Evaluation the Presence of SERPINA5 (Exon 3) and FTO rs9939609 Polymorphisms in Papillary Thyroid Cancer Patients.

BACKGROUND: A few researches evaluated the association of polymorphisms at SERPINA5 and fat mass and obesity-associated protein (FTO) genes with papillary thyroid cancer (PTC) globally. Here, we examined the presence of genetic variations within coding exon 3 of SERPINA5 gene and FTO rs9939609 polymorphism in Iranian PTC patients. METHODS: A total of 122 patients (42 cases for SERPINA5 and 80 cases for FTO gene) and 120 healthy subjects (40 subjects or SERPINA5 and 80 subjects for FTO gene) were recruited. The genetic variation within coding exon 3 of SERPINA5 gene was evaluated by reaction-single-strand conformation polymorphism (PCR-SSCP) and FTO rs9939609 polymorphism was evaluated by RFLP-PCR assay. RESULTS: The PCR-SSCP technique detected two rs6115G>A and rs6112T>C genetic variations within coding exon 3 of SERPINA5 gene and approved also by direct sequencing. For rs6112T>C polymorphism seven patients was heterozygous and for rs6115G>A seven PTC patients were heterozygous and two patients were homozygous. CONCLUSION: This study indicated that SERPINA5 rs6115G>A and rs6112T>C polymorphisms might be a novel susceptibility locus for PTC in Iranian patients. However, our findings do not support an association between FTO rs9939609 polymorphism and PTC risk.

Plasma SerpinA5 in conjunction with uterine artery pulsatility index and clinical risk factor for the early prediction of preeclampsia.

OBJECT: This study aimed to combine plasma protein SerpinA5 with uterine artery doppler ultrasound and clinical risk factor during the first trimester for prediction of preeclampsia. METHODS AND MATERIALS: This study was a nested cohort study and was divided into the screening set and developing set. The plasma was collected during the first trimester (11+0-13+6 weeks), at the same time, UtA-PI was detected and recorded with four-dimensional color Doppler ultrasound. These pregnancies were followed up until after delivery. The plasma proteins were examined using ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) and enzyme linked immunosorbent assay (ELISA). Placental samples preserved after delivery were analysed by immunohistochemistry. Clinical risk factors were obtained from medical records or antenatal questionnaires. Upregulation or downregulation of SerpinA5 expression in TEV-1 cells was performed to investigate the role of SerpinA5 in trophoblasts invasion. RESULTS: We demonstrated that SerpinA5 levels were greater not only in preeclampsia placental tissue but also in plasma (both p<0.05), and we found that SerpinA5 may interfere with trophoblastic cell invasion by inhibiting MSP. SerpinA5 may be a potential predictor of preeclampsia. What is more, the sensitivity and specificity of predictive power were strengthened when plasma SerpinA5 was combined with UtA-PI and pre-pregnancy BMI & family history of PE for prediction of preeclampsia. CONCLUSION: These findings showed that placenta-derived plasma SerpinA5 may be a novel biomarker for preeclampsia, which together with uterine artery Doppler ultrasound and clinical risk factor can more effectively predict preeclampsia.

Targeted inhibition of activated protein C by a non-active-site inhibitory antibody to treat hemophilia.

Activated protein C (APC) is a plasma serine protease with antithrombotic and cytoprotective functions. Based on the hypothesis that specific inhibition of APC's anticoagulant but not its cytoprotective activity can be beneficial for hemophilia therapy, 2 types of inhibitory monoclonal antibodies (mAbs) are tested: A type I active-site binding mAb and a type II mAb binding to an exosite on APC (required for anticoagulant activity) as shown by X-ray crystallography. Both mAbs increase thrombin generation and promote plasma clotting. Type I blocks all APC activities, whereas type II preserves APC's cytoprotective function. In normal monkeys, type I causes many adverse effects including animal death. In contrast, type II is well-tolerated in normal monkeys and shows both acute and prophylactic dose-dependent efficacy in hemophilic monkeys. Our data show that the type II mAb can specifically inhibit APC's anticoagulant function without compromising its cytoprotective function and offers superior therapeutic opportunities for hemophilia.

Identification of novel diagnostic biomarkers for deep venous thrombosis.

The combination of a negative D-dimer and a Wells score can rule out, but not confirm, a diagnosis of deep venous thrombosis (DVT). We aimed to identify new diagnostic biomarkers for DVT and to investigate their relationship with hypercoagulability markers [D-dimer and activated protein C-protein C inhibitor (APC-PCI) complex]. We screened 92 cardiovascular-specific proteins in plasma samples from 45 confirmed DVT patients and 45 age- and sex-matched non-DVT patients selected from a prospective multicentre diagnostic management study (SCORE) by Proseek Multiplex CVDIII96×96 . Plasma levels of 30 proteins were significantly different between DVT and non-DVT patients. After Bonferroni correction, plasma levels of seven proteins: P-selectin, transferrin receptor protein 1, von Willebrand factor, tissue factor pathway inhibitor, osteopontin (OPN), bleomycin hydrolase and ST2 protein remained significantly different. The area under curve (AUC) for these proteins ranged from 0·70 to 0·84. Furthermore, all seven identified proteins were significantly associated with markers of hypercoagulability. A combination of OPN and APC-PCI had the best ability to discriminate DVT from non-DVT patients (AUC = 0·94; sensitivity = 89% and specificity = s84%). In conclusion, we identified multiple proteins associated with markers of hypercoagulability and with a potential to become novel diagnostic biomarkers for DVT.

Diagnostic potential of plasma microRNA signatures in patients with deep-vein thrombosis.

For excluding deep-vein thrombosis (DVT), a negative D-dimer and low clinical probability are used to rule out DVT. Circulating microRNAs (miRNAs) are stably present in the plasma, serum and other body fluids. Their diagnostic function has been investigated in many diseases but not in DVT. The aims of present study were to assess the diagnostic ability of plasma miRNAs in DVT and to examine their correlation with known markers of hypercoagulability, such as D-dimer and APC-PCI complex. Plasma samples were obtained from 238 patients (aged 16-95 years) with suspected DVT included in a prospective multicentre management study (SCORE). We first performed miRNA screening of plasma samples from three plasma pools containing plasma from 12 patients with DVT and three plasma pools containing plasma from 12 patients without DVT using a microRNA Ready-to-use PCR Panel comprising 742 miRNA primer sets. Thirteen miRNAs that differentially expressed were further investigated by quantitative real-time (qRT)-PCR in the entire cohort. The plasma level of miR-424-5p (p=0.01) were significantly higher, whereas the levels of miR-136-5p (p=0.03) were significantly lower in DVT patients compared to patients without DVT. Receiver-operating characteristic curve analysis showed the area under the curve (AUC) values of 0.63 for miR-424-5p and 0.60 for miR-136-5p. The plasma level of miR-424-5p was associated with both D-dimer and APC-PCI complex levels (p<0.0001 and p=0.001, respectively). In conclusions, these findings indicate that certain miRNAs are associated with DVT and markers of hypercoagulability, though their diagnostic abilities are probably too low.

Effects of a Terrified-Sound Stress on Serum Proteomic Profiling in Mice.

The serum proteomic profiles of mice exposed to terrified-sound-induced stress and after stress release were investigated. Serum samples from 32 mice were divided into four groups (n = 8 each) and analyzed using matrix-assisted laser desorption and ionization time-of-flight mass spectrometry techniques (MALDI-TOF MS) combined with magnetic bead-based weak cation-exchange chromatography. ClinProTools software identified several distinct markers that differed between the stressed and control groups and between the stress released and stressed released controls. Of 33 m/z peaks that differed among the four groups, 17 were significantly different (P < 0.05). Five peaks (m/z: 2793.37, 2924.86, 1979.90, 3492.49, 3880.24) showed significant differences in expression after exposure to terrified-sound stress and returned to control levels after stress release. These were sequence identified as peptide regions of dimethylaniline monooxygenase, myosin-9, uncharacterized protein in Rattus norvegicus, apolipoprotein C-I, and plasma serine protease inhibitor (Serpina 5). Our study provides the first evidence of significant changes in serum proteomic profiles in mice exposed to terrified-sound stress, which suggests that protein expression profiles are affected by the stress. Normal expression levels were restored after stress release, suggesting the activation of self-adjustment mechanisms for the recovery of protein expression levels altered by this stress.

Host protein C inhibitor inhibits tumor growth, but promotes tumor metastasis, which is closely correlated with hypercoagulability.

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.

Quantification of a proteotypic peptide from protein C inhibitor by liquid chromatography-free SISCAPA-MALDI mass spectrometry: application to identification of recurrence of prostate cancer.

BACKGROUND: Biomarker validation remains one of the most challenging constraints to the development of new diagnostic assays. To facilitate biomarker validation, we previously developed a chromatography-free stable isotope standards and capture by antipeptide antibodies (SISCAPA)-MALDI assay allowing rapid, high-throughput quantification of protein analytes in large sample sets. Here we applied this assay to the measurement of a surrogate proteotypic peptide from protein C inhibitor (PCI) in sera from patients with prostate cancer. METHODS: A 2-plex SISCAPA-MALDI assay for quantification of proteotypic peptides from PCI and soluble transferrin receptor (sTfR) was used to measure these peptides in 159 trypsin-digested sera collected from 51 patients with prostate cancer. These patients had been treated with radiation with or without neoadjuvant androgen deprivation. RESULTS: Patients who experienced biochemical recurrence of prostate cancer showed decreased serum concentrations of the PCI peptide analyte within 18 months of treatment. The PCI peptide concentrations remained increased in the sera of patients who did not experience cancer recurrence. Prostate-specific antigen concentrations had no predictive value during the same time period. CONCLUSIONS: The high-throughput, liquid chromatography-free SISCAPA-MALDI assay is capable of rapid quantification of proteotypic PCI and sTfR peptide analytes in complex serum samples. Decreased serum concentrations of the PCI peptide were found to be related to recurrence of prostate cancer in patients treated with radiation with or without hormone therapy. However, a larger cohort of patients will be required for unequivocal validation of the PCI peptide as a biomarker for clinical use.

APC-PCI complex levels for screening of AAA in patients with peripheral atherosclerosis.

To evaluate the use of activated protein C-protein C inhibitor (APC-PCI) complex levels for detection of abdominal aortic aneurysm (AAA) in patients with peripheral atherosclerotic disease (PAD). APC-PCI levels and aortic diameter evaluated in 511 PAD patients without previously known AAA followed-up concerning survival for 4.8(0.5) years. AAA was found in 13% of patients. Aortic diameter correlated (r = 0.138; p = 0.002) with APC-PCI levels which were higher (0.40[0.45] vs. 0.30[0.49] µg/l; p = 0.004) in patients with AAA. This difference persisted in multivariate analysis (p = 0.029). A threshold value of APC-PCI ≥0.15 µg/L showed a specificity of 11%, a sensitivity of 97% and a negative predictive value of 96% for an AAA diagnosis. APC-PCI levels were higher in patients with AAA, and showed high sensitivity but low specificity for the diagnosis and can therefore not be considered as a screening tool in PAD patients. An AAA prevalence of 13% in patients with PAD indicates a need for AAA screening within this population.

Toxicological biomarkers of 2,3,4,7,8-pentachlorodibenzofuran in proteins secreted by HepG2 cells.

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 48h. MTT and comet assays were then conducted for determination of cytotoxicity and genotoxicity, respectively. Results of an MTT assay showed that 1nM 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 5nM, 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.

Precision of heavy-light peptide ratios measured by maldi-tof mass spectrometry.

We have investigated the precision of peptide quantitation by MALDI-TOF mass spectrometry (MS) using six pairs of proteotypic peptides (light) and same-sequence stable isotope labeled synthetic internal standards (heavy). These were combined in two types of dilution curves spanning 100-fold and 2000-fold ratios. Coefficients of variation (CV; standard deviation divided by mean value) were examined across replicate MALDI spots using a reflector acquisition method requiring 100 000 counts for the most intense peak in each summed spectrum. The CV of light/heavy peptide centroid peak area ratios determined on four replicate spots per sample, averaged across 11 points of a 100-fold dilution curve and over all six peptides, was 2.2% (ranging from 1.5 to 3.7% among peptides) at 55 fmol total (light + heavy) of each peptide applied per spot, and 2.5% at 11 fmol applied. The average CV of measurements at near-equivalence (light = heavy, the center of the dilution curve) for the six peptides was 1.0%, about 17-fold lower CV than that observed when five peptides were ratioed to a sixth peptide (i.e., a different-sequence internal standard). Response curves across the 100-fold range were not completely linear but could be closely modeled by a power law fit giving R(2) values >0.998 for all peptides. The MALDI-TOF MS method was used to determine the endogenous level of a proteotypic peptide (EDQYHYLLDR) of human protein C inhibitor (PCI) in a plasma digest after enrichment by capture on a high affinity antipeptide antibody, a technique called stable isotope standards and capture by anti-peptide antibodies (SISCAPA). The level of PCI was determined to be 770 ng/mL with a replicate measurement CV of 1.5% and a >14 000-fold target enrichment via SISCAPA-MALDI-TOF. These results indicate that MALDI-TOF technology can provide precise quantitation of high-to-medium abundance peptide biomarkers over a 100-fold dynamic range when ratioed to same-sequence labeled internal standards and enriched to near purity by specific antibody capture. The robustness and throughput of MALDI-TOF in comparison to conventional nano-LC-MS technology could enable currently impractical large-scale verification studies of protein biomarkers.

The APC-PCI concentration as an early marker of activation of blood coagulation: a study of women on combined oral contraceptives.

BACKGROUND: The risk of venous tromboembolism (VTE) in women taking combined oral contraceptives (COCs) is attributed to changes in coagulation and fibrinolysis. The impact of the COCs may be greater in women with preexisting thrombophilic defects. Nevertheless most women who suffer from venous thrombosis do not have any of the well known hereditary or acquired risk factors. A simple and sensitive marker of"thrombogenicity" has not been identified. OBJECTIVES: To investigate the effects of two different monophasic combined oral contraceptives (COCs) on the plasma concentrations of activated protein C-inhibitor of protein C ( APC-PCI) and on comparable hemostatic factors in fertile women. METHOD: Forty four healthy nulliparous women with regular menstrual periods were included and randomly assigned to start with a monophasic preparation containing 30µg ethinylestradiol and 150µg levonogestrel (LNG/EE) or a preparation containing 30µg ethinylestradiol and 150 ug desogestrel (DG/EE). After a wash out period of two months, treatment with the alternate preparation was initiated and continued for two more cycles. RESULTS: The plasma concentration of the APC-PCI complex and thrombin-antithrombin complex (TAT) increased during treatment with the two COCs. During DG/EE treatment the APC-PCI complex increased significantly more than during LNG/EE (p<0,01).The plasma concentration of D-dimer did not increase during OC treatment. CONCLUSION: The APC-PCI complex concentration, which serves as a marker for thrombin generation and indicates hypercoagulability, was increased during COC treatment compared to baseline. The method is a sufficiently sensitive marker to detect even small differences in the activation of coagulation.

N-glycans of human protein C inhibitor: tissue-specific expression and function.

Protein C inhibitor (PCI) is a serpin type of serine protease inhibitor that is found in many tissues and fluids in human, including blood plasma, seminal plasma and urine. This inhibitor displays an unusually broad protease specificity compared with other serpins. Previous studies have shown that the N-glycan(s) and the NH2-terminus affect some blood-related functions of PCI. In this study, we have for the first time determined the N-glycan profile of seminal plasma PCI, by mass spectrometry. The N-glycan structures differed markedly compared with those of both blood-derived and urinary PCI, providing evidence that the N-glycans of PCI are expressed in a tissue-specific manner. The most abundant structure (m/z 2592.9) had a composition of Fuc3Hex5HexNAc4, consistent with a core fucosylated bi-antennary glycan with terminal Lewis(x). A major serine protease in semen, prostate specific antigen (PSA), was used to evaluate the effects of N-glycans and the NH2-terminus on a PCI function related to the reproductive tract. Second-order rate constants for PSA inhibition by PCI were 4.3±0.2 and 4.1±0.5 M⁻¹ s⁻¹ for the natural full-length PCI and a form lacking six amino acids at the NH2-terminus, respectively, whereas these constants were 4.8±0.1 and 29±7 M⁻¹ s⁻¹ for the corresponding PNGase F-treated forms. The 7-8-fold higher rate constants obtained when both the N-glycans and the NH2-terminus had been removed suggest that these structures jointly affect the rate of PSA inhibition, presumably by together hindering conformational changes of PCI required to bind to the catalytic pocket of PSA.

Protein C inhibitor.

Protein C inhibitor (PCI) is a serine protease inhibitor and was originally identified as an inhibitor of activated protein C (APC). However, PCI is not specific for APC and also inhibits several proteases involved in coagulation, fibrinolysis, cancer, wound healing, and fertility. The biological function of PCI is unknown due to broad enzyme specificity, its wide tissue distribution, and the lack of a suitable animal model. This review highlights the specific roles of PCI in the areas of hemostasis and thrombosis and fertilization, and it also describes the latest information on the fascinating participation of the protein in intracellular processes, phospholipid binding, and killing of bacteria.

Identifying serum biomarkers for TACE therapy efficiency of hepatocellular carcinoma.

Transcatheter Arterial Chemoembolization (TACE) is the first line of treatment in inoperable hepatocellular carcinoma. Magnetic affinity beads can be used to extract peptides from un-fractionated serum samples. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) can detect the presence and the molecular mass of peptides. In this study, we used a highly optimized ClinProt-matrix-assisted laser desorption/ionization time-of flight mass spectrometer (MALDI-TOF-MS) to screen hepatocellular carcinoma markers for TACE. 40 sera from 20 patients, including before and after TACE to explore those biomarkers, might be related with therapy efficiency, and some of the patients who received another therapy were analyzed as well. The spectra were analyzed statistically using FlexAnalysis™ and Clin-Prot™ bioinformatic software. The seven most significant differential peaks (p < 0.0.5) were selected out by ClinProTool software to identify hepatocellular carcinoma markers for TACE therapy. Furthermore, the differential peptide of 3883Da was identified as plasma serine protease inhibitor precursor (Protein C inhibitor). This study provides a direct link between peptide marker profiles and TACE therapy, and the markers may have clinical utility for monitoring efficiency of therapy.

Activated protein C-protein C inhibitor complex in peripheral arterial disease.

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, Malmö 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] microg/L vs. 0.140 [0.190-0.220] microg/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.

Hepatocyte growth factor activator (HGFA): its regulation by protein C inhibitor.

Protein C inhibitor (PCI; SERPINA5) is a plasma serine protease inhibitor, and a potent inhibitor of activated protein C (APC), which plays a critical role in the anticoagulant protein C pathway. Recently, PCI was also found to form a complex with the serine protease hepatocyte growth factor activator (HGFA), inhibiting the HGFA-catalyzed activation of the single-chain hepatocyte growth factor precursor. In vivo studies using human PCI-transgenic (hPCI-Tg) mice, which mimic PCI expression in humans, showed that the regeneration rate of the liver after partial hepatectomy was significantly impaired as compared with wild-type mice. The decreased liver regeneration in hPCI-Tg mice was restored by pretreatment with antibody against human PCI. Furthermore, APC protected hepatic nonparenchymal cells from thrombin-induced inflammation in vitro, suggesting that plasma PCI may inhibit the cytoprotective action of APC on hepatic cells in hPCI-Tg mice. It was shown that the levels of HGFA-PCI are increased in plasma of patients who have been subjected to hepatectomy, as compared with complex levels in the plasma of normal individuals. Thus, PCI may play a role as a potent inhibitor of HGFA and APC in plasma and/or at the sites of tissue injury in the regulation of tissue regeneration.

Markers of proteolysis, fibrinolysis, and coagulation in relation to size and growth rate of abdominal aortic aneurysms.

UNLABELLED: We evaluated whether matrix metalloproteinases (MMPs) 2 and 9, their inhibitors, markers for fibrinolysis, and thrombin activation are associated with diameter and growth of abdominal aortic aneurysms (AAAs). MATERIAL AND METHODS: Matrix metalloproteinases 2 and 9, tissue inhibitor of MMPs (TIMP-1), serpine-1, tPa-serpine-1, and activated protein C- protein C inhibitor (APC-PCI) complex were analyzed in 353 patients with AAA grouped according to AAA size, and 219 gender- and age-matched healthy individuals. Follow-up of AAA growth for up to 7 years was possible in 178 of 353 patients. RESULTS: At baseline, all groups of patients with AAA showed lower levels of MMP-2 and -9, and higher levels of TIMP-1, serpine-1, and t-Pa-serpine-1 than controls. Matrix metalloproteinase 2 correlated inversely and APC-PCI complex correlated directly with AAA diameter. We found no correlations between markers for proteolysis, fibrinolysis, coagulation, and yearly AAA growth. CONCLUSION: Matrix metalloproteinase 2 is lower and APC-PCI higher in patients with larger AAA, but the relevance of the markers for AAA growth is far from clarified.

The diagnostic performance of APC-PCI complex determination compared to D-dimer in the diagnosis of deep vein thrombosis.

UNLABELLED: D-dimer testing is widely used as part of the diagnostic algorithm for the exclusion of deep vein thrombosis (DVT) but is considered of limited in value for ruling DVT in. Since D-dimers are poorly defined, there is no standardization of the assays and this makes reliable comparisons between clinical studies difficult. We report on a performance evaluation of a new marker of activated coagulation (Activated Protein-C in complex with Protein-C inhibitor, APC-PCI complex) compared to two quantitative D-dimer assays (Vidas D-dimer Exclusion and Autodimer). The post-hoc comparison was made on 350 frozen plasma samples from consecutive outpatients suspected of DVT in a multicenter management study including clinical probability score, D-dimer testing, venous ultrasound and contrast venography as part of the diagnostic algorithm. RESULTS: The APC-PCI complex performed inferior to the D-dimer assays in terms of sensitivity: 74 vs. >93%, negative predictive value: 91 vs. >96% and area under the curve: 0.82 vs. 0.9, but showed a significantly higher specificity: 80 vs. 40-60%. Specificity for the APC-PCI complex did not decrease with higher clinical probability score and the positive predictive value was significantly higher than that of the D-dimer assays in the intermediate/high probability cohort (66 vs. <52%). In this probability cohort, high levels of the APC-PCI complex and to a lesser extent, D-dimers, can give positive predictive values of >90% in up to 20% of the patients which indicates important clinical implications. However, for the exclusion of DVT at the pre-specified cut-off level, the APC-PCI complex perform inferior to the D-dimer assays in this study.

Coagulation activation and ultrasound characteristics in patients with carotid artery disease.

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.