Search

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.

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., Activated protein C (APC) is a plasma serine protease with antithrombotic and cytoprotective functions. Here, the authors develop a monoclonal antibody that specifically inhibits APC’s anticoagulant function without compromising its cytoprotective function, and shows efficacy in animal models.

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.

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.

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.

Introduction Activated protein C (APC) is the central enzyme of the anticoagulant protein C pathway. Low concentrations of APC circulate in plasma and are believed to contribute to the maintenance of a normal haemostatic balance. Materials and Methods We have used a structure-based virtual screening approach to discover small drug-like molecules that inhibit the interaction between APC and its substrate FVa through inhibition of a predominant APC exosite, known to be involved in FVa substrate binding. We have combined in silico selection with functional screening and direct binding analysis to identify novel molecules and to ascertain and characterize the inhibition of the interaction between APC and FVa. Results We have identified a number of novel molecules that bind to APC and protein C with Kd values in the range of 10 -3 – 10 -5 M. Inhibition by these molecules is incomplete, which most likely reflects the extended surface that is involved in the interaction between APC and its substrates. Direct binding of hit molecules to variant APC molecules that were mutated in the targeted binding site revealed that several of the molecules presented a 100–500 fold lower affinity for the variant molecule, suggesting that these molecules indeed bind the exosite of APC. Conclusions The protein-protein interaction inhibitors discovered here, could function as starting molecules for further development of small molecules with anti-APC properties. Such molecules may be of clinical interest, in particular in individuals where thrombin formation is compromised and the haemostatic balance is tipped towards bleeding tendencies, such as in haemophilia A.

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.

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.

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.

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.

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.

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.

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.

Introduction: Direct oral anticoagulants are as effective as vitamin K antagonists for the treatment of venous thromboembolism and are associated with less bleeding. We have reported that a direct oral factor Xa inhibitor edoxaban exerts a thrombus resolution effect in a rat model of venous thrombosis and enhances tissue plasminogen activator (t-PA)-induced clot lysis in human plasma. However, the mechanism underlying the thrombus resolution effect and fibrinolysis enhancement by edoxaban remains to be determined. Purposes: To evaluate the effect of edoxaban on plasmin generation during clot lysis in human plasma in vitro. To determine the role of thrombin-activatable fibrinolysis inhibitor (TAFI) in the enhancement of fibrinolysis by edoxaban. Methods: Pooled human normal plasma or TAFI-deficient plasma (both containing 180 ng/mL t-PA and 0.1 nM thrombomodulin) was mixed with edoxaban, an activated TAFI (TAFIa) inhibitor (a fibrinolysis enhancer) potato tuber carboxypeptidase inhibitor (PCI), or vehicle. Clot was induced by adding 2.5 pM tissue factor and 4 µM phospholipids. To monitor the clot formation and lysis, the absorbance of plasma at 405 nm was measured every 30 sec. Clot lysis time was defined as the interval between the time of the midpoint of the clear to maximum turbidity transition and the midpoint of the maximum turbidity to clear transition. Plasmin-α2 antiplasmin complex (PAP) concentration was measured by ELISA as an indicator of plasmin generation. Results: In normal plasma, PCI accelerated clot lysis and increased plasma PAP concentration. There was a correlation between plasma PAP concentration and percent of clot lysis, indicating that plasma PAP concentration is an appropriate marker of fibrinolysis. Edoxaban at clinically relevant concentrations (75, 150, and 300 ng/mL) significantly shortened clot lysis time and elevated plasma PAP concentration. The additive combined effect of edoxaban and PCI was observed. In TAFI-deficient plasma, clot lysis time was shortened and plasma PAP concentration increased compared with normal plasma. In these samples, the effects of edoxaban and PCI on clot lysis and plasma PAP concentration were markedly diminished as compared with normal plasma. Conclusions: Edoxaban at clinically relevant concentrations enhanced t-PA-induced clot lysis with increasing plasma PAP concentration in human plasma. The effects of edoxaban on clot lysis and plasmin generation were diminished in TAFI-deficient plasma. A TAFIa inhibitor PCI exerted similar effects. These data suggest that edoxaban enhanced fibrinolysis via inhibition of TAFI activation and enhancement of plasmin generation. Disclosures Morishima: Daiichi Sankyo Co., Ltd.: Employment. Honda:Daiichi Sankyo Co., Ltd.: Employment. Furugohri:Daiichi Sankyo Co., Ltd.: Employment.

BACKGROUND: Tissue Factor Pathway inhibitor (TFPI) is a plasma serine protease inhibitor that modulates the initiation of coagulation by directly binding and inhibiting the Tissue Factor (TF)/Factor VIIa/Factor Xa complex. TFPI is a multi-Kunitz domain protein that directly binds to and inhibits both activated Factor Xa (FXa) and FVIIa. Blocking TFPI can act as a bypass therapy by facilitating hemostasis initiated by tissue factor/FVIIa, thereby, compensating for loss of Factor VIII or Factor IX (in hemophilia A or B). PF-06741086, a fully human antibody engineered to inhibit TFPI, exhibits broad cross reactivity to TFPI from numerous species, including mouse. PF-06741086 is being developed as a potential treatment for bleeding disorders including hemophilia A and hemophilia B with and without inhibitors. aPCC (activated Prothrombin complex concentrates or FEIBA, Factor Eight Inhibitor Bypass Agent) is a bypass agent for to control bleed in Hemophilia patients with inhibitors. Since it is a plasma-derived concentrate containing various prothrombin complex coagulation factors in their enzymatic or zymogen form, it is possible that FEIBA could potentially impact the activity of PF-06741086. AIMS: Here, we directly compare the hemostatic effect of PF-06741086 alone, and in combination with aPCC in Hemophilia A mouse model using severe tail vein transection. METHODS: Male hemophilia A mice were dosed with a single intravenous dose of PF-06741086 (0.5, 1, 2 or 6 mg/kg) 30 minutes prior to a 3mm tail clip, or aPCC (50, 100 or 200 U/kg) was administered 5 minutes before the tail clip. Mice were also treated with a combined dose of 0.5 mg/kg anti-TFPI PF-06741086 and aPCC at 50, 100 or 200 U/kg. Blood was collected for 10 minutes and quantified against a standard curve of hemoglobin as volume blood loss. RESULTS: PF-06741086 demonstrated a dose dependent response in improving hemostasis in Hemophilia A mice after tail clip. PF-06741086 was able to restore hemostasis at 1 mg/kg (49%), and higher doses further improved hemostasis at 2 mg/kg (63%), and 6 mg/kg (78%). aPCC also demonstrated a dose dependent reduction in blood loss and improved hemostasis with all tested doses of 50 U/kg (25%), 100 U/kg (23%) and 200 U/kg (66%). At a dose of 0.5 mg/kg, PF-06741086 did not show any improvement in hemostasis over vehicle control. We used this dose for all combination studies with aPCC. Combined use of low dose PF-06741086 (0.5 mg/kg) and 100 U/kg aPCC shows a trend towards improvement in hemostasis compared to either drug alone. A higher dose of aPCC (200 U/kg) combined with low dose PF-06741086 (0.5 mg/kg) significantly reduces blood loss (86%) in Hemophilia A mice in tail clip model compared to saline, TFPI or aPCC alone used at the same dose of 0.5 mg/kg or 200 U/kg respectively. CONCLUSIONS: Prophylactic administration of PF-06741086 exhibits a dose response and improves hemostasis in an injury model in Hemophilia A mice. The addition of aPCC alone restores hemostasis at 200 U/kg and this effect was enhanced in combination with PF-06741086 in this mouse model. Disclosures Barakat: Pfizer: Employment. Jasuja:Pfizer: Employment. Murphy:Pfizer: Employment. Pittman:Pfizer: Employment.

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

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.

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.

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

ObjectiveChildren who undergo surgery for complex congenital cardiac disease are reported to be at increased thrombotic risk. Our aim was to evaluate long-term changes in the haemostatic system after surgery, to compare markers of activated coagulation in children having surgery with those in a healthy control population, and to relate them to adverse clinical outcome.Patients and methodsWe studied, prior to surgery, the coagulation profiles of a cohort of 28 children admitted for a modified Fontan operation, studying them again after a period of mean follow-up of 9.6 years. Median age at the time of final surgery was 18.5 months, with a range from 12 to 76 months. We compared generation of thrombin, and levels of the activated protein C-protein C inhibitor complex to controls at follow-up. Thrombophilia and clinical outcome were evaluated.ResultsAt long-term follow-up, a lower incidence of procoagulant abnormalities was observed compared to that before surgery. Of 27 patients, 3 (11%), but none of 45 controls, had levels of activated protein C-protein C inhibitor complex above the reference range. There were no significant differences in generation of thrombin between patients and controls. No thrombotic events were recorded, and the patients were generally in good clinical condition.ConclusionsOverall, haemostasis appeared to be in balance, and less prothrombotic, after surgery. A subset of the cohort did show indications of activated coagulation. The current therapeutic approach seems to be sufficient to protect the majority of patient. New tests of global coagulation, nonetheless, may be helpful in improving identification of individuals at increased thrombotic risk.

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

Increased concentration of activated protein C (APC) has been observed in patients with thromboembolic disorders, but whether the level of APC in patients with bleeding disorders is decreased remains unknown. Seventy patients with haemophilia A or B with mild, moderate or severe form were studied. Detailed information on bleeding, arthropathy and factor consumption was collected during a 10-year period. The clinical severity of the disease was expressed as the Hemophilia Severity Score (HSS). Plasma concentration of APC was measured as APC in complex with protein C inhibitor. The median concentration of APC-PCI complex in patients with haemophilia was 0.14 mu g L-1 and it did not differ between the types and forms. In 16 patients with severe haemophilia A and the inversion mutation in intron 22, there was no correlation between clinical severity and the concentration of APC-PCI complex. Patients with haemophilia appear to generate normal concentrations of APC during basal conditions. APC does not seem to be an important modulator of the phenotypic expression of haemophilia. (Less)

Background: Vascular access failure is a common cause of morbidity in patients with end-stage renal failure on hemodialysis (HD). Activation of the coagulation system and formation of a thrombus play important roles in recurrent arteriovenous fistula/graft (AVFG) failure. Thrombin in complex with thrombomodulin (TM) activates the anticoagulant protein C and creates activated protein C (APC), which is subsequently inactivated by the protein C inhibitor (PCI). The plasma concentration of the complex between APC and PCI (P-APC-PCI complex) is increased in hypercoagulable states and is therefore a sensitive indicator of the degree of activation of blood coagulation. Methods: Thirty-five HD patients dialyzed through a functioning AVFG were studied. The period of patency of AVFGs was recorded. Blood was drawn before and after the HD session for the analysis of the APC-PCI complex, soluble TM concentration and activity, von Willebrand factor antigen and homocysteine. Results: Patients with frequent AVFG failures (n = 8) had a significantly lower level of P-APC-PCI complex (median 0.09 µg/l) than those with less frequent AVFG failures (median 0.18 µg/l; n = 27; p = 0.04). No other significant differences were found between the groups. Conclusion: Thus, a low level of P-APC-PCI complex may be associated with an increased risk of AVFG failure in HD patients. Further prospective studies are needed to confirm these results and to evaluate the possibility of prophylactic measures.

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

Increased thrombin activation was documented in patients with abdominal aortic aneurysm (AAA). Activated protein C—protein C inhibitor (APC—PCI) complex, a new biological marker of thrombin generation, was measured in a population of 232 patients with AAA and a control group, and the association between aneurysm size, growth rate, and APC—PCI was studied. The patients were divided into cohorts according to AAA diameter and compared with a control group. APC—PCI was significantly higher in all AAA cohorts (n = 232; median, 0.36 µg/L; 10th to 90th percentile, 0.18-1.01) compared with the control group (n = 41; median, 0.19 µg/L; 10th to 90th percentile, 0.11-0.31; P ≤ .001). APC—PCI correlated with AAA diameter ( r = .22; P = .001), body mass index ( r = −.19; P = .004), and age ( r = .19; P = .004). APC—PCI did not correlate with AAA growth rate ( r = .11; P = .14).

Protein C inhibitor (PCI) is a serpin with many roles in biology, including a dual role as pro- and anticoagulant in blood. The protease specificity and local function of PCI depend on its interaction with cofactors such as heparin-like glycosaminoglycans (GAGs) and thrombomodulin (TM). Both cofactors significantly increase the rate of thrombin inhibition, but GAGs serve to promote the anticoagulant activity of PCI, and TM promotes its procoagulant function. To gain insight into how PCI recognition of thrombin is aided by these cofactors, we determined a crystallographic structure of the Michaelis complex of PCI, thrombin, and heparin to 1.6 Å resolution. Thrombin interacts with PCI in an unusual fashion that depends on the length of PCI's reactive center loop (RCL) to align the heparin-binding sites of the two proteins. The principal exosite contact is engendered by movement of thrombin's 60-loop in response to the unique P2 Phe of PCI. This mechanism of communication between the active site of thrombin and its recognition exosite is previously uncharacterized and may relate to other thrombin substrate–cofactor interactions. The cofactor activity of heparin thus depends on the formation of a heparin-bridged Michaelis complex and substrate-induced exosite contacts. We also investigated the cofactor effect of TM, establishing that TM bridges PCI to thrombin through additional direct interactions. A model of the PCI–thrombin–TM complex was built and evaluated by mutagenesis and suggests distinct binding sites for heparin and TM on PCI. These data significantly improve our understanding of the cofactor-dependent roles of PCI in hemostasis.

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

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

Aims Activated protein C (APC) is a key regulator of the clotting system and immune responses. We studied the relationship between the degree of atherosclerosis as measured by the intima-media thickness (IMT) of carotid artery and APC generation in Type 2 diabetic patients. Methods Eighty-seven Type 2 diabetic patients and 35 control subjects participated. APC generation was assessed by the plasma APC–protein C inhibitor complex (APC-PCI) levels and the mean IMT of carotid artery was measured by ultrasonography. The plasma levels of the thrombin–anti-thromobin complex (TAT) and platelet-derived growth factor (PDGF) were measured by enzyme-linked immunoassays. Results Plasma TAT levels were significantly higher in diabetic patients [2.03 (1.12, 2.56) ng/ml, median (25th, 75th percentile)] compared with control subjects [0.85 (0.55, 2.08) ng/ml, P

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

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

Protein C inhibitor (PCI) is a serpin with affinity for heparin and phosphatidylethanolamine (PE). We analyzed the interaction of PCI with different phospholipids and their oxidized forms. PCI bound to oxidized PE (OxPE), and oxidized and unoxidized phosphatidylserine (PS) immobilized on microtiter plates and in aqueous suspension. Binding to OxPE and PS was competed by heparin, but not by the aminophospholipid-binding protein annexin V or the PCI-binding lipid retinoic acid. PS and OxPE stimulated the inhibition of activated protein C (aPC) by PCI in a Ca++-dependent manner, indicating that binding of both, aPC (Ca++ dependent) and PCI (Ca++ independent), to phospholipids is necessary. A peptide corresponding to the heparin-binding site of PCI abolished the stimulatory effect of PS on aPC inhibition. No stimulatory effect of phospholipids on aPC inhibition was seen with a PCI mutant lacking the heparin-binding site. A heparin-like effect of phospholipids (OxPE) was not seen with antithrombin III, another heparin-binding serpin, suggesting that it is specific for PCI. PCI and annexin V were found to be endogenously colocalized in atherosclerotic plaques, supporting the hypothesis that exposure of oxidized PE and/or PS may be important for the local regulation of PCI activity in vivo.

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

d email: ycy201388@163.com Abstract. The influence of glycosaminoglycan from Urechis unicinctus on the protein C system and relevant factors expression of human umbilical vein endothelial cells (HUVECs) were studied with enzyme linked immunosorbent and reverse transcript-polymerase chain reaction (RT-PCR). Results indicated that: glycosaminoglycan could significantly decrease content of activated protein C, activated protein S in blood of rats (P

Protease nexin-1 (PN-1), also known as SERPINE2, is the phylogenetically closest relative of plasminogen activator type 1 (PAI-1). Unlike PAI-1, antithrombin (AT), protein C inhibitor (PCI) or heparin cofactor II (HCII), other members of the serpin family that share its ability to inhibit thrombin, PN-1 is not detectable in the circulating blood. In contrast, PN-1 is found within diverse organs, including the brain, the blood vessel wall and the lungs, among many others. PN-1 expression is tightly and finely regulated in different organs at distinct stages of development, suggesting a dynamic role in vivo depending on the cell type, the tissue and the stage of development. PN-1 is now recognized as a central regulator of vascular thrombosis and appears to play a protective role in the different tissues where it is expressed, by preventing pathological states induced by excessive proteolytic activities. In this chapter, we examine current knowledge of PN-1 in the pathophysiology of the nervous, vascular, respiratory, renal and reproductive systems, as well as in the development of tumours.

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

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

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