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This paper discusses the evaluation of two 3D simulated practice learning environments, Tiny Oaks and Play2Do, focused on supporting people working with children, families, and vulnerable people in special educational settings. Pre-test/post-test evaluation methodology was employed consisting of a questionnaire with 16 questions covering knowledge and understanding, professional practice, and transferable skills. Tiny Oaks had 530 participants pre-test and 423 post-test from six European countries, and results show a significant increase in learning across all questions. Play2Do had 318 participants pre-test and 301 participants post-test from the UK and Bulgaria, and again results show a significant increase in learning across all questions. The system usability scale (SUS) questionnaire was also used to measure the usability of the two environments, and usability was found to be excellent. Findings suggest that 3D simulated practice environments can provide a valuable learning experience and can provide practice learning scenarios that may be difficult to encounter in real-life.

Artificial intelligence (AI) and machine learning (ML) offer significant opportunities in healthcare for innovation with its ability to solve cognitive problems normally requiring human intelligence. However, the potential of ML in healthcare has not been realised to date, with limited existing reports of the clinical and cost benefits that have arisen from their real-world in clinical practice. This is due to the lack of understanding about how some ML models operate and ultimately the way they come to make decisions. Explainable AI (XAI) has emerged as a response to this problem investigating methods and techniques that provide insights into the outcome of an ML model and present it in qualitative understandable terms or visualisations to the stakeholders of the model. This chapter introduces XAI and provides some examples of its use within healthcare.

Machine learning (ML) applications hold significant promise for innovation within healthcare; however, their full potential has not yet been realised, with limited reports of their clinical and cost benefits in clinical practice. This is due to complex clinical, ethical, and legal questions arising from the lack of understanding about how some ML models operate and come to make decisions. eXplainable AI (XAI) is an approach to help address this problem and make ML models understandable. This chapter reports on a systematic literature review investigating the use of XAI in healthcare within the last six years. Three research questions identified as issues in the literature were examined around how bias was dealt with, which XAI techniques were used, and how the applications were evaluated. Findings show that other than class imbalance and missing values, no other types of bias were accounted for in the shortlisted papers. There were no evaluations of the explainability outputs with clinicians and none of the shortlisted papers used an interventional study or RCT.

Assessment is a crucial aspect of any teaching and learning process. New tools such as educational games offer promising advantages: they can personalize feedback to students and save educators time by automating the assessment process. However, while many teachers agree that educational games increase motivation, learning, and retention, few are ready to fully trust them as an assessment tool. A likely reason behind this lack of trust is that educational games are distributed as black boxes, unmodifiable by educators and not providing enough insight about the gameplay. This chapter presents three systematic literature reviews looking into the integration of assessment, feedback, and learning analytics in educational games. It then proposes a framework and present a fully developed engine. The engine is used by both developers and educators. Designed to separate game and assessment, it allows teachers to modify the assessment after distribution and visualize gameplay data via a learning analytics dashboard.

7α-hydroxy-4-cholesten-3-one (C4) is an oxidative enzymatic product of cholesterol metabolism via cholesterol 7α-hydroxylase, an enzyme also known as cholesterol 7-alpha-monooxygenase or cytochrome P450 7A1 (CYP7A1). C4 is a stable intermediate in the rate limiting pathway of bile acid biosynthesis. Previous studies showed that plasma C4 levels correlated with CYP7A1 enzymatic activity and could serve as a biomarker for bile acid synthesis. Here we developed and qualified a simple and robust high-throughput method using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) to quantify C4 in rat and monkey plasma. As C4 being an endogenous compound, this method used calibration standards in 50/50: acetonitrile/water (v/v). In order to mimic the incurred samples, quality control samples were prepared in the authentic plasma. Stable isotope labeled C4 (C4-d7) was used as the internal standard. The sample volume for analysis was 20μL and the sample preparation method was protein precipitation with acetonitrile. The average endogenous C4 concentrations, from 10 different lots of rat and monkey plasma, were 53.0±16.5ng/mL and 6.8±5.6ng/mL, respectively. Based on these observed endogenous C4 levels, the calibration curve ranges were established at 1-200ng/mL and 0.5-100ng/mL for rat assay and monkey assay, respectively. The method was qualified with acceptable accuracy, precision, linearity, and specificity. Matrix effect, recovery, and plasma stability of bench-top, freeze-thaw, and long-term frozen storage were also evaluated. The method has been successfully applied to pre-clinical studies.

AimsJNJ-64179375 (hereafter JNJ-9375) is a first-in-class, highly specific, large molecule, exosite 1 thrombin inhibitor. In preclinical studies, JNJ-9375 demonstrated robust antithrombotic protection with a wider therapeutic index when compared to apixaban. The purpose of the present study was to examine for the first time the antiplatelet, anticoagulant and antithrombotic effects of JNJ-9375 in a translational model of ex vivo human thrombosis.Methods and resultsFifteen healthy volunteers participated in a double-blind randomized crossover study of JNJ-9375 (2.5, 25, and 250 μg/mL), bivalirudin (6 μg/mL; positive control), and matched placebo. Coagulation, platelet activation, and thrombus formation were determined using coagulation assays, flow cytometry, and an ex vivo perfusion chamber, respectively.JNJ-9375 caused concentration-dependent prolongation of all measures of blood coagulation (prothrombin time, activated partial thromboplastin time, and thrombin time; P ConclusionExosite 1 inhibition with JNJ-9375 caused prolongation of blood coagulation, selective inhibition of thrombin-mediated platelet activation, and reductions in ex vivo thrombosis driven by a decrease in fibrin-rich thrombus formation. JNJ-9375 represents a novel class of anticoagulant with potential therapeutic applications.

Aims The aim of this study was to investigate the effects of liver X receptors (LXRs)-β preferential activation by LXR-623 (WAY-252623), a novel LXRs agonist, on plaque progression/regression in a rabbit model of advanced atherosclerosis. Methods and results Advanced atherosclerosis was induced in New Zealand White Rabbits ( n = 41). At the end of atherosclerosis induction, animals underwent a baseline magnetic resonance imaging (MRI) and were randomized to receive LXR-623 (1.5, 5, or 15 mg/kg/day), simvastatin (5 mg/kg/day), or placebo. The combination of LXR-1.5/simvastatin was also tested. After a final MRI, animals were euthanized and their aortas processed for further analysis. Simvastatin significantly reduced lesion progression (−25%; P < 0.01) in comparison with the placebo group. A similar effect was observed in the LXR-1.5 and -5 groups. A significant regression (16.5%; P < 0.01) of existing atherosclerosis was observed in the LXR-1.5/simvastatin group. Histological and molecular analysis showed plaque stabilization in the animals treated with the LXR-1.5 and -5, and LXR-1.5/simvastatin. The effects of LXR-623 were observed in the presence of a non-significant effect on total-cholesterol, low-density lipoproteins-cholesterol, and triglyceride levels. Conclusion The results of the present study show that LXR-623 significantly reduces the progression of atherosclerosis and induces plaque regression in combination with simvastatin. These observations could drive future development of novel anti-atherosclerotic therapeutic approaches.

The Badimon chamber is a clinical ex vivo model of thrombosis that mimics flow conditions within the coronary circulation of man. The aims of this study were to characterise thrombus formation in the chamber and evaluate its reproducibility.Using blood from 24 healthy human volunteers, thrombus formation was assessed at low and high shear rates with porcine aortic tunica media as the thrombogenic substrate. Thrombus area was measured histomorphometrically. Reproducibility was assessed by paired measurements made both within and between days. Platelet activation was assessed before and at selected points within the extracorporeal circuit using flow cytometry, and fibrin content and distribution within the thrombus were assessed by immunohistochemistry.Total thrombus area was highly reproducible within and between days in the low shear ([mean thrombus area, mean difference ± SEM] 8,018μm(2), 58±204μm(2) and 8,177μm(2), -154±168μm(2) respectively) and high shear chambers (11,802μm(2), -52±175μm(2) and 11,877μm(2), 220±181μm(2) respectively). Total thrombus area was greater in the high compared to the low shear chamber (11,970±285μm(2)versus 7,892±298μm(2); P0.0001). Transit through the extracorporeal circuit did not result in platelet activation which was only detected after blood passed across the perfusion chambers (P=0.02 for platelet-monocyte aggregate formation and P=0.05 for P-selectin expression). Thrombus in the low shear chamber contained a greater proportion of fibrin (25.0±6.0% versus 8.3±1.6%, P0.001).The Badimon chamber provides a highly reproducible technique for the assessment of ex vivo platelet-rich thrombus formation in man.

Summary. Background: The principal inhibitor of fibrinolysis in vivo is plasminogen activator inhibitor-1 (PAI-1). PAI-749 is a small molecule inhibitor of PAI-1 with proven antithrombotic efficacy in several preclinical models. Objective: To assess the effect of PAI-749, by using an established ex vivo clinical model of thrombosis and a range of complementary in vitro human plasma-based and whole blood-based models of fibrinolysis. Methods: In a double-blind, randomized, crossover study, ex vivo thrombus formation was assessed using the Badimon chamber in 12 healthy volunteers during extracorporeal administration of tissue-type plasminogen activator (t-PA) in the presence of PAI-749 or control. t-PA-mediated lysis of plasma clots and of whole blood model thrombi were assessed in vitro. The role of vitronectin was examined by assessing lysis of fibrin clots generated from purified plasma proteins. Results: There was a dose-dependent reduction in ex vivo thrombus formation by t-PA (P

Heart failure (HF) is a global health burden despite effective therapies, and patients hospitalized for HF are particularly at risk. Identifying genetic associates of death or rehospitalization may identify new targets for therapy or especially high risk patient subgroups. The Acute Study of

d -serine is present in the mammalian central nervous system, where it acts as one of the co-activators of N-methyl-D aspartate receptors. Synthesis of d -serine is catalyzed by the serine racemase enzyme. The current studies report on the isolation of a cDNA encoding a human serine racemase (SRR) from the human neuronal like cell line, NT2N. The SRR gene was localized on chromosome 17q13. The full-length cDNA has 1020 nucleotides which encode for a protein of 340 amino acids. The human protein shares 89% sequence identity with the mouse serine racemase. Human embryonic kidney 293 cells transiently transfected with this SRR gene were able to produce d -serine, indicating that the sequence encodes for an active enzyme. In Northern blot analysis the SRR mRNA was expressed in human brain, heart, skeletal muscle, kidney and liver tissues. Different splice forms of SRR were present in the peripheral tissues. Transcripts of at least three different sizes were present in heart and kidney, while in Western blot analysis multiple bands of different sizes were observed. Immunohistochemical studies, using a polyclonal anti-human serine racemase antibody, revealed a peripheral expression of serine racemase protein in human cardiac myocytes and convoluted tubules of the kidney. Experiments in non-human primate brain demonstrated the localization of SRR in amygdala nuclei, cortex, thalamus and hippocampus. Co-localization studies in the hippocampus demonstrated the exclusive expression of serine racemase in glial cells. The cloning of a functional human serine racemase and its expression in central nervous system of primates support a role for d -serine in neuronal activity. Furthermore, its presence in human periphery such as in heart and kidney suggest a potential biological role for d -serine in the regulation of N-methyl- d -aspartate (NMDA) receptor activity in these peripheral organs as well.

The current studies describe a new, robust cell-based functional assay useful to characterize L-type voltage-dependent calcium channels and their antagonists. The basis of this assay is measurement in plate format of Ca2+ influx through the L-type Ca2+ channel complex (alpha1C, alpha2delta, and beta2a subunits) in response to potassium-mediated depolarization; EC(50)=11 mM [K+](o). The Ca2+ influx was inhibited by the L-type Ca2+ channel antagonist, nimodipine; IC(50)=59 nM. These cells were also transfected with the Kir2.3 inward rectifier K(+) channel, which allows for changing the cell membrane potential by modulation of extracellular [K](o); -65 mV in physiological [K](o) and -28 mV in 30 mM [K](o) containing buffer. The conformational state of the voltage-sensitive Ca2+ channel is altered under these different conditions. Under the depolarized condition, nimodipine was a more potent antagonist, inhibiting Ca2+ influx with an IC(50) value of 3 nM. The results demonstrate that the interaction of nimodipine and other antagonists with the channel is modulated by changes in membrane potential and thus the state of the channel. Overall, this novel assay can be used to identify state-dependent calcium channel antagonists and should be useful for evaluating state-dependent inhibitory potency of a large number of samples.

In the search for a readily available source of native cardiac cells, we investigated the molecular and pharmacological properties of the immortalized cardiac atrial myocyte cell line, HL-1 cells. This work focused on the expression pattern of voltage-gated Ca2+ channels (VGCC). Reverse transcription-polymerase chain reaction analysis revealed that HL-1 cells have mRNA for several types of Ca2+ channels including the L-types, alpha1C and alpha1D, as well as T-types, alpha1H and alpha1G, but are lacking N-type, alpha1B and the T-type, alpha1I. Western blot analysis demonstrated significant alpha1C protein subunit expression, with less alpha1D subunit apparent, while alpha1A, alpha1B and alpha1E subunit expression was undetectable. Immunocytochemical staining showed that the alpha1C protein subunit is expressed predominantly on the cell surface, whereas the alpha1D protein is expressed mostly intracellularly. Whole-cell patch-clamp measurements demonstrated the presence of low (ICa,T) and high (ICa,L) voltage-activated Ca2+ currents, with preferential sensitivity to mibefradil and nimodipine, respectively. Addition of increasing external Ca2+ concentrations, [Ca2+]o, resulted in Ca2+ influx measured by fluorometric imaging with an EC50 of 0.8 mM [Ca2+]o. At a fixed [Ca2+]o of 0.125 mM, Ca2+ influx was also triggered by increasing the extracellular K+ concentration, [K+]o, with an EC50 of 3.7 mM [K+]o. As increasing [K+]o depolarizes the cell, this latter result is consistent with Ca2+ influx through a voltage-dependent mechanism. L-type (nimodipine and verapamil) and T-type (mibefradil and pimozide) Ca2+ channel blockers inhibited Ca2+ influx with IC50s of 1, 2, 0.4 and 0.2 microM, respectively. Antagonists of N-type (omega-conotoxins GVIA) and P/Q-type (MVIIC or omega-agatoxin IVA) did not inhibit Ca2+ influx, consistent with the lack of expression of N-, P-, or Q-type channels observed in the molecular studies. Taken together, these findings indicate that HL-1 cells express L- and T-subtypes of VGCC and are a unique in vitro model system for the study of native, mammalian cardiac Ca2+ channels.

Establishment of stable cell lines that constitutively express Ca(2+) channels at high density and that are useful for in vitro studies may be complicated by problems with seal quality and duration during whole-cell patch-clamp electrophysiology. The current studies describe the generation and characterization of cells that express the human alpha1H T-type Ca(2+) channel under the control of a tetracycline-inducible expression system. Western blot and immunostaining studies revealed that expression of the alpha1H protein occurred only in the presence of tetracycline. Using the whole-cell patch-clamp method, the cells displayed peak inward currents of 1.15 +/- 0.14 nA in response to voltage-clamp steps. The T-type Ca(2+) current was inhibited by the T-type Ca(2+) channel antagonist, mibefradil, with an IC(50) of 160 nM. This cell line, with inducible channel expression, sealed with longer duration during whole-cell patch-clamp recording when compared with a cell line that constitutively expresses the alpha1H Ca(2+) channel. Ca(2+) influx through this channel could also be detected after the addition of extracellular Ca(2+). The amount of Ca(2+) influx was dependent on the [Ca](o) with an EC(50) of 4 mM. The Ca(2+) influx was also inhibited by mibefradil with a potency (IC(50) = 183 nM) similar to that observed in the voltage-clamp studies. Overall, this inducible alpha1H Ca(2+) channel-expressing cell line is useful for the study of human T-type Ca(2+) channel function, and offers advantages over a similar cell line that constitutively expresses the channel.

A novel series of benzamidines was synthesized and shown to exhibit NR2B-subtype selective NMDA antagonist activity. Compound 31 is orally active in a carrageenan-induced rat hyperalgesia model of pain and shows no motor coordination side effects.

We established HEK-293 cell lines that stably express functional canine ether-à-go-go-related gene (cERG) K+ channels and examined their biophysical and pharmacological properties with whole cell patch clamp and35S-labeled MK-499 ([35S]MK-499) binding displacement. Functionally, cERG current had the hallmarks of cardiac delayed rectifier K+ current ( I Kr). Channel opening was time- and voltage dependent with threshold near −40 mV. The half-maximum activation voltage was −7.8 ± 2.4 mV at 23°C, shifting to −31.9 ± 1.2 mV at 36°C. Channels activated with a time constant of 13 ± 1 ms at +20 mV, showed prominent inward rectification at depolarized potentials, were highly K+ selective (Na+-to-K+permeability ratio = 0.007), and were potently inhibited by I Kr blockers. Astemizole, terfenadine, cisapride, and MK-499 inhibited cERG and human ERG (hERG) currents with IC50 values of 1.3, 13, 19, and 15 nM and 1.2, 9, 14, and 21 nM, respectively, and competitively displaced [35S]MK-499 binding from cERG and hERG with IC50 values of 0.4, 12, 35, and 0.6 nM and 0.8, 5, 47, and 0.7 nM, respectively. cERG channels had biophysical properties appropriate for canine action potential repolarization and were pharmacologically sensitive to agents known to prolong QT. A novel MK-499 binding assay provides a new tool to detect agents affecting ERG channels.

Leech anti-platelet protein (LAPP) from the leech Haementeria officinalis is a collagen-binding protein that inhibits the collagen-mediated adhesion of blood platelets. The crystal structure of recombinant LAPP has been determined using single isomorphous replacement with anomalous scattering combined with solvent flattening and threefold molecular averaging. The model of LAPP has been refined to 2.2 A resolution (R factor 21.5%; free R factor 24.0%). LAPP contains an 89-residue C-terminal domain consisting of a central six-stranded antiparallel beta-sheet flanked on one side by an alpha-helix and on the other side by two extended loops with little secondary structure. A 36-residue N-terminal region is not visible in the electron-density map. This region is rich in glycine and lacks hydrophobic residues. It probably does not have a compact globular fold, but instead has an extended conformation and is flexible. The crystal packing suggests that LAPP may form tightly interacting dimers. The fold of the C-terminal domain of LAPP closely resembles that of the N-domain of hepatocyte growth factor (HGF), which classifies LAPP as a PAN domain. However, no significant sequence homology exists between LAPP and other PAN domains. Common structural features between LAPP and the HGF N-domain include two disulfide bonds that link the alpha-helix to the central region of the protein and five residues with a conserved hydrophobic nature that are located in the core of the domain. These conserved structural features may be an important determinant of the PAN-domain type of fold.

Abstract Salivary glands of the leech Haementeria officinalis contain a protein, leech antiplatelet protein (LAPP). This protein was cloned and expressed in yeast and blocks collagen-mediated platelet aggregation and the adhesion of platelets to collagen-coated plates under static conditions. In the current study we investigated the effect of rLAPP on platelet deposition to collagen and collagen-rich surfaces under flow conditions. rLAPP completely inhibited platelet adhesion on collagen types I, III, and IV with IC 50 values of 70, 600, and 90 nmol/L, respectively (shear rate=1600 s −1 ). Approximately 10-fold more rLAPP was required to obtain a similar inhibition at a low shear rate of 375 s −1 . rLAPP caused a concentration-dependent inhibition of binding of 125 I–von Willebrand factor (vWF) to collagen type III and was able to displace prebound vWF even after 24 hours. Since platelet adhesion at low shear rate is less dependent on vWF than at high shear rate, this property of rLAPP may explain why less rLAPP is needed at high shear rate than at low shear rate to produce the same effect. Platelet adhesion to collagen type VI was only partially inhibited by rLAPP (maximal 44% with 3 μmol/L rLAPP). rLAPP also caused a pronounced inhibition of platelet deposition to cross sections of human atherosclerotic coronary arteries but had no effect on matrices of cultured human umbilical vein endothelial cells. rLAPP is a potent platelet adhesion inhibitor at high shear rate, which binds to collagen and works by inhibiting binding of vWF to collagen.

The N-terminal thrombin receptor peptide H-Ser-Phe-Leu-Leu-Arg-Asn-Pro-Asn-Asp-Lys-Tyr-Glu-Pro-Phe-OH (1) fully activates the thrombin receptor with an EC50 of 10 microM. Structural features in the tetradecapeptide which are responsible for receptor activation have been elucidated. Agonist potency has been enhanced 1000-fold with the design of the shortened peptide H-Ala-Phe(p-F)-Arg-Cha-HArg-Tyr-NH2 (56). This analog exhibits an EC50 of 0.01 microM and is the most potent agonist for receptor activation reported to date. The monoiodinated derivative H-Ala-Phe(p-F)-Arg-Cha-HArg-Tyr(3-I)-NH2 (59) exhibits an EC50 of 0.03 microM, a level sufficient for development of a radioligand.

Optimal conditions for a method to simultaneously measure aggregation in 96 samples using a microplate reader were developed. The temperature of the assay was set at 25 degrees C, the optimal platelet concentration range was determined to be from 1-3 x 10(8) per mL, the assay volume was determined to be best at 100 microL and an agitation rate of setting #5 on the vortex was found to yield the most reliable aggregation response. After these initial assay parameters were established, EC50 values for standard platelet agonists including ADP, thrombin, collagen and thrombin receptor activating peptides were determined using the plate assay and compared to those obtained by measuring light transmittance in an aggregometer. The results were quantitatively similar, and qualitatively the shapes of the aggregations as monitored by both methods were characteristic of those expected for each agonist. The use of this assay was then extended to quantitate the inhibition of aggregation by antagonists of the fibrinogen receptor as well as by an inactive thrombin receptor peptide and by antibodies against the thrombin receptor. This method provided useful data for characterization of both platelet agonists and antagonists and should be useful for future platelet aggregation studies.

Acute thrombotic reocclusion and restenosis after successful coronary angioplasty are limitations of the procedure. Although the restenotic process is not completely understood, acute platelet deposition and thrombosis are considered important initiating mechanisms. The effort to identify pharmacologic agents capable of modifying acute platelet action following mechanical injury requires an animal model mimicking the clinical pathophysiology as closely as possible. We developed a model of angioplasty-induced injury in atherosclerotic rabbit femoral arteries. Acute 111indium-labelled platelet deposition and thrombosis were assessed four hours after balloon-injury in arteries subjected to prior endothelial damage (air desiccation) and cholesterol supplementation (one month). The effects of recombinant tick anticoagulant peptide (rTAP), a blood coagulation factor Xa (fXa) inhibitor and of recombinant leech antiplatelet protein (rLAPP), a platelet adhesion inhibitor, were compared to heparin (HEP) and aspirin (ASA). Recombinant TAP and HEP, but not rLAPP or ASA, successfully prevented thrombus formation and reduced platelet deposition in balloon-injured vessel segments to levels not significantly different from those observed in the contralateral atherosclerotic non-balloon-injured vessels. Therefore, this model, incorporating balloon catheter dilation of arteries exhibiting neointimal growth and atherosclerotic plaque formation, may be useful for evaluation of possible adjunctive therapies during angioplasty.

A ruptured atherosclerotic plaque leads to exposure of deeper layers of the plaque to flowing blood and subsequently to thrombus formation. In contrast to the wealth of data on the occurrence of thrombi, little is known about the reasons why an atherosclerotic plaque is thrombogenic. One of the reasons is the relative inaccessibility of the atherosclerotic plaque. We have circumvented this problem by using 6-microns cryostat cross sections of human coronary arteries. These sections were mounted on coverslips that were exposed to flowing blood in a rectangular perfusion chamber. In normal-appearing arteries, platelet deposition was seen on the luminal side of the intima and on the adventitia. In atherosclerotic arteries, strongly increased platelet deposition was seen on the connective tissue of specific parts of the atherosclerotic plaque. The central lipid core of an advanced plaque was not reactive towards platelets. The results indicate that the atherosclerotic plaque by itself is more thrombogenic than the normal vessel wall. To study the cause of the increased thrombus formation on the atherosclerotic plaque, perfusion studies were combined with immunohistochemical studies. Immunohistochemical studies of adhesive proteins showed enrichment of collagen types I, III, V, and VI, vitronectin, fibronectin, fibrinogen/fibrin, and thrombospondin in the atherosclerotic plaque. Laminin and collagen type IV were not enriched. von Willebrand Factor (vWF) was not present in the plaque. The pattern of increased platelet deposition in serial cross sections corresponded best with areas in which collagen types I and III were enriched, but there were also areas in the plaque where both collagens were enriched but no increased reactivity was seen. Inhibition of platelet adhesion with a large range of antibodies or specific inhibitors showed that vWF from plasma and collagen types I and/or III in the plaque were involved. Fibronectin from plasma and fibronectin, fibrinogen, laminin, and thrombospondin in the vessel wall had no effect on platelet adhesion. We conclude that the increased thrombogenicity of atherosclerotic lesions is due to changes in quantity and nature of collagen types I and/or III.

SummaryThe aggregation of platelets from a variety of animal species in response to thrombin receptor-derived activating peptides was evaluated. A series of 14-(SFLLRNPNDKYEPF), 7-(SFLLRNP-NH2), 6-(SFLLRN-HN2) or 5-(SFLLR-NH2) residue peptides, the structures of which were based on the deduced amino acid sequence of the human thrombin receptor, promoted full aggregation of platelets in plasma from humans, African Green and Rhesus monkeys, baboons and guinea pigs at 4-50 μM depending on the peptide used. Platelets in plasma from rabbit, dog, pig, and hamster underwent a shape change but failed to aggregate in response to these peptides over 3 log units of peptide up to 800 μM, despite being fully responsive to human thrombin. However, because the receptor peptides induced shape change in the platelets from these non-aggregating species, they apparently can activate some of the intracellular signaling system(s) usually initiated by thrombin in these platelets. In contrast, platelets from rats did not undergo shape change or aggregate in response to the peptides. A 7-residue receptor-derived peptide based on the deduced amino acid sequence of the clone of the hamster thrombin receptor (SFFLRNP-N2) was nearly as efficacious as the corresponding human receptor-derived 7-residue peptide to promote aggregation of human platelets. However, the hamster peptide could not promote aggregation of hamster platelets in plasma at up to 800 μM peptide, while a shape change response was elicited. Platelets from rats, rabbits and pigs also did not aggregate in response to this peptide derived from the hamster thrombin receptor, but all species except the rat underwent a shape change. Longer 17-residue peptides derived from the sequences of the hamster or mouse thrombin receptors elicited aggregation of human platelets but no aggregation of the hamster platelets. In contrast, the human 14- and 5-residue and the hamster 7-residue thrombin receptor-derived peptides promoted mitogenesis of CCL39 cells, a hamster fibroblast cell line. Finally, the human 6-residue thrombin receptor-derived peptide promoted contraction of normal and de-endothelialized canine coronary artery rings, despite having no pro-aggregatory effect on canine platelets. Taken together, these results demonstrate that the thrombin receptor-derived peptides are able to mimic many, but not all, of the activating effect of thrombin in different tissues from multiple species. The heterogeneity of responsiveness to these peptides should be taken into account in future experiments designed to elucidate the mechanism of thrombin stimulation of platelets and other cells.

Hydrophobic affinity chromatography on p -chlorobenzylamido-agarose ( p -CBA-agarose) was used to characterize various modified forms of human thrombin. Native α-thrombin bound tightly to the column and was eluted with either acetonitrile or 1,4-dioxane, while the catalytically inactive prethrombin 2 did not bind to the matrix. Site-specific chemical modification with pyridoxal 5′-phosphate resulted in the loss of at least 80% of fibrinogen clotting activity but did not influence the binding of thrombin to p -CBA agarose. Modification of thrombin with pyridoxal 5-phosphate is thought to occur at the fibrinogen-binding site and the heparin-binding site. In contrast, binding of thrombin to p -CBA agarose was eliminated by modification of the active site histidine using either H-D-phenylalanyl-L-prolyl-L-arginine chloromethylketone or dansyl-L-glutamyl-glycyl-L-arginine chloromethylketone but not with tosyl-L-lysine chioromethylketone. The presence of either hirudin or heparin blocked the binding of thrombin to p -CBA-agarose but dansyl-arginine- N - (3-ethyl-1,5-pentanediyl)amide had no effect. These results indicate that p -CBA agarose binds to thrombin outside of the enzyme active site and its use should be valuable in characterizing site-specific modified thrombins obtained by either protein engineering or chemical modification.

In cardiovascular disease rupture of a vulnerable atherosclerotic plaque is the major causative factor of acute coronary syndromes, myocardial infarction and stroke, and can ultimately lead to death. Identification of biomarkers that could accurately predict the risk of plaque rupture would be a significant advance in guiding treatment of patients with this disease, The use of these biomarkers would also facilitate the development of new drugs to treat cardiovascular disease, particularly those that act through novel mechanisms. There is currently a lack of specific biomarkers for vulnerable plaque, and thus, it is an area of intense research including the concepts of live detection versus retrospective characterization, molecular imaging, and biochemical biomarker discovery. This review will focus on recent advances on both imaging and circulating molecular biomarkers in atherosclerosis. The use of combinations of different imaging modalities (such as molecular, functional, and anatomical) and modalities with circulating/biochemical markers is the current trend and will likely provide the most useful information for the assessment of the vulnerability of atherosclerotic plaques.

1. Abstract In cardiovascular disease rupture of a vulnerable atherosclerotic plaque is the major causative factor of acute coronary syndromes, myocardial infarction and stroke, and can ultimately lead to death. Identification of biomarkers that could accurately predict the risk of plaque rupture would be a significant advance in guiding treatment of patients with this disease. The use of these biomarkers would also facilitate the development of new drugs to treat cardiovascular disease, particularly those that act through novel mechanisms. There is currently a lack of specific biomarkers for vulnerable plaque, and thus, it is an area of intense research including the concepts of live detection versus retrospective characterization, molecular imaging, and biochemical biomarker discovery. This review will focus on recent advances on both imaging and circulating molecular biomarkers in atherosclerosis. The use of combinations of different imaging modalities (such as molecular, functional, and anatomical) and modalities with circulating/biochemical markers is the current trend and will likely provide the most useful information for the assessment of the vulnerability of atherosclerotic plaques.

A protein that blocks collagen-stimulated platelet aggregation has been identified and isolated from the soluble fraction of salivary glands from Haementeria officinalis leeches. We have named this protein leech antiplatelet protein (LAPP). LAPP was isolated from soluble crude salivary gland extract by heparin-agarose, size exclusion, and C18 reverse phase high-performance chromatography. Its molecular weight is approximately 16,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under both reduced and nonreduced conditions. The sequences of peptides generated by V8 digestion of LAPP as well as its amino acid composition suggested no homology to other known proteins. The IC50 for LAPP to inhibit platelet aggregation was approximately 60 nM. This inhibitory activity is specific for collagen-induced aggregation. Platelet aggregation in response to ADP, arachidonic acid, U46619, thrombin, and ionophore A23187 was not inhibited by LAPP at a concentration that blocked platelet aggregation to collagen by 100%. In contrast, crude salivary gland-soluble extract contained activity(ies) which inhibited aggregation to all these agonists except thrombin at 1 unit/ml and 2 microM A23187. Thus, the H. officinalis leech has evolved multiple mechanisms to prevent hemostasis, including an inhibitor of collagen-stimulated platelet aggregation. The identification and isolation of LAPP demonstrates the existence of a new type of platelet inhibitor that should be useful to better understand the mechanism of collagen stimulation of platelets.

Salivary glands of the leech Haementeria officinalis contain a protein, leech antiplatelet protein (LAPP), that specifically blocks collagen-mediated platelet aggregation (Connolly, T. M., Jacobs, J. W., and Condra, C. (1992) J. Biol. Chem. 267, 6893-6898). Degenerate oligonucleotides whose sequences were derived from two short peptides from V8 digests of the native LAPP were used as primers to generate a polymerase chain reaction (PCR) product which contains the cDNA region coding for the sequence between these two peptides. Using this PCR product as a hybridization probe, phage containing cDNA clones were isolated containing the entire deduced amino acid sequence for LAPP. Computer analysis of the amino acid sequence predicts a peptidase cleavage site between a 21-residue pre-peptide and a mature protein of 126 amino acids. A DNA insert to express the predicted mature LAPP protein was generated by PCR amplification using phage-derived cDNA clones as a substrate. This insert encoded a fusion protein with the leader sequence of the yeast alpha mating factor and the mature LAPP cDNA. These PCR products were cloned into the yeast expression vector pKH4 alpha 2. A KEX 2 Lys-Arg endopeptidase cleavage site was placed NH2-terminal to the predicted mature protein. This vector transfected into the yeast Saccharomyces cerevisiae directs expression of a secreted mature protein at levels up to 200 mg of LAPP/liter of culture medium. The recombinant protein was comparable to native LAPP in its electrophoretic mobility, its reactivity with anti-LAPP antisera, and its biological activity including inhibition of collagen-stimulated platelet aggregation and the adhesion of platelets to collagen. Availability of significant quantities of recombinant LAPP opens the way to further biochemical structure/function studies and to studies on the effects of an inhibitor of collagen-stimulated platelet aggregation in vivo.

SummaryWe compared a selective thrombin inhibitor (MCI-9038; Argatroban), a thromboxane A2 (TXA2) receptor antagonist (L-670,596) and a serotonin-2 receptor antagonist (ketanserin) for their ability to hasten clot lysis and delay reocclusion in a canine model of femoral arterial thrombosis. Occlusive thrombosis was induced by insertion of a thrombogenic copper coil. Femoral arterial blood flow velocity (FABFV) was monitored directly and continuously by Doppler flowmetry. Thrombolysis was induced with tissue plasminogen activator (t-PA; 0.8 mg/kg, i.v.), starting 60 min after thrombotic occlusion and continued for 90 min. Ten minutes after occlusion, dogs received an intravenous infusion of either vehicle, MCI-9038 (10 Μ;g kg−1 min−1), ketanserin (0.1 mg/ kg bolus plus 5 pg kg−1 min−1), D670,596 (1 mg/kg bolus plus 17 pg kg−1 min−1) or a combination of D670,596 and ketanserin. All infusions were discontinued 1 h after stopping the t-PA, and were followed by a 30 min observation period. The times to thrombolysis were similar for all treatments (mean ± SEM = 47 ± 3; all groups). MCI-9038 prevented reocclusion, defined as permanent cessation of FABFV during the hour after stopping the t-PA. All dogs receiving MCI-9038 reoccluded within 30 min after stopping its infusion (71 ± 3 min). Reocclusion occurred in all other dogs, except one vehicle-treated dog and a second dog that received L-670,596 plus ketanserin. Vehicle-treated dogs reoccluded within 23 ± 8 min. Reocclusion was not delayed significantly by ketanserin, L-670,596 or the combination of the two. Thus, in this model, MCI-9038 prevented reocclusion, suggesting that thrombin may be an important mediator of arterial rethrombosis after t-PA-induced thrombolysis.

A novel series of cyclic benzamidines was synthesized and shown to exhibit NR2B-subtype selective NMDA antagonist activity. Compound 29 is orally active in a carrageenan-induced rat hyperalgesia model of pain.

The synthesis of the first high specific activity S-35-labeled hERG radioligand, [(35)S]MK-0499, for use in HTS assays of drug candidates for hERG interaction is described. The radioligand is prepared by [(35)S]sulfonylation of a high diastereomeric excess (de) aniline precursor prepared from unlabeled MK-0499.

The thrombin receptor on human platelets is the first member identified of a new family of G-protein coupled receptors referred to as protease activated receptors (PARs). These receptors are activated by a unique mechanism involving proteolytic cleavage of a portion of the extracellular domain to generate a new N-terminus which then acts as a tethered or intramolecular ligand (agonist) for the receptor. The hexapeptide SFLLRN-NH2 comprising the new N-terminus is referred to as the Thrombin Receptor Activating Peptide, or "TRAP" Thrombin is the most potent agonist for platelet aggregation and selective blockade of the intramolecular activation step without effecting the proteolytic activity of thrombin should result in moderation of platelet activation and aggregation without interfering with the other coagulation cascade effects of thrombin. Screening of combinatorial libraries identified a novel, non-peptide PAR-1 thrombin receptor antagonist. Examination of structure-activity relationships revealed that portions of the molecule could be replaced resulting in simpler molecules of lower molecular weight that were at the same time more potent. Molecules in this series were effective antagonists of TRAP-stimulated platelet activation, but had limited activity when thrombin was the agonist. Additional directed screening and subsequent lead refinement resulted in a second series of isoxazole based compounds. Some of the resultant molecules were potent PAR-1 antagonists that were effective against both TRAP- and thrombin-stimulated receptor activation. These compounds do not inhibit the proteolytic effects of thrombin but rather interfere with the intramolecular binding of the tethered ligand (SFLLRN) to the transmembrane portion of the thrombin receptor. They represent promising leads for future explorations of antithrombotic activity of thrombin receptor antagonists.

The mineralocorticoid aldosterone plays an important role in the regulation of plasma electrolyte homeostasis. Exposure of acutely isolated rat adrenal zona glomerulosa cells to elevated K(+) activates voltage-gated calcium channels and initiates a calcium-dependent increase in aldosterone synthesis. We developed a novel 96-well format aldosterone secretion assay to rapidly evaluate the effect of known T- and L-type calcium channel antagonists on K(+)-stimulated aldosterone secretion and better define the role of voltage-gated calcium channels in this process. Reported T-type antagonists, mibefradil and Ni(2+), and selected L-type antagonist dihydropyridines, inhibited K(+)-stimulated aldosterone synthesis. Dihydropyridine-mediated inhibition occurred at concentrations which had no effect on rat alpha1H T-type Ca(2+) currents. In contrast, below 10 microM, the L-type antagonists verapamil and diltiazem showed only minimal inhibitory effects. To examine the selectivity of the calcium channel antagonist-mediated inhibition, we established an aldosterone secretion assay in which 8Br-cAMP stimulates aldosterone secretion independent of extracellular calcium. Mibefradil remained inhibitory in this assay, while the dihydropyridines had only limited effects. Taken together, these data demonstrate a role for the L-type calcium channel in K(+)-stimulated aldosterone secretion. Further, they confirm the need for selective T-type calcium channel antagonists to better address the role of T-type channels in K(+)-stimulated aldosterone secretion.

Two classes of 5-substituted benzimidazoles were identified as potent antagonists of the NR2B subtype of the N-methyl-d-aspartate (NMDA) receptor. Selected compounds show very good selectivity versus the NR2A, NR2C, and NR2D subtypes of the NMDA receptor as well as versus hERG-channel activity and alpha(1)-adrenergic binding. Benzimidazole 37a shows excellent activity in the carrageenan-induced mechanical hyperalgesia assay in rats as well as good pharmacokinetic behavior in dogs.

The synthesis and biological evaluation of a series of nonpeptidic small molecule antagonists of the human platelet thrombin receptor (PAR-1) are described. Optimization of the 5-amino-3-arylisoxazole lead resulted in an approximate 100-fold increase in potency. The most potent of these compounds (54) inhibits platelet activation with IC50s of 90 nM against the thrombin receptor agonist peptide (TRAP) and 510 nM against thrombin as the agonist. Further, antagonist 54 fully blocks platelet aggregation stimulated by 1 nM thrombin for 10 min.

Thrombin is the most potent agonist of platelet activation, and its effects are predominantly mediated by platelet thrombin receptors. Therefore, antagonists of the thrombin receptor have potential utility for the treatment of thrombotic disorders. Screening of combinatorial libraries revealed 2 to be a potent antagonist of the thrombin receptor. Modifications of this structure produced 11k, which inhibits thrombin receptor stimulated secretion and aggregation of platelets.

Introduction Stem cell and other cell-based therapies have emerged as promising novel treatment options for acute myocardial infarction and heart failure. Ascertaining whether cells reach and remain in the target site is essential to monitor the success of these therapeutic strategies. We have developed a Good Manufacturing Practice (GMP)-compliant protocol for labelling monocytes with an MRI contrast agent (Endorem, Guerbet) containing superparamagnetic particles of iron oxide (SPIO), and evaluated the potential for its use for human cell tracking studies. Method Up to 10 9 human peripheral blood mononuclear cells were labelled with SPIO using protamine sulphate as a transfection agent. In vitro labelling efficiency, viability and migratory capacity were evaluated. Six healthy volunteers each received intramuscular thigh injections of labelled cells (10 7 ), unlabelled cells (10 7 ) and SPIO alone, and underwent T2-weighted (T2W) MRI (1.5T) immediately and 7 days later. Safety of intravenous infusion was determined using a phased-dosing protocol in which two volunteers each received six doses of cells (10 4 –10 9 cells). Six further volunteers received 5×10 7 SPIO-labelled cells intravenously, and underwent serial T2 and multiecho (TE 4.1–22.1 ms) T2*-weighted imaging (3 T) before and serially after administration of cells. Imaging focused on the liver and spleen since this was where cells were expected to accumulate in healthy volunteers in the absence of an inflammatory focus. Results An optimised labelling protocol maximised SPIO uptake without affecting viability or migratory capacity. SPIO-labelled cells were visualised on T2W imaging following intramuscular administration (Abstract 74 Figure 1). Intravenous administration of up to 109 labelled cells was well tolerated with no effect on coagulation parameters. Reduction in signal intensity was seen in the liver and spleen on T2W imaging (Abstract 74 Figure 2) and a significant reduction in T2* value (Abstract 74 Figure 3) was observed in the liver (p Conclusions We have shown that up to 10 9 human mononuclear cells can be labelled with SPIO under GMP-compliant conditions without affecting cell viability or migratory capacity. Intramuscular and intravenous administration of up to 10 9 SPIO-labelled cells is safe, and we have demonstrated for the first time in humans that SPIO-labelled cells can be imaged at a target site following local or systemic administration. Key to the success of cell-based studies, we have demonstrated the operational capacity of our institution to deliver a study of this nature. This pilot work enables us to progress to clinical cell tracking studies in patients with advanced atherosclerotic disease.

Disagregin, a 6 kDa protein isolated from salivary glands of the tick Ornithodoros Moubata, is a potent and selective inhibitor of fibrinogen dependent platelet aggregation and of the adhesion of platelets to fibrinogen (Karczewski et al. (1994) J. Biol. Chem. 269, 6702-6708). In the current study the interaction of disagregin with purified glycoprotein IIb-IIIa (GPIIb-IIIa) was examined. Biotin-labeled disagregin (b-disagregin) bound to GPIIb-IIIa immobilized on the surface of the ELISA plate. This binding was specific, dependent on divalent cations, and was blocked by the peptides fibrinogen gamma-chain fg gamma (400-411), GPIIb(296-306) and by the RGD-containing peptide, GRGDSP. Disagregin also bound to soluble GPIIb-IIIa as demonstrated in studies using the chemical crosslinker, BS3. This binding was inhibited by the peptides fg gamma (400-411) and GPIIb(296-306). In contrast to the results in the solid phase, peptide GRGDSP had no effect on the binding of b-disagregin to soluble GPIIb-IIIa. These data demonstrate that disagregin binds to GPIIb-IIIa through a mechanism distinct from that used by RGD-containing disintegrins. Further analysis of the region(s) of disagregin which bind to GPIIb-IIIa should provide useful information for molecular modeling of the fibrinogen binding site on GPIIb-IIIa and for the design of a new class of potent fibrinogen receptor antagonists.

Background Thrombin inhibitors have been shown to be efficacious in animal models of thrombosis and in initial human clinical trials. It is unknown if their efficacy is due to their prevention of thrombin-mediated fibrin formation or to an inhibitory effect on thrombin-stimulated platelet activation. Appropriate tools to address this question have not been available. Therefore, to evaluate the role of the platelet thrombin receptor in intravascular thrombus formation, a polyclonal antibody was raised against a peptide derived from the thrombin-binding exosite region of the cloned human thrombin receptor. This antibody serves as a selective inhibitor of the thrombin receptor for in vivo evaluation. Methods and Results The immune IgG (IgG 9600) inhibited thrombin-stimulated aggregation and secretion of human platelets. In contrast, it had no effect on platelet activation induced by other agonists including ADP, collagen, or the thrombin receptor–derived peptide SFLLR-NH 2 . IgG 9600 also inhibited thrombin-induced aggregation of African Green monkey (AGM) platelets. By Western blot analysis, the IgG identified a protein of ≈64 kD in homogenates of both human and AGM platelets. The effect of thrombin receptor blockade by this antibody on arterial thrombosis was evaluated in an in vivo model of platelet-dependent cyclic flow reductions (CFRs) in the carotid artery of the AGM. The intravenous administration of IgG 9600 (10 mg/kg) abolished CFRs in three monkeys and reduced CFR frequency by 50% in a fourth monkey. Ex vivo platelet aggregation in response to up to 100 nmol/L thrombin was completely inhibited during the 120-minute postbolus observation period in all four animals. There was a twofold increase in bleeding time, which was not statistically different from baseline, and ex vivo clotting time (APTT) was not changed. The glycoprotein IIb/IIIa receptor antagonist MK-0852 and the thrombin inhibitor recombinant hirudin also demonstrated inhibitory effects on CFRs at doses that did not significantly prolong template bleeding time. Control IgG had no effect on CFRs, ex vivo platelet aggregation, bleeding time, or APTT. Conclusions These results demonstrate that blockade of the platelet thrombin receptor can prevent arterial thrombosis in this animal model without significantly altering hemostatic parameters and suggest that the thrombin receptor is an attractive antithrombotic target.

An inhibitor of the adhesion of human platelets to collagen was identified in soluble extracts of the soft tick Ornithodoros Moubata and purified by four chromatographic steps. The isolated inhibitor, TAI (Tick Adhesion Inhibitor), is a approximately 15-kDa protein that completely blocks the adhesion of platelets to collagen-coated microtiter plates with an IC50 of 8 nM. In the same concentration range it does not inhibit collagen-induced platelet aggregation or platelet adhesion to fibrinogen and has a partial inhibitory effect on platelet adhesion to fibronectin. TAI also blocks the adhesion of human endothelial cells to collagen, thus its inhibitory effect is not limited to platelets. TAI competes for the binding to platelets of a radiolabeled monoclonal antibody against the platelet glycoprotein Ia-IIa integrin complex. Based on its selective activity and small size, TAI is a promising new molecule for exploring cell-collagen interactions.

Leech antiplatelet protein (LAPP) is a specific inhibitor of collagen-induced human platelet aggregation and adhesion to collagen under static conditions. Recombinant LAPP (rLAPP) and L-366,763 (acetylated-Cys-Asn-Pro-Arg-Gly-Asp-Cys-NH2), a peptidyl fibrinogen receptor antagonist, were evaluated in an anesthetized baboon thrombosis model using a collagen-coated graft segment of an arteriovenous shunt to elicit thrombus formation. Animals were randomized to receive systemic intravenous administration of rLAPP (100 micrograms.kg-1 x min-1; n = 5), L-366,763 (8.5 micrograms.kg-1 x min-1; n = 3), or saline (n = 3). Despite complete and selective inhibition of type I collagen-induced ex vivo aggregation of platelets, rLAPP had no significant effect on the rate or the extent of 111-In-labeled platelet deposition onto the collagen graft and no effect on template bleeding time. In contrast, L-366,763 completely prevented platelet deposition, maintained blood flow, and significantly prolonged bleeding time at the dosage that inhibited ex vivo aggregation in response to all agonists studied. In this study, the absence of an antithrombotic benefit of rLAPP contrasted sharply with the efficacy of the fibrinogen receptor antagonist. These results demonstrate that specific inhibition of collagen-mediated platelet aggregation alone is not sufficient to prevent platelet-dependent thrombosis in this baboon model.

The role of the thrombin receptor tethered ligand hypothesis in mediating the mitogenic responses of cells to thrombin was explored. We have found that small (5-14 amino acid) peptides corresponding to the proposed amino terminus of thrombin activated human and hamster thrombin receptors are mitogenic for the Chinese hamster fibroblast cell line, CCL39. Hirudin and hirugen block the mitogenic effects of thrombin but not the activity of the agonist peptides. Pertussis toxin treated cells do not respond to either alpha-thrombin or the agonist peptides. The data support the idea that the thrombin receptor on CCL39 cells, which is homologous to the thrombin receptor on human platelets, is capable of transmitting mitogenic signals by a mechanism consistent with the tethered ligand hypothesis.

This paper describes experiments showing that one of the pathways of sodium transport across the red-cell membrane, sodium-lithium countertransport, is faster in patients with essential hypertension than in control subjects. This transport system accepts only sodium or lithium and is not inhibited by ouabain. The maximum rate of transport shows inherited differences. The mean maximum rate of sodium-lithium countertransport was found to be 0.55 +/- 0.02 (mean +/- S.E.M.) mmol (liter of red cells X hour)(-1) in a group of 36 patients with essential hypertension and 0.24 +/- 0.02 in 26 control subjects (P less than 0.001). The first-degree relatives of eight patients with essential hypertension and 10 control subjects had mean maximum rates of sodium-lithium countertransport of 0.54 +/- 0.05 and 0.23 +/- 0.02, respectively. Five patients with secondary hypertension had normal mean maximum rates of sodium-lithium countertransport. The relation between heritability of red-cell sodium-lithium countertransport and essential hypertension should be investigated further.