Usefulness of Pilot Stations and Plants

Applied Incustrial Research can be, and generally is done either in the works which undertake production or in laboratories. PRODUCTION PLANT RESEARCH : It has the advantage of being very close to production site and, if crowned with success , its final adjustment will evidently be easy to make. This, however, has nume-rous disadvantages. Production disturbances: First of all, research causes oarturbations in the normal fabricating operations of the plant, having as a result lower, and less unif orm production. In point of fact, adjusting new devices sometimes causes many changes to be made. While doing so, the prod-uction units used for the trials often stand idle, thus giving a negligible yield. The same situation arises when the trials cannot be carried out consecutively but only at rather long interr-uptions. This is the case, for instance, when a study of the products resulting from a test takes a long time

MMP-13 binds to platelet receptors αIIbβ3 and GPVI and impairs aggregation and thrombus formation.

BACKGROUND: Acute thrombotic syndromes lead to atherosclerotic plaque rupture with subsequent thrombus formation, myocardial infarction and stroke. Following rupture, flowing blood is exposed to plaque components, including collagen, which triggers platelet activation and aggregation. However, plaque rupture releases other components into the surrounding vessel which have the potential to influence platelet function and thrombus formation. OBJECTIVES: Here we sought to elucidate whether matrix metalloproteinase-13 (MMP-13), a collagenolytic metalloproteinase up-regulated in atherothrombotic and inflammatory conditions, affects platelet aggregation and thrombus formation. RESULTS: We demonstrate that MMP-13 is able to bind to platelet receptors alphaIIbbeta3 (αIIbβ3) and platelet glycoprotein (GP)VI. The interactions between MMP-13, GPVI and αIIbβ3 are sufficient to significantly inhibit washed platelet aggregation and decrease thrombus formation on fibrillar collagen. CONCLUSIONS: Our data demonstrate a role for MMP-13 in the inhibition of both platelet aggregation and thrombus formation in whole flowing blood, and may provide new avenues of research into the mechanisms underlying the subtle role of MMP-13 in atherothrombotic pathologies

Citalopram inhibits platelet function independently of SERT-mediated 5-HT transport.

Citalopram prevents serotonin (5-HT) uptake into platelets by blocking the serotonin reuptake transporter (SERT). Although some clinical data suggest that selective serotonin reuptake inhibitors (SSRIs) may affect haemostasis and thrombosis, these poorly-characterised effects are not well understood mechanistically and useful in vitro data is limited. We sought to determine whether the inhibitory effects of citalopram on platelets are mediated via its pharmacological inhibition of 5-HT transport. We quantified the inhibitory potency of (RS)-, (R)- and (S)-citalopram on platelet function. If SERT blockade is the primary mechanism for citalopram-mediated platelet inhibition, these potencies should show quantitative congruence with inhibition of 5-HT uptake. Our data show that citalopram inhibits platelet aggregation, adhesion and thromboxane production with no difference in potency between (R)- and (S)-isomers. By contrast, citalopram had a eudysmic ratio of approximately 17 (S > R) for SERT blockade. Furthermore, nanomolar concentrations of citalopram inhibited 5-HT uptake into platelets but had no effect on other platelet functions, which were inhibited by micromolar concentrations. Our data indicate that citalopram-induced inhibition of platelets in vitro is not mediated by blockade of 5-HT transport. This raises a new question for future investigation: by what mechanism(s) does citalopram inhibit platelets

Supramaximal calcium signaling triggers procoagulant platelet formation.

Procoagulant platelets promote thrombin generation during thrombosis. Platelets become procoagulant in an all-or-nothing manner. We investigated how distinct Ca2+ signaling between platelet subpopulations commits some platelets to become procoagulant, using the high-affinity Ca2+ indicator Fluo-4, which may become saturated during platelet stimulation, or low-affinity Fluo-5N, which reports only very high cytosolic Ca2+ concentrations. All activated platelets had high Fluo-4 fluorescence. However, in Fluo-5N-loaded platelets, only the procoagulant platelets had high fluorescence, indicating very high cytosolic Ca2+. This finding indicates a novel, "supramaximal" Ca2+ signal in procoagulant platelets (ie, much higher than normally considered maximal). Supramaximal Ca2+ signaling and the percentage of procoagulant platelets were inhibited by cyclosporin A, a mitochondrial permeability transition pore blocker, and Ru360, an inhibitor of the mitochondrial Ca2+ uniporter, with no effect on Fluo-4 fluorescence. In contrast, Synta-66, an Orai1 blocker, reduced Fluo-4 fluorescence but did not directly inhibit generation of the supramaximal Ca2+ signal. Our findings show a distinct pattern of Ca2+ signaling in procoagulant platelets and provide a new framework to interpret the role of platelet signaling pathways in procoagulant platelets. This requires reassessment of the role of different Ca2+ channels and may provide new targets to prevent formation of procoagulant platelets and limit thrombosis

Zinc is a transmembrane agonist that induces platelet activation in a tyrosine phosphorylation-dependent manner

Following platelet adhesion and primary activation at sites of vascular injury, secondary platelet activation is induced by soluble platelet agonists, such as ADP, ATP, thrombin and thromboxane. Zinc ions are also released from platelets and damaged cells and have been shown to act as a platelet agonist. However, the mechanism of zinc-induced platelet activation is not well understood. Here we show that exogenous zinc gains access to the platelet cytosol and induces full platelet aggregation that is dependent on platelet protein tyrosine phosphorylation, PKC and integrin αIIbβ3 activity and is mediated by granule release and secondary signalling. ZnSO4 increased the binding affinity of GpVI, but not integrin α2β1. Low concentrations of ZnSO4 potentiated platelet aggregation by collagen-related peptide (CRP-XL), thrombin and adrenaline. Chelation of intracellular zinc reduced platelet aggregation induced by a number of different agonists, inhibited zinc-induced tyrosine phosphorylation and inhibited platelet activation in whole blood under physiologically relevant flow conditions. Our data are consistent with a transmembrane signalling role for zinc in platelet activation during thrombus formation

Coronary-Pulmonary Fistulas Involving All Three Major Coronary Arteries Co-Existing With Myocardial Infarction

We report the case of a man who presented with acute anterior myocardial infarction and in whom the coronary angiogram showed tight stenosis of the left anterior descending coronary artery and the right coronary artery associated with substantial coronary-pulmonary fistulas involving all three major coronary arteries. We discuss the possible links between coronary artery fistulas and myocardial infarction

Cleavage by MMP-13 renders VWF unable to bind to collagen but increases its platelet-reactivity

Background Atherosclerotic plaque rupture and subsequent thrombosis underpin thrombotic syndromes. Under inflammatory conditions in the unstable plaque, perturbed endothelial cells secrete von Willebrand Factor (VWF) which, via its interaction with GpIbα, enables platelet rolling across and adherence to the damaged endothelium. Following plaque rupture, VWF and platelets are exposed to subendothelial collagen which supports stable platelet adhesion, activation and aggregation. Plaque‐derived matrix metalloproteinase (MMP)‐13 is also released into the surrounding lumen where it may interact with VWF, collagen and platelets. Objectives We sought to discover whether MMP‐13 can cleave VWF and whether this might regulate its interaction with both collagen and platelets. Methods We have used platelet adhesion assays and whole blood flow experiments assess the effects of VWF cleavage by MMP‐13 on platelet adhesion and thrombus formation. Results Unlike the shear‐dependent cleavage of VWF by ADAMTS13, MMP‐13 is able to cleave VWF under static conditions. Following cleavage by MMP‐13, immobilised VWF cannot bind to collagen but interacts more strongly with platelets, supporting slower platelet rolling in whole blood under shear. Compared with intact VWF, the interaction of cleaved VWF with platelets results in greater GpIbα up‐regulation and P‐selectin expression, and the thrombi formed on cleaved VWF–collagen co‐coatings are larger and more contractile than platelet aggregates on intact VWF‐collagen co‐coatings or on collagen alone. Conclusions Our data suggest a VWF‐mediated role for MMP‐13 in the recruitment of platelets to the site of vascular injury and may provide new insights into the association of MMP‐13 in atherothrombotic and stroke pathologies

Modulating hESC-derived cardiomyocyte and endothelial cell function with triple-helical peptides for heart tissue engineering.

In this study, we investigated the role of cardiomyocyte (CM) and endothelial cell (EC) specific interactions with collagen in the assembly of an operational myocardium in vitro. Engineered cardiac patches represent valuable tools for myocardial repair following infarction and are generally constituted of a suitable biomaterial populated by CMs and supportive cell types. Among those, ECs are required for tissue vascularization and positively modulate CM function. To direct the function of human embryonic stem cell (hESC)-derived CM and EC seeded on biomaterials, we replicated cell-collagen interactions, which regulate cellular behaviour in the native myocardium, using triple-helical peptides (THPs) that are ligands for collagen-binding proteins. THPs enhanced proliferation and activity of CMs and ECs separately and in co-culture, drove CM maturation and enabled coordinated cellular contraction on collagen films. These results highlight the importance of collagen interactions on cellular response and establish THP-functionalized biomaterials as novel tools to produce engineered cardiac tissues

Platelet surface receptor glycoprotein VI-dimer is overexpressed in stroke: The Glycoprotein VI in Stroke (GYPSIE) study results.

OBJECTIVES: Platelet activation underpins thrombus formation in ischemic stroke. The active, dimeric form of platelet receptor glycoprotein (GP) VI plays key roles by binding platelet ligands collagen and fibrin, leading to platelet activation. We investigated whether patients presenting with stroke expressed more GPVI on their platelet surface and had more active circulating platelets as measured by platelet P-selectin exposure. METHODS: 129 ischemic or hemorrhagic stroke patients were recruited within 8h of symptom onset. Whole blood was analyzed for platelet-surface expression of total GPVI, GPVI-dimer, and P-selectin by flow cytometry at admission and day-90 post-stroke. Results were compared against a healthy control population (n = 301). RESULTS: The platelets of stroke patients expressed significantly higher total GPVI and GPVI-dimer (P<0.0001) as well as demonstrating higher resting P-selectin exposure (P<0.0001), a measure of platelet activity, compared to the control group, suggesting increased circulating platelet activation. GPVI-dimer expression was strongly correlated circulating platelet activation [r2 = 0.88, P<0.0001] in stroke patients. Furthermore, higher platelet surface GPVI expression was associated with increased stroke severity at admission. At day-90 post-stroke, GPVI-dimer expression and was further raised compared to the level at admission (P<0.0001) despite anti-thrombotic therapy. All ischemic stroke subtypes and hemorrhagic strokes expressed significantly higher GPVI-dimer compared to controls (P<0.0001). CONCLUSIONS: Stroke patients express more GPVI-dimer on their platelet surface at presentation, lasting at least until day-90 post-stroke. Small molecule GPVI-dimer inhibitors are currently in development and the results of this study validate that GPVI-dimer as an anti-thrombotic target in ischemic stroke.British Heart Foundation, SP/13/7/30575, Dr Stephanie M Jung British Heart Foundation, RE/13/6/30180, Dr Isuru Induruwa NIHR CL to Dr Isuru Induruw

Selecting the correct cellular model for assessing of the biological response of collagen-based biomaterials.

UNLABELLED: Accurate evaluation of the biological performance of biomaterials requires the correct assessment of their native-like cell ligation properties. However, cell attachment studies often overlook the details of the substrate-cell binding mechanisms, be they integrin-mediated or non-specific, and ignore the class- and species-specificities of the cell adhesion receptor involved. In this work we have used different collagen (Col) substrates (fibrillar collagens I, II and III and network-forming Col IV), containing different affinity cell-recognition motifs, to establish the influence of the receptor identity and species-specificity on collagen-cell interactive properties. Receptor expression was varied by using cells of different origin, or transfecting collagen-binding integrins into integrin-null cells. These include mouse C2C12 myoblasts transfected with human α1, α2, α10 or α11; human fibrosarcoma HT1080 cells which constitutively express only human α2β1, and rat glioma Rugli cells, with only rat α1β1. Using these lines, the nature of integrin binding sites was studied in order to delineate the bioactivity of different collagen substrates. Integrin ligation was studied on collagen coatings alongside synthetic (GFOGER/GLOGEN) and Toolkit (Col II-28/Col III-7) triple-helical peptides to evaluate (1) their affinity towards different integrins and (2) to confirm the activity of the inserted integrin in the transfected cells. Thin films of dermal and tendon Col I were used to evaluate the influence of the carbodiimide (EDC)-based treatment on the cellular response on Col of different origin. The results showed that the binding properties of transfected C2C12 cells to collagens depend on the identity of inserted integrin. Similar ligation characteristics were observed using α1+ and α10+ cells, but these were distinct from the similar binding features of α2+ and α11+ cells. Recombinant human and rat-α1 I domain binding to collagens and peptides correlated with the cell adhesion results, showing receptor class- and species-specificities. The understanding of the physiologically relevant cell anchorage characteristics of bio-constructs may assist in the selection of (1) the optimum collagen source for cellular supports and (2) the correct cellular model for their biological assessment. This, in turn, may allow reliable prediction of the biological performance of bio-scaffolds in vivo for specific TE applications. STATEMENT OF SIGNIFICANCE: Integrins play a vital role in cellular responses to environmental cues during early-stage cell-substrate interaction. We describe physiologically relevant cell anchorage to collagen substrates that present different affinity cell-recognition motifs, to provide experimental tools to assist in understanding integrin binding. Using different cell types and recombinant integrin α1-I-domains, we found that cellular response was highly dependent on collagen type, origin and EDC-crosslinking status, as well as on the integrin class and species of origin. This comprehensive study establishes selectivity amongst the four collagen-binding integrins and species-specific properties that together may influence choice of cell type and receptor in different experimental settings. This work offers key guidance in selecting of the correct cellular model for the biological testing of collagen-based biomaterials