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Metastasis is still the major cause of endometrial cancer (EC)-related death. Because of their biological function and regenerative properties, exosomes have been applied to therapeutic regimens. SERPINA5 expression is downregulated in several tumors and linked to tumor cell migration and invasion. However, the expression and biological functions of SERPINA5 in EC remain unclear.The levels of SERPINA5 in plasma exosomes were determined with ELISAs. SERPINA5 expression in EC and its relationship with survival outcomes were analyzed using the TCGA database and clinical EC tissue samples. The effect of SERPINA5 overexpression or exosomal SERPINA5 on EC metastasis was examined by cell migration and invasion assays in vitro. Mechanistically, overexpression of SERPINA5 or high exosomal SERPINA5 levels mediated the regulation of the integrin β1/FAK signaling pathway in EC cell lines. The therapeutic effect of exosomal SERPINA5 was determined with xenograft models.This study revealed that the level of exosomal SERPINA5 was increased in the circulating plasma of EC patients. In addition, the expression of SERPINA5 was decreased in EC patients with distant metastasis, and low expression of SERPINA5 indicated worse survival. In addition, SERPINA5 was elevated in normal tissues adjacent to EC tumors. Moreover, overexpression of SERPINA5 inhibited metastatic potential of EC cell lines in vitro. Furthermore, SERPINA5 loaded on secreted exosomes reduced the metastatic ability of EC cells. Notably, overexpression of SERPINA5 or high exosomal SERPINA5 levels suppressed EC metastatic potential by suppressing integrin β1/FAK signaling pathway activation. Finally, exosomal SERPINA5 impeded tumor growth and metastasis in xenograft models.Our findings revealed that a low level of SERPINA5 expression indicated poor survival outcomes in EC and that exogenous SERPINA5 loading of exosomes may be a novel therapeutic strategy for metastatic EC.

Obtaining high-quality sperm is key to improving the success rate of assisted reproductive technology (ART). Although cytokines secreted by cumulus-oocyte complexes (COCs) bind to sperm surface receptors to improve sperm quality, the effects of adding mouse COCs to human tubal fluid (HTF) medium on sperm capacitation have not yet been explored. Eight-week-old ICR mouse COCs were added to HTF medium and crushed to obtain the post-modified HTF medium. Compared with using HTF medium, the fertilisation rate and number of sperm combined with the zona pellucida significantly increased after in vitro capacitation using the post-modified HTF medium (P 0.01). Proteomic and Western blotting analyses showed that the level of SERPINA5 in sperm increased significantly following in vitro capacitation with the post-modified HTF medium (P 0.05). Immunohistochemical staining analysis demonstrated that SERPINA5 protein was expressed in mouse cumulus cells. A SERPINA5 antibody was added in the post-modified HTF medium to block the effects of SERPINA5 after in vitro capacitation, which significantly decreased the fertilisation rate and the number of sperm combined with the zona pellucida (P 0.05). Recombinant mouse SERPINA5 protein (1 ~ 2 μg/ml) was added to HTF medium and the fertilisation rate and the number of sperm combined with the zona pellucida significantly increased (P 0.01). Moreover, recombinant human SERPINA5 protein (5 μg/ml) was added before human semen freezing. Compared with adding no SERPINA5 protein, the percentage of normal sperm morphology and the intact acrosome significantly increased (P 0.05). Our study provides a reference method for optimising sperm quality in the process of in vitro capacitation.

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.

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.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.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).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.Цель. Изучить профиль биохимических маркеров системы гемостаза для уточнения их роли и взаимоотношений в патогенезе развития тромботических осложнений (ТО) ишемического инсульта (ИИ) и связанной с этим оценки возможностей их диагностического применения. Материалы и методы. В группу исследования включены 302 пациента (164 мужчины, 138 женщин), поступивших в стационар с диагнозом ИИ в течение 24 ч от начала развития заболевания. Диагноз подтвержден методом компьютерной томографии. Средний возраст пациентов составлял 69 (5088) лет. У всех пациентов в 1-е сутки заболевания взята кровь для определения профиля аналитов, предположительно ассоциированных с патогенезом развития ТО. В сыворотке крови методом иммуноферментного анализа определены уровни гомоцистеина, ингибитора протеина С, тромбомодулина, плазминогена, тканевого активатора плазминогена, урокиназы, ингибитора активатора плазминогена 1-го типа, комплекса t-PA/PAI-1, витронектина, комплекса плазмин-2-антиплазмин, D-димера, фибронектина. Результаты. ТО в остром периоде ИИ (до 21 сут болезни) зафиксированы у 32 (10,6%, 95% доверительный интервал ДИ 7,3714,3) пациентов, из них тромбоэмболия легочной артерии отмечалась у 27 (8,94%, 95% ДИ 5,9812,4) пациентов, тромбоз глубоких вен у 5 (1,66%, 95% ДИ 0,473,47) пациентов. Исследование панели специфических белков, участвующих в патогенетических процессах, сопровождающих некроз ткани мозга при ИИ, продемонстрировало, что из всего выбранного для исследования перечня биохимических маркеров системы гемостаза наиболее значимы: концентрация фибронектина в прогнозе отсутствия ТО с пороговым значением более 61 мкг/мл и отношением шансов 4,4 (95% ДИ 1,512,9, р=0,011) и концентрация комплекса t-PA/PAI-1 в прогнозе развития ТО с пороговым значением более 14 нг/мл и отношением шансов 11,3 (95% ДИ 1,18109,3, р=0,03). Заключение. Значимость комплекса t-PA/PAI-1 и фибронектина как маркеров ТО при ИИ может быть обусловлена нарушением процессов активации фибринолитического звена гемостаза и накоплением неопсонированных соединений, повреждающих сосудистую стенку.

This study aims to evaluate the expression of genes associated with the fertilisation potential and embryo development, sperm DNA fragmentation (SDF), and acrosome reaction in male partners of infertile couples with different sperm parameters compared to fertile men. First, male partners of infertile couples with abnormal (N = 25) and normal sperm parameters (N = 25), and fertile men (N = 10) were included in experimental groups I, II, and controls respectively. The mRNA levels of the Annexin A2 (ANXA2), Sperm protein 17 (SP17), Plasma serine protease inhibitor (SERPINA5), and Peroxiredoxin-2 (PRDX2) genes and SDF were evaluated. To evaluate the maturity of the sperm and oxidative stress, the acrosome reaction, the lipid peroxidation, and total antioxidant were measured. As result, SP17 showed a significantly lower expression in both experimental groups. SERPINA5 was significantly down-regulated in experimental group I that was aligned with the low rate of acrosome reaction. Significant overexpression of PRDX2 was found between experimental group II and controls. Significant higher rates of SDF were seen in both experimental groups compared to the controls. Finally, our data suggest that differentially gene expression of SP17 is a potential diagnostic biomarker in infertile men either with normal or abnormal sperm parameters. SDF is one of the causes of male infertility, independent of the sperm parameters.

SERPINA5 belongs to the serine protease inhibitor superfamily and has been reported to be lowly expressed in a variety of malignancies. However, few report of SERPINA5 in gastric cancer has been found. The purpose of this study was to determine the role of SERPINA5 in GC and to investigate potential tumorigenic mechanisms. We performed qPCR to determine the level of SERPINA5 expression in GC. We used public databases to evaluate whether SERPINA5 could be utilized to predict overall survival and disease-free survival in GC patients. We also knocked down the expression of SERPINA5 and evaluated its effect on cell proliferation and migration. Furthermore, we explored the signal pathways and regulatory mechanisms related to SERPINA5 functions. According to our findings, SERPINA5 was shown to exhibit high expression in GC. Notably, SERPINA5 was prognostic in GC with high expression being unfavourable. SERPINA5 was further observed to promote GC tumorigenesis by modulating GC cell proliferation ability. Mechanically, SERPINA5 could inhibit CBL to regulate the PI3K/AKT/mTOR signalling pathway, thereby promoting GC carcinogenesis progression. These results highlight the important role of SERPINA5 in GC cell proliferation and suggest that SERPINA5 could be a novel target for GC treatment and a predictor for GC prognosis.

Preeclampsia (PE) is the leading cause of maternal and fetal morbidity or mortality but lacks reliable methods for early diagnosis. In a previous study, serum SERPINA5 levels were higher in women with PE before the clinical manifestation of the disease. This study aimed to evaluate the efficacy of SERPINA5 in predicting PE and investigate its role in trophoblast cell biology. A multicenter, 2-stage observational case-control study was performed to develop and validate an early predictive PE model based on SERPINA5, maternal characteristics, and inflammatory factors. To further understand the relationship between SERPINA5 and PE, SERPINA5 was overexpressed or knocked down in extravillous trophoblast cells (EVT) and a pregnant rat model. After development and initial validation, a model that combined SERPINA5 and inflammatory factors had a high predictive ability for PE before 20 weeks gestation with an AUC of 0.90 (95% CI 0.83-0.96). It also demonstrated that SERPINA5 inhibited primary EVT cell invasion by disrupting the urokinase-type plasminogen activator/urokinase-type plasminogen activator receptor (uPA/uPAR) pathway, in turn, is involved in the development of PE. In vivo experiments also proved that overexpression of SERPINA5 induced a PE-like syndrome (hypertension and proteinuria) in pregnant rats. Therefore, serum SERPINA5 is a promising early biomarker of PE, suggesting that it may be involved in placental development through its action on the uPA/uPAR system prior to the clinical manifestation of PE.

Protein C inhibitor is a non-specific serine protease inhibitor with extensive protease reactivity. Protein C inhibitor (SERPINA5, plasminogen activator inhibitor-3/PAI-3) is a secreted, extracellular clade A serpin. SERPINA5/PCI 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. PCI plays role at thrombosis and fibrinolysis, regulation of fertilization, tissue regeneration, vascular permeability, tumor development, invasion, metastasis and angiogenesis. In this review; aimed to provide information about the functions of PCI and to provide guidance for studies.

Introduction and hypothesis Previous studies have indicated a hereditary component of stress urinary incontinence; however, evidence on candidate genes or single-nucleotide polymorphisms (SNPs) is scarce. We hypothesize a genetic association of female stress urinary incontinence based on significant differences of the urinary and serum proteomic pattern in the identical study population. Methods Case-control study of 19 patients and 19 controls. We searched for known SNPs of SUI candidate genes (COL1A1, MMP1, SERPINA5, UMOD) in the database of short genetic variations and PubMed. Genomic DNA was isolated using QIAamp DNA Blood Midi Kit (Qiagen). We performed Sanger sequencing of selected exons and introns. Results The rs885786 SNP of the SERPINA5 gene was identified in 15 cases and 10 controls (p = 0.09). The rs6113 SNP of the SERPINA5 gene was present in 4 controls compared to 0 cases (p = 0.105). The rs4293393, rs13333226 and rs13335818 SNPs of the UMOD gene were identified in five cases and two controls (p = 0.20), the rs1800012 SNP of the COL1A1 gene in five cases versus four controls (p = 0.24) and the homozygous rs1799750 SNP of the MMP1 gene in eight cases versus five controls (p = 0.18). The combination of the rs885786 SNP of the SERPINA5 gene and rs179970 SNP of the MMP1 gene was detected in ten cases versus five controls (p = 0.072). Conclusions We found nonsignificant trends toward associations of SNPs on the SERPINA5, UMOD and MMP1 gene and SUI. Electronic supplementary material The online version of this article (10.1007/s00192-019-03878-0) contains supplementary material, which is available to authorized users.

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 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.

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.

Background The protein plasminogen activator inhibitor-1 (PAI-1), an inhibitor specific for urokinase plasminogen activator (uPA) and tissue plasminogen activator (tPA), has been shown to have a key role in cancer metastases. Currently, it is unknown as to whether the exocellular inhibition of PAI-1 can inhibit the migration of cancer cells. Methods By fusing the mutated serine protease domain (SPD) of uPA and human serum albumin (HSA), PAItrap3, a protein that traps PAI-1, was synthesized and experiments were conducted to determine if exocellular PAItrap3 attenuates PAI-1-induced cancer cell migration in vitro. Results PAItrap3 (0.8 μM) significantly inhibited the motility of MCF-7, MDA-MB-231, HeLa and 4T1 cancer cells, by 90%, 50%, 30% and 20%, respectively, without significantly altering their proliferation. The PAI-1-induced rearrangement of F-actin was significantly inhibited by PAItrap3, which produced a decrease in the number of cell protrusions by at least 20%. Conclusions In vitro, PAItrap3 inhibited PAI-1-induced cancer cell migration, mainly through inhibiting the rearrangement of F-actin. Overall, these results, provided they can be extrapolated to humans, suggest that the PAItrap3 protein could be used as an exocellular inhibitor to attenuate cancer metastases.

Thrombin, a procoagulant protease, cleaves and activates protease-activated receptor-1 (PAR1) to promote inflammatory responses and endothelial dysfunction. In contrast, activated protein C (APC), an anticoagulant protease, activates PAR1 through a distinct cleavage site and promotes anti-inflammatory responses, prosurvival, and endothelial barrier stabilization. The distinct tethered ligands formed through cleavage of PAR1 by thrombin versus APC result in unique active receptor conformations that bias PAR1 signaling. Despite progress in understanding PAR1 biased signaling, the proteins and pathways utilized by thrombin versus APC signaling to induce opposing cellular functions are largely unknown. Here, we report the global phosphoproteome induced by thrombin and APC signaling in endothelial cells with the quantification of 11,266 unique phosphopeptides using multiplexed quantitative mass spectrometry. Our results reveal unique dynamic phosphoproteome profiles of thrombin and APC signaling, an enrichment of associated biological functions, including key modulators of endothelial barrier function, regulators of gene transcription, and specific kinases predicted to mediate PAR1 biased signaling. Using small interfering RNA to deplete a subset of phosphorylated proteins not previously linked to thrombin or APC signaling, a function for afadin and adducin-1 actin binding proteins in thrombin-induced endothelial barrier disruption is unveiled. Afadin depletion resulted in enhanced thrombin-promoted barrier permeability, whereas adducin-1 depletion completely ablated thrombin-induced barrier disruption without compromising p38 signaling. However, loss of adducin-1 blocked APC-induced Akt signaling. These studies define distinct thrombin and APC dynamic signaling profiles and a rich array of proteins and biological pathways that engender PAR1 biased signaling in endothelial cells.

Background Accumulating evidence suggests that plasminogen activator inhibitor-1 (PAI-1) plays an important role in bladder tumorigenesis by regulating cell cycle. However, it remains unclear whether and how inhibition of PAI-1 suppresses bladder tumorigenesis. Methods To elucidate the therapeutic effect of PAI-1 inhibition, we tested its tumorigenicity in PAI-1 knockout (KO) mice exposed to a known bladder carcinogen. Results PAI-1 deficiency did not inhibit carcinogen-induced bladder cancer in mice although carcinogen-exposed wild type mice significantly increased PAI-1 levels in bladder tissue, plasma and urine. We found that PAI-1 KO mice exposed to carcinogen tended to upregulate protein C inhibitor (PAI-3), urokinase-type plasminogen activator (uPA) and tissue-type PA (tPA), and significantly increased PAI-2, suggesting a potential compensatory function of these molecules when PAI-1 is abrogated. Subsequent studies employing gene expression microarray using mouse bladder tissues followed by post hoc bioinformatics analysis and validation experiments by qPCR and IHC demonstrated that SERPING1 is further downregulated in PAI-1 KO mice exposed to BBN, suggesting that SERPING1 as a potential missing factor that regulate PAI-2 overexpression (compensation pathway). Conclusions These results indicate that serpin compensation pathway, specifically PAI-2 overexpression in this model, supports bladder cancer development when oncoprotein PAI-1 is deleted. Further investigations into PAI-1 are necessary in order to identify true potential targets for bladder cancer therapy.

Nowadays, there is no clinically applicable biomarker for osteoarthritis (OA). Therefore, the aim of the study is to discover a potential biomarker for OA.We performed a proteomics of eight cartilage samples (four damaged cartilage and four macroscopically intact cartilage) from four OA patients without any comorbidities to search for valuable OA biomarkers. Four rats underwent bilateral ovariectomy to induce the OA (OVX-OA) model, while another four underwent a sham procedure wherein the ovaries were exteriorized but not removed (SHAM). Selected candidate proteins were further verified in the patients and the OVX-OA animal model.A comprehensive cartilage proteome profile of patients with OA was constructed. Additionally, the complement and coagulation cascades were found to be significantly altered, and serpinA5 was chosen as a protein of interest based on biological information analysis. The reduction of serpinA5 in locally damaged cartilage and serum of patients with OA compared to the control group was determined. Furthermore, we found that serpinA5 was decreased in OVX-OA rats compared to that in SHAM rats.Our results suggest that there is dyscoagulation in the OA process and that serpinA5 can serve as a potentially valuable OA biomarker.

Hemophilia is a bleeding disorder caused by deficiency in factors VIII or IX, the two components of the intrinsic Xase complex. Treatment with replacement factor can lead to the development of inhibitory antibodies, requiring the use of bypassing agents such as factor VIIa and factor concentrates. An alternative approach to bypass the Xase complex is to inhibit endogenous anticoagulant activities. Activated protein C (APC) breaks down the complex that produces thrombin by proteolytically inactivating factor Va. Defects in this mechanism (eg, factor V Leiden) are associated with thrombosis but result in less severe bleeding when co-inherited with hemophilia. Selective inhibition of APC might therefore be effective for the treatment of hemophilia. The endogenous inhibitors of APC are members of the serpin family: protein C inhibitor (PCI) and α1-antitrypsin (α1AT); however, both exhibit poor reactivity and selectivity for APC. We mutated residues in and around the scissile P1-P1' bond in PCI and α1AT, resulting in serpins with the desired specificity profile. The lead candidate was shown to promote thrombin generation in vitro and to restore fibrin and platelet deposition in an intravital laser injury model in hemophilia B mice. The power of targeting APC was further demonstrated by the complete normalization of bleeding after a severe tail clip injury in these mice. These results demonstrate that the protein C anticoagulant system can be successfully targeted by engineered serpins and that administration of such agents is effective at restoring hemostasis in vivo.

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.

Although suspected conceptually in the 60 s, Protein C and Protein S activities in hemostasis were investigated and reported from the mid-80 s, followed by the discovery of Thrombomodulin, an endothelial cell membrane associated protein, playing the most important heamostatic role. These 3 proteins act in regulating thrombogenesis and protecting against thrombo-embolic events. When blood is activated, any trace of circulating thrombin is captured by Thrombomodulin in the microcirculation, making thrombin become an anticoagulant through its capacity to activate Protein C to Activated Protein C, which operates as a sentinel in blood coagulation, in the form of a complex with free Protein S, to block any new blood activation site, and more especially circulating activated Factors V and VIII. Protein S not only acts as the Activated Protein C cofactor, but also as the cofactor of Tissue Factor Pathway Inhibitor. In addition, it has some functions in the complement pathway through its binding to C4b-BP. Another capability of activated protein C is to lower fibrinolytic activity, as the Activated Protein C Inhibitor is also known as Plasminogen Activator Inhibitor 3. The Protein C-Protein S system becomes less efficient in the presence of mutated Factor V (Factor V-Leiden or other variants), which is resistant to its inactivating effect. Other pathologies linked to this system concern the development of allo- or auto-antibodies to Protein S or to thrombin, which can generate severe thrombotic complications in affected patients. Some antithrombotic drugs have originated from this regulatory system. Protein C or Protein S concentrates are used for treating deficient patients. Activated Protein C (especially in patients with sepsis) or Thrombomodulin are proposed as antithrombotic medications. Most importantly, congenital or acquired Protein C or Protein S deficiencies are associated with severe recurrent thrombotic events. From the clinical standpoint most of the patients are heterozygous, as homozygosity is almost incompatible with life in the absence of a continuous and efficient treatment. Laboratory investigation of this highly complex system involves many different specialized assays for measuring these 3 proteins’ activities, their antigenic content or their genetic sequence. The Protein S in-vitro anticoagulant activity is weak and contrasts with its high antithrombotic role in-vivo, showing that diagnostic assays have not yet succeeded in reproducing all the natural mechanisms for evidencing the anticoagulant role of Protein S. This paradoxal notion is discussed and illustrated in this manuscript as well is a revisit of the major characteristics and pathophysiological functions of the Protein C-Protein S-Thrombomodulin system; the associated pathologies; and the main laboratory tools available for clinical diagnosis. In respect to future perspectives, we also focused on developing more significant and relevant assays, especially for Protein S, thanks to the understanding of its biological roles.

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

SummaryFor 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 (pSupplementary Material to this article is available online at www.thrombosis-online.com.

The aim of this study was to examine whether glycosaminoglycan (GAG) from Mactra veneriformis had an impact on the protein C (PC) system in rats and expression of relevant factors in human umbilical vein endothelial cells. Isotonic saline, heparin sodium (4 mg/kg), and GAG (1, 4, and 16 mg/kg) were administered to Wistar rats through caudal vein at the total volume of 0.5 ml for each injection, and the anticoagulant blood was prepared. The activities of PC and protein S, and the concentrations of activated protein C inhibitor (APCI) and thrombomodulin were detected according to the kit instructions. The expression levels of tissue factor, tissue factor pathway inhibitor and thrombomodulin genes in human umbilical vein endothelial cells were detected by the reverse transcription polymerase chain reaction method. GAG could significantly reduce the activity of PC (P < 0.05, P < 0.01) and slightly increase the concentration of APCI, and high concentration of GAG could significantly decrease the activity of protein S (P < 0.05) and significantly increase the concentration of thrombomodulin (P < 0.05). GAG could significantly downregulate the expression level of tissue factor gene (P < 0.05, P < 0.01) and significantly upregulate the expression levels of tissue factor pathway inhibitor and thrombomodulin genes (P < 0.05, P < 0.01). GAG showed the inhibitory effect on PC system in vivo. GAG might play an important role in the resistance to extrinsic coagulation pathway.

Background: Inflammation is a known activator of coagulation, and COPD is a chronic inflammatory disease. Yet, the role of coagulation in COPD is underexplored. Methods: The study sample included 413 COPD patients and 49 controls from the 3-yr longitudinal Bergen COPD Cohort Study. 148 COPD patients were also examined during an acute COPD exacerbation (AECOPD). The three markers thrombin-antithrombin (TAT) complex, activated protein C –protein C inhibitor (APC-PCI), and D-dimer were measured in plasma at baseline and during exacerbation by enzyme inhibition essays (manufacturer). Median levels of the markers between stable COPD and controls, and in COPD between stable state and AECOPD were examined. We examined the association between the coagulation markers and later AECOPD and mortality by Cox and negative binomial regression. Results: TAT was significantly lower in stable COPD 1.03 ng/mL (0.76-1.44), than in controls 1.28 (1.04-1.49); p=0.002, whereas ACP-PCI and D-dimer trended higher in COPD (p=0.07 and 0.11 respectively). During AECOPD, all markers were higher than in the stable state; for TAT 2.56 versus 1.43; for APC-PCI 489.3 versus 416; and for D-dimer 763.5 versus 479.7 (p Conclusion: All coagulation markers were activated during AECOPD, and could be biomarkers for later events.

Background Activated protein C (APC) is a major regulator of thrombin formation. Two major plasma inhibitors form complexes with APC, protein C inhibitor (PCI) and α1-antitrypsin (α1AT), and these complexes have been quantified by specific enzyme-linked immunosorbent assays (ELISAs). Also, complexes of APC with α2-macroglobulin (α2M) have been observed by immunoblotting. Here, we report an ELISA for APC:α2M complexes in plasma. Methods Plasma samples were pre-treated with dithiothreitol and then with iodoacetamide. The detection range of the newly developed APC:α2M assay was 0.031 to 8.0 ng/mL of complexed APC. Following infusions of APC in humans and baboons, complexes of APC with α2M, PCI and α1AT were quantified. These complexes as well as circulating APC were also measured in 121 patients with a history of venous thromboembolism (VTE) and 119 matched controls. Results In all the in vivo experiments, α2M was a significant APC inhibitor. The VTE case–control study showed that VTE patients had significantly lower APC:α2M and APC levels than the controls (p Conclusion The APC:α2M assay reported here may be useful to help monitor the in vivo fate of APC in plasma. In addition, our results show that a low APC:α2M level is associated with increased VTE risk.

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. (Less)

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.

Serine protease inhibitors (serpin) have therapeutic potential in a variety of pathogenic processes, ranging from thrombosis and altered immune response to liver cirrhosis. To investigate the physiological effects of protein C inhibitor (PCI, serpinA5), its gene was inactivated in a mouse model, resulting in male infertility. In the present report, 2D differential gel electrophoresis was utilized to investigate the molecular mechanisms for PCI involvement in male reproduction. Comparing the testes proteomes of three PCI-knockout mice with three wild types demonstrated similar patterns with the exception of a massive upregulation of prostaglandin reductase 1 (tenfold; p < 0.002) and the complete shifts in the molecular weights of serpinA1C and serpinA3K. All these PCI-dependent proteome changes were immunologically verified. Unbiased proteome analysis indicated that inactivation of serpinA5 strongly influenced both the protein species pattern of other A-clade serpins as well as prostaglandin metabolism in the testes.

The membrane-anchored serine proteases are a unique group of trypsin-like serine proteases that are tethered to the cell surface via transmembrane domains or glycosyl-phosphatidylinositol-anchors. Overexpressed in tumors, with pro-tumorigenic properties, they are attractive targets for protease-activated prodrug-like anti-tumor therapies. Here, we sought to engineer anthrax toxin protective antigen (PrAg), which is proteolytically activated on the cell surface by the proprotein convertase furin to instead be activated by tumor cell-expressed membrane-anchored serine proteases to function as a tumoricidal agent. PrAg's native activation sequence was mutated to a sequence derived from protein C inhibitor (PCI) that can be cleaved by membrane-anchored serine proteases, to generate the mutant protein PrAg-PCIS. PrAg-PCIS was resistant to furin cleavage in vitro, yet cytotoxic to multiple human tumor cell lines when combined with FP59, a chimeric anthrax toxin lethal factor-Pseudomonas exotoxin fusion protein. Molecular analyses showed that PrAg-PCIS can be cleaved in vitro by several serine proteases including the membrane-anchored serine protease testisin, and mediates increased killing of testisin-expressing tumor cells. Treatment with PrAg-PCIS also potently attenuated the growth of testisin-expressing xenograft tumors in mice. The data indicates PrAg can be engineered to target tumor cell-expressed membrane-anchored serine proteases to function as a potent tumoricidal agent.

Neuroinflammation is thought to contribute to cell death in neurodegenerative disorders, but the factors involved in the inflammatory process are not completely understood. Proteinase-activated receptor-2 (PAR2) expression in brain is increased in Alzheimer's disease and multiple sclerosis, but the status of PAR2 in Parkinson's disease is unknown. This study examined expression of PAR2 and endogenous proteinase activators (trypsin-2, mast cell tryptase) and proteinase inhibitors (serpin-A5, serpin-A13) in areas vulnerable and resistant to neurodegeneration in Parkinson's disease at different Braak α-synuclein stages of the disease in post-mortem brain. In normal aged brain, expression of PAR-2, trypsin-2, and serpin-A5 and serpin-A13 was found in neurons and microglia, and alterations in the amount of immunoreactivity for these proteins were found in some brain regions. Namely, there was a decrease in neurons positive for serpin-A5 in the dorsal motor nucleus, and serpin-A13 expression was reduced in the locus coeruleus and primary motor cortex, while expression of PAR2, trypsin-2 and both serpins was reduced in neurons within the substantia nigra. There was an increased number of microglia that expressed serpin-A5 in the dorsal motor nucleus of vagus and elevated numbers of microglia that expressed serpin-A13 in the substantia nigra of late Parkinson's disease cases. The number of microglia that expressed trypsin-2 increased in primary motor cortex of incidental Lewy body disease cases. Analysis of Parkinson's disease cases alone indicated that serpin-A5 and serpin-A13, and trypsin-2 expression in midbrain and cerebral cortex was different in cases with a high incidence of L-DOPA-induced dyskinesia and psychosis compared to those with low levels of these treatment-induced side effects. This study showed that there was altered expression in brain of PAR2 and some proteins that can control its function in Parkinson's disease. Given the role of PAR2 in neuroinflammation, drugs that mitigate these changes may be neuroprotective when administered to patients with Parkinson's disease.

Background Thrombosis in patients with acute lymphocytic leukemia (ALL) can develop after treatment with L-asparaginase (asp) and is often localized to the central nervous system (CNS). We hypothesize that changes in the cerebrospinal fluid (CSF) proteome will occur after asp therapy and will anticipate CNS clots. Methods Five newly diagnosed patients, ages 1–11 years, with ALL (n = 4) or lymphoblastic lymphoma (LL) (n = 1) underwent serial lumbar punctures during induction. CSF was depleted of abundant plasma proteins and analyzed by gel-free, label-free quantitative proteomics. Results More than 600 proteins were quantified across all CSF samples. In four subjects, the expression of proteins involved in coagulation such as protein C Inhibitor (SERPINA5) and heparin cofactor II (SERPIND1) changed over the course of asp therapy. Antithrombin III (ATIII) and plasminogen (PLMN) levels were shown to have decreased expression over time in one child who developed a CNS thrombosis, compared to other subjects. Conclusions CSF proteomics is feasible and reproducible in ALL and LL. CSF ATIII and PLMN should be further investigated as predictive markers of CNS thrombosis. Pediatr Blood Cancer 2015;62:1190–1194. © 2015 Wiley Periodicals, Inc.

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.

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.

AB002 is a recombinant thrombin analog that generates endogenous activated protein C (APC) on intravascular surfaces in a thrombomodulin-dependent manner but does not promote thrombosis. Endogenously generated APC downregulates thrombin generation, thereby reducing pathological clot formation. During hemodialysis (HD), thrombi can form within the dialyzer filter and circuit, reducing dialyzer efficiency, and increasing blood loss. Heparin administration reduces clotting within the filter and circuit, but increases bleeding risk and is not tolerated in all patients. Since end-stage renal disease (ESRD) patients undergoing HD are at higher risk of both thromboembolism and bleeding, a safe, short-term and self-limiting anticoagulant alternative to heparin is currently an unmet need for this population. This is a phase 2, randomized, double-blind, placebo-controlled, single-dose study designed to evaluate the safety and efficacy of AB002 at two dose levels when administered to ESRD patients during heparin-free HD. A total of 36 adult patients are planned to be enrolled into two cohorts and dosed sequentially. Within each cohort, patients will be randomized to active drug (12) or placebo (6). Dose levels (1.5 µg/kg or 3 µg/kg; iv infusion) were based on safety data from a completed phase 1 study performed in healthy adult subjects (NCT03453060), which demonstrated that a single dose of AB002 was well tolerated, as well as based on preclinical efficacy studies performed in a baboon vascular thrombosis model. The primary objective of this phase 2 proof-of-concept study is safety and tolerability assessed as the number and severity of adverse events (AE), changes in baseline physical and lab parameters and immunogenicity of AB002. The number and severity of AE will be tabulated and summary statistics evaluated. The secondary objectives are to assess 1) pharmacodynamics using activated protein C/protein C inhibitor complexes in plasma as a surrogate marker for drug exposure, and 2) efficacy via pre-post dialyzer pressures, visual inspection of thrombus accumulation in the HD filter and dialysis efficiency based on urea kinetics. The study population includes ESRD patients on a stable outpatient HD regimen at least 3 times per week and excludes patients with history of acute vaso-occlusive thrombotic events, concomitant use of anticoagulant/antiplatelet agents immediately prior to and during the study, active cancer and bleeding disorders. This study is currently recruiting participants at one study site (Orlando Clinical Research Center) in Orlando, FL and is estimated to complete in October 2020 (NCT03963895). Disclosures Verbout: Aronora, Inc.: Employment, Equity Ownership. Lorentz:Aronora, Inc.: Employment. Markway:Aronora, Inc.: Employment. Shatzel:Aronora, Inc.: Consultancy. Tucker:Aronora, Inc.: Employment, Equity Ownership. Gruber:Aronora, Inc.: Employment, Equity Ownership.

Up to 30% of men with normal semen parameters suffer from infertility and the reason for this is unknown. Altered expression of sperm proteins may be a major cause of infertility in these men. Proteomic profiling was performed on pooled semen samples from eight normozoospermic fertile men and nine normozoospermic infertile men using LC-MS/MS. Furthermore, key differentially expressed proteins (DEPs) related to the fertilization process were selected for validation using Western blotting. A total of 1139 and 1095 proteins were identified in normozoospermic fertile and infertile men, respectively. Of these, 162 proteins were identified as DEPs. The canonical pathway related to free radical scavenging was enriched with upregulated DEPs in normozoospermic infertile men. The proteins associated with reproductive system development and function, and the ubiquitination pathway were underexpressed in normozoospermic infertile men. Western blot analysis revealed the overexpression of annexin A2 (ANXA2) (2.03 fold change, P = 0.0243), and underexpression of sperm surface protein Sp17 (SPA17) (0.37 fold change, P = 0.0205) and serine protease inhibitor (SERPINA5) (0.32 fold change, P = 0.0073) in men with unexplained male infertility (UMI). The global proteomic profile of normozoospermic infertile men is different from that of normozoospermic fertile men. Our data suggests that SPA17, ANXA2, and SERPINA5 may potentially serve as non-invasive protein biomarkers associated with the fertilization process of the spermatozoa in UMI.

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.

Protein C inhibitor is a heparin dependent serine protease inhibitor found in human plasma, urine and other body fluids. It was originally identified as an inhibitor of activated protein C. Stroke is an important cause of morbidity and mortality in the pediatric age group. In this study we analyzed the protein C inhibitor gene mutations in Turkish pediatric stroke patients. We found a missense mutation of G to A at nucleotide 6760 in exon 2, resulting in a transition serine to asparagine (p.Ser188Asp) and in a child and his father and also we found same alteration in exon 2 in an another pediatric stroke case following bone marrow transplantation.

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.

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.

Pro-coagulant, anti-coagulant and fibrinolytic pathways are responsible for maintaining hemostatic balance under physiological conditions. Any deviation from these pathways would result in hypercoagulability leading to life threatening diseases like myocardial infarction, stroke, portal vein thrombosis, deep vein thrombosis (DVT) and pulmonary embolism (PE). Angiogenesis is the process of sprouting of new blood vessels from pre-existing ones and plays a critical role in vascular repair, diabetic retinopathy, chronic inflammation and cancer progression. Serpins; a superfamily of protease inhibitors, play a key role in regulating both angiogenesis and coagulation. They are characterized by the presence of highly conserved secondary structure comprising of 3 β-sheets and 7-9 α-helices. Inhibitory role of serpins is modulated by binding to cofactors, specially heparin and heparan sulfate proteoglycans (HSPGs) present on cell surfaces and extracellular matrix. Heparin and HSPGs are the mainstay of anti-coagulant therapy and also have therapeutic potential as anti-angiogenic inhibitors. Many of the heparin binding serpins that regulate coagulation cascade are also potent inhibitors of angiogenesis. Understanding the molecular mechanism of the switch between their specific anti-coagulant and anti-angiogenic role during inflammation, stress and regular hemostasis is important. In this review, we have tried to integrate the role of different serpins, their interaction with cofactors and their interplay in regulating coagulation and angiogenesis.

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

The fibrinolytic system, also known as the plasminogen-plasmin (PP) system, is composed of plasminogen activators (PAs) which converts plasminogen to the proteolytic enzyme plasmin. It is maintained in a state of balance by inhibitors of PAs and of plasmin (Fig. 1). There are four known PA inhibitors (PAIs): PAI-1, PAI-2, activated protein C inhibitor (also known as PAI-3), and thrombin activatable fibrinolytic inhibitor (TAFI), of which PAI-1 has the most influence on many physiologic and pathologic functions.

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

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

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

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

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) values0.998 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 a14 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.

SummaryRecent in vitro studies have shown that the zymogen and activated form of factor (F)VII bind to endothelial cell protein C receptor (EPCR). At present, there is no evidence that FVIIa binds to EPCR on vascular endothelium in vivo in the presence of circulating protein C, a primary ligand for EPCR. The present study was carried out to investigate the interaction of murine and human ligands with murine EPCR both in vivo and in vitro. Measurement of endogenous plasma levels of FVII in wild-type, EPCR-deficient and EPCR-over expressing mice showed slightly lower levels of FVII in EPCR-over expressing mice. However, infusion of high concentrations of competing ligands, either human APCi or FVIIai, to EPCR-over expressing mice failed to increase plasma levels of mouse FVII whereas they increased the plasma levels of protein C by two- to three-fold. Examining the association of exogenously administered mouse FVIIa or human FVIIa by immunohistochemistry revealed that human, but not murine FVIIa, binds to the murine endothelium in an EPCR-dependent manner. In vitro binding studies performed using surface plasmon resonance and endothelial cells revealed that murine FVIIa binds murine EPCR negligibly. Human FVIIa binding to EPCR, particularly to mouse EPCR, is markedly enhanced by availability of Mg2+ ions. In summary, our data show that murine FVIIa binds poorly to murine EPCR, whereas human FVIIa binds efficiently to both murine and human EPCR. Our data suggest that one should consider the use of human FVIIa in mouse models to investigate the significance of FVIIa and EPCR interaction.

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

Heparin-induced thrombocytopenia (HIT) is caused by antibodies that recognize complexes between platelet factor 4 (PF4) and heparin or glycosaminoglycan side chains. These antibodies can lead to a limb- and life-threatening prothrombotic state. We now show that HIT antibodies are able to inhibit generation of activated protein C (aPC) by thrombin/thrombomodulin (IIa/TM) in the presence of PF4. Tetrameric PF4 potentiates aPC generation by formation of complexes with chondroitin sulfate (CS) on TM. Formation of these complexes occurs at a specific molar ratio of PF4 to glycosaminoglycan. This observation and the finding that the effect of heparin on aPC generation depends on the concentration of PF4 suggest similarity between PF4/CS complexes and those that bind HIT antibodies. HIT antibodies reduced the ability of PF4 to augment aPC formation. Cationic protamine sulfate, which forms similar complexes with heparin, also enhanced aPC generation, but its activity was not blocked by HIT antibodies. Our studies provide evidence that complexes formed between PF4 and TM's CS may play a physiologic role in potentiating aPC generation. Recognition of these complexes by HIT antibodies reverses the PF4-dependent enhancement in aPC generation and may contribute to the prothrombotic nature of HIT.

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