Investigating the role of HSP47 in megakaryocyte and platelet function

HSP47, a collagen specific chaperone protein, has been identified within the platelet and has been shown to influence the ability of platelets to respond to collagen and initiate haemostasis. Megakaryocytes, which produce platelets, mature in the bone marrow (BM) but also secrete extracellular matrix factors that support the BM environment. We aimed to understand the role HSP47 plays in megakaryocyte maturation, proplatelet formation and collagen synthesis. We also aimed to determine whether HSP47 acts as a platelet surface receptor. Using a megakaryocyte lineage-specific transgenic HSP47 gene-deficient mouse, we identified that megakaryocyte ploidy and proplatelet formation was not altered in MKs from HSP47 deficient mice (P>0.05) however collagen production from the MKs was reduced (P<0.05) suggesting that HSP47 plays no role in MK maturation but is involved in the maintenance of the BM environment. Using peptide sequences identified to enable HSP47-collagen interactions, we identified that human platelets are able to adhere and spread on these sequences, however they do not support granule release, aggregation or thrombus formation. We also identified, using the HSP47 binding sequences, that HSP47 is able to modulate platelet-collagen interactions that result in platelet aggregation and activation but not thrombus formation. Finally, we confirm the presence of additional chaperone proteins within the platelet, and identify using inhibitors, that Endoplasmin may be involved in collagen receptor-mediated aggregation and thrombus formation on collagen, and that BiP may be implicated in TRAP6 stimulated aggregation and thrombus formation on collagen. Deletion of HSP47 from MKs suggests that HSP47 may be a therapeutic target to prevent fibrosis of the BM. Investigations using HSP47 binding sequences identified that HSP47 has dual functionality within the platelet, both supporting platelet adhesion in addition to modulating platelet collagen receptor-collagen interactions. Confirmation of additional chaperone proteins add to the increasing understanding that ER proteins regulate platelet function and can be targeted using inhibitors

Recent advances in the generation of nitrilium betaine 1,3-dipoles

Nitrilium betaine 1,3-dipoles are ubiquitous reagents in organic chemistry, with applications ranging from natural product synthesis to materials science. Given the high reactivity of these zwitterionic motifs, they are invariably generated in situ from a suitable precursor, prior to use. This short review summarises the recent progress in the development of modern approaches towards the formation of these 1,3-dipoles, and their applications within a diverse range of fields

Mind the gap: connexins and pannexins in platelet function

Connexins are a family of gap junction forming proteins widely expressed by mammalian cells. They assemble into hexameric hemichannels, which can either function independently or dock with opposing hemichannels on apposite cells, forming a gap junction. Pannexins are structurally related to the connexins but extensive glycosylation of these channels prevents docking to form gap junctions and they function as membrane channels. Platelets express pannexin-1 and several connexin family members (Cx37, Cx40 and Cx62). These channels are permeable to molecules up to 1,000 Daltons in molecular mass and functional studies demonstrate their role in non-vesicular ATP release. Channel activation is regulated by (patho)physiological stimuli, such as mechanical stimulation, making them attractive potential drug targets for the management of arterial thrombosis. This review explores the structure and function of platelet pannexin-1 and connexins, the mechanisms by which they are gated and their therapeutic potential

Critical items for assessing risk of lung and colorectal cancer in primary care: a Delphi study

Background Patients with lung or colorectal cancer often present late and have a poor prognosis. Identifying diagnostic indicators to optimally assess the risk of these cancers in primary care would support early identification and timely referral for patients at increased risk. Aim To obtain consensus regarding potential diagnostic indicators that are important for assessing the risk of lung or colorectal cancer in primary care consulters presenting with lung or abdominal symptoms. Design and setting A Delphi study was conducted with 28 participants from primary and secondary care and academic settings in the UK and Europe. Method Indicators were obtained from systematic reviews, recent primary studies and consultation with experts prior to the Delphi study being conducted. Over three rounds, participants rated each diagnostic indicator in terms of its importance, ranked them in order of importance, and rated each item as crucial or not crucial to assess during a GP consultation. Results The final round resulted in 25 items remaining for each type of cancer, including established cancer symptoms such as rectal bleeding for colorectal cancer and haemoptysis for lung cancer, but also less frequently used indicators such as patients’ concerns about cancer. Conclusion This study highlights the items clinicians feel would be most crucial to include in the clinical assessment of primary care patients, a number of which have rarely been noted in the previous literature. Their importance in assessing the risk of lung or colorectal cancer will be tested as part of a large prospective cohort study (CANDID)

Remote videolink observation of model home sampling and home testing devices to simplify usability studies for point-of-care diagnostics

Both home sample collection and home testing using rapid point-of-care diagnostic devices can offer benefits over attending a clinic/hospital to be tested by a healthcare professional. Usability is critical to ensure that in-home sampling or testing by untrained users does not compromise analytical performance. Usability studies can be laborious and rely on participants attending a research location or a researcher visiting homes; neither has been appropriate during COVID-19 outbreak control restrictions. We therefore developed a remote research usability methodology using videolink observation of home users. This avoids infection risks from home visits and ensures the participant follows the test protocol in their home environment. In this feasibility study, volunteers were provided with models of home blood testing and home blood sampling kits including a model lancet, sampling devices for dried blood spot collection, and model lateral flow device. After refining the study protocol through an initial pilot (n = 7), we compared instructions provided either as written instructions (n = 5), vs addition of video instructions (n = 5), vs written and video instructions plus videolink supervision by the researcher (n = 5). All users were observed via video call to define which test elements could be assessed remotely. All 22 participants in the study accessed the video call and configured their videolink allowing the researcher to clearly observe all testing tasks. The video call allowed the researcher to assess distinct errors during use including quantitative (volume of blood) and qualitative (inaccurate interpretation of results) errors many of which could compromise test accuracy. All participants completed the tasks and returned images of their completed tests (22/22) and most returned completed questionnaires (20/22). We suggest this remote observation via videolink methodology is a simple, rapid and powerful methodology to assess and optimise usability of point-of-care testing methods in the home setting

Protein disulphide isomerase and NADPH oxidase 1 cooperate to control platelet function and are associated with cardiometabolic disease risk factors

Background: Protein disulphide isomerase (PDI) and NADPH oxidase 1 (Nox- 1) regulate platelet function and reactive oxygen species (ROS) generation, suggesting potentially interdependent roles. Increased platelet reactivity and ROS production have been correlated with cardiometabolic disease risk factors. Objectives: To establish whether PDI and Nox-1 cooperate to control platelet function. Methods: Immunofluo- rescence microscopy was utilised to determine expression and localisation of PDI and Nox-1. Platelet aggregation, fibrinogen binding, P-selectin exposure, spreading and cal- cium mobilization were measured as markers of platelet function. A cross-sectional population study (n=136) was conducted to assess the relationship between platelet PDI and Nox-1 levels and cardiometabolic risk factors. Results: PDI and Nox-1 co-localized upon activation induced by the collagen receptor GPVI. Co-inhibition of PDI and Nox-1 led to additive inhibition of GPVI-mediated platelet aggregation, activation and calcium flux. This was confirmed in murine Nox-1-/- platelets treated with PDI inhibitor be- pristat, without affecting bleeding. PDI and Nox-1 together contributed to GPVI signal- ling that involved the phosphorylation of p38 MAPK, p47phox, PKC and Akt. Platelet PDI and Nox-1 levels were upregulated in obesity, with platelet Nox-1 also elevated in hypertensive individuals. Conclusions: We show that PDI and Nox-1 cooperate to con- trol platelet function and are associated with cardiometabolic risk factors

Identification of HSP47 Binding Site on Native Collagen and Its Implications for the Development of HSP47 Inhibitors

HSP47 (heat shock protein 47) is a collagen-specific molecular chaperone that is essential for procollagen folding and function. Previous studies have shown that HSP47 binding requires a critical Arg residue at the Y position of the (Gly-Xaa-Yaa) repeats of collagen; however, the exact binding sites of HSP47 on native collagens are not fully defined. To address this, we mapped the HSP47 binding sites on collagens through an ELISA binding assay using collagen toolkits, synthetic collagen peptides covering the entire amino acid sequences of collagen types II and III assembled in triple-helical conformation. Our results showed that HSP47 binds to only a few of the GXR motifs in collagen, with most of the HSP47 binding sites identified located near the N-terminal part of the triple-helical region. Molecular modelling and binding energy calculation indicated that residues flanking the key Arg in the collagen sequence also play an important role in defining the high-affinity HSP47 binding site of collagen. Based on this binding mode of HSP47 to collagen, virtual screening targeting both the Arg binding site and its neighboring area on the HSP47 surface, and a subsequent bioassay, we identified two novel compounds with blocking activity towards HSP47 binding of collagen. Overall, our study revealed the native HSP47 binding sites on collagen and provided novel information for the design of small-molecule inhibitors of HSP47

Platelet factor XIII-A regulates platelet function and promotes clot retraction and stability.

Factor XIII (FXIII) is an important proenzyme in the hemostatic system. The plasma-derived enzyme activated FXIII cross-links fibrin fibers within thrombi to increase their mechanical strength and cross-links fibrin to fibrinolytic inhibitors, specifically α2-antiplasmin, to increase resistance to fibrinolysis. We have previously shown that cellular FXIII (factor XIII-A [FXIII-A]), which is abundant in the platelet cytoplasm, is externalized onto the activated membrane and cross-links extracellular substrates. The contribution of cellular FXIII-A to platelet activation and platelet function has not been extensively studied. This study aims to identify the role of platelet FXIII-A in platelet function. We used normal healthy platelets with a cell permeable FXIII inhibitor and platelets from FXIII-deficient patients as a FXIII-free platelet model in a range of platelet function and clotting tests. Our data demonstrate that platelet FXIII-A enhances fibrinogen binding to the platelet surface upon agonist stimulation and improves the binding of platelets to fibrinogen and aggregation under flow in a whole-blood thrombus formation assay. In the absence of FXIII-A, platelets show reduced sensitivity to agonist stimulation, including decreased P-selectin exposure and fibrinogen binding. We show that FXIII-A is involved in platelet spreading where a lack of FXIII-A reduces the ability of platelets to fully spread on fibrinogen and collagen. Our data demonstrate that platelet FXIII-A is important for clot retraction where clots formed in its absence retracted to a lesser extent. Overall, this study shows that platelet FXIII-A functions during thrombus formation by aiding platelet activation and thrombus retraction in addition to its antifibrinolytic roles

Rationale, design and organization of the delayed antibiotic prescription (DAP) trial: a randomized controlled trial of the eficacy and safety of delayed antibiotic prescribing strategies in the non-complicated acute respiratory tract infections in general practice

Background: Respiratory tract infections are an important burden in primary care and it's known that they are usually self-limited and that antibiotics only alter its course slightly. This together with the alarming increase of bacterial resistance due to increased use of antimicrobials calls for a need to consider strategies to reduce their use. One of these strategies is the delayed prescription of antibiotics. Methods: Multicentric, parallel, randomised controlled trial comparing four antibiotic prescribing strategies in acute non-complicated respiratory tract infections. We will include acute pharyngitis, rhinosinusitis, acute bronchitis and acute exacerbation of chronic bronchitis or chronic obstructive pulmonary disease (mild to moderate). The therapeutic strategies compared are: immediate antibiotic treatment, no antibiotic treatment, and two delayed antibiotic prescribing (DAP) strategies with structured advice to use a course of antibiotics in case of worsening of symptoms or not improving (prescription given to patient or prescription left at the reception of the primary care centre 3 days after the first medical visit). Discussion: Delayed antibiotic prescription has been widely used in Anglo-Saxon countries, however, in Southern Europe there has been little research about this topic. The DAP trial wil evaluate two different delayed strategies in Spain for the main respiratory infections in primary care