AMPK-ACC signaling modulates platelet phospholipids and potentiates thrombus formation.

AMP-activated protein kinase (AMPK) α1 is activated in platelets on thrombin or collagen stimulation, and as a consequence, phosphorylates and inhibits acetyl-CoA carboxylase (ACC). Because ACC is crucial for the synthesis of fatty acids, which are essential for platelet activation, we hypothesized that this enzyme plays a central regulatory role in platelet function. To investigate this, we used a double knock-in (DKI) mouse model in which the AMPK phosphorylation sites Ser79 on ACC1 and Ser212 on ACC2 were mutated to prevent AMPK signaling to ACC. Suppression of ACC phosphorylation promoted injury-induced arterial thrombosis in vivo and enhanced thrombus growth ex vivo on collagen-coated surfaces under flow. After collagen stimulation, loss of AMPK-ACC signaling was associated with amplified thromboxane generation and dense granule secretion. ACC DKI platelets had increased arachidonic acid-containing phosphatidylethanolamine plasmalogen lipids. In conclusion, AMPK-ACC signaling is coupled to the control of thrombosis by specifically modulating thromboxane and granule release in response to collagen. It appears to achieve this by increasing platelet phospholipid content required for the generation of arachidonic acid, a key mediator of platelet activation.

Comparison of the GPVI inhibitors losartan and honokiol.

Losartan and honokiol are small molecules which have been described to inhibit aggregation of platelets by collagen. Losartan has been proposed to block clustering of GPVI but not to affect binding of collagen. Honokiol has been reported to bind directly to GPVI but only at a concentration that is three orders of magnitude higher than that needed for inhibition of aggregation. The mechanism of action of both inhibitors is so far unclear. In the present study, we confirm the inhibitory effects of both agents on platelet aggregation by collagen and show that both also block the aggregation induced by the activation of CLEC-2 or the low affinity immune receptor FcγRIIa at similar concentrations. For GPVI and CLEC-2, this inhibition is associated with a reduction in protein tyrosine phosphorylation of multiple proteins including Syk. In contrast, on a collagen surface, spreading of platelets and clustering of GPVI (measured by single molecule localisation microscopy) was not altered by losartan or honokiol. Furthermore, in flow whole-blood, both inhibitors suppressed the formation of multi-layered platelet thrombi at arteriolar shear rates at concentrations that hardly affect collagen-induced platelet aggregation in platelet rich plasma. Together, these results demonstrate that losartan and honokiol have multiple effects on platelets which should be considered in the use of these compounds as anti-platelet agents.

Does fibrin(ogen) bind to monomeric or dimeric GPVI, or not at all?

GPVI is the major signalling receptor for collagen on platelets. Dimerization of GPVI is required for collagen binding and initiation of signalling through the associated FcR-γ chain. Recently, fibrin and fibrinogen have been identified as ligands for GPVI and shown to induce signalling in support of thrombus formation and stabilization. Contrasting observations have been reported on whether fibrin binds to monomeric or dimeric GPVI, or to neither form. In this article, we discuss reasons for the contradictory results and how to reconcile these. We conclude that a lack of structural knowledge regarding the GPVI constructs that are being used, along with the use of non-standardized reagents, might be the main cause of the discrepant results. This article aims to highlight some of the key areas that need to be addressed.

Immobilized fibrinogen activates human platelets through glycoprotein VI.

Glycoprotein VI, a major platelet activation receptor for collagen and fibrin, is considered a particularly promising, safe antithrombotic target. In this study, we show that human glycoprotein VI signals upon platelet adhesion to fibrinogen. Full spreading of human platelets on fibrinogen was abolished in platelets from glycoprotein VI- deficient patients suggesting that fibrinogen activates platelets through glycoprotein VI. While mouse platelets failed to spread on fibrinogen, human-glycoprotein VI-transgenic mouse platelets showed full spreading and increased Ca2+ signaling through the tyrosine kinase Syk. Direct binding of fibrinogen to human glycoprotein VI was shown by surface plasmon resonance and by increased adhesion to fibrinogen of human glycoprotein VI-transfected RBL-2H3 cells relative to mock-transfected cells. Blockade of human glycoprotein VI with the Fab of the monoclonal antibody 9O12 impaired platelet aggregation on preformed platelet aggregates in flowing blood independent of collagen and fibrin exposure. These results demonstrate that human glycoprotein VI binds to immobilized fibrinogen and show that this contributes to platelet spreading and platelet aggregation under flow.

Reduction of lower-limb edema in patients with chronic venous disease by micronized purified flavonoid fraction: a systematic literature review and meta-analysis.

INTRODUCTION: The prevalence of lower limb edema is high among patients with chronic venous disease (CVD). Several clinical studies with various designs have assessed the effect of micronized purified flavonoid fraction (MPFF) on edema. The aim of this work was to provide a comprehensive and accurate evaluation of the reduction in ankle and calf circumference as an indicator of lower limb edema reduction in patients with CVD treated with MPFF by combining studies that use different designs in a single group meta-analysis. EVIDENCE ACQUISITION: We conducted a systematic literature review in April 2022 based on the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) criteria to identify prospective studies investigating the effect of oral MPFF treatment 1000 mg/day on ankle and calf circumference in patients with CVD. Studies with population including at least one patient with an ulcer were excluded. All prospective studies irrespectively of design (i.e., interventional and non-interventional studies, randomized controlled trials (RCTs), non-randomized studies, studies without a control or reference treatment) were eligible. The Medline, Embase and Cochrane databases were searched. Endpoints were ankle and calf circumference measurements and their overall mean change from baseline estimated with random-effects meta-analysis methods. The evaluation criterion feeling of swelling was also analyzed as a standardized mean change (SMC) with 95% confidence intervals after combination of quantitative scales. EVIDENCE SYNTHESIS: Among 861 articles identified, eight studies (five RCTs including one placebo-controlled, three non-comparative studies) met the criteria. The overall population consisted of 1635 patients, predominantly female (89% ranging from 64% to 94%) with a mean age of 47 years ranging from 41 to 48 years. Mean reduction in ankle circumference was 6.0 mm (95%CI: 3.6 to 8.4; P<0.001) and 7.0 mm (95%CI: 0.9 to 13.1; P=0.024) after two and at least six months of treatment respectively. The results were similar when considering the study type RCTs and non-RCTs. Mean reduction in calf circumference was 5.7 mm (95%CI: 2.8 to 8.6; P<0.001) and 6.7 mm (95%CI: 5.2 to 8.1; P<0.001), at two months and at the last post-baseline evaluation respectively. Heterogeneity among studies was statistically significant (degree of consistency I2=93.5%; P<0.001 and I2=81.1%, P<0.01 for ankle and calf circumference, respectively). In the three studies reporting the effect on feeling of swelling a significant standardized mean change (SMC) reduction of 2.2 (95%CI: 0.2 to 4.2; P=0.028) on a quantitative scale was observed after two months of treatment with MPFF. CONCLUSIONS: MPFF appeared to be effective in reducing ankle and calf circumference as well as feeling of swelling irrespective of study design. The circumference reduction is present at short and long term, suggesting that benefit occurs early and is maintained overtime. Despite the observed heterogeneity among included studies, this meta-analysis supports the significant therapeutic efficacy of MPFF in reducing lower-limb edema in patients with CVD. The complete video presentation of the work is available online at www.minervamedica.it (Supplementary Digital Material 1: Supplementary Video 1, 5 min, 192 MB).

Platelet Acetyl-CoA Carboxylase Phosphorylation: A Risk Stratification Marker That Reveals Platelet-Lipid Interplay in Coronary Artery Disease Patients.

Adenosine monophosphate-activated protein kinase (AMPK) acetyl-CoA carboxylase (ACC) signaling is activated in platelets by atherogenic lipids, particularly by oxidized low-density lipoproteins, through a CD36-dependent pathway. More interestingly, increased platelet AMPK-induced ACC phosphorylation is associated with the severity of coronary artery calcification as well as acute coronary events in coronary artery disease patients. Therefore, AMPK-induced ACC phosphorylation is a potential marker for risk stratification in suspected coronary artery disease patients. The inhibition of ACC resulting from its phosphorylation impacts platelet lipid content by down-regulating triglycerides, which in turn may affect platelet function.

Fibrin and D-dimer bind to monomeric GPVI.

Fibrin has recently been shown to activate platelets through the immunoglobulin receptor glycoprotein VI (GPVI). In the present study, we show that spreading of human platelets on fibrin is abolished in patients deficient in GPVI, confirming that fibrin activates human platelets through the immunoglobulin receptor. Using a series of proteolytic fragments, we show that D-dimer, but not the E fragment of fibrin, binds to GPVI and that immobilized D-dimer induces platelet spreading through activation of Src and Syk tyrosine kinases. In contrast, when platelets are activated in suspension, soluble D-dimer inhibits platelet aggregation induced by fibrin and collagen, but not by a collagen-related peptide composed of a repeat GPO sequence or by thrombin. Using surface plasmon resonance, we demonstrate that fibrin binds selectively to monomeric GPVI with a KD of 302 nM, in contrast to collagen, which binds primarily to dimeric GPVI. These results establish GPVI as the major signaling receptor for fibrin in human platelets and provide evidence that fibrin binds to a distinct configuration of GPVI. This indicates that it may be possible to develop agents that selectively block the interaction of fibrin but not collagen with the immunoglobulin receptor. Such agents are required to establish whether selective targeting of either interaction has the potential to lead to development of an antithrombotic agent with a reduced effect on bleeding relative to current antiplatelet drugs.