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Context.--Rivaroxaban causes a false increase in activated protein C resistance (APCR) ratios and protein S activity.Objective.--To investigate whether this increase masks a diagnosis of factor V Leiden (FVL) or protein S deficiency in a 'real-world' population of patients undergoing rivaroxaban treatment and hypercoagulation testing.Design.--During a 2.5-year period, we compared 4 groups of patients (n = 60): FVL heterozygous (FVL-HET)/ taking rivaroxaban, wild-type/taking rivaroxaban, FVLHET/no rivaroxaban, and normal APCR/no rivaroxaban. Patients taking rivaroxaban were tested for protein S functional activity and free antigen (n = 32).Results.--The FVL-HET patients taking rivaroxaban had lower APCR ratios than wild-type patients (P < .001). For FVL-HET patients taking rivaroxaban, mean APCR was 1.75 [+ or -] 0.12, versus 1.64 [+ or -] 0.3 in FVL-HET patients not taking rivaroxaban (P = .005). Activated protein C resistance in FVL-HET patients fell more than 3 SDs below the cutoff of 2.2 at which the laboratory reflexes FVL DNA testing. No cases of FVL were missed despite rivaroxaban. In contrast, rivaroxaban falsely elevated functional protein S activity, regardless of the presence or absence of FVL (P < .001). A total of 4 of 32 patients (12.5%) had low free protein S antigen (range, 58%-67%), whereas their functional protein S activity appeared normal (range 75%-130%). Rivaroxaban would have caused a missed diagnosis of all cases of protein S deficiency during the study if testing relied on the protein S activity assay alone.Conclusions.--Despite rivaroxaban treatment, APCR testing can distinguish FVL-HET from normal patients, rendering indiscriminate FVL DNA testing of all patients on rivaroxaban unnecessary. Free protein S should be tested in patients taking rivaroxaban to exclude hereditary protein S deficiency.(Arch Pathol Lab Med. 2018;142:70-74; doi: 10.5858/arpa.2016-0616-OA), Rivaroxaban is a direct, antithrombin-independent factor Xa inhibitor, which inhibits not only free factor Xa, but also clot-bound factor Xa and the prothrombinase complex. It is used for treatment and [...]

In skinned myocardium, cyclic AMP--dependent protein kinase A (PKA)-catalyzed phosphorylation of cardiac myosin--binding protein C (cMyBP-C) and cardiac troponin I (cTnI) is associated with a reduction in the [Ca.sup.2+] responsiveness of myofilaments and an acceleration in the kinetics of cross-bridge cycling, although the respective contribution of these two proteins remains controversial. To further examine the relative roles that cTnI and cMyBP-C phosphorylation play in altering myocardial function, we determined the [Ca.sup.2+] sensitivity of force ([pCa.sub.50]) and the activation dependence of the rate of force redevelopment ([k.sub.tr]) in control and PKA-treated mouse myocardium (isolated in the presence of 2,3-butanedione monoxime) expressing: (a) phosphorylatable cTnI and cMyBP-C (wild type [WT]), (b) phosphorylatable cTnI on a cMyBP-C--null background ([cMyBP-C.sup.-/-]), (c) nonphosphorylatable cTnI with [serines.sup.23/24/43/45] and [threonine.sup.144] mutated to alanines ([cTnI.sub.Ala5]), and (d) nonphosphorylatable cTnI on a cMyBP-C-null background ([cTnI.sub.Ala5]/[cMyBP-C.sup.-/-]). Here, PKA treatment decreased [pCa.sub.50] in WT, [cTnI.sub.Ala5], and [cMyBP-C.sup.-/-] myocardium by 0.13, 0.08, and 0.09 pCa units, respectively, but had no effect in [cTnI.sub.Ala5]/[cMyBP-C.sup.-/-] myocardium. In WT and [cTnI.sub.Ala5] myocardium, PKA treatment also increased [k.sub.tr] at submaximal levels of activation; however, PKA treatment did not have an effect on [k.sub.tr] in [cMyBP-C.sup.-/-] or [cTnI.sub.Ala5]/[cMyBP-C.sup.-/-] myocardium. In addition, reconstitution of [cTnI.sub.Ala5]/[cMyBP-C.sup.-/-] myocardium with recombinant cMyBP-C restored the effects of PKA treatment on [pCa.sub.50] and [k.sub.tr] reported in [cTnI.sub.Ala5] myocardium. Collectively, these results indicate that the attenuation in myofilament force response to PKA occurs as a result of both cTnI and cMyBP-C phosphorylation, and that the reduction in [pCa.sub.50] mediated by cMyBP-C phosphorylation most likely arises from an accelerated cross-bridge cycling kinetics partly as a result of an increased rate constant of cross-bridge detachment. 10.1085/jgp.201010448

The coagulation system is central to the pathophysiology of acute lung injury. We have previously demonstrated that the anticoagulant activated protein C (APC) prevents increased endothelial permeability in response to edemagenic agonists in endothefial cells and that this protection is dependent on the endothelial protein C receptor (EPCR). We currently investigate the effect of APC in a mouse model of ventilator-induced lung injury (VILI). C57BL/6J mice received spontaneous ventilation (control) or mechanical ventilation (MV) with high ([HV.sub.T]; 20 ml/kg) or low ([LV.sub.T]; 7 ml/kg) tidal volumes for 2 h and were pretreated with APC or vehicle via jugular vein 1 h before MV. In separate experiments, mice were ventilated for 4 h and received APC 30 and 150 min after starting MV. Indices of capillary leakage included bronchoalveolar lavage (BAL) total protein and Evans blue dye (EBD) assay. Changes in pulmonary EPCR protein and Rho-associated kinase (ROCK) were assessed using SDS-PAGE. Thrombin generation was measured via plasma thrombin-antithrombin complexes. HVT induced pulmonary capillary leakage, as evidenced by significant increases in BAL protein and EBD extravasation, without significantly increasing thrombin production. [HV.sub.T] also caused significant decreases in pulmonary, membrane-bound EPCR protein levels and increases in pulmonary ROCK-1. APC treatment significantly decreased pulmonary leakage induced by MV when given either before or after initiation of MV. Protection from capillary leakage was associated with restoration of EPCR protein expression and attenuation of ROCK-1 expression. In addition, mice overexpressing EPCR on the pulmonary endothelium were protected from [HV.sub.T]-mediated injury. Finally, gene microarray analysis demonstrated that APC significantly altered the expression of genes relevant to vascular permeability at the ontology (e.g., blood vessel development) and specific gene (e.g., MAPK-associated kinase 2 and integrin-[[beta].sub.6]) levels. These findings indicate that APC is barrier-protective in VILI and that EPCR is a critical participant in APC-mediated protection. endothelial protein C receptor; mechanical stress; vascular barrier function; ventilator-induced lung injury

To investigate the potential role of the local expression of alternative complement factor B (hBf) in human sepsis, we examined the induction of Bf gene expression in human peripheral blood monocytes (PBMCs) from patients with septic shock and the mechanisms of hBf gene regulation by tumor necrosis factor (TNF)-[alpha], interferon (IFN)-[gamma], and lipopolysaccharide (LPS) in human monocytes. PBMCs from septic shock patients showed increased hBf mRNA expression when compared with control patients. Costimulation with TNF-[alpha] and IFN-[gamma] or stimulation with LPS demonstrated a time-and dose-dependent induction of hBf mRNA expression in human PBMCs. A region of the hBf promoter between -735 and + 128 bp was found to mediate IFN-[gamma], TNF-[alpha], and LPS responsiveness as well as the synergistic effect of IFN-[gamma]/TNF-[alpha] on hBf promoter activity. Site-directed mutagenesis of a IFN-[gamma]-activation site (GAS) cis element (-90 to -82 bp) abrogated IFN-[gamma] responsiveness. Mutagenesis of a nuclear factor (NF)-[kappa]B cis element at -466 to -456 bp abrogated TNF-[alpha] and LPS responsiveness of the Bf promoter. Thus hBf gene expression is induced in PBMCs from septic shock patients, and the induction of hBf by IFN-[gamma] TNF-[alpha], and LPS is through GAS and NF-[kappa]B cis-binding sites on the hBf promoter. Furthermore, activated protein C (APC) inhibited LPS-stimulated hBf promoter activity and protein expression in human monocytes suggesting that the beneficial effect of APC therapy in sepsis may in part be due to inhibition of complement induction and/or activation via the alternative pathway. septic shock; monocytes/macrophages; complement factor B; cytokines

Byline: Fabio Baratto (1), Flavio Michielan (1), Muzio Meroni (1), Antonella Dal Palu (1), Annalisa Boscolo (1), Carlo Ori (1) Keywords: Sepsis; Therapy; Shock; Septic; Protein C Abstract: Objective To describe the efficacy and safety of protein C (PC) concentrate to restore physiological values in adult septic patients having clinical contraindications to activated PC. Design Case series (pilot study). Setting Three adult ICUs of a University Hospital. Patients and participants Twenty adult patients affected by severe sepsis or septic shock with plasma values of PC < 50%. Interventions Patients were treated with PC concentrate (Ceprotin .sup.(r)----Baxter) with a starting bolus followed by a continuous infusion for 72 h [3 IU/(kg h)]. Measurements and results PC activity, WBC, platelets, d-Dimer, fibrinogen, PT, aPTT, AT III, lactate, Sepsis-related Organ Failure Assessment (SOFA), Disseminated Intravascular Coagulation (DIC) score, adverse events, and mortality were measured. Baseline plasma PC activity was 34.5 +- 9.1%. PC concentrate normalized the PC activity in all patients within 48 h, and then remained stable for the following days. At baseline, several patients showed abnormal PT, aPTT, platelets values, and lactate levels. During the study period, there was a significant increase of platelets, fibrinogen, PT, AT III, and a significant decrease of d-Dimer, aPTT, DIC score, and lactate. No adverse reactions (hemorrhage or thrombosis) were observed. Mortality at 28 days was 35%. Conclusions Our pilot study shows that the administration of PC concentrate to patients having contraindications to the treatment with activated PC was safe and possibly useful to control the coagulopathy triggered and sustained by sepsis. A randomized, double blind study in patients with severe sepsis and contraindications to activated PC administration would be advisable to state the safety and the possible role of this product in the treatment of severe sepsis. Author Affiliation: (1) Department of Pharmacology and Anesthesiology, University of Padua, Padua, Italy Article History: Registration Date: 21/04/2008 Received Date: 19/03/2007 Accepted Date: 17/04/2008 Online Date: 06/05/2008

Endothelial protein C receptor (EPCR) and thrombomodulin (TM) are expressed at high levels in the resting microvasculature and convert protein C (PC) into its activated form, which is a potent [...]

Ever-increasing evidence in the literature suggests that the anti-inflammatory and cytoprotective properties of activated protein C (APC) are mediated through its endothelial protein C receptor (EPCR)-dependent cleavage of protease-activated receptor 1 (PAR-1) on endothelial cells. However, recent results monitoring the cleavage rate of PAR-1 on human umbilical vein endothelial cells, transfected with an alkaline phosphatase--PAR-1 fusion reporter construct, have indicated that the catalytic activity of thrombin toward PAR-1 is several orders of magnitude higher than that of APC. Because thrombin is required for generation of APC, and because it also functions in the proinflammatory pathways through the activation of PAR-1, it has been difficult to understand how APC can elicit protective cellular responses through the activation of PAR-1 when thrombin is present. In this study we provide a plausible answer to this question by demonstrating that the critical receptors required for both protein C activation (thrombomodulin and EPCR) and APC cellular signaling (EPCR and PAR-1) pathways are colocalized in the membrane lipid rafts in endothelial cells. We further show that the APC cleavage of PAR-1 on cells transfected with a PAR-1 cleavage reporter construct is not sensitive to the cofactor function of EPCR. Thus, the colocalization of EPCR and PAR-1 in lipid rafts is a key requirement for the cellular signaling activity of APC. Thrombomodulin colocalization with these receptors on the same membrane microdomain can also recruit thrombin to activate the EPCR-bound protein C, thereby eliciting PAR-1 signaling events that are involved in the APC protective pathways. endothelial protein C receptor | protease-activated receptor 1 | thrombin | thrombomodulin

Crystalline silica has long been well established as a fibrogenic agent, and recent evidence has implicated it as a potential human carcinogen. However, the mechanisms of silica-induced disease development and progression are not well understood. Our previo us studies demonstrated that crystalline silica is able to activate activator protein-1 (AP-1) through mitogen-activated protein kinase (MAPK) pathways. The present study investigates the possible involvement of protein kinase C (PKC) in silica-induced activation of the MAPK/AP-1 signal transduction pathway. Treatment of mouse epidermal cells (JB6 cell line) with freshly fractured silica stimulated translocation of PKC[alpha] and PKC [member of] from the cytosol to the membrane and activated AP-1 transcription activity. Pretreatment of cells with PKC inhibitors, including RO-32-0432, calphostin C, and bisindolylmaleimide I, inhibited silica-induced AP-1 activation and phosphorylation of ERKs and p38 kinase. These inhibitory effects by PKC inhibitors were dose dependent. Furthermore, overexpression of dominant negative mutant (DNM) of PKC [alpha] or PKC [member of] markedly blocked AP-1 activation as well as phosphorylation of ERKs and p38 kinase induced by freshly fractured silica. These results demonstrate that PKC [alpha] and PKC [member of] are essential in silica-induced AP-1 activation through the MAP kinase (ERKs and p38 kinases) pathway. protein kinase C; mitogen-activated protein kinase; activator protein-1; silica

Mechanical forces regulate lung maturation in the fetus by promoting type II epithelial differentiation. However, the cell surface receptors that transduce these mechanical cues into cellular responses remain largely unknown. When distal lung type II epithelial cells isolated from embryonic day 19 rat fetuses were cultured on flexible plates coated with laminin, fibronectin, vitronectin, collagen, or elastin and exposed to a level of mechanical strain (5%) similar to that observed in utero, transmembrane signaling responses were induced under all conditions, as measured by ERK activation. However, mechanical stress maximally increased expression of the type II cell differentiation marker surfactant protein C when cells were cultured on laminin substrates. Strain-induced alveolar epithelial differentiation was inhibited by interfering with cell binding to laminin using soluble laminin peptides (IKVIV or YIGSR) or blocking antibodies against integrin [[beta].sub.1], [[alpha].sub3], or [[alpha].sub.6]. Additional studies were carried out with substrates coated directly with different nonactivating anti-integrin antibodies. Blocking integrin [[beta].sub.1] and [[alpha].sub.6] binding sites inhibited both cell adhesion and differentiation, whereas inhibition of [[alpha].sub.3] prevented differentiation without altering cell attachment. These data demonstrate that various integrins contribute to mechanical control of type II lung epithelial cell differentiation on laminin substrates. However, they may act via distinct mechanisms, including some that are independent of their cell anchoring role. laminin; surfactant protein C

Surfactant protein B (SP-B) is required for function of newborn and adult lung, and partial deficiency has been associated with susceptibility to lung injury. In the present study, transgenic mice were produced in which expression of SP-B in type II epithelial cells was conditionally regulated. Concentrations of SP-B were maintained at 60-70% of that normally present in control. Immunostaining for SP-B demonstrated cellular heterogeneity in expression of the protein. In subsets of type II cells in which SP-B staining was decreased, immunostaining for pro-SP-C was increased and lamellar body ultrastructure was disrupted, consistent with focal SP-B deficiency. Fluorescence-activated cell sorting analyses of freshly isolated type II cells identified a population of cells with low SP-B content and a smaller population with increased SP-B content. confirming nonuniform expression of the SP-B transgene. Focal air space enlargement, without cellular infiltration or inflammation, was observed. Pressure-volume curves indicated that maximal tidal volume was unchanged: however, hysteresis was modestly altered and residual volumes were significantly decreased in the SP-B-deficient mice. Chronic, nonuniform SP-B deficiency perturbed pulmonary function and caused air space enlargement. transgenic mice; surfactant protein B; surfactant protein C

The term sepsis is derived from the Greek word sepien meaning 'to make rotten.' (1) In 2001, 1.3% of all deaths in the United States were attributed to sepsis, and [...]

Tetranectin (TN) is a homotrimeric protein, which is correlated with higher survival rates in several types of cancer, and it is found in human and several animal species. The structure and dynamics of the calcium-free, K4-binding form of the third exon of the TN gene (TN3) is addressed to gain further insight into the interaction between TN and plasminogen.

Proteins have emerged as viable treatments for a broad range of diseases, and their therapeutic applications have expanded considerably during the past decade. Although most biologics are administered in their native form to patients, engineering efforts to improve the properties of therapeutic proteins are becoming more prevalent. A recent study has shown that substantial improvements in the pharmacological profile of Xigris [R] can be achieved by mutations that diminish its inactivation by serpins, underscoring the importance of optimization efforts to create biologics with improved clinical profiles.

Byline: Francois Fourrier (1), Francis Leclerc (2), Karl Aidan (1), Ahmed Sadik (2), Merce Jourdain (1), Antoine Tournoys (3), Odile Noizet (2) Keywords: Meningococcemia; Purpura fulminans; Antithrombin; Protein C; Protein S; Plasma concentrate pharmacokinetics Abstract: Purpose To document in patients with meningococcal purpura fulminans (PF), the effects of a combined supplementation with antithrombin (AT) and protein C (PC) plasma concentrates and to estimate the pharmacokinetics and dose requirements of each inhibitor. Design Retrospective study of 15 patients. Setting One paediatric and one adult ICU in a university hospital. Interventions In addition to standard intensive care, all patients received a 100 IU/kg loading dose of AT and PC concentrates, followed by a continuous infusion (AT: 100--150 IU*kg*day PC: 100 IU*kg*day in adults, and 400 IU/kg in infants). Measurements Clinical data, coagulation, and fibrinolysis parameters, AT and PC activities, and free protein S (PS) levels were sequentially measured. Restitution ratio, median increment after supplementation, and half-life of clearance from plasma were calculated for the two plasma substitutes. Results At admission, all patients had a severe decrease in AT, PC, and PS levels. The supplementation regimen induced a substantial increase in AT and PC activities, peaking at H18 and H48, respectively. The supplementation procedure did not modify free PS levels. The median values of AT and PC restitution ratio, increment in plasma activity observed after 100 IU/kg concentrate, and apparent half-life of clearance from plasma were 0.85 U*ml*U*kg and 0.59 U*ml*U*kg, 23% and 21%, 16 h and 6 h, respectively. Conclusion If AT and PC concentrates are to be given in fulminant meningococcemia, the doses of supplementation should be at least 150 IU/kg AT and 250 IU/kg PC as loading dose and 150 IU/kg AT and 200 IU/kg PC as daily maintenance therapy. Taking into account the individual variability in inhibitor deficiency and restitution ratio, repeated measurements of plasma levels are mandatory to obtain a patient-based adjustment of the supplementation. Author Affiliation: (1) Service de Reanimation, Polyvalente Hopital Roger Salengro, CHRU, 59037, Lille, France (2) Service de Reanimation, Pediatrique Hopital Jeanne de Flandre, CHRU, Lille, France (3) Laboratoire d'Hematologie B, Hopital Roger Salengro, CHRU, Universite de Lille, Lille, France Article History: Received Date: 06/09/2002 Accepted Date: 27/03/2003 Online Date: 22/05/2003

Byline: Zoltan Szolnoki (2), Ferenc Somogyvari (1), Andras Kondacs (2), Mihaly Szabo (2), Lajos Fodor (1) Keywords: Key words Leiden V mutation; Stroke; ACE I/D polymorphism; Genetics; Risk factors Abstract: Objective The Leiden V mutation, which causes activated protein C resistance and thrombophilia, has been found to be a risk factor for venous thrombosis. The angiotensin converting enzyme (ACE) D allele indirectly exerts an unfavourable effect on the vasoregulatory system. In this study, the frequency of these mutations was analysed in different subtypes of ischaemic stroke. Method and material According to the clinical and radiological features 664 Hungarian patients who had suffered acute ischaemic stroke were divided into 3 subtypes: small and large vessel infarcts and a mixed type. In all 664 patients, the Leiden V mutation and ACE I/D polymorphism were examined by means of the PCR technique. The frequencies of the different genotypes for the Leiden V mutation and ACE I/D polymorphism in the 3 subgroups of stroke were compared with 199 stroke-free control subjects whose MRI findings were normal. Results No significant associations were found between the overall group of cerebral infarctions and the Leiden V, ACE I/D and ACE D/D genotypes. The ACE D/D genotype was significantly more common in the patients with small deep infarcts (40.3 % p &lt 0.0005 OR 2.31, 95 % CI 1.49--3.57) than in the control group (22.6 %). The Leiden V mutation was significantly more common in patients with large infarcts (13.6 % p &lt 0.025 OR 2.25, CI 1.16--4.34) than in the stroke-free control subjects (6.5 %). Conclusions The ACE D/D genotype possibly contributes to the occurrence of small-vessel infarcts rather than large vessel infarcts. The Leiden V mutation might predispose to large brain infarcts. Neither the Leiden V factor nor the ACE D/D genotype has been proved to be a risk factor for ischaemic stroke as a whole. Author Affiliation: (1) Central Laboratory, Pandy Kalman County Hospital, Gyula, Hungary, HU (2) Department of Neurology and Neurophysiology, Pandy Kalman County Hospital, 5700 Gyula, Semmelweis 1, Hungary, HU (3) 5600 Bekescsaba, Pipacs koz 9, Hungary, Tel.: +0036-66442904, e-mail: szolnoki99@hotmail.com, HU Article note: Received: 22 June 2000 / Received in revised form: 5 February 2001 / Accepted: 3 March 2001

Byline: Peter Zunker (1), Christian Hohenstein (4), Hans-Jorg Plendl (1), Jorn A. Zeller (1), Valeria Caso (2), Dimitrios Georgiadis (3), Arne Allardt (1), Gunther Deuschl (1) Keywords: Key words Ischaemic stroke; Thrombophilia; APC-resistance; TOAST criteria Abstract: Objectives Resistance to activated protein C (APC) is the most frequent cause of thrombophilia and a well known risk factor for deep and cerebral vein thrombosis. Its causative role in ischaemic stroke is still a matter of debate. We undertook this study to determine the prevalence of APC-resistance in a cohort of consecutive patients with acute ischaemic stroke, especially with respect to patients' age and the underlying stroke causation. Materials and methods 489 patients with proven ischaemic stroke were included in this study. Subtypes of stroke were classified according to the TOAST criteria, i. e. large artery artherosclerosis (LAA), small vessel occlusion (SVO), cardioembolism (CE), stroke of other etiology (SOE), and stroke of undetermined etiology (SUE). APC-resistance was determined with a functional method with high sensitivity and specificity for the factor V Leiden mutation. The results were compared with the prevalence of APC-resistance in healthy volunteers, all born in the same area. Results APC-resistance was found in 24 of 489 patients (4.9 %) and in 6 of the 112 (5.4 %) control subjects. In the stroke patients, APC-resistance was distributed as follows: LAA 6.5 % (9/138), SVO 3,9 % (4/104), CE 6.7 % (7/104), SOE 3.6 % (1/28), SUE 2.6 % (3/115). Prevalence of APC-resistance was not significantly different between young stroke patients (6--45 years) and older patients (7.7 % [5/65] versus 4.5 % [19/424]). Conclusions Prevalence of APC-resistance is not increased in patients with ischaemic stroke. Additionally, no significant differences in the prevalence of APC-resistance are evident within the various stroke subtypes. Author Affiliation: (1) Department of Neurology and Institute of Human Genetics, Kiel University Hospital, Niemannsweg 147, 24105 Kiel, Germany, DE (2) Department of Neurology, University Hospital Perugia, 06123 Perugia, Italy, IT (3) Department of Neurology, Heidelberg University Hospital, Im Neuenheimer Feld 400, 69120 Heidelberg, Germany, DE (4) Department of Surgery, Ludwig-Heilmeyer Str. 1, 89312 Guenzburg, Germany Tel.: +49-8221-96-01 Fax: +49-8221-96-1114 e-mail: hohensteinc@gmx.net, DE Article note: Received: 11 September 2000 / Received in revised form: 2 February 2001 / Accepted: 3 March 2001

Research has been conducted on the activated protein C-mediated inactivation of factor Va. The effect of the C4b-binding protein on this factor in the absence and presence of protein S has been investigated.

Research has been conducted on the activated protein C. The use of molecular modeling and site-directed mutagenesis in creating a human protein C resistant to alpha1-antitrypsin inhibition is described.

Research was conducted to examine the variation in the affinity of propeptide amino acids involved in the binding of gamma-glutamyl carboxylase to its protein substrates by using a synthesized propeptide containing high affinity residues. Results show that the reduced binding affinity is due to residues at 15, 10, 6, and 9.

The article characterizes functional determinants of nitrogen regulatory protein C (NtrC). Mutagenesis experiments to scan the central domain of Salmonella typhimurium NtrC protein for amino acid residues are discussed.

A study was conducted to analyze the phospholipid composition requirements for maximum prothrombin activation and factor Va inactivation by activated protein C anticoagulant. A three-stage assay was utilized to examine factor Va inactivation. Results indicated that changes in the phospholipid composition of cells can influence procoagulant and anticoagulant reactions under normal and pathological environments.

A study was conducted to determine if recombinant human soluble thrombomodulin can stem the increase of rat pulmonary vascular permeability induced by endotoxin through protein C activation. Results show that recombinant human soluble thrombomodulin treated with monoclonal antibody F2H5 cannot inhibit endotoxin-induced coagulation. Results also indicate that endotoxin-induced intravascular coagulation is inhibited by recombinant human soluble thrombomodulin through thrombin binding.

Nuclear magnetic resonance spectroscopic study was utilized to describe the solution structure and backbone dynamics of the human alpha3-chain type VI collagen. Results have shown that the global fold configuration retained its form in the solution phase and assumed the same form as in the crystal phase and C5 domain core. It was indicated that the additional six residues in the N-terminal and the 12 residues in the C-terminal affected the function of the C5 domain.

The specificity of thrombin toward protein C was optimized by individually substituting thrombin residues W50, K52, E229 and R233 with all 19 naturally occurring amino acids. Screening of this collection of all 76 possible substitutions revealed a mutant, E229K, with improved specificity for protein C and improved anticoagulant potency in vivo which may be attributable to decreased clearance by antithrombin III, the main physiological inhibitor of thrombin.

Centromere proteins are localized within the centromere-kinetochore complex, which can be proven by means of immunofluorescence microscopy and immunoelectron microscopy. In consequence, their putative functions seem to be related exclusively to mitosis, namely to the interaction of the chromosomal kinetochores with spindle microtubules. However, electron microscopy using immune sera enriched with specific antibodies against human centromere protein C (CENP-C) showed that it occurs not only in mitosis but during the whole cell cycle. Therefore, we investigated the cell cycle-specific expression of CENP-C systematically on protein and mRNA levels applying HeLa cells synchronized in all cell cycle phases. Immunoblotting confirmed protein expression during the whole cell cycle and revealed an increase of CENP-C from the S phase through the [G.sub.2] phase and mitosis to highest abundance in the [G.sub.1] phase. Since this was rather surprising, we verified it by quantifying phase-specific mrnA levels of CENP-C, paralleled by the amplification of suitable internal standards, using the polymerase chain reaction. The results were in excellent agreement with abundant protein amounts and confirmed the cyclic behavior of CENP-C during the cell cycle. In consequence, we postulate that in addition to its role in mitosis, CENP-C has a further role in the [G.sub.1] phase that may be related to cell cycle control.

Protein C activation on the surface of the endothelium is critical to the negative regulation of blood coagulation. We now demonstrate that monoclonal antibodies that block protein C binding to the endothelial cell protein C receptor (EPCR) reduce protein C activation rates by the thrombin-thrombomodulin complex on endothelium, but that antibodies that bind to EPCR without blocking protein C binding have no effect. The kinetic result of blocking the EPCR-protein C interaction is an increased apparent [K.sub.m] for the activation without altering the affinity of thrombin for thrombomodulin. Activation rates of the protein C derivative lacking the [Gamma]-carboxyglutamic acid domain, which is required for binding to EPCR, are not altered by the anti-EPCR antibodies. These data indicate that the protein C activation complex involves protein C, thrombin, thrombomodulin, and EPCR. These observations open new questions about the control of coagulation reactions on vascular endothelium.

The disulfide bonding pattern of the fourth and fifth epidermal growth factor (EGF)-like domains within the smallest active fragment of thrombomodulin have been determined. In previous work, this fragment was expressed and purified to homogeneity, and its cofactor activity, as measured by [k.sub.cat] for thrombin activation of protein C, was the same as that for full-length thrombomodulin. CNBr cleavage at the single methionine in the connecting region between the domains and subsequent deglycosylation yielded the individual EGF-like domains. The disulfide bonds were mapped by partial reduction with tris(2-carboxyethyl)phosphine according to the method of Gray [Gray, W. R. (1993) Protein Sci. 2, 1732-1748], which provides unambiguous results. The disulfide bonding pattern of the fourth EGF-like domain was (1-3, 2-4, 5-6), which is the same as that found previously in EGF and in a synthetic version of the fourth EGF-like domain. Surprisingly, the disulfide bonding pattern of the fifth domain was (1-2, 3-4, 5-6), which is unlike that found in EGF or in any other EGF-like domain analyzed so far. This result is in line with an earlier observation that the (1-2, 3-4, 5-6) isomer bound to thrombin more tightly than the EGF-like (1-3, 2-4, 5-6) isomer. The observation that not all EGF-like domains have an EGF-like disulfide bonding pattern reveals an additional element of diversity in the structure of EGF-like domains.

Endoproteolytic processing of the human protein C (HPC) precursor to its mature form involves cleavage of the propeptide after amino acids [Lys.sup.-2]-[Arg.sup.-1] and removal of a [Lys.sup.156]-[Arg.sup.157] dipeptide connecting the light and heavy chains. This processing was inefficient in the mammary gland of transgenic mice and pigs. We hypothesized that the protein processing capacity of specific animal organs may be improved by the coexpression of selected processing enzymes. We tested this by targeting expression of the human proprotein processing enzyme, named paired basic amino acid cleaving enzyme (PACE)/furin, or an enzymatically inactive mutant, PACEM, to the mouse mammary gland. In contrast to mice expressing HPC alone, or to HPC/PACEM bigenic mice, coexpression of PACE with HPC resulted in efficient conversion of the precursor to mature protein, with cleavage at the appropriate sites. These results suggest the involvement of PACE in the processing of HPC in vivo and represent an example of the engineering of animal organs into bioreactors with enhanced protein processing capacity.

A molecular model of protein C inhibitor (PCI) is produced to identify the intermolecular interactions responsible for the inhibition of multiple plasma serine proteases by PCI. This molecular model uses thrombin with energy minimization to find a lowest energy complex. PCI positions P3 and P3' show reactive site mutations, and their kinetic data fits the docked complex models.

The binding of Ca2+ to the Gla domain of protein C affects the tryptophans at the 231st (Trp231) and 234th (Trp234) position. In protein C mutants, which do not contain the Gla domain (GDPC), the replacement of Trp231 with Phe removes the quenching of the emission while that of Trp234 increases the quenching. The activation of protein C by the thrombin-thrombomodulin complex requires a structural change caused by the binding of Ca2+ to the GDPC. However, this effect of Trp231 and Trp234 is seen only in the zymogen and not the enzyme.

The hydrophobic residues at the amino terminus of the gamma-carboxyglutamic acid-rich region of human protein C are required for phospholipid (PL) binding and other PL-based activities. Leusine residues at positions 5 and 8 are important, as substitutions in these residues bring about changes in binding activity and are probably required for correct orientation of the enzymes on the PL surface. Human protein C belongs to a family of blood coagulation proteins that require vitamin K for activity.

The functional properties of individual gamma-carboxyglutamic acid (Gla) residues of human protein C (PC) and activated protein C (APC) are studied using site-directed mutagenesis. Ca2+- and phospholipid-dependent anticoagulant properties of PC and APC do not require Gla14 and Gla19. Ca2+ coordination is facilitated by a single carboxylate of the Gla6 residue which is a crucial Ca2+ binding site.

The properties of gamma-carboxyglutamic acid (Gla) containing activated protein C and chimeric human protein C are studied by substituting helical stack (HS) domains of protein C with human coagulation factor IX. Gla and HS domains of the proteins are influenced by calcium ions, with homologous properties detected in factor IX. The calcium sites located outside protein domains is independent of the native Gla/HC domains within the protein.

Protein C inhibitor (PCI) inhibits acrosin, and heparin stimulates this reaction. Heparin is an allosteric modulator of the proteolytic activity of acrosin. Kinetic studies suggest that inhibition of acrosin, rather than the inhibition of the activated protein C, is the main physiological function of PCI. High concentration of PCI in seminal plasma suggests the involvement of serpin in the control of the activity of acrosin.

Abundant serpin inhibitor alpha-1-anti-trypsin (AAT) inhibits human activated protein C (APC). The inhibition is physiologically susceptible to AAT. The rates of inactivation can be compared to the half life of APC function in animals. This indicates the complex formation of the physiologic regulation processes for APC. The bovine APC and the human APC are different in their physiological function.

Coagulation factor V manifests coagulant characteristics following its activation by thrombin, and is crucial to the anticoagulant system as cofactor to activated protein C (APC). A selective error in the anticoagulant action of factor V is responsible for APC resistance or the fault in anticoagulant response to APC.

A mechanism for familial thromboembolic disease that is characterized by a poor anticoagulant response to activated protein C (APC) was described. A new coagulation-based assay was developed to measure anticoagulant response in plasma to added APC. Based on experimental data and observations made in an individual patient with thromboembolic disease, it was hypothesized that the inherited poor anticoagulant response to APC is due to an unknown APC cofactor.

A transgenic construct consisting of the human protein C gene and the whey acidic protein gene was developed and used to generate transgenic swine expressing the chimeric protein. The results showed that transgenic swine produced human protein C in their milk at a concentration of up to one gram per liter. The expressed human protein C possessed anticoagulant activity equivalent to plasma-derived protein C. These results demonstrate that pig mammary glands support gamma-carboxylation.

The circulating zymogen human protein C is activated at endothelial cell surfaces by thrombin in complex with an integral membrane protein. The activated form of protein C has several important properties including strong anticoagulant activity. However, protein C has only been therapeutically useful when administered in activated form and then it has a short biologic half-life. Genetic alteration results in a zymogen protein C with 60 times the half-life of the activated protein which can be activated by thrombin generated in clotting plasma alone, thus representing a site-activated agent.

The interactions between protein C, thrombin and the elastase fragment of rabbit thrombomodulin (elTM) were examined by analytical ultracentrifugation. The role of the protein C Gla domain in the interaction ofprotein C with thrombin and elTM was also examined by direct binding experiments and by competition studies. It was demonstrated that Ca2+ is not required for complex formation and that activated protein C is a potent inhibitor of protein C activation. Moreover, the results suggest that the Gla domain may be involved directly in the binding of protein C to both thrombin and elTM.

A modulator of the enzymatic activity of human thrombin, designated LY254603, was identified that enhances the thrombin-catalyzed generation of the anticoagulant factor activated protein C, yet inhibits thrombin-dependent fibrinogen clotting. By means of mutant substrates, it was shown that LY254603 mediates the change in enzymatic substrate specificity through an alteration in thrombin's S3 substrate recognition site, a mechanism that appeared to be independent of allosteric changes induced by either sodium ions or by thrombomodulin. This compound may represent the prototype of a class of agents that specifically modulates the balance between thrombin's procoagulant and anticoagulant functions., The serine protease thrombin is the pivotal controlling factor of all thrombosis, a leading cause of death as well as of morbidity associated with vascular disease. This enzyme plays a [...]

Byline: Deven. Juneja, S. Mohan, Vivek. Veturi, Palepu. Gopal Results of the PROWESS trial suggested that heparin may reduce the efficacy of recombinant human activated protein C (rhAPC) and the [...]

Three spatially distant surface loops were found to mediate the interaction of the coagulation protein factor X with the leukocyte integrin Mac-1. This interacting region, which by computational modeling defines a three-dimensional macromotif in the catalytic domain, was also recognized by glycoprotein C (gC), a factor X receptor expressed on herpes simplex virus (HSV)-infected endothelial cells. Peptidyl mimicry of each loop inhibited factor X binding to Mac-1 and gC, blocked monocyte generation of thrombin, and prevented monocyte adhesion to HSV-infected endothelium. These data link the ligand recognition of Mac-1 to established mechanisms of receptormediated vascular injury., INITIATION OF COAGULATION ON VASCULAR cells in implicated in various immune and inflammatory reactions and contributes to vascular injury and atherogenesis [1]. Leukocytes, platelets, and endothelial cells each interact with [...]

The molecular and structural characterization of a C-terminal fragment of xeroderma pigmentousum (XPC) implicated in centrin 2 and TFIIH binding is presented. The physical interaction between C-XPC and centrin 2 inducing minor conformational changes into XPC, localized at the 17-mer segment is found to be crucial for centrin binding.

* Context.--Factor V Leiden (FVL) is the most common heritable cause of venous thrombosis. It is caused by a single nucleotide substitution resulting in an R506Q missense mutation, resulting in factor V resistance to activated protein C (APC) inactivation. Carriers of FVL have an increased susceptibility to venous thrombosis, which is further increased in the presence of other genetic or environmental risk factors. Objective.--To review the biology, clinical findings, laboratory detection methods, and screening recommendations for patients with the FVL mutation. Data Sources.--PubMed review of published literature and online information. Conclusions.--FVL remains an important heritable cause of hypercoagulability since its discovery more than 10 years ago. Clinical suspicion should be high in cases of unexplained venous thrombosis. APC resistance and FVL mutation can be diagnosed with high sensitivity and specificity with use of clotting time-based functional assays and genetic assays, respectively, allowing for evidence-guided clinical decision making regarding the benefit of long-term anticoagulation. (Arch Pathol Lab Med. 2007;131:866-871), Factor V Leiden (FVL) describes a G1691A nucleotide transition resulting in an R506Q amino acid missense mutation. Early papers showed that the resulting factor V protein was resistant to proteolysis [...]

A theoretical and practical basis for measurements of homonuclear (super 13)C-(super 13)C residual dipolar couplings (RDCs) stemming from deuterated side chains is provided. Table showing experimental and theoretical Dcc values derived from 13 C doublets is presented.

Angiogenesis and morphogenesis of murine fetal distal lung in an allograft model. Am J Physiol Lung Cell Mol Physiol 278: L1000-L1007, 2000.--Neovascularization is crucial to lung morphogenesis; however, factors determining vessel growth and formation are poorly understood. The goal of our study was to develop an allograft model that would include maturation of the distal lung, thereby ultimately allowing us to study alveolar development, including microvascular formation. We transplanted 14-day gestational age embryonic mouse lung primordia subcutaneously into the back of nude mice for 3.5-14 days. Lung morphogenesis and neovascularization were evaluated by light microscopy, in situ hybridization, and immunohistochemical techniques. Embryonic 14-day gestational age control lungs had immature structural features consistent with pseudoglandular stage of lung development. In contrast, 14 days after subcutaneous transplantation of a 14-day gestational age lung, the allograft underwent significant structural morphogenesis and neovascularization. This was demonstrated by continued neovascularization and cellular differentiation, resulting in mature alveoli similar to those noted in the 2-day postnatal neonatal lung. Confirmation of maturation of the allograft was provided by progressive type II epithelial cell differentiation as evidenced by enhanced local expression of mRNA for surfactant protein C and a threefold (P [is less than] 0.008) increase in vessel formation as determined by immunocytochemical detection of platelet endothelial cell adhesion molecule-1 expression. Using the tyrosine kinase Flk-1 receptor (flk-1) LacZ transgene embryos, we determined that the neovascularization within the allograft was from the committed embryonic lung endothelium. Therefore, we have developed a defined murine allograft model that can be used to study distal lung development, including neovascularization. The model may be useful when used in conjunction with an altered genetic background (knockout or knock in) of the allograft and has the further decided advantage of bypassing placental barriers for introduction of pharmacological agents or DNA directly into the lung itself. pulmonary microvasculature; lung development; embryo; vasculogenesis; surfactant protein C; platelet endothelial cell adhesion molecule-1