3-, and 8-wk-old mice in crude and enriched germ cell fractions, mouse Leydig tumor cells (mLTC-1), and primary cultures of 3- and 8-wk-old enriched fractions of Leydig cells and 3-wk-old Sertoli cells. New members were identified in the testis protease repertoire. Within the Leydig repertoire, a PCR product was found for plasminogen activators urokinase plasminogen activator (uPA) and tissue plasminogen activator (8-wk-old cells), matriptase-2 (mLTC-1), kallikrein-21, SERPINA5, SERPINB2 (primary cultures), and serine peptidase inhibitor Kunitz type 2 (SPINT2). The gonadotropin regulation was explored by semiquantitative RT-PCR, using steroidogenic acute regulatory protein (StAR) as a positive control. Matriptase-2, kallikrein-21, SPINT2, and SERPINA5 were down-regulated, whereas uPA and its receptor were up-regulated by human chorionic gonadotropin (hCG) via cAMP in the mLTC-1 cells. Positive effects were observed transiently after 1-8 h of hCG exposure, and negative effects, first evidenced after 6 h, lasted 48 h. The hCG-induced effects were confirmed in primary cultures. In addition, SERPINB2 was augmented by hCG in primary cultures. Addition of either trypsin or protease inhibitors did not alter the hCG-induced surge of StAR. Because hCG regulated proteases and SERPINs (whereas testosterone did not), it could alter the proteolytic balance of Leydig cells and consequently the metabolism of extracellular matrix components. Therefore, even though a direct interplay between the early hCG-induced surge of uPA and StAR is unlikely, our data together with the literature suggest that extracellular matrix proteins alter Leydig cell steroidogenesis.

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 alpha-helices that sit upon an 8-stranded beta-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 alpha-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 blood plasma serine proteinase inhibitors (serpins) are glycoproteins whose activities are involved in many important homeostatic reactions. The heparin-dependent plasma serpins, antithrombin, heparin cofactor II and protein C inhibitor, regulate the proteinases of blood coagulation. Heparin and some other glycosaminoglycans increase the rate of proteinase inhibition by these three plasma serpins. Proteinases recognize a specific peptide, termed the reactive site, near the carboxyl-terminus of serpins (for antithrombin and protein C inhibitor this is Arg-Ser and for heparin cofactor II this is Leu-Ser). Additionally, these three serpins contain unique structural elements that confer glycosaminoglycan binding activities. The therapeutic anticoagulant action of the glycosaminoglycan heparin is believed to depend partially on heparin-accelerated inhibition of proteinases by antithrombin. The physiological importance of specific proteoglycans has been attributed to their recognition of these serpins and their biological 'activation' of these proteinase inhibitors.

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 (España 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 alpha 1-antitrypsin (alpha 1 AT), 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 alpha 1AT 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 alpha 1AT, the level of PCI activity measured by method B increased parallel to the increase in alpha 1AT 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:alpha 1AT complex increased parallel to the increase in the amount of alpha 1AT 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 alpha 1AT and are not reliable for the determination of functional PCI levels in patients with altered levels of alpha 1AT.(ABSTRACT TRUNCATED AT 400 WORDS)

Activation and inactivation of protein C during the clinical course of disseminated intravascular coagulation (DIC) was studied in three patients by qualitative (Western blotting) and quantitative (ELISA) analysis and the intensity of procoagulant activity monitored by the measurement of thrombin and factor Xa antithrombin III complexes. In one patient, inhibitor complexes of APC with protein C inhibitor (PCI) and alpha 1-antitrypsin (alpha 1-AT) were observed and the latter predominated at presentation. Both disappeared during the development of remission but the loss of alpha 1-AT complexes preceded PCI complexes which on Western blotting appeared to increase in intensity prior to disappearance. The two other patients bled to death from uncontrollable haemorrhage. In both cases, APC/inhibitor complexes with alpha 2-macroglobulin (alpha 2-M) in addition to PCI and alpha 1-AT were detected and persisted until death. Although PCI appeared to be the primary inhibitor in all three cases, alpha 1-antitrypsin and particularly alpha 2-macroglobulin appeared to assume greater roles in the two fatal cases. These data are similar to previous findings in an experimental animal model of DIC that suggested that alpha 2-macroglobulin and alpha 1-antitrypsin become more important inhibitors of APC as the primary inhibitor PCI is consumed in the face of a sustained procoagulant challenge.

The region and mechanism of urinary protein C inhibitor (PCI) binding to fibrin(ogen) were examined using fibrin(ogen)-Sepharose and ligand blotting. Urinary PCI bound to fibrin(ogen)-Sepharose in a heparin-dependent manner at a level about 1.6-fold higher to fibrin-Sepharose than to fibrinogen-Sepharose. Scatchard analysis of the binding between urinary PCI and fibrin(ogen)-Sepharose showed that the Kd for fibrin-Sepharose and fibrinogen-Sepharose were 4.0 nM and 5.7 nM respectively. Ligand blotting using urinary PCI and an enzyme-linked immunoassay showed that urinary PCI bound to fibrinogen, fibrinogen degradation products (X, Y, D and E) and the A alpha-, B beta- and gamma-chains of fibrinogen. Binding between urinary PCI and fibrin(ogen)-Sepharose was slightly suppressed (16%) by alpha-methylmannose and largely suppressed (42%) by EACA, which indicates that the carbohydrate chain and the lysine binding site participate in the binding. These findings suggest that urinary PCI binds to fibrin(ogen) via the A alpha-, B beta- and gamma-chains and its binding is partly mediated by carbohydrate and the lysine binding site.

Alpha 1-antitrypsin (alpha 1AT; protease inhibitor [PI] locus), alpha 1-antichymotrypsin (alpha 1ACT; AACT locus), corticosteroid-binding globulin (CBG; CBG locus), and protein C inhibitor (PCI; PCI locus) are members of the serine protease inhibitor (serpin) superfamily. A noncoding PI-like (PIL) gene has been located 12 kb 3' of the PI gene. The PI, PIL, and AACT loci have been localized to 14q32.1, the CBG locus has been localized to 14q31-14q32.1, and PCI has been mapped to chromosome 14. Genetic linkage analysis suggests tight linkage between PI and AACT. We have used pulsed-field gel electrophoresis to generate a physical map linking these five serpin genes. The order of the genetic loci is AACT/PCI-PI-PIL-CBG, with a maximum distance of about 220 kb between the AACT/PCI and PI genes. These genes form a PI cluster at 14q32.1, similar to that of the homologous genes on murine chromosome l2. The close proximity of these genes has implications for disease-association studies.

Activated protein C (APC), a serine protease, is regulated in plasma by protease inhibitors. This study was undertaken to determine the role of the major plasma inhibitors in regulating APC in plasma. Kinetic analysis and specific immunoassays for APC-inhibitor complexes were used to determine the inhibitors that form complexes with APC. Of the eight plasma inhibitors investigated, four interact with APC: protein C inhibitor (PCI), alpha 1-proteinase inhibitor (PI), alpha 2-antiplasmin (AP) and C1 esterase inhibitor (C1 Inh). The second order rate constants are: 1.3 x 10(4) M-1 s-1 (PCI); 15 M-1 s-1 (PI); 410 M-1 s-1 (AP); and < 6 M-1 s-1 (C1 Inh), with a relative effectiveness of each inhibitor to inactivate APC in plasma: 49:36:15: < 1, respectively. PCI, PI and AP are the major inhibitors of APC in plasma. Low concentrations of APC will be inhibited by PCI with PI and AP playing a secondary role. However, as increasing APC is generated, PI and AP begin to play more important roles as the PCI is consumed.

Protein C inhibitor (PCI), a glycosaminoglycan (GAG) dependent serine protease inhibitor, inhibits its target proteases by forming SDS-stable 1:1 complexes. GAGs alter target enzyme specificity of PCI in such a way that e.g. urokinase (uPA) is the preferred target enzyme in the presence of GAGs while in their absence preferentially tissue kallikrein (TK) complexes are formed. The effect of the GAG-binding adhesive glycoprotein vitronectin (Vn) on the GAG-stimulated inhibition of uPA by PCI was studied using an amidolytic assay. In the presence of heparin, Vn protected uPA from inhibition by PCI in a dose-dependent manner with respect to both, Vn- and heparin-concentration. Vn also was active when heparin was replaced by low-molecular weight heparin or heparan sulfate, respectively. In the absence of GAGs, Vn had no effect on the inhibition of uPA by PCI. In a similar system, Vn was far less effective in modifying the inhibitory function of heparin on the inhibition of TK by PCI. When equimolar concentrations of radiolabelled uPA and TK were incubated with PCI in the presence of heparin, only complexes of PCI with uPA were detectable. Addition of Vn reduced this complex formation, whereas, in contrast, complexes of PCI and TK appeared. These results indicate that Vn modulates both, the activity and specificity of PCI and suggest different structural heparin-requirements for the PCI/uPA versus PCI/TK interaction.

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

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.

The objectives of this study were to determine the genetic basis of the electrophoretic differences of human plasma protein C inhibitors (PCI) from 977 individuals. Three discrete antibodies were produced against the PCI purified from human plasma and peptides that corresponded to the N-terminal 15 amino acid residues and the C-terminal 15 residues of human PCI, the chemical structures of which were determined by cDNA sequence analysis. The combined techniques of polyacrylamide gel isoelectric focusing and immunoblotting with these three different antibodies resolved the plasma PCI into several isoprotein bands, with a pH range of 6-7. These PCI isoproteins, however, were not stained by anti-human kallikrein, anti-human protein C or anti-human urokinase antibodies. Therefore, each of the PCI bands, which were detected by immunoblotting with the anti-PCI antibody and the two different anti-peptide antibodies, were derived from free PCI, and not an inactive PCI species. Two common phenotypes, designated PCI 1 and 1-2, were recognized, and family studies showed that they represented homozygosity or heterozygosity for two autosomal codominant alleles, PCI*1 and PCI*2. A population study of plasma samples collected from 977 Japanese individuals indicated that the frequencies of the PCI*1 and PCI*2 alleles were 0.988 and 0.012, respectively.

We studied the inhibition of tissue kallikrein by protein C inhibitor (PCI), a relatively unspecific heparin-dependent serine protease inhibitor present in plasma and urine. PCI inhibited the amidolytic activity (cleavage of H-D-valyl-L-leucyl-arginine-p-nitroaniline) of urinary kallikrein with an apparent second order rate constant of 2.3 x 10(4) M-1 s-1 and formed stable complexes (85 kDa) with urinary kallikrein as judged from silver-stained sodium dodecyl sulfate-polyacrylamide gels. Complex formation was time-dependent and was paralleled by a decrease in the intensity of the main PCI protein band (Mr = 57,000) and an increase in the intensity of the lower Mr (54,000) PCI form (cleaved inhibitor). Heparin interfered with the inhibition of tissue kallikrein by PCI and with the formation of tissue kallikrein-PCI complexes in a dose-dependent fashion and completely abolished PCI-tissue kallikrein interaction at 300 micrograms/ml. This is in contrast to findings on the interaction of PCI with all other target proteases studied so far (i.e. stimulation of inhibition by heparin) but is similar to the reaction pattern of 125I-labeled tissue kallikrein with so called kallikrein binding protein described in serum and other systems. To study a possible relationship between PCI and this kallikrein binding protein we incubated 125I-labeled urinary kallikrein in serum and in PCI-immunodepleted serum in the absence and presence of heparin and analyzed complex formation using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In normal serum, formed complexes co-migrated with complexes of purified PCI and 125I-kallikrein and were less intense in the presence of heparin. No complex formation at all was seen in PCI-depleted serum. Our data indicate that PCI may be a physiologically important endogenous inhibitor of tissue kallikrein and provide evidence that PCI may be identical to the previously described kallikrein binding protein.

Effects of zinc and calcium ions on the heparin-neutralizing abilities of histidine-rich glycoprotein (HRG) and platelet factor 4 (PF4) were examined. Both HRG and PF4 effectively neutralized the ability of heparin to accelerate the activated protein C (APC) and the thrombin inhibitions by protein C inhibitor (PCI). the heparin-neutralizing ability of HRG in the APC inhibition by PCI, however, was decreased in a Ca(2+)-dependent manner and apparently lost at 1 mM Ca2+, while it was enhanced by Zn2+ regardless of the presence or absence of Ca2+. The heparin-neutralizing ability of HRG in the thrombin inhibition by PCI was not affected by Ca2+. In contrast to HRG, there was no significant difference in the heparin-neutralizing ability of PF4 in the presence or absence of 1 mM Ca2+. These results strongly suggest additional physiological functions of HRG and PF4 as modulators of PCI.

Protein C inhibitor (PCI) inhibits tissue kallikrein by forming stable 1:1 complexes (k1 = 2.3 x 10(4)M-1s-1). Heparin inhibits the tissue kallikrein/PCI-interaction and complex formation of 125I-tissue kallikrein in serum. 125I-tissue kallikrein complexes formed in plasma can be immunoprecipitated with monoclonal anti-PCI IgG suggesting that PCI might be identical to the kallikrein binding protein described previously (J. Chao et al. 1986, Biochem. J. 239, 325-331).

Protein C inhibitor (PCI) is a heparin-dependent serpin present in a native form in plasma at concentrations of 5 micrograms/mL. In vitro, PCI inhibits activated protein C (APC), thrombin, plasma kallikrein (KK) and urokinase-(uPA) and tissue-type plasminogen activator (tPA), and we have shown in vivo inhibition of APC, uPA and KK by PCI. In order to further characterize the physiological role of PCI, we have measured the level of PCI in several biological fluids. PCI antigen was assayed by ELISA and PCI activity was measured by its capability to form complexes with APC in the presence of heparin. Seminal plasma from voluntary donors had PCI levels (160 +/- 20 micrograms/mL, mean +/- SD) about 30 or 40 times higher than those found in blood plasma. Patients under a fertilization program had significantly reduced PCI seminal levels (110 +/- 35 micrograms/mL). Seminal plasma PCI retained about 45% of its activity immediately after ejaculation, and the activity rapidly decreased following incubation of seminal plasma at 37 degrees C, in parallel with the appearance of complexes of PCI with prostate-specific antigen (PSA). PCI was present in seminal vesicle secretion, obtained by autopsy, at concentration similar to that observed in semen, was mostly active and was not inactivated by incubation of secretion at 37 degrees C. The mean functional and antigen levels of PCI in urine from normal donors were 0.58 and 0.25 micrograms/mL, respectively, whereas in saliva these levels were 20 and 0.8 ng/mL, respectively. Amniotic fluid contained PCI antigen levels of 2.1 +/- 0.2 microgram/mL. These results show that PCI is secreted in the seminal vesicles in a functional form, and suggest that PSA, a major secretory component of the prostate, is responsible for its inactivation. They also suggest a physiological role of PCI in reproduction, and show that PCI is present in various biological fluids.

Three types of antibody, which were specific to the purified human protein C inhibitor (PCI) and to two synthetic peptides, which corresponded to the N-terminal 15 amino acid residues (PCI-N) and C-terminal 15 residues (PCI-C) of PCI, were produced. Plasma PCI patterns, which were shown by means of polyacrylamide gel isoelectric focusing followed by immunoblotting with the antibody to purified PCI, were almost identical to those obtained with the antibodies to the peptides, PCI-N and PCI-C. The latter antibodies against the synthetic peptides proved very useful for biochemical or genetic analysis of human plasma PCI, as such peptide antigens could be produced without any of the tedious procedures necessary to obtain purified PCI as an immunogen.

Protein C inhibitor (plasminogen activator inhibitor-3) is a plasma glycoprotein and a member of the serine proteinase inhibitor superfamily. In the present study, the human gene for protein C inhibitor was isolated and characterized from three independent phage that contained overlapping inserts coding for the entire gene. The genomic DNA was isolated and studied by restriction mapping, polymerase chain reaction analysis, and DNA sequencing. The gene was 11.5 kilobases in length and consisted of five exons separated by four introns. In addition, 0.8 kilobases of DNA from the 5'-flanking region were sequenced. The exon-intron boundaries all observed the "GT-AG" rule. The gene for protein C inhibitor was assigned to chromosome 14 by polymerase chain reaction analysis of human/hamster hybrid cell lines. The organization of the gene for protein C inhibitor is similar to the genes coding for alpha 1-antitrypsin and alpha 1-antichymotrypsin. The genes for these two proteins are also localized on chromosome 14 suggesting a recent evolution of the genes for these three proteins from a common ancestor.

Mediterranean spotted fever (MSF) is a rickettsiosis that induces widespread microvascular injury. To obtain quantitative information on the in vivo activation and inactivation of the protein C system during the acute phase of endothelial damage, several components of the protein C pathway were studied in 28 MSF patients. Upon admission (day 1), patients showed clear evidence of endothelial damage as reflected by the significant decrease in the ratio VIII:C/vWF:Ag (0.36 +/- 0.14, mean +/- SD) compared with normals (0.98 +/- 0.14), and clinical and laboratory signs of hemostatic alterations such as decreased platelet count, positive fibrinogen/fibrin degradation products, and increased thrombin:antithrombin-III complex levels. Antigenic protein C (72% +/- 18%) and protein C inhibitor (PCI) (41% +/- 20%) were significantly decreased (P less than .001). Complexes of activated protein C (APC) with PCI or with alpha 1-antitrypsin (alpha 1AT) and of plasma kallikrein with PCI (KK:PCI) were measured using sandwich enzyme-linked immunosorbent assays. APC:alpha 1AT complex levels were increased in patients at day 1 (27 +/- 13 ng/mL) compared with controls (7 +/- 2 ng/mL), and APC:PCI and KK:PCI complexes, which were not detectable in any of the controls, were present in 57% and 75% of the 28 MSF patients, with mean levels of 11 +/- 5 and 46 +/- 16 ng/mL, respectively. After remission of the disease (day 30), a trend toward normal values in the majority of the parameters studied was found. This study shows that, in the course of endothelial injury, MSF patients experience a generalized activation of the protein C pathway, resulting in consumption of protein C and PCI, and in the appearance of APC:inhibitor complexes. Moreover, these data provide the evidence that KK:PCI circulating complexes occur in vivo.

Protein C inhibitor (PCI), also known as plasminogen activator inhibitor 3, inhibits a variety of serine proteases by forming sodium dodecyl sulfate-stable 1:1 complexes. In purified systems PCI is only a weak inhibitor of urokinase. Nevertheless, complexes between PCI and urokinase are found in appreciable amounts in native human urine. Since PCI activity is stimulated by heparin and other glycosaminoglycans, we investigated the presence of stimulating glycosaminoglycans on cells lining the urinary tract. We chose the epithelial kidney tumor cell line TCL-598 as a model and isolated metabolically labeled glycosaminoglycans. TCL-598 incorporated [35S] sulfate into high Mr components (Mr greater than 200,000 and approximately 75,000) as judged from sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of cell extracts; the Mr greater than 200,000 component bound specifically to PCI-Sepharose 4B and was eluted either with heparin (5 mg/ml) or with NaCl (2.0 M). Treatment of this PCI-binding material with chondroitinase ABC, but not with chondritinase AC or heparitinase, abolished binding to PCI-Sepharose, confirming the glycosaminoglycan nature of this material and suggesting the involvement of dermatan sulfate in binding. These glycosaminoglycans eluted from PCI-Sepharose stimulated urokinase inhibition by PCI in a dose-dependent way and enhanced complex formation of 125I-urokinase and PCI as did in control experiments dermatan sulfate from porcine skin and from bovine mucosa. Our results suggest that PCI activity might be regulated also in vivo by the presence or absence of stimulating glycosaminoglycans; dermatan sulfate-containing glycosaminoglycans associated with kidney cells might be responsible for stimulation of the urokinase inhibitory activity of PCI in the urinary tract; the type of glucosaminoclycans might furthermore regulate enzyme specificity of PCI.

Protein C is a vitamin K-dependent regulator of blood coagulation. Activated protein C is regulated in plasma in large part by two inhibitors, protein C inhibitor and alpha 1-antitrypsin. Complexes of activated protein C with both inhibitors in plasma samples from subjects with normal or pathologic pregnancy were measured. In normal pregnancy we observed a progressive and significant increase in activated protein C/alpha 1-antitrypsin complex levels, from 9 +/- 3 ng/ml in the first trimester to 16 +/- 3 ng/ml in the third trimester, as well as an increase in alpha 1-antitrypsin plasma levels. In severe preeclampsia, but not in chronic hypertension with superimposed severe preeclampsia, there was a greater increase in activated protein C/alpha 1-antitrypsin levels (25 +/- 10 ng/ml) (p less than 0.001) and a decrease in protein C and protein C inhibitor levels as compared with normal pregnant women at similar gestational ages. These data show an increase in the activation of the protein C pathway in both normal and pathologic pregnancy and provide evidence for an enhancement of thrombin generation in severe preeclampsia compared with chronic hypertension with superimposed severe preeclampsia.

In vivo complex formation of activated protein C with protein C inhibitor (APC-PCI) and with alpha 1-antitrypsin (APC-alpha 1AT) following infusion of 0.25 or 1.0 mg APC/kg in 1 hour into baboons was studied using immunoblotting and sandwich enzyme-linked immunosorbent assay (ELISA)s. Before APC infusion, detectable plasma levels (about 30 ng/mL) of APC-alpha 1AT complex were found in the baboon plasma. At the lower APC dose, APC-PCI and APC-alpha 1AT complex levels were 1.4 +/- 0.3 (mean +/- SD) and 0.8 +/- 0.1 microgram/mL after 1 hour of infusion. At the higher APC dose, the APC-PCI level was similar to the APC-alpha 1AT level during the first 30 minutes, but after 1 hour of infusion the APC-alpha 1AT level was higher than the APC-PCI level, reaching 4.1 +/- 1.2 and 2.9 +/- 1.2 microgram/mL, respectively. After 24 hours, complex levels had returned to basal conditions. During infusion of protein C (1.0 mg/kg in 1 hour), both complexes were detected in low concentrations. Following bolus injection of APC, half-lives (t1/2) for APC and APC-PCI and APC-alpha 1AT complexes of 10, 40, and 140 minutes, respectively, were observed. After 1-hour incubation with 2.5 micrograms/mL APC, baboon plasma contained 1.0 +/- 0.2 and 0.8 +/- 0.1 microgram/mL of APC-PCI and APC-alpha 1AT, respectively. Addition of 10 micrograms/mL APC to baboon plasma yielded 2.5 and 2.4 micrograms/mL APC-PCI and APC-alpha 1AT after 1 hour, respectively. Immunoblotting analysis also showed in vivo formation of complexes of APC with an auxilliary inhibitor but not in vitro in citrated plasma. These data show that both PCI and alpha 1AT are physiologic inhibitors of APC and suggest that when PCI is depleted by a high dose of APC, alpha 1AT becomes the major inhibitor of APC.

The interaction between plasma kallikrein (KK) and protein C inhibitor (PCI) and the influence of KK on the complex formation between activated protein C (APC) and PCI was studied in purified systems as well as in plasma in order to assess the significance of these reactions in the plasma milieu. PCI complexed to KK (KK:PCI) or to APC (APC:PCI) was measured by sandwich ELISA's using antibodies directed against each protein in the complexes. The formation of KK:PCI complexes assayed by this method paralleled the inhibition of KK amidolytic activity by PCI in purified system. Incubation of normal plasma (NHP) at 4 degrees C, which can induce prekallikrein activation due to cold activation, resulted in PCI inactivation and appearance of KK:PCI complexes. PCI activity fell to 35% of the NHP and 1.2 micrograms/ml of KK:PCI complex was formed. However, incubation of NHP at room temperature or of prekallikrein deficient plasma at 4 degrees C did not result in significant decrease of PCI activity. Thus the PCI inactivation was associated with prekallikrein activation and complexation to PCI following cold activation. Incubation of exogenous purified KK with NHP resulted in PCI inactivation and complexation with KK in a temperature-dependent manner. Addition of 2.8 micrograms/ml KK to plasma at 4 degrees C resulted in the inactivation of 55% of plasma PCI and the formation of 0.9 microgram/ml KK:PCI which represents 21% of the KK added, whereas at 37 degrees C PCI was inactivated to 30% and only 0.30 microgram/ml KK:PCI complexes were measured. These results indicate that PCI is a major KK inhibitor at 4 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

The rates of clearance and catabolism of human protein C inhibitor (PCI) and human alpha 1-antitrypsin (alpha 1-AT) and their complexes with human activated protein C (APC) were studied in the rabbit. The radioiodinated-free inhibitors had biologic half-lives of 23.4 and 62.1 hours, respectively, while the corresponding *I-labeled activated-protein C complexes were cleared with half-lives of 19.6 +/- 3.1 and 72.2 +/- 6.1 minutes. Complex clearances were linked to their catabolism as shown by a correlation between clearance and the appearance of free radioiodine in the plasma. Thus, the difference in the rates of catabolism would result in a fivefold greater amount of alpha 1-AT-APC complex than PCI-APC complex 1 hour after the formation of equal amounts of these in vivo. These results lead to the conclusion that the relative contribution of PCI and alpha 1-AT to the physiologic inhibition of APC cannot be determined only from the rates of the formation of these complexes in vitro, or from measurement of their levels in plasma. The APC-PCI complex is unstable as compared with the APC-alpha 1-AT complex, compounding the problem of estimating rates of complex formation from their levels in plasma.

Glycosaminoglycans (GAGs) including heparin accelerate the inhibition of serine proteases by serine protease inhibitors (serpins), an essential process in regulating blood coagulation. to analyze the molecular basis for GAG recognition by the plasma serpin protein C inhibitor (PCI; also known as plasminogen activator inhibitor 3), we have constructed a complete, energy-minimized, three-dimensional model of PCI by using the structure of homologous alpha 1-antitrypsin as a template. Sequence analysis, hydrogen-bonding environment, and shape complementarity suggested that the N-terminal residues of PCI, which are not homologous to those of alpha 1-antitrypsin, form an amphipathic alpha-helix, here designated A+ since it precedes the alpha 1-antitrypsin A helix. Electrostatic calculations revealed a single, highly positive surface region arising from both the A+ and H helices, suggesting that this two-helix motif is required for GAG binding by PCI. The dominant role of electrostatic interactions in PCI-heparin binding was confirmed by the strong ionic strength dependence of heparin stimulation. The involvement of the A+ helix in heparin binding was verified by demonstrating that an anti-PCI antibody that specifically binds the A+ peptide blocks heparin binding.

Components of the natural anticoagulant system (NAS) and anticardiolipin antibodies were examined in 21 patients with lupus anticoagulant (LA), 13 of whom had past histories of thrombotic episodes. No relationship could be shown between the antigenic levels of protein C and S (PC, PS) and a history of thrombosis. Inhibition of the anticoagulant activity of activated protein C (APC) was observed using plasma from 20/21 patients when phospholipid vesicles were used as the surface for the coagulation reaction. This effect was not affected by the addition of PS. When platelet membranes were employed only 2/21 patients demonstrated inhibition of APC. Under the latter condition, PS functional activity was inhibited in 7/21 patients, six of whom had a past history of thrombosis. Reduced antithrombin III or heparin cofactor II levels were observed in a total of 4/21 patients and may have contributed to the development of thrombosis in three of these patients. Antibodies specifically directed against these proteins were not detected suggesting the possibility of an associated constitutional deficiency. Anticardiolipin antibodies, though elevated in 17/21 patients, did not serve as a useful marker for an increased risk of thrombosis, and the level did not correlate with inhibition of the activity of APC or PS. We conclude that the mechanism of thrombosis in patients with LA is multi-factorial. A subset of patients in whom LA specifically inhibits PS function may represent patients who are at significant risk from thrombosis.

We developed an ELISA to quantitate complexes of activated protein C (APC) with a major plasma APC inhibitor, alpha 1-antitrypsin (alpha 1AT) in human plasma based on the sandwich principle using two different antibodies directed towards protein C and alpha 1AT, respectively. This ELISA test was specific for APC:alpha 1AT complexes and sensitive to greater than or equal to 150 pg complex. Fifty-one of 56 healthy donors had APC:alpha 1AT complex levels above the detection limit (3 ng/ml) ranging from 4 to 14 ng/ml (mean value +/- SD: 7.6 +/- 2.5 ng/ml). Patients (n = 10) with disseminated intravascular coagulation (DIC) had detectable levels of APC:alpha 1AT complex ranging from 21 to 125 ng/ml (median: 69 ng/ml). Complexes of APC with plasma protein C inhibitor (PCI) were also measured using an ELISA sandwich assay. None of the 30 healthy donors had detectable levels (greater than or equal to 5 ng/ml) of APC:PCI complex, and plasma samples from 9 of 10 DIC patients had detectable concentrations of APC:PCI complex ranging from 10 to 63 ng/ml (median: 22 ng/ml). APC:alpha 1AT complex was detected in 25 of 26 patients with deep venous thrombosis (DVT), with levels ranging from 5 to 136 ng/ml (median: 23 ng/ml), whereas APC:PCI was detected in only 6 DVT patients, with levels between 11 and 105 ng/ml. PCI antigen levels in 70 normals ranged from 56 to 175% (mean +/- SD: 99.1% +/- 24.2%). PCI antigen levels were decreased in DIC patients, in patients with cerebral arterial thrombosis, and in DVT patients undergoing heparin therapy, but not in patients with myocardial infarction. PCI antigen levels were decreased much further in DVT patients receiving heparin compared to those not receiving heparin, showing that heparin therapy is associated with a decrease in PCI levels. The detection in normal subjects and in thrombotic patients of circulating APC:inhibitor complexes supports the view that the protein C pathway is activated during DIC and DVT. Moreover, it emphasizes that both PCI and alpha 1AT are physiologic inhibitors of APC. Thus, measurement of APC complexes may provide sensitive parameters for specific detection of activation of the clotting and protein C pathways.

The protein C inhibitor (PCI) concentration and other parameters of the protein C pathway were studied in 19 patients with symptomatic acute deep vein thrombosis before and during the first 5 days of heparin treatment. The mean levels of PCI antigen and activity decreased rapidly and significantly during the first day of heparin therapy from 83 and 84% to 60 and 59% of the pooled normal human plasma (p less than 0.01), respectively, and to 56 and 54% after 5 days of treatment (p less than 0.01). In contrast, antithrombin III decreased progressively 25% during 5 days of heparin treatment. Protein C antigen and activity and total protein S remained unchanged during heparin treatment. Free protein S was decreased before heparin treatment (83%, p less than 0.05) and increased to normal values after 5 days of treatment. C4b-binding protein was significantly increased before and during heparin treatment (p less than 0.01). Activated protein C (APC) complexed to its two major plasma inhibitors, PCI and alpha 1-antitrypsin (alpha 1AT) were measured by specific ELISA's. Before treatment, 18 of the 19 patients studied had increased levels of APC:alpha 1AT complexes with a mean value of 27 +/- 22 ng/ml (range, 6-86 ng/ml) compared to normal values (8 +/- 2 ng/ml) and 12 of the patients also had detectable APC:PCI complex levels with a mean value of 11 +/- 17 ng/ml (range, 5-68 ng/ml). Both APC:inhibitor complexes decreased significantly during heparin treatment.

A 38-year-old patient with cerebral P. falciparum malaria was admitted 12 days after a short trip to Kenya. The serum level of tumor necrosis factor (TNF-alpha) was elevated (251 pg/ml). In contrast, Protein C (plasma activity 36.1%; antigen concentration 31.7%) and protein C inhibitor 1 (activity 0.55 U/ml) levels were decreased. This suggested a state of functional activation of the clotting system which was confirmed by elevated levels (4.8 ng/ml) of circulating thrombin-antithrombin-III-complexes (TAT). Protein S (total and free) and coagulation factor IX levels were within normal range. Under successful antiparasitic therapy, TNF-alpha as well as protein C and protein C inhibitor 1 levels returned to baseline within one week. In the context of other studies that demonstrate procoagulant effects of TNF-alpha, it is remarkable that in the case of complicated P. falciparum malaria, an elevated concentration of TNF-alpha can be paralleled by a decreased plasma level of protein C and an increase in TAT suggesting a procoagulant state.

Bovine plasma protein C inhibitor was purified; it was then characterized in comparison with human protein C inhibitor. The specific inhibitory activity of the purified inhibitor for bovine activated protein C was 8,500 times that of the inhibitor in plasma. The purified inhibitor showed a single band with Mr 56,000 by SDS-PAGE at pH 7.0, and two bands at pH 8.8, a major one with Mr 56,000 and a minor one with Mr 105,000, under both unreduced and reduced conditions. The pI range of the inhibitor was between 4.4 and 6.1. The Mr of the inhibitor was reduced by treatment with neuraminidase, O-glycanase, and also with glycopeptidase-A, suggesting that the inhibitor has both Asn-linked and Ser/Thr-linked carbohydrate chains. Twenty-seven of the NH2-terminal 49 amino acid residues of the bovine inhibitor, which lacks the first 4 residues from the NH2-terminal amino acid sequence of human inhibitor, were identical to those of the human inhibitor. The bovine inhibitor inhibited bovine and human activated protein C, human thrombin, Factor Xa, Factor XIa, and plasma kallikrein with Ki = 1.0, 5.2, 2.6, 3.0, 1.3 X 10(-8) M, and 4.5 X 10(-9) M, respectively. The inhibitory rates for activated protein C and thrombin were accelerated significantly in the presence of heparin or negatively charged dextran sulfate. However, the acceleration by heparin or dextran sulfate for the inhibition of Factor Xa, Factor XIa, and plasma kallikrein was not significant. The bovine inhibitor did not inhibit human Factor XIIa or plasmin.(ABSTRACT TRUNCATED AT 250 WORDS)

An assay system for protein C (PC) activity and PC-inhibitor in plasma was developed. The assay was based on: (1) binding of PC to wells of a microtiter plate coated with a murine monoclonal anti-PC antibody (C3) that did not interfere with the activity or activation of PC; (2) activation of immobilized PC with Protac C; (3) incubation with or without a source of activated PC inhibitor; and (4) measurement of amidolytic activity using the substrate S-2366. The activity assay was specific for PC and sensitive to less than 1 microliter of plasma or 4 ng PC. Inhibition of activated PC by plasma followed pseudo first order kinetics. Heparin caused a dose dependent increase in the inhibition rate with half maximal stimulation at approximately 3 U/ml and maximal stimulation at heparin concentrations greater than or equal to 10 U/ml. This assay is suitable not only for determination of functional plasma levels of PC and PC inhibitor activities but also for kinetic studies of inhibition of activated PC in complex systems, such as plasma. Studies showed that urokinase interfered with the inhibition of APC by plasma inhibitor(s).

Activated protein C is an anticoagulant plasma protease enzyme that inactivates Factors V and VIII in plasma. Normal plasma contains a protein that inhibits activated protein C and that is distinct from previously described plasma protease inhibitors. Protein C inhibitory activity is not detectable in plasmas from four unrelated patients with combined Factor V/VIII deficiency but is present in normal amounts in plasmas from patients with simple factor V deficiency or Factor VIII deficiency. It is suggested that the molecular basis for combined Factor V/VIII deficiency that exhibits simple autosomal recessive inheritance is a deficiency of protein C inhibitor.

A cDNA library in lambda-phage lambda gt11 containing DNA inserts prepared from human liver mRNA was screened with monoclonal antibodies to human protein C inhibitor. Six positive clones were isolated from 6 X 10(6) phages and plaque purified. The cDNA in the phage containing the largest insert, which hybridized to a DNA probe prepared on the basis of the amino-terminal amino acid sequence of the mature inhibitor, was sequenced. This cDNA insert contained 2106 base pairs coding for a 5'-noncoding region, a 19-amino acid signal peptide, a 387-amino acid mature protein, a stop codon, and a long 3'-noncoding region of 839 base pairs. Based on the amino acid sequence of the carboxyl-terminal peptide released by cleavage of protein C inhibitor by activated protein C as well as by thrombin, the reactive site peptide bond of protein C inhibitor is Arg354-Ser355. Five potential carbohydrate-binding sites were found in the mature protein. The high homology of the amino acid sequence of protein C inhibitor to the other known inhibitors clearly demonstrates that protein C inhibitor is a member of the superfamily of serine protease inhibitors including alpha 1-antichymotrypsin, alpha 1-antitrypsin, antithrombin III, ovalbumin, and angiotensinogen. Based on the difference matrices for these proteins, we present possible phylogenetic trees for these proteins.

Enzyme-linked immunosorbent assays (ELISA's) were developed for the measurement of protein C inhibitor (PCI) antigen and activity and for its complexes with activated protein C (APC) in plasma. For PCI activity and antigen, APC or anti-PCI, respectively, was immobilized to microtiter plates and PCI bound was detected with labelled anti-PCI antibodies. For APC:PCI complexes, two different antibodies directed against protein C and PCI were used. The assays for PCI were calibrated with pooled normal human plasma (NHP) and with purified PCI, and for APC:PCI complexes with known concentrations of purified pre-formed complexes added to buffer or to plasma. The lower limit of sensitivity of the PCI activity and antigen assays was 10 ng/ml and 0.5 ng/ml, respectively and for plasma APC:PCI complexes 12 ng/ml. Mean coefficients of variation of 1.5% to 5.8% (intra-assay) and 2.1% to 9.8% (inter-assay) were found for the assays. For PCI antigen, a range of 56% to 162% of the NHP value was obtained in samples from 70 healthy donors (mean +/- SD = 98.6% +/- 23.1%). For PCI activity, the range was 59% to 148% (94.3% +/- 20.2). A good correlation (0.92) was obtained when both assays were compared. Plasma levels of APC:PCI complexes in 30 normals were under the detection limit (less than 12 ng/ml). In plasma samples from 10 patients with disseminated intravascular coagulation (DIC) PCI antigen concentrations were decreased (55.6% +/- 20%) and 8 of the patients had APC-PCI complex levels between 32 and 240 ng/ml (median, 35 ng/ml). After addition of 20 micrograms/ml APC to NHP or to protein C depleted plasma, 6.1 micrograms/ml complexes were recovered after 90 min incubation. Incubation of 10 micrograms/ml APC with NHP in the presence of 10 U/ml heparin yielded 11 micrograms/ml complexes after 90 min, which represent more than 90% of the maximum possible value. Thus, the method should be adequate to study complexes of APC in vivo in clinical conditions in which activation of protein C pathway may occur.