Enhanced Platelet Aggregation Research Articles

A review: the therapeutic power of natural extracts in wound recovery

Natural extracts from plants, herbs, and other biological sources have demonstrated substantial potential in wound healing through their diverse biochemical activities, primarily attributed to active compounds such as alkaloids, flavonoids, phenols, and terpenoids. These compounds facilitate wound healing across various stages, including hemostasis, inflammation, proliferation, and remodeling, by modulating cellular responses, promoting tissue regeneration, and minimizing scar formation. During the hemostasis phase, natural extracts enhance platelet aggregation and fibrin formation, creating an initial matrix conducive to cell migration. In the inflammation phase, these extracts exhibit antioxidant and anti-inflammatory effects, reducing the production of pro-inflammatory cytokines and reactive oxygen species (ROS) and contributing to chronic inflammation. This anti-inflammatory activity accelerates the transition to the proliferative phase, where active compounds stimulate fibroblast and keratinocyte proliferation, promoting collagen synthesis and angiogenesis critical for new tissue formation. During the remodeling phase, natural extracts aid in collagen reorganization and maturation, improving tissue strength and elasticity while minimizing excessive scarring. However, despite the benefits, challenges such as limited bioavailability, stability, and inconsistent dosing hinder clinical applications. Advanced delivery systems, including nanofibers and hydrogels, are being explored to improve the stability and efficacy of these bioactive compounds. This review synthesizes recent research on the biochemical mechanisms of natural extracts in wound healing, outlines the clinical challenges, and highlights future directions for enhancing their therapeutic utility.

A study of platelet function in patients with chronic immune thrombocytopenia treated with thrombopoietin receptor agonists

BackgroundThrombopoietin receptor agonists (TPO-RAs) are widely used in immune thrombocytopenia (ITP) and are associated with increased thrombotic risk. However, data regarding their impact on platelet function is scarce. MethodsPlatelet function was evaluated in chronic ITP patients enrolled over one year, using light transmission aggregometry and platelet-derived microparticle (PMP) levels measurement with flow cytometry. Aggregation responses to various concentrations of ADP, collagen, ristocetin, and PMP levels were compared between TPO-RA-treated patients, patients treated with other agents and healthy individuals. ResultsTPO-RA-treated patients (n = 24) displayed significantly reduced aggregation responses to 2.5 μM, 5 μM, and 10 μM of ADP and collagen compared to 15 healthy individuals (59.5 % vs. 87.6 %, p < .0001, 43.6 % vs. 79.9 %, p < .0001, 26.1 % vs. 75.2 %, p < .0001, 67.2 % vs. 86 %, p < .0001, respectively). Reduced responses to ADP and collagen were also recorded in patients treated with other agents (n = 16) compared to healthy controls but without difference between the two treatment groups. Aggregation response to ristocetin was normal in all three groups. None of the patients yielded enhanced platelet aggregation. In TPO-RA-treated patients, a strong positive correlation between platelet counts and aggregation response to ristocetin was observed (rs = 0.65, p = .0005). PMP levels were significantly elevated in TPO-RA-treated patients compared to patients treated with other agents (49.5 vs 4.5 events/uL, p < .0001) and healthy controls (5 events/uL, p < .0001). ConclusionsThese results suggest that TPO-RAs may not enhance platelet aggregation responses, whereas impaired responses may be a disease feature. Furthermore, TPO-RAs may increase PMP levels and thus be implicated in the modulation of platelet function in ITP patients.

The Relationship between Plasminogen Activator Inhibitors and the Severity of COVID-19

The fibrinolysis process is assisted by plasminogen activators and inhibitors by converting plasminogen into plasmin, which later will promote the fibrinolysis process. Incomplete fibrinolysis increases the risk of thrombosis in patients with COVID-19. Plasminogen Activator Inhibitor (PAI-1) plays an important role as acute phase reactants to be used as a marker to assess the prognosis and mortality of COVID-19 patients. This study aimed to elaborate on whether fibrinolysis shutdown occurs in COVID-19 patients using PAI-1 as a marker of fibrinolysis. This was a cross-sectional analytical study from November 2022 to May 2023. This research consisted of a total of 39 patients with COVID-19, hospitalized at Haji Adam Malik General Hospital, Medan. PAI-test in COVID-19 patients was carried out with the ELISA method using Chemwell Analyzer. The severity of COVID-19 measured by clinical examination was divided into moderate, severe, and critical. The association between the two variables was subjected to a comparative test followed by a correlation test to explore the association between the two variables with an independent T-test. In this study, the median PAI-1 level was 1.77 ng/mL (0.71–11.49 ng/mL). The highest PAI-1 levels were observed in the critical severity group, followed by the severe and moderate group of 8.54 ng/mL (5.76–10.84), 2.45 ng/mL (0.71–11.49), and 1.29 ng/mL (0.73–3.77), respectively. There was a significant relationship between PAI-1 levels and the severity of COVID-19 patients (p=0.003). PAI-1 cut-off value of 1.89 ng/mL may predict the degree of COVID-19 severity with sensitivity of 88.9%, specificity of 90.5%, and accuracy of 89.7%. This study classified the severity of COVID-19 into two categories, which are moderate (n=21) and severe-critical (n=18) to obtain the AUC value of PAI-1. PAI-1 can be used to predict the severity of COVID-19 disease with a moderate level (AUC &gt;70–80%). This phenomenon can be secondary to enhanced platelet aggregation, inflammation micro thrombosis, and impaired fibrinolysis. Fibrinolysis disorders lead to a fibrin buildup and increased levels of PAI-1 in the circulation.

A novel role for thioredoxin-related transmembrane protein TMX4 in platelet activation and thrombus formation

BackgroundThe functions of critical platelet proteins are controlled by thiol-disulfide exchanges, which are mediated by the protein disulfide isomerase (PDI) family. It has been shown that some PDI family members are important in platelet activation and thrombosis with distinct functions. TMX4, a membrane-type PDI family member, is expressed in platelets, but whether it has a role in platelet activation remains unknown. ObjectivesTo determine the role of TMX4 in platelet activation and thrombosis. MethodsThe phenotypes of TMX4-deficient mice were evaluated in tail bleeding time assay and laser-induced and FeCl3-induced arterial injury models. The functions of TMX4 in platelets were assessed in vitro using TMX4-null platelets, recombinant TMX4 protein, and anti-TMX4 antibody. ResultsCompared with the control mice, Tie2-Cre/TMX4fl/fl mice deficient of hematopoietic and endothelial TMX4 exhibited prolonged tail bleeding times and reduced platelet thrombus formation. Pf4-Cre/TMX4fl/fl mice deficient of platelet TMX4 also had prolonged tail bleeding times and decreased thrombus formation, which was rescued by injection of recombinant TMX4 protein. Consistently, TMX4 deficiency inhibited platelet aggregation, integrin αIIbβ3 activation, P-selectin expression, phosphatidylserine exposure, and thrombin generation, without affecting tyrosine phosphorylation of intracellular signaling molecules Syk, LAT, PLCγ2 and calcium mobilization. Recombinant TMX4 protein enhanced platelet aggregation and reduced integrin αIIbβ3 disulfide bonds, and TMX4 deficiency decreased free thiols of integrin αIIbβ3, consistent with a potent reductase activity of TMX4. In contrast, an inactive TMX4 protein and a specific anti-TMX4 antibody inhibited platelet aggregation. ConclusionTMX4 is a novel PDI family member that enhances platelet activation and thrombosis.

SARS-CoV-2 spike protein potentiates platelet aggregation via upregulating integrin αIIbβ3 outside-in signaling pathway.

Platelet hyperreactivity is one of the crucial causes of coagulative disorders in patients with COVID-19. Few studies have indicated that integrin αIIbβ3 may be a potential target for spike protein binding to platelets. This study aims to investigate whether spike protein interacts with platelet integrin αIIbβ3 and upregulates outside-in signaling to potentiate platelet aggregation. In this study, we found that spike protein significantly potentiated platelet aggregation induced by different agonists and platelet spreading in vitro. Mechanism studies revealed that spike protein upregulated the outside-in signaling, such as increased thrombin-induced phosphorylation of β3, c-Src. Moreover, using tirofiban to inhibit spike protein binding to αIIbβ3 or using PP2 to block outside-in signaling, we found that the potentiating effect of spike protein on platelet aggregation was abolished. These results demonstrate that SARS-CoV-2 spike protein directly enhances platelet aggregation via integrin αIIbβ3 outside-in signaling, and suggest a potential target for platelet hyperreactivity in patients with COVID-19. HIGHLIGHTS: •Spike protein potentiates platelet aggregation and upregulates αIIbβ3 outside-in signaling. •Spike protein interacts with integrin αIIbβ3 to potentiate platelet aggregation. •Blocking outside-in signaling abolishes the effect of spike protein on platelets.

Investigation of the impact of thirdhand e-cigarette exposure on platelet function: A pre-clinical study.

The use of e-cigarettes (ECs) has reached unprecedented levels, due to a variety of reasons, including the misconception regarding their safety. Thus, there have been efforts to characterize the effects of EC exposure, including in the context of thirdhand EC (THEC) on a host of disorders, such as cardiovascular disease (CVD). To address this issue, we sought to characterize the effects of THEC on platelet function and thrombus formation, using a novel mouse exposure protocol that resembles real life scenarios. To assess these effects, a host of related in vivo (i.e. tail bleeding time, and ferric chloride injury induced thrombosis model) assays and in vitro platelet specific (e.g. aggregation, and dense granule secretion) investigative assays were conducted. Our in vivo characterization demonstrated that THEC exposed mice exhibited a prothrombotic phenotype reflected by their shortened tail bleeding (THEC: 37 ± 15 seconds, versus clean air: 183 ± 56 s) and occlusion times (THEC: 188 ± 39 s, versus clean air: 519 ± 70 s), relative to those exposed to clean air. Importantly, we found no difference in the platelet counts between the THEC and clean air mice. As for the underlying mechanism, separate experiments revealed significantly enhanced platelet aggregation, dense and alpha granule secretion, as well as integrin/GPIIb-IIIa activation and phosphatidylserine exposure in response to thrombin and ADP agonist stimulation. Taken together, these results provide evidence that THEC does have the capacity to increase the risk of thrombotic disease, which should increase awareness regarding its underappreciated negative health effects.

Serum podoplanin expression and frequency of thrombosis in adult de novo acute myeloid leukemia patients: An Egyptian single center study

Background Cancer-associated thrombosis although represents a significant disease worse prognosis, understanding its underlying pathophysiology remains obscure. Podoplanin and its platelet ligand C-type lectin receptor type-2 have been investigated in the past few years for their potential role as procoagulant substances as it has been shown in many studies that binding of podoplanin with platelet C-type lectin receptor type-2 results in enhancement of platelet aggregation and thrombotic insults. Aim In this study, serum podoplanin was measured in newly diagnosed acute myeloid leukemia (AML) patients and its relation to the incidence of thrombosis in these patients in addition to its relation to other clinical, laboratory, and prognostic outcomes. Patients and methods This study included 80 participants (60 newly diagnosed adult AML patients and 20 age-matched and sex-matched healthy controls). Blood counts and flow cytometry using an acute leukemia panel were performed in addition to serum podoplanin, and cytogenetic studies including conventional karyotyping and fluorescence in-situ hybridization were performed to determine the risk group. The clinical status of the patients, particularly the incidence of thrombotic events in the following 6 months, as well as the D28 outcome and 6-month survival, were determined and reported. Results Serum podoplanin was highly expressed in AML patients compared with control samples, with a highly significant difference (P=0.001). There was a significant inverse relation between serum podoplanin level, hemoglobin level, and white blood cell count (P=0.006 and 0.036, respectively). Those with thrombotic events during the course of the disease did not show statistically significant results when compared with the control group with regard to serum podoplanin levels (P=0.546). Conclusion Serum podoplanin was much higher in AML patients than in the control group (P=0.000), and it was inversely related to platelet level (P=0.036) and total leukocyte count (P=0.006). However, there was no statistically significant link between podoplanin level and the incidence of thrombotic events (P=0.546).

The Association Between the Gut Microbiome and COVID-19 Severity: The Potential Role of TMAO Produced by the Gut Microbiome

Context: The COVID-19 pandemic has had profound impacts on public health, resulting in nearly 1 million deaths. Emerging evidence suggests an association between certain metabolites produced by gut microbiota and potential alterations in the severity of infection. Trimethylamine N-oxide (TMAO) is a waste metabolite generated by gut microbes from dietary choline and betaine. Evidence Acquisition: Several investigations have indicated an association between serum TMAO concentrations and the development of inflammation and thrombosis. Trimethylamine N-oxide, produced by the gut microbiome in a state of dysbiosis, upregulates various molecular mechanisms, such as the nuclear factor kappa (NF-kB) molecular pathway, and promotes the expression of scavenger receptors (SRs) on the surfaces of macrophages, leading to foam cell formation and inflammation. High levels of TMAO have been shown to induce the expression of pro-inflammatory cytokines like tumor necrosis factor-alpha (TNF-α) and interleukin 1β (IL-1β) while reducing the expression of anti-inflammatory cytokines such as interleukin-10 (IL-10). Additionally, gut-derived TMAO enhances platelet aggregation and adhesion to collagen, increasing the risk of thrombosis. Conclusions: Understanding the association between gut microbiome compositions such as gut TMAO and their effects on SARS-CoV-19 infection progression helps to control disease severity. In this review, we presented a hypothesis that the gut TMAO has the potential to increase COVID-19 disease severity.

Coagulation and complement crosstalk: molecular mechanisms of complement-mediated diseases

The complement and coagulation systems are ancestrally related mechanisms of serine protease-induced protein activation. Recent studies have shown that the complement system enhances platelet aggregation by activating platelets and vascular endothelial cells. This system is also involved in the expression of tissue factor, which induces the coagulation reaction. Activated platelets and coagulation factors are also known to activate the complement system. In diseases involving the complement system, such as paroxysmal nocturnal hemoglobinuria, autoimmune hemolytic anemia, and atypical hemolytic uremic syndrome, excessive activation of this system contributes to complement-mediated thrombosis. The anti-C5 antibody eculizumab has shown a remarkable thromboprophylactic effect in these complement diseases. The recent surge in development of new anti-complement agents has raised expectations for the advancement of treatments and preventive measures for thrombosis associated with complement disorders. This review outlines the crosstalk between these two systems, and describes the mechanisms of several diseases featuring both thrombosis and complement activation.

Restrained glycoprotein VI-induced platelet signaling by tyrosine protein phosphatases independent of phospholipase Cγ2

The platelet collagen receptor glycoprotein VI (GPVI) signals to activation of phospholipase Cγ2 (PLCγ2) and phosphoinositide 3-kinases (PI3K), causing platelet activation and aggregation. The non-receptor Src homology tyrosine phosphatases Shp1/2 modulate GPVI signaling in partly opposite ways, both of which are targeted by the potential drug NSC87877. Effect measurements of the Shp1/2 inhibitor NSC87877 on platelet activation via GPVI using light transmission aggregometry, Ca2+ flux assay, western blotting and flow cytometry. Effect measurements of selective PI3K inhibitor TGX221. Inhibition of Shp1/2 with NSC87877 enhanced platelet aggregation induced by the GPVI agonist, collagen-related peptide (CRP). Furthermore, NSC87877 antagonized the effects of PI3Kb inhibition, but not of Btk inhibition. Both NSC87877 and TGX221 suppressed the CRP-induced phosphorylation of PLCγ2 at activation site Tyr759. These findings indicate that drug interference of the two phosphatases Shp1/2 subtly enhances GPVI-induced platelet responses via a mechanism not involving PLCγ2 activation, even upon PI3K inhibition.

Loss of zinc transporters ZIP1 and ZIP3 augments platelet reactivity in response to thrombin and accelerates thrombus formation in vivo.

Zinc (Zn2+) is considered as important mediator of immune cell function, thrombosis and haemostasis. However, our understanding of the transport mechanisms that regulate Zn2+ homeostasis in platelets is limited. Zn2+ transporters, ZIPs and ZnTs, are widely expressed in eukaryotic cells. Using mice globally lacking ZIP1 and ZIP3 (ZIP1/3 DKO), our aim was to explore the potential role of these Zn2+ transporters in maintaining platelet Zn2+ homeostasis and in the regulation of platelet function. While ICP-MS measurements indicated unaltered overall Zn2+ concentrations in platelets of ZIP1/3 DKO mice, we observed a significantly increased content of FluoZin3-stainable free Zn2+, which, however, appears to be released less efficiently upon thrombin-stimulated platelet activation. On the functional level, ZIP1/3 DKO platelets exhibited a hyperactive response towards threshold concentrations of G protein-coupled receptor (GPCR) agonists, while immunoreceptor tyrosine-based activation motif (ITAM)-coupled receptor agonist signalling was unaffected. This resulted in enhanced platelet aggregation towards thrombin, bigger thrombus volume under flow ex vivo and faster in vivo thrombus formation in ZIP1/3 DKO mice. Molecularly, augmented GPCR responses were accompanied by enhanced Ca2+ and PKC, CamKII and ERK1/2 signalling. The current study thereby identifies ZIP1 and ZIP3 as important regulators for the maintenance of platelet Zn2+ homeostasis and function.

The Impact of Convertase Subtilisin/Kexin Type 9 Monoclonal Antibodies with and without Apheresis on Platelet Aggregation in Familial Hypercholesterolemia

Background and aimsIt is well known that elevated cholesterol is associated with enhanced platelet aggregation and patients suffering from familial hypercholesterolemia (FH) have a high risk of thrombotic cardiovascular events. Although decreasing cholesterol level is associated with attenuation of platelet hyperactivity, there are currently no data on the effect of convertase subtilisin/kexin type 9 monoclonal antibodies (PCSK9ab) on platelet reactivity in FH. The aim of the study was to analyse the impact of different therapies including PCSK9ab on platelet aggregation in FH.MethodsThis study enrolled all 15 patients treated in the University Hospital Hradec Králové for FH. PCSK9ab have been administered in 12 of 15 patients while 8 patients were also undergoing lipid apheresis. Blood samples from all patients including pre- and post-apheresis period were tested for platelet aggregation triggered by 7 inducers, and the effect of 3 clinically used drugs (acetylsalicylic acid, ticagrelor and vorapaxar) was compared as well.ResultsAlthough apheresis decreased the reactivity of platelets in general, platelet responses were not different between non-apheresis patients treated with PCSK9ab and apheresis patients (post-apheresis values) with the exception of ristocetin. However, when compared to age-matched healthy population, FH patients had significantly lower platelet aggregation responses to 4 out of 7 used inducers and higher profit from 2 out of 3 used antiplatelet drugs even after exclusion of FH patients regularly receiving conventional antiplatelet treatment.ConclusionThis study showed for the first time the suitability of PCSK9ab treatment for reduction of platelet reactivity in FH patients.

TRIMETHYLAMINE OXIDE – FACTOR IN THE DEVELOPMENT OF ATHEROSCLEROSIS AND A POTENTIAL TARGET FOR DIETARY AND PHARMACOLOGICAL INTERVENTIONS

Atherosclerotic Cardiovascular Diseases (ASCVD) are the most common cause of death worldwide. Among the well-known con¬tributors to atherosclerosis are less common ones, such as trimethylamine oxide (TMAO). This substance is formed from the oxida¬tion of trimethylamine (TMA) with the participation of flavin oxidases in the liver. TMA is produced with the involvement of the intestinal microbiota from foods rich in choline and carnitine. TMAO promotes the production of foam cells, enhances platelet aggregation, affects endothelial inflammation, and promotes atherosclerosis development. However, further research is needed to determine the effect of dietary changes on reducing TMAO levels and thus reducing incident ASCVD.

Coagulation status and determinants of possible aspirin resistance in patients with essential thrombocythemia.

The currently recommended aspirin regimen appears inadequate for thromboprophylaxis in essential thrombocythemia (ET). This study aimed not only to evaluate the curative effect of aspirin but also to explore the coagulation status and determinants of aspirin resistance (AR) of ET patients. A total of 80 ET patients who underwent coagulation tests, thromboelastography (TEG), and next-generation sequencing (NGS) were involved in the study. Patients were divided into the aspirin sensitivity (AS) group and AR group according to the arachidonic acid inhibition rate. Their clinical features and coagulation function were analyzed. The incidence of AR was 53.75% (43/80) in 80 ET patients. Fbg was significantly higher in coagulation tests in AR patients compared with AS patients (P < 0.05), while the differences in other variables (D-D, PT, PTA, INR, APTT, TT, FDP, and AT-III) were not statistically significant (P > 0.05). Compared with AS patients, the K values, α angles, MA values, and CI values of TEG in AR patients were statistically smaller (P < 0.05), but there was no significant difference in R value between them (P > 0.05). Univariate and multivariate logistic regression analysis showed that age, irregular use of aspirin, smoking, dyslipidemia, and hypertension increased the risk of AR (P < 0.05). In the routine NGS, the driver gene and non-driver gene had no effect on AR in ET patients. Compared with AS patients, AR patients have enhanced platelet aggregation function, are in a relatively hypercoagulable state, and haveelevated fibrinogen function/levels, all of which cause a worse coagulation status. ET patients with increasing age, irregular use of aspirin, smoking, dyslipidemia, and hypertension are possibly at higher risk of AR. The routine NGS may not be helpful for the prediction of AR, therefore we recommend adding relevant drug-resistance genes to NGS.

763 ACTIVATION OF SIRT1 ATTENUATES VASCULAR DYSFUNCTION AND THROMBOSIS IN MTHFR DEFICIENCY

Abstract Beyond well-assessed risk factors, cardiovascular events could be also associated with the presence of epigenetic and genetic alterations, such as the methylenetetrahydrofolate-reductase (MTHFR) C677T polymorphism. This gene variant is related to increased circulating levels of homocysteine (Hcy) and cardiovascular risk. However, heterozygous carriers have an augmented risk of cardiovascular accidents independently from normal Hcy levels, suggesting the presence of additional deregulated processes in MTHFR C677T carriers. Here we hypothesize that targeting Sirtuin 1 (SIRT1) could be an alternative mechanism to control the cardiovascular risk associated to MTHFR deficiency condition. Flow Mediated Dilatation (FMD) and light transmission aggregometry assay were performed in subjects carrying MTHFR C677T allele after administration of resveratrol, the most powerful natural clinical usable compound that owns SIRT1 activating properties. MTHFR C677T carriers with normal Hcy levels revealed endothelial dysfunction and enhanced platelet aggregation associated with SIRT1 downregulation. SIRT1 activity stimulation by resveratrol intake was able to override these abnormalities without affecting Hcy levels. Impaired endothelial function, bleeding time, and wire-induced thrombus formation were rescued in a heterozygous Mthfr-deficient (Mthfr+/-) mouse model after resveratrol treatment. Using a cell-based high-throughput multiplexed screening (HTS) assay, a novel selective synthetic SIRT1 activator, namely ISIDE11, was identified. Ex vivo and in vivo treatment of Mthfr+/- mice with ISIDE11 rescues endothelial vasorelaxation and reduces wire-induced thrombus formation, effects that were abolished by SIRT1 inhibitor. Moreover, platelets from MTHFR C677T allele carriers treated with ISIDE11 showed normalization of their typical hyper-reactivity. These results candidate SIRT1 activation as a new therapeutic strategy to contain cardio and cerebrovascular events in MTHFR carriers.

Importance of catecholamine signaling in the development of platelet exhaustion after traumatic injury

BackgroundImpaired ex vivo platelet aggregation is common in trauma patients. The mechanisms driving these impairments remain incompletely understood, but functional platelet exhaustion due to excessive in vivo activation is implicated. Given platelet adrenoreceptors and known catecholamine surges after injury, impaired ex vivo platelet aggregation in trauma patients may be linked to catecholamine‐induced functional platelet exhaustion. ObjectiveTo determine the relationship of catecholamines with platelet‐dependent hemostasis after injury and to model catecholamine‐induced functional platelet exhaustion in healthy donor platelets. Patients/MethodsWhole blood was collected from 67 trauma patients as part of a prospective cohort study. Platelet aggregometry and rotational thromboelastometry were performed, and plasma epinephrine (EPI) and norepinephrine (NE) concentrations were measured. The effect of catecholamines on healthy donor platelets was examined in a microfluidic model, with platelet aggregometry, and by flow cytometry examining surface markers of platelet activation. ResultsIn trauma patients, EPI and NE were associated with impaired platelet aggregation (both p < 0.05), and EPI was additionally associated with decreased viscoelastic clot strength, increased fibrinolysis, and mortality (all p < 0.05). In healthy donors, short duration incubation with EPI enhanced platelet aggregation, platelet adhesion under flow, and increased glycoprotein IIb/IIIa activation, while weaker effects were observed with NE. Compared with short incubation, longer incubation with EPI resulted in decreased platelet adhesion, platelet aggregation, and surface expression of glycoprotein IIb/IIIa. ConclusionsThese findings suggest sympathoadrenal activation in trauma patients contributes to impaired ex vivo platelet aggregation, which mechanistically may be explained by a functionally exhausted platelet phenotype under prolonged exposure to high plasma catecholamine levels.

When Will Fondaparinux Induce Thrombocytopenia?

The pentasaccharide Fondaparinux, a synthetic selective factor Xa inhibitor, is one of the safest anticoagulants in the heparin family that is recommended as an alternative drug for patients with hypersensitivity to other drugs such as heparin-induced thrombocytopenia (HIT). However, some observations of Fondaparinux-induced thrombocytopenia (FIT) have been reported while others claimed that FIT does not occur in patients with fondaparinux therapy, indicating that the mechanism of FIT remains controversial. Here, we utilized different methodologies including dynamic light scattering, immunosorbent and platelet aggregation assays, confocal laser scanning microscopy, and flow cytometry to gain insights into FIT. We found that at a certain concentration, Fondaparinux formed sufficient large and stable complexes with PF4 that facilitated binding of the HIT-like monoclonal KKO antibody and enhanced platelet aggregation and activation. We proposed a model to describe the role of Fondaparinux concentration in the formation of complexes with platelet factor 4 and how it promotes the binding of KKO. Our results clarify controversial observations of FIT in patients as each contains a dissimilar PF4:Fondaparinux concentration ratio.

SIRT1 pharmacological activation rescues vascular dysfunction and prevents thrombosis in MTHFR deficiency

Beyond well-assessed risk factors, cardiovascular events could be also associated with the presence of epigenetic and genetic alterations, such as the methylenetetrahydrofolate-reductase (MTHFR) C677T polymorphism. This gene variant is related to increased circulating levels of homocysteine (Hcy) and cardiovascular risk. However, heterozygous carriers have an augmented risk of cardiovascular accidents independently from normal Hcy levels, suggesting the presence of additional deregulated processes in MTHFR C677T carriers. Here, we hypothesize that targeting Sirtuin 1 (SIRT1) could be an alternative mechanism to control the cardiovascular risk associated to MTHFR deficiency condition. Flow Mediated Dilatation (FMD) and light transmission aggregometry assay were performed in subjects carrying MTHFR C677T allele after administration of resveratrol, the most powerful natural clinical usable compound that owns SIRT1 activating properties. MTHFR C677T carriers with normal Hcy levels revealed endothelial dysfunction and enhanced platelet aggregation associated with SIRT1 downregulation. SIRT1 activity stimulation by resveratrol intake was able to override these abnormalities without affecting Hcy levels. Impaired endothelial function, bleeding time, and wire-induced thrombus formation were rescued in a heterozygous Mthfr-deficient (Mthfr+/–) mouse model after resveratrol treatment. Using a cell-based high-throughput multiplexed screening (HTS) assay, a novel selective synthetic SIRT1 activator, namely ISIDE11, was identified. Ex vivo and in vivo treatment of Mthfr+/– mice with ISIDE11 rescues endothelial vasorelaxation and reduces wire-induced thrombus formation, effects that were abolished by SIRT1 inhibitor. Moreover, platelets from MTHFR C677T allele carriers treated with ISIDE11 showed normalization of their typical hyper-reactivity. These results candidate SIRT1 activation as a new therapeutic strategy to contain cardio and cerebrovascular events in MTHFR carriers.

Protein tyrosine phosphatase PTPN22 negatively modulates platelet function and thrombus formation

AbstractProtein tyrosine phosphatase nonreceptor type 22 (PTPN22) is a protein tyrosine phosphatase that negatively regulates T-cell signaling. However, whether it is expressed and functions in platelets remains unknown. Here we investigated the expression and role of PTPN22 in platelet function. We reported PTPN22 expression in both human and mouse platelets. Using PTPN22−/− mice, we showed that PTPN22 deficiency significantly shortened tail-bleeding time and accelerated arterial thrombus formation without affecting venous thrombosis and the coagulation factors VIII and IX. Consistently, PTPN22-deficient platelets exhibited enhanced platelet aggregation, granule secretion, calcium mobilization, lamellipodia formation, spreading, and clot retraction. Quantitative phosphoproteomic analysis revealed the significant difference of phosphodiesterase 5A (PDE5A) phosphorylation in PTPN22-deficient platelets compared with wild-type platelets after collagen-related peptide stimulation, which was confirmed by increased PDE5A phosphorylation (Ser92) in collagen-related peptide–treated PTPN22-deficient platelets, concomitant with reduced level and vasodilator-stimulated phosphoprotein phosphorylation (Ser157/239). In addition, PTPN22 interacted with phosphorylated PDE5A (Ser92) and dephosphorylated it in activated platelets. Moreover, purified PTPN22 but not the mutant form (C227S) possesses intrinsic serine phosphatase activity. Furthermore, inhibition of PTPN22 enhanced human platelet aggregation, spreading, clot retraction, and increased PDE5A phosphorylation (Ser92). In conclusion, our study shows a novel role of PTPN22 in platelet function and arterial thrombosis, identifying new potential targets for future prevention of thrombotic or cardiovascular diseases.

Influence of cardiopulmonary exercise test on platelet function in patients with coronary artery diseases on antiplatelet therapy

BackgroundCardiac rehabilitation reduces mortality and morbidity rate of patients with coronary artery diseases (CAD); however, acute exercise stimulation may also increase the thrombotic risk through platelet activation. Studies on the effects of cardiac rehabilitation on platelet function have been sparse.MethodsA total of 28 patients (24 men and 4 women; average age = 54.6 ± 8 years old) with stable CAD were enrolled in this study and divided into Aspirin-treated (n = 11; Aspirin group) and dual-antiplatelet-treated group (DAPT group; n = 17). Symptom-limited cardiopulmonary exercise test (CPET) with a cycle ergometer was performed on all the patients. Before and after CPET, platelet function was evaluated using light transmission aggregometry and whole blood flow cytometry.ResultsAll patients completed the CPET without provoked cardiac events, and the mean value of peak oxygen uptake (Peak Vo2) was 19.3 ± 3 ml/(kg min). Prior to CPET, platelet aggregation was significantly suppressed in DAPT group compared to Aspirin group (43.0 ± 21.5 vs. 72.9 ± 7.5, p < 0.001). CPET promoted platelet aggregation in Aspirin group (72.9 ± 7.5 vs. 80.9 ± 7.6, p = 0.005) and DAPT group (43.0 ± 21.5 vs. 50.1 ± 20.9, p = 0.010), and platelet count was increased in Aspirin (210.9 ± 54.6 vs. 227.5 ± 58.1, p = 0.001) and DAPT group (217.5 ± 63.8 vs. 229.7 ± 63.7, p = 0.001). However, the expression levels of CD62p and PAC-1 were not affected by CPET in both groups.ConclusionSymptom-limited CPET enhanced platelet aggregation in patients with CAD despite treatment with antiplatelet, mainly via platelet count augmentation, but not through single platelet activation.Trial registration: Effects of high intensity interval training versus moderate intensity continue training in cardiac rehabilitation on platelet function of patients with coronary heart diseases: a exploratory randomized controlled trial.ChiCTR-INR-17010717. Registered 23 February 2017, https://www.chictr.org.cn/edit.aspx?pid=18206&htm=4.