Platelet Receptor Glycoprotein VI in Ischaemic Stroke

Platelet activation, thrombus growth and subsequent thromboembolism underpins the pathophysiology of ischaemic stroke. At sites of atherothrombotic plaque rupture, it is established that platelet surface receptor glycoprotein (GP) VI-dimer binds to exposed sub-endothelial collagen, initiating the signalling required to cause platelet activation. Then, separate platelet-fibrin interactions cause the thrombus to grow, culminating in distal tissue ischaemia; crucial in thrombotic diseases such as ischaemic stroke. The work in this thesis demonstrates that GPVI-dimer binds to fibrin also, causing platelet activation through a mechanism independent of collagen. In ischaemic stroke this is important as it not only implicates GPVI-dimer in the large-artery atherosclerotic stroke subtype, it suggests a key role in cardioembolic stroke, where fibrin, rather than collagen, is the key platelet ligand. We compared the platelet surface expression of GPVI-dimer in a control, pre-stroke atrial fibrillation (AF), and a stroke population admitted to hospital using flow cytometry. We also measured platelet activation by platelet P-selectin exposure in all the cohorts. The results demonstrate that both AF and stroke patients have more ‘active’ circulating platelets compared to the controls. Furthermore, both AF and stroke patients – irrespective of AF type or stroke aetiology, express more GPVI-dimer on their platelets compared to controls. In the stroke cohort, GPVI-dimer expression was significantly higher at day-90 post-stroke than at admission. The ability of GPVI to interact with the two main ligands that drive thrombosis, collagen and fibrin, cements its role as a key platelet receptor in human thromboembolic disease. These results intimate an important role for GPVI-dimer in driving thrombotic risk pre-stroke, as well as after having a stroke, suggesting that the direct inhibition of GPVI-dimer could be a promising future antithrombotic target.British Heart Foundation Addenbrooke's Charitable Trust Cambridge BR

The impact of misdiagnosing Bell's palsy as acute stroke

Idiopathic Bell’s Palsy (BP) can lead to a serious, and sometimes permanently disfiguring and emotionally challenging facial palsy. Early diagnosis and treatment with corticosteroids are vital, as it has shown to significantly improve recovery rates. BP is a benign condition that should be diagnosed and managed in primary care. Patients who self-present to the emergency department should be managed and discharged without the need for admission. In this study, we looked at all patients referred urgently to our hospital with facial weakness and discharged with diagnosis of BP, to explore whether clinicians were confident in making diagnoses of BP at initial assessment, and if not, how often they sought a specialist opinion. Furthermore, we assessed the impact of over-investigation and mis-treatment of this condition on healthcare resources and the patients.II would like to thank the Cambridge British Heart Foundation Centre of Research Excellence for supporting his work through a Clinical Research Training Fellowship

Beyond antiplatelets: The role of glycoprotein VI in ischemic stroke.

BACKGROUND: Platelets are essential to physiological hemostasis or pathological thrombus formation. Current antiplatelet agents inhibit platelet aggregation but leave patients at risk of systemic side-effects such as hemorrhage. Newer therapeutic strategies could involve targeting this cascade earlier during platelet adhesion or activation via inhibitory effects on specific glycoproteins, the thrombogenic collagen receptors found on the platelet surface. AIMS: Glycoprotein VI (GPVI) is increasingly being recognized as the main platelet-collagen receptor involved in arterial thrombosis. This review summarizes the crucial role GPVI plays in ischemic stroke as well as the current strategies used to attempt to inhibit its activity. SUMMARY OF REVIEW: In this review, we discuss the normal hemostatic process, and the role GPVI plays at sites of atherosclerotic plaque rupture. We discuss how the unique structure of GPVI allows for its interaction with collagen and creates downstream signaling that leads to thrombus formation. We summarize the current strategies used to inhibit GPVI activity and how this could translate to a clinically viable entity that may compete with current antiplatelet therapy. CONCLUSION: From animal models, it is clear that GPVI inhibition leads to an abolished platelet response to collagen and reduced platelet aggregation, culminating in smaller arterial thrombi. There is now an increasing body of evidence that these findings can be translated into the development of a bleeding free pharmacological entity specific to sites of plaque rupture in humans.British Heart FoundationThis is the final version of the article. It first appeared from SAGE via https://doi.org/ 10.1177/174749301665453

Computed Tomography Perfusion Can Guide Endovascular Therapy in Bilateral Carotid Artery Dissection

Carotid artery dissection (CAD) is a major cause of stroke in those under age 45, accounting for around 20% of ischaemic events[1,2]. In the absence of known connective tissue disorders, most dissections are traumatic[2]. First-line management is comprised of antiplatelet or anticoagulation therapy, but many traumatic dissections progress despite this and carry the risk of long-term complications from embolism or stenosis[3]. We report a case of traumatic bilateral carotid dissection leading to progressive neurological symptoms and hypoperfusion on computed tomography perfusion (CTP), despite escalation in anticoagulation, which led to emergency carotid stenting

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

Identifying the optimal time period for detection of atrial fibrillation after ischaemic stroke and TIA, an updated systematic review and meta-analysis of randomised control trials

Background: Atrial fibrillation (AF) is a major risk-factor for ischaemic stroke (IS) and transient ischaemic attack (TIA). The timely detection of first-diagnosed or ‘new’ AF (nAF) would prompt a switch from antiplatelets to anticoagulation to reduce the risk of stroke recurrence, however, the optimal timing and duration of rhythm monitoring to detect nAF remains unclear. Aims: We searched MEDLINE, PubMed, Cochrane database, and Google Scholar to undertake a systematic review and meta-analysis of randomised controlled trials (RCT) between 2012-2023 investigating nAF detection after IS and TIA. Outcome measures were overall detection of nAF (control; (usual care) compared to intervention; (continuous cardiac monitoring >72h)) and the time period in which nAF detection is highest (0-14 days, 15-90 days, 91-180 days or 181-365 days). A random-effects model with generic inverse variance weights was used to pool the most adjusted effect measure from each trial. Summary of review: A total of eight RCTs investigated rhythm monitoring after IS, totaling 5820 patients. The meta-analysis of the studies suggested that continuous cardiac monitoring was associated with a pooled odds ratio of 3.81 (95% CI 2.14 to 6.77), compared to usual care (control), for nAF detection. In the time period analysis, the odds ratio for nAF detection at 0-14 days, 15-90 days, 91-180 days, 181-365 days were 1.79 (1.24-2.58); 2.01 (0.63-6.37); 0.98 (0.16-5.90) and 2.92 (1.30-6.56) respectively. Conclusion: There is an almost 4-fold increase in nAF detection with continuous cardiac monitoring, compared to usual care. The results also demonstrate two statistically significant time periods in nAF detection; at 0-14 days and 6-12 months following monitoring commencement. These data support the utilisation of different monitoring methods to cover both time periods and a minimum of 1 year of monitoring to maximise nAF detection in patients after IS and TIA.NIHR Academic Clinical Lectureship (RG85316

Dimers of the platelet collagen receptor glycoprotein VI bind specifically to fibrin fibers during clot formation, but not to intact fibrinogen.

OBJECTIVE: The platelet collagen receptor glycoprotein VI (GPVI) has an independent role as a receptor for fibrin produced via the coagulation cascade. However, various reports of GPVI binding to immobilized fibrin(ogen) are not consistent. As a collagen receptor, GPVI-dimer is the functional form, but whether GPVI dimers or monomers bind to fibrin remains controversial. To resolve this, we analyzed GPVI binding to nascent fibrin clots, which more closely approximate physiological conditions. METHODS AND RESULTS: ELISA using biotinyl-fibrinogen immobilized on streptavidin-coated wells indicated that GPVI dimers do not bind intact fibrinogen. Clots were formed by adding thrombin to a mixture of near-plasma level of fibrinogen and recombinant GPVI ectodomain: GPVI dimer (GPVI-Fc2 or Revacept) or monomer (GPVI-His: single chain of Revacept GPVI domain, with His tag). Clot-bound proteins were analyzed by SDS-PAGE/immunoblotting. GPVI-dimer bound to noncrosslinked fibrin clots with classical one-site binding kinetics, with µM-level KD , and to crosslinked clots with higher affinity. Anti-GPVI-dimer (mFab-F) inhibited the binding. However, GPVI-His binding to either type of clot was nonsaturable and nearly linear, indicating very low affinity or nonspecific binding. In clots formed in the presence of platelets, clot-bound platelet-derived proteins were integrin αIIbβ3, present at high levels, and GPVI. CONCLUSIONS: We conclude that dimeric GPVI is the receptor for fibrin, exhibiting a similar KD to those obtained for its binding to fibrinogen D-fragment and D-dimer, suggesting that fibrin(ogen)'s GPVI-binding site becomes exposed after fibrin formation or cleavage to fragment D. Analysis of platelets bound to fibrin clots indicates that platelet GPVI binds to fibrin fibers comprising the clot

Factor XIII is a newly identified binding partner for platelet collagen receptor GPVI‐dimer—An interaction that may modulate fibrin crosslinking

Abstract: Background: In the fibrin‐forming process, thrombin cleaves fibrinogen to fibrin, which form fibrils and then fibers, producing a gel‐like clot. Thrombin also activates coagulation factor XIII (FXIII), which crosslinks fibrin γ‐chains and α‐chains, stabilizing the clot. Many proteins bind to fibrin, including FXIII, an established regulation of clot structure, and platelet glycoprotein VI (GPVI), whose contribution to clot function is largely unknown. FXIII is present in plasma, but the abundant FXIII in platelet cytosol becomes exposed to the surface of strongly activated platelets. Objectives: We determined if GPVI interacts with FXIII and how this might modulate clot formation. Methods: We measured interactions between recombinant proteins of the GPVI extracellular domain: GPVI‐dimer (GPVI‐Fc2) or monomer (GPVIex) and FXIII proteins (nonactivated and thrombin‐activated FXIII, FXIII subunits A and B) by ELISA. Binding to fibrin clots and fibrin γ‐chain crosslinking were analyzed by immunoblotting. Results: GPVI‐dimer, but not GPVI‐monomer, bound to FXIII. GPVI‐dimer selectively bound to the FXIII A‐subunit, but not to the B‐subunit, an interaction that was decreased or abrogated by the GPVI‐dimer–specific antibody mFab‐F. The GPVI‐dimer–FXIII interaction decreased the extent of γ‐chain crosslinking, indicating a role in the regulation of clot formation. Conclusions: This is the first report of the specific interaction between GPVI‐dimer and the A‐subunit of FXIII, as determined in an in vitro system with defined components. GPVI‐dimer–FXIII binding was inhibitory toward FXIII‐catalyzed crosslinking of fibrin γ‐chains in fibrin clots. This raises the possibility that GPVI‐dimer may negatively modulate fibrin crosslinking induced by FXIII, lessening clot stability

Higher incidence of new atrial fibrillation in hospitalised COVID-19 patients compared to lower respiratory tract infection, however, less patients anticoagulated at discharge.

Abstract Infection contributes to developing cardiac arrhythmias such as atrial fibrillation (AF), which causes over 25% of ischaemic stroke. We analysed a hospital coding database of patients hospitalised with COVID-19 +/-AF or a lower respiratory tract infection (LRTI) +/-AF, to compare the incidence of first-diagnosed or ‘new’ AF (nAF) between COVID-19 and LRTI, as well as risk-factors associated with developing nAF during COVID-19. 2243 LRTI and 488 COVID-19 patients were included. nAF was diagnosed in significantly more COVID-19 patients compared to LRTI (7.0% vs 3.6%, P=0.003), however, significantly less COVID-19 patients were discharged on anticoagulation (26.3% vs 56.4%, P=0.02). Patients who developed nAF during COVID-19 were older (P<0.001), had congestive cardiac failure (P=0.004), ischaemic heart disease (IHD) or peripheral vascular disease (PVD) (P<0.001) and a higher CHA2DS2-VASc score (P=0.02), compared COVID-19 patients who didn’t. Older age (Odds ratio (OR) 1.03, P=0.007) and IHD/PVD (OR 2.87, P=0.01) increased odds of developing nAF with COVID-19

Sepsis-driven atrial fibrillation and ischaemic stroke. Is there enough evidence to recommend anticoagulation?

Sepsis can lead to cardiac arrhythmias, of which the most common is atrial fibrillation (AF). Sepsis is associated with up to a six-fold higher risk of developing AF, where it occurs most commonly in the first 3 days of hospital admission. In many patients, AF detected during sepsis is the first documented episode of AF, either as an unmasking of sub-clinical AF or as a newly developed arrhythmia. In the short term, sepsis that is complicated by AF leads to longer hospital stays and an increased risk of inpatient mortality. Sepsis-driven AF can also increase an individual's risk of inpatient stroke by nearly 3-fold, compared to sepsis patients without AF. In the long-term, it is estimated that up to 50% of patients have recurrent episodes of AF within 1-year of their episode of sepsis. The common perception that once the precipitating illness is treated or sinus rhythm is restored the risk of stroke is removed is incorrect. For clinicians, there is a paucity of evidence on how to reduce an individual's risk of stroke after developing AF during sepsis, including whether to start anticoagulation. This is pertinent when considering that more patients are surviving episodes of sepsis and are left with post-sepsis sequalae such as AF. This review provides a summary on the literature available surrounding sepsis-driven AF, focusing on AF recurrence and ischaemic stroke risk. Using this, pragmatic advice to clinicians on how to better detect and reduce an individual's stroke risk after developing AF during sepsis is discussed