Thrombin Receptor Research Articles

The factor XI/XIa antibody abelacimab combined with enoxaparin inhibits filter clotting in hemodialysis circuits ex vivo.

Drugs targeting factor XI may offer an alternative to heparin for preventing blood clotting in extracorporeal circulation. We investigated the effects of abelacimab, a novel monoclonal antibody targeting factor XI. We collected whole blood samples into two bags (each 240ml, control group: enoxaparin 1.2mg, treatment group: enoxaparin 1.2mg plus abelacimab 5mg) and circulated in a hemodialysis device for up to 3h. We performed whole blood aggregation and thromboelastometry at several time points. Time to filter clotting was the primary endpoint. We included 10 volunteers. Each volunteer's blood was split into two bags (containing enoxaparin +/- abelacimab) and used simultaneously on two hemodialysis devices. The treatment group's time to filter clotting was significantly prolonged (treatment: 180min, IQR 180-180 vs. control: 120min, IQR 97-147, p < 0.001), and the transmembrane pressure was significantly lower at the end of the circuit flow (treatment: 13 mmHg vs. control: 65 mmHg, p = 0.001). Fibrinogen levels and median platelet counts were preserved. Platelet aggregation was better preserved in the treatment group for ristocetin (p = 0.015), thrombin receptor activating peptide (p = 0.015), and arachidonic acid (p = 0.001). Thromboelastometry showed prolonged clotting times in the treatment group at the end of the experiment (INTEM, p < 0.001; HEPTEM, p = 0.001). Abelacimab prolonged the time to filter clotting in this ex vivo model of hemodialysis. This is an aggressive model due to the frequent re-circulation of blood and a lack of endothelial cells. These data provide support for testing abelacimab in patients on hemodialysis.

Elderly patients are hyperresponsive to potent P2Y12 inhibitors

Abstract Background Aging has recently been associated with increased basal platelet activation and adenosine diphosphate (ADP) hyperreactivity on the one hand, but decreased platelet response to thrombin receptor stimulation on the other hand in individuals without antiplatelet therapy. In the current study, we investigated platelet response to agonist stimulation in elderly patients (70 years or older) on dual antiplatelet therapy with potent P2Y12 inhibitors. Methods Platelet aggregation in response to arachidonic acid (AA), ADP, collagen, the protease-activated receptor (PAR)-1 agonist SFLLRN and the PAR-4 agonist AYPGKF was assessed by multiple electrode aggregometry in 79 prasugrel- and 77 ticagrelor-treated patients 3 days after acute percutaneous coronary intervention. Results In the overall study population (n=156), patients aged ≥70 years (n=33) had lower platelet aggregation in response to AA, ADP and SFLLRN than younger patients (all p&amp;lt;0.05). In prasugrel-treated patients (n=79), those aged ≥70 years (n=13) showed significantly lower platelet aggregation in response to all agonists compared to younger patients (all p&amp;lt;0.05). In contrast, in ticagrelor-treated patients (n=77), those aged ≥70 years (n=20) only had significantly lower ADP-inducible platelet aggregation than younger patients (p=0.03), whereas platelet aggregation in response to AA, collagen, SFLLRN and AYPGKF was similar between elderly and younger patients on ticagrelor (all p&amp;gt;0.05). Among patients aged ≥70 years, prasugrel-treated patients showed significantly lower platelet aggregation in response to AA, collagen and AYPGKF than those receiving ticagrelor (all p&amp;lt;0.05). Conclusion Patients aged ≥70 years on potent P2Y12 inhibitors exhibit increased inhibition of ADP-inducible platelet aggregation. In addition, elderly patients on prasugrel show a significantly lower response to AA, collagen, SFLLRN and AYPGKF than younger patients.Platelet aggregation prasugrel patientsPlatelet aggregation ticagrelor patients

Direct IL-6 inhibition prevents arterial thrombosis by reducing collagen-mediated platelets activation

Abstract Introduction Interleukin 6 (IL-6) has a pivotal role in atherothrombosis. Yet, targeting IL-6 to prevent atherothrombotic events still requires validation of efficacy and safety. The recent phase 2 RESCUE trial reported that direct inhibition of IL-6 ligand by the monoclonal antibody (mAb) Ziltivekimab is safe in patients with elevated cardiovascular risk. Purpose This project investigated the effects of direct IL-6 inhibition on arterial thrombosis, and assessed the underlying cellular mechanisms. Methods Three month old C56Bl/6 male mice received very-low dose LPS i.p. for 4 weeks. During the last week they were randomized to receive in addition either anti-mouse IL-6 mAb 200 μg i.p. every other day or IgG1 isotype control. Then thrombosis of left common carotid artery (LCCA) was induced by laser injury under anesthesia. Platelets of treated mice were isolated and stimulated with either adenosine di-phosphate (ADP), collagen-related peptide (CRP) or thrombin receptor activating peptide (TRAP). Platelets activation was measured by flow-cytometry, as expression of P-selectin and JON/A.The effect of direct IL-6 inhibition was confirmed in patients with stable coronary artery disease (sCAD) by ex-vivo incubation of isolated platelets with either anti-human IL-6 mAb 1.5 μg/mL or IgG1 isotype control. Platelet activation was measured by flow-cytometry, as expression of P-selectin. Results Mice treated with anti-IL6 antibody displayed a significantly longer time-to-occlusion after the LCCA (Figure 1), without any significant difference in coagulation parameters. After stimulation with CRP, platelets were significantly activated in control mice, but not in mice treated with anti-IL6 mAb. Similarly, activation of isolated platelets from patients with sCAD was significantly reduced (Figure 2) by the ex-vivo treatment with anti-IL6 mAb. Conclusion The direct inhibition of IL-6 reduces platelets reactivity to collagen, thereby blunting arterial thrombosis upon endothelial damage. These results provide a potential mechanism to explain the reduction of cardiovascular risk associated to direct IL-6 inhibition. Conclusion The direct inhibition of IL-6 reduces platelets reactivity to collagen, thereby blunting arterial thrombosis upon endothelial damage. These results provide a potential mechanism to explain the reduction of cardiovascular risk associated to direct IL-6 inhibition.Figure 1Figure 2

From bench to bedside and back to bench: investigating the role of platelet heterogeneity in coronary artery disease

Abstract Background/Introduction Platelets exhibit considerable heterogeneity in RNA content, size, and thrombogenicity. Our preliminary investigation of the transcriptome of the RNA-rich reticulated platelets(RPs) in healthy donors revealed an enrichment of prothrombotic transcripts compared to mature platelets(MPs). Recently, we validated the prognostic role of elevated RPs at clinical level in a modern cohort of ACS patients treated with potent P2Y12 inhibitors. However, the pathophysiology of RP hyperreactivity remains unclear, particularly its correlation with adverse events and cardiovascular death. Purpose To study the biology of RPs in patients with chronic coronary syndrome(CCS) and to elucidate their role in disease progression. Methods RPs were isolated from peripheral blood of CCS patients(N=20) and compared with MPs. Cell viability and reactivity of RPs were quantified by FACS. Deep transcriptomic profiling was performed using post-sorting bulk RNA sequencing of RPs and MPs and analyzed with ad-hoc bioinformatic pipelines. Proteomic profiling using mass cytometry(CyTOF) was used to validate the transcriptomic findings with single cell resolution. Results FACS analysis confirmed RPs hyperreactivity showing increased P-Selectin expression upon activation with TRAP, ADP and Collagen(Fig. 1A). Total RNA-sequencing detected 2213 differentially regulated genes with an enrichment in RPs of key functional transcript such as, the von Willebrand Factor VWF(p=3.06*10-33), the thromboxane receptor TBXA2R(p=2.07*10-29) and the collagen receptor GP6(p=1.29*10-38, Fig. 1B-C). Gene ontology and gene set enrichment analyses detected an upregulation of relevant categories as "Regulation of platelet activation" and "Platelet aggregation"(Fig. 1D-F). In addition, we detected a novel alternative splicing event on the collagen receptor transcript GP6 upregulated in RPs(Fig. 1G). We detected 33 differentially regulated miRNAs, including miR-409, miR-134 and miR-432, which have been already linked to prothrombotic phenotypes(Fig. 1H). Additionally, we detected an enrichment of circular RNAs in RPs compared to MPs with several circular RNA upregulated in RPs, that have not been described before(Fig. 1I-K). Mass cytometry detected a significant upregulation in RPs of relevant proteins including the collagen receptor GPVI(p=8.98*10-12, Fig.2) and the thrombin receptor PAR1(p=5.21*10-11). Validation assays in an independent cohort detected an upregulation of the PI3K/AKT pathway in RPs which is downstream of the receptor PAR1 and GP6. Conclusion This study represents the first comprehensive multiomic characterization of RPs in CCS patients, providing insights into their prothrombotic phenotype and into their correlation with cardiovascular events. These findings suggest potential avenues for tailored therapeutic interventions targeting the identified upregulated pathways in patients with elevated RPs, warranting further investigation at the clinical level.

Platelet Function, Platelet Size and Content of Reticulated Platelets: Interactions in Patients Receiving Dual Antiplatelet Therapy.

Increased platelet activity is a risk factor of thrombotic events in cardiovascular patients. We studied the relationship between platelet function, platelet size, and the content of reticulated platelets (RP) in patients with coronary heart disease (CHD, n = 55) and acute coronary syndrome (ACS, n = 95) receiving acetylsalicylic acid + clopidogrel or ticagrelor, respectively. The control group consisted of patients with risk factors for CHD, but with no CHD/ACS and free of antiplatelet drugs (n = 66). Platelet function was evaluated by the exposure of activated glycoprotein (GP) IIb-IIIa and P-selectin. In the control group, platelets were activated by TRAP (Thrombin Receptor Activating Peptide) 10 µM, and ADP 20, 5, 2.5 µM, and in the CHD/ACS groups, by TRAP 10 µM, and ADP 20 5 µM (±epinephrine 20 µM). Platelet size was assessed by the mean volume, % large forms, and forward scattering. RP were stained by thiazole orange. In the control group, activated GP IIb-IIIa and P-selectin correlated with platelet size and RP content after platelet activation by all agonists. Despite the decrease in platelet activity by antiplatelet drugs, most correlations (primarily for activated GP IIb-IIIa) were preserved in the CHD/ACS patients. In conclusion, increased platelet size and RP content are associated with increased platelet activity and the reduced efficacy of antiplatelet therapy.

Acetaminophen Overdose Reveals PAR4 as a Low-Expressing but Potent Receptor on the Hepatic Endothelium in Mice.

The protease thrombin, which elicits multiple physiological and pathological effects on vascular endothelial cells (ECs), can signal through PARs (protease-activated receptors) 1 and 4. PAR1 is a high-affinity thrombin receptor known to signal on ECs, whereas PAR4 is a low-affinity thrombin receptor, and evidence for its expression and function on ECs is mixed. This study aims to exploit the high levels of thrombin generation and hepatic vascular dysfunction that occur during acetaminophen (APAP) overdose to determine (1) whether hepatic endothelial PAR4 is a functional receptor, and (2) the endothelial-specific functions for PAR1 and PAR4 in a high thrombin and pathological setting. We generated mice with conditional deletion of Par1/Par4 in ECs and overdosed them with APAP. Hepatic vascular permeability, erythrocyte accumulation in the liver, thrombin generation, and liver function were assessed following overdose. Additionally, we investigated the expression levels of endothelial PARs and how they influence transcription in APAP-overdosed liver ECs using endothelial translating ribosome affinity purification followed by next-generation sequencing. We found that mice deficient in high-expressing endothelial Par1 or low-expressing Par4 had equivalent reductions in APAP-induced hepatic vascular instability, although mice deficient for both receptors had lower vascular permeability at an earlier timepoint after APAP overdose than either of the single mutants. Additionally, mice with loss of both endothelial Par1 and Par4 had reduced thrombin generation after APAP overdose, suggesting decreased hypercoagulability. Last, we found that endothelial PAR1-but not PAR4-can regulate transcription in hepatic ECs. Low-expressing PAR4 can react similarly to high-expressing PAR1 in APAP-overdosed hepatic ECs, demonstrating that PAR4 is a potent thrombin receptor. Additionally, these receptors are functionally redundant but act divergently in their expression and ability to influence transcription in hepatic ECs.

Thrombin receptor PAR4 cross-activates the tyrosine kinase c-met in atrial cardiomyocytes.

Thrombin supports coagulation-independent inflammation via protease-activated receptors (PAR). PAR4 is specifically increased in obese human atria, correlating with NLRP3 inflammasome activation. PAR4-mediated NLRP3 inflammasome activation in atrial cardiomyocytes is not known, nor have signaling partners been identified. Thrombin transactivates the hepatocyte growth factor receptor in some cancer cells, so we examined PAR4/c-met cross-talk in atrial cardiomyocytes and its possible significance in obesity. Cardiomyocytes from right atrial appendages (RAA) of obese patients expressed more PAR1 and PAR4 compared to non-obese. In HL-1 atrial cardiomyocytes, thrombin induced caspase-1 auto-activation and IL-1β maturation; IL-1β secretion was evoked by PAR4-activating peptide (AP), but not PAR1-AP. PAR4-AP additionally increased phosphorylated CaMKII-Thr287, mTOR-Ser2481, and Akt-Ser473 while suppressing AMPK-Thr172 phosphorylation. Total kinase levels were largely unaltered. PAR4AP rapidly increased phosphorylated c-met in HL-1 cells and over time also transcriptionally upregulated c-met. The c-met inhibitor SGX-523 abrogated the effects of PAR4-AP on CaMKII/AKT/mTOR phosphorylation but did not affect PAR4-stimulated IL-1β production. Obese human RAA contained more IL-1β, phospho-c-met, and phospho-mTOR than non-obese RAA; CamKII phosphorylation was not modified. Atria from high-fat diet (HFD) versus chow-fed mice also contained more IL-1β, together with higher myeloperoxidase activity, Acta2 mRNA total and phosphorylated c-met; these increases were blunted in PAR4-/- HFD-fed mice. Thrombin cross-activates c-met via PAR4 in atrial cardiomyocytes. Transactivated c-met contributes partially to PAR4-mediated signaling, but NLRP3 inflammasome activation appears to be largely independent of c-met. Abundance of PAR4 and activated c-met increases with obesity, providing therapeutic targets for management of adiposity-driven AF.

Physicochemical Features of Thrombin Binding to Platelet Membrane

Thrombin is a key enzyme of the blood coagulation system, which has been actively studied since the beginning of the last century. The formation of thrombin from prothrombin in the area of vessel injury leads not only to the formation of fibrin – an important structural component of the hemostatic clot – but also to the activation of platelets, endothelium and immune system cells. The binding of thrombin to the platelet surface is thought to play a critical role in the process of platelet activation and may also ensure the maintenance of a high concentration of thrombin within the thrombus due to the concentration of protease on the platelet surface. To date, all major thrombin receptors on platelets have been thoroughly characterized: through various experimental methods, the physicochemical parameters of the corresponding intermolecular interactions have been established. Since the interaction of thrombin with platelets leads to their activation, which includes changes in the number of receptors as a result of granule secretion, the interpretation of the observed kinetic binding curves faces a number of difficulties. It is known that some receptors as a result of platelet activation are able to redistribute on the membrane and form dimers and clusters, which makes the kinetics of thrombin binding to platelets an extremely complex process depending on many factors, such as activator concentrations, platelet state, and other local parameters of the system. This review aims to describe the current understanding of the interaction of thrombin with the platelet membrane and to outline important unresolved issues in this area of research. The survey provides not only information on structural and kinetic features of thrombin binding to individual platelet membrane proteins, but also analyzes the relationship between the relevant interaction parameters and previously obtained data on the integral kinetics of protease binding to the platelet surface.

Reduced platelet activation and thrombus formation in male transgenic model mice of Alzheimer's disease suggests early sex-specific differences in platelet pathophysiology

Alzheimer's disease (AD) is the most common form of dementia and characterized by extracellular amyloid-β (Aβ) plaques, intracellular neurofibrillary tau tangles and neurodegeneration. Over 80 % of AD patients also exhibit cerebral amyloid angiopathy (CAA). CAA is a cerebrovascular disease caused by deposition of Aβ in the walls of cerebral blood vessels leading to vessel damage and impairment of normal blood flow. To date, different studies suggest that platelet function, including activation, adhesion and aggregation, is altered in AD due to vascular Aβ deposition. For example, the transgenic AD model mice APP23 mice that exhibit CAA and parenchymal Aβ plaques, show pre-activated platelets in the blood circulation and increased platelet integrin activation leading to a pro-thrombotic phenotype in these mice late stages of AD. However, it is still an open question whether or not platelets exhibit changes in their activation profile before they are exposed to vascular Aβ deposits. Therefore, the present study examined platelets from middle-aged transgenic APP23 mice at the age of 8–10 months. At this age, APP23 mice show amyloid plaques in the brain parenchyma but not in the vasculature. Our analyses show that these APP23 mice have unaltered platelet numbers and size, and unaltered surface expression of glycoproteins. However, the number of dense granules in transgenic platelets was increased while the release was unaltered. Male, but not female APP23 mice, exhibited reduced platelet activation after stimulation of the thrombin receptor PAR4 and decreased thrombus stability on collagen under flow conditions ex vivo compared to control mice. In an arterial thrombosis model in vivo, male APP23 mice showed attenuated occlusion of the injured artery compared to controls. These findings provide clear evidence for early changes in platelet activation and thrombus formation in male mice before development of overt CAA. Furthermore, reduced platelet activation and thrombus formation suggest sex-specific differences in platelet physiology in AD that has to be considered in future studies of platelets and their role in AD.

An αIIbβ3 monoclonal antibody traps a semiextended conformation and allosterically inhibits large ligand binding

AbstractMonoclonal antibodies (mAbs) have provided valuable information regarding the structure and function of platelet αIIbβ3. Protein disulfide isomerase (PDI) has been implicated in αIIbβ3 activation and binds to thrombin-activated αIIbβ3. Using human platelets as the immunogen, we identified a new mAb (R21D10) that inhibits the binding of PDI to platelets activated with thrombin receptor–activating peptide (T6). R21D10 also partially inhibited T6-induced fibrinogen and PAC-1 binding to platelets, as well as T6- and adenosine 5'-diphosphate–induced platelet aggregation. Mutual competition experiments showed that R21D10 does not inhibit the binding of mAbs 10E5 (anti-αIIb cap domain) or 7E3 (anti-β3 β-I domain), and immunoblot studies indicated that R21D10 binds to β3. The dissociation of αIIbβ3 by EDTA had a minimal effect on R21D10 binding. Cryogenic electron microscopy of the αIIbβ3-R21D10 Fab complex revealed that R21D10 binds to the β3 integrin-epidermal growth factor 1 (I-EGF1) domain and traps an intermediate conformation of αIIbβ3 with semiextended leg domains. The binding of R21D10 produces a major structural change in the β3 I-EGF2 domain associated with a new interaction between the β3 I-EGF2 and αIIb thigh domains, which may prevent the swing-out motion of the β3 hybrid domain required for high-affinity ligand binding and protect αIIbβ3 from EDTA-induced dissociation. R21D10 partially reversed the ligand binding priming effect of eptifibatide, suggesting that it could convert the swung-out conformation into a semiextended conformation. We concluded that R21D10 inhibits ligand binding to αIIbβ3 via a unique allosteric mechanism, which may or may not be related to its inhibition of PDI binding.

Protamine and Heparin Interactions: A Narrative Review.

Protamine, first isolated from salmon fish sperm and now produced through recombinant biotechnology, is an antidote that neutralizes the anticoagulant properties of heparin. Protamine function is based on the capacity to dissociate the heparin-antithrombin III (AT III) complex (an important link that promotes blood fluidification by inhibiting coagulation), forming the inactive heparin-protamine complex. Protamine has itself dose-dependent anticoagulant properties: It interferes with coagulation factors and platelet function; it stimulates fibrinolysis; it can lead to thrombocytopenia and reduction in thrombin-related platelet aggregation; it decreases platelet response to thrombin receptor agonist in a dose-dependent manner. In this review, we will focus on protamine and its interaction with heparin. Notably, protamine is able to antagonize not only unfractionated heparin (UFH) but also low molecular weight heparins to various degrees. Protamine-allergic and anaphylactoid systemic reactions may affect up to 1 in 10 people and should be prevented and treated early.

STING Contributes to Pulmonary Hypertension by Targeting IFN and BMPR2 Signaling through Regulating of F2RL3.

Pulmonary hypertension (PH) is an incurable disease characterized by pulmonary vascular remodeling. Endothelial injury and inflammation are the key triggers of disease initiation. Recent findings suggest that STING (stimulator of IFN genes) activation plays a critical role in endothelial dysfunction and IFN signaling. Here, we investigated the involvement of STING in the pathogenesis of PH. Patients with PH and rodent PH model samples, a Sugen 5416/hypoxia PH model, and pulmonary artery endothelial cells (PAECs) were used to evaluate the hypothesis. We found that the cyclic guanosine monophosphate-AMP synthase-STING signaling pathway was activated in lung tissues from rodent PH models and patients with PH and in TNF-α-induced PAECs invitro. Specifically, STING expression was significantly elevated in the endothelial cells in PH disease settings. In the Sugen 5416/hypoxia mouse model, genetic knockout or pharmacological inhibition of STING prevented the progression of PH. Functionally, knockdown of STING reduced the proliferation and migration of PAECs. Mechanistically, STING transcriptionally regulates its binding partner F2RL3 (F2R-like thrombin or trypsin receptor 3) through the STING-NF-κB axis, which activated IFN signaling and repressed BMPR2 (bone morphogenetic protein receptor 2) signaling both invitro and invivo. Further analysis revealed that F2RL3 expression was increased in PH settings and identified negative feedback regulation of F2RL3/BMPR2 signaling. Accordingly, a positive correlation of expression amounts between STING and F2RL3/IFN-stimulated genes was observed invivo. Our findings suggest that STING activation in PAECs plays a critical role in the pathobiology of PH. Targeting STING may be a promising therapeutic strategy for preventing the development of PH.

Acetaminophen Overdose Reveals Protease-Activated Receptor 4 as a Low-Expressing but Potent Receptor on the Hepatic Endothelium.

Hepatic endothelial cell (EC) dysfunction and centrilobular hepatocyte necrosis occur with acetaminophen (APAP) overdose. The protease thrombin, which is acutely generated during APAP overdose, can signal through protease-activated receptors 1 and 4 (PAR1/PAR4). PAR1 is a high-affinity thrombin receptor that is known to signal on ECs, whereas PAR4 is a low-affinity thrombin receptor, and evidence for its expression and function on ECs is mixed. This study aims to exploit the high levels of thrombin generated during APAP overdose to determine (1) if hepatic endothelial PAR4 is a functional receptor, and (2) endothelial-specific functions for PAR1 and PAR4 in a high thrombin setting. We generated mice with conditional deletion(s) of Par1/Par4 in ECs and overdosed them with APAP. Hepatic vascular permeability, erythrocyte congestion/bleeding, and liver function were assessed following overdose. Additionally, we investigated the expression levels of endothelial PARs and how they influence transcription in APAP-overdosed liver ECs using endothelial Translating Ribosome Affinity Purification followed by next-generation sequencing (TRAPseq). We found that mice deficient in high-expressing endothelial Par1 or low-expressing Par4 had equivalent reductions in APAP-induced hepatic vascular instability but no effect on hepatocyte necrosis. Additionally, mice with loss of endothelial Par1 and Par4 had reduced permeability at an earlier time point after APAP overdose when compared to mice singly deficient in either receptor in ECs. We also found that endothelial PAR1-but not PAR4-can regulate transcription in hepatic ECs. Low-expressing PAR4 can react similarly to high-expressing PAR1 in APAP-overdosed hepatic ECs, demonstrating that PAR4 is a potent thrombin receptor. Additionally, these receptors are functionally redundant but act divergently in their expression and ability to influence transcription in hepatic ECs.

The thrombin receptor (PAR1) is associated with microtubules, mitosis and process formation in glioma cells

The cell surface protease-activated receptor 1 (PAR1) is overexpressed in glioblastoma multiforme (GBM). We studied the function and structure of intracellular microtubule (MT) and PAR1 in a tubulin-mediated process. We found that exposure to thrombin increased the percentage of proliferative, S, and M phases cells, affected morphology, and increased process elongation. PAR1 antagonist inversely affects these measures, increases tubulin end-binding protein 3 (EB3) mRNA expression in C6 cells, and reduces EB3 comet length, track length, and duration in neuroblastoma cells. In addition, immunofluorescence staining suggests that PAR1 is in close association with the MT α-tubulin and with coagulation cascade proteins during cell division stages. Our findings support PAR1 involvement in MT dynamics.

Unraveling the transcriptomic landscape of platelets: RPs vs. MPs in CCS patients

Abstract Funding Acknowledgements Type of funding sources: Private grant(s) and/or Sponsorship. Main funding source(s): Else Kröner Fresenius Stiftung Background Reticulated platelets (RPs), characterized by youth, hyper-reactivity, and RNA abundance, signify pro-thrombotic potential and serve as predictors for inadequate antiplatelet therapy response post-myocardial infarction. Their pivotal role in chronic coronary syndrome (CCS) and high on-treatment platelet reactivity highlights their significance as promising biomarkers for adverse cardiovascular events in diverse pathological settings. Purpose We aimed to compare for the first time the transcriptomic profiles of RPs and MPs in CCS patients. Methods Using fluorescent activated cell sorting (FACS), RPs and MPs were isolated based on RNA content from peripheral blood of 19 CCS patients. RNA assessment involved the Tapestation 4200 platform (Agilent), and totalRNA libraries were sequenced on a NextSeq 500 Illumina platform. RNA sequencing analysis, with a cut-off of p &amp;lt;0.005 and a log2fc &amp;gt;1, and alternative splicing event detection using MAJIQ, were performed. Circular RNA (circRNA) analysis employed CIRCexplorer. Results Total RNA-sequencing identified 1589 differentially expressed genes, with 1100 transcripts upregulated in RPs and 489 enriched in MPs (Figure 1 A, B and D). Notably, collagen receptor GP6 (log2FC 1.12, p=6.89x10-41), thrombin receptor PAR4 (F2RL3, log2FC 1.1, p=3.54*10-21) and Von Willebrand Factor (log2FC 1.2, p=1.26*10-38) transcripts were significantly enriched in RPs. Gene Ontology identified a higher enrichment of relevant biological categories in RPs compared to MPs, such as "platelet activation", "platelet degranulation" and "blood coagulation" (Figure 1C). These findings are consistent with our previously established discovery and support the hypothesis that RPs in cardiovascular disease are hyper-reactive and pro-thrombotic due to their different RNA content compared to other platelets. Several splicing events were detected as differentially regulated: there is an upregulation of an alternative splicing on the collagen receptor transcript GP6 in RPs (Figure 1E); by the transcript of the G-protein GNAQ, one alternative splicing event was found upregulated in RPs, whilst two others were upregulated in MPs. Moreover, we found an enrichment of the total level of circular RNAs in MPs as compared to RPs (Figure 1F). Several previously undescribed circular RNAs were discovered to be enriched in RPs, as shown in Figure 1F. Conclusion This groundbreaking transcriptomic profiling of RPs and MPs in CCS patients elucidates the biological basis for RPs' hyperreactivity, emphasizing the differential enrichment of platelet activation transcripts. The pronounced upregulation of prothrombotic signaling in RPs provides insight into their hyperactivity and correlation with cardiovascular events. These findings illuminate a novel therapeutic niche in CCS patients, although further investigations are essential to comprehend the detrimental role of RPs in coronary artery disease.

Phenolic profile, cheminformatics, and antiplatelet aggregation activity of orange and purple sweet potato (Ipomoea batatas L.) storage roots

Sweet potatoes are rich in cardioprotective phytochemicals with potential anti-platelet aggregation activity, although this benefit may vary among cultivars/genotypes. The phenolic profile [HPLC-ESI(−)-qTOF-MS2], cheminformatics (ADMET properties, affinity toward platelet proteins) and anti-PA activity of phenolic-rich hydroalcoholic extracts obtained from orange (OSP) and purple (PSP) sweet potato storage roots, was evaluated. The phenolic richness [Hydroxycinnamic acids> flavonoids> benzoic acids] was PSP > OSP. Their main chlorogenic acids could interact with platelet proteins (integrins/adhesins, kinases/metalloenzymes) but their bioavailability could be poor. Just OSP exhibited a dose-dependent anti-platelet aggregation activity [inductor (IC50, mg.ml−1): thrombin receptor activator peptide-6 (0.55) > Adenosine-5′-diphosphate (1.02) > collagen (1.56)] and reduced P-selectin expression (0.75–1.0 mg.ml−1) but not glycoprotein IIb/IIIa secretion. The explored anti-PA activity of OSP/PSP seems to be inversely related to their phenolic richness. The poor first-pass bioavailability of its chlorogenic acids (documented in silico) may represent a further obstacle for their anti-PA in vivo.

Glyphosate modifies the gene expression and migration of trophoblastic cells without altering the process of angiogenesis or the implantation of blastocysts in vitro

Adverse pregnancy outcomes have been associated with the presence of glyphosate (G) in umbilical cord, serum, and urine samples from pregnant women. Our aim was to study the effect of G on blastocyst implantation using an in vitro mouse model, and the migration and acquisition of endothelial phenotype of the human trophoblastic HTR8/SVneo (H8) cells. In mouse blastocysts, no differences in attachment time and implantation outgrowth area were observed after G exposure. H8 cell migration was stimulated by 0.625 μM G without cytotoxicity. After 6 h, the mRNA expression of vascular endothelial growth factor (VEGF) and C–C motif chemokine ligand 2 (CCL2) was upregulated in H8 cells exposed to 1.25 μM G when compared vehicle-treated cells (p ≤ 0.05). No differences were observed in interleukin 11, VEGF receptor 1, and coagulation factor II thrombin receptor in H8 cells exposed to different concentrations of G for 6 h compared to the vehicle. Interestingly, exposure to G did not alter angiogenesis as measured by a tube formation assay. Taken all together, these results suggest that G exposure may contribute as a risk factor during pregnancy, due to its ability to alter trophoblast migration and gene expression.

Binding of coagulation factor IXa to procoagulant platelets revisited: Low affinity and interactions with other factors

Binding of activated factor IX (fIXa) to the phosphatidylserine-expressing procoagulant platelets is a critical step in blood coagulation, which is necessary for the membrane-dependent intrinsic tenase complex assembly and factor X activation. However, the nature and parameters of the fIXa binding sites on the procoagulant platelet surface remain unclear. We used flow cytometry to elucidate the quantitative details of the fluorescently labeled fIXa binding to gel-filtered activated platelets. FIXa bound to the procoagulant platelet subpopulation only, with the parameters (maximal number of binding sites at 58900 ± 3400, Kd at 1000 ± 170 nM) similar to binding observed with phospholipid vesicles. No specific high-affinity binding sites for fIXa were detected, and binding proceeded similarly for different methods of procoagulant platelet production (thrombin, thrombin receptor activation peptide, collagen-related peptide, their combinations, or calcium ionophore A23187). Factor VIII, known to form a high affinity complex with fIXa, enhanced fIXa binding to platelets. In contrast, only competition effects were observed for factor X, which binds fIXa with much lower affinity. Unexpectedly, fIXa itself, fIX, and prothrombin also dose-dependently enhance fIXa binding at concentrations below 1000 nM, suggesting the formation of membrane-bound fIXa dimers and fIXa-prothrombin complexes on platelets. These findings provide a novel perspective on the fIXa binding site on procoagulant platelets, which does not have any major differences from pure phospholipid-based model membranes, exhibits inherently low affinity (3–5 orders of magnitude below the physiologically relevant fIXa concentration) but is significantly enhanced by its cofactor VIII, and regulated by previously unknown membrane interactions.

F2RL3 Regulates Epithelial-Mesenchymal Transition and Angiogenesis in Gastric Cancer through the Rap1/MAPK Signaling Pathway.

Gastric cancer (GC) is frequently diagnosed at advanced stages, when cancer cells have already metastasized. Therefore, patients with GC have a low survival rate and poor prognosis even after treatment. We downloaded GC-related RNA sequencing (RNA-Seq) data, copy number variation (CNV) data, and clinical data for bioinformatics analysis to screen prognostic genes of GC. Single-sample gene set enrichment analysis and survival analyses were performed on the RNA-Seq data, and differential and correlation analyses were conducted on the CNV data to obtain CNV-driven differentially expressed genes (DEGs). Prognostic genes were identified through univariate Cox analyses of the CNV-driven DEGs, combined with the clinical data. F2R like thrombin or trypsin receptor 3 (F2RL3) was finally selected for verification after functional and survival analyses of the prognostic genes. F2RL3 expression was lower in paracancer tissue than in GC tissue, and lower in GES-1 gastric epithelial cells than in GC cells. The cell culture supernatants from F2RL3-knockdown GC cells were collected and used to culture human umbilical vein endothelial cells (HUVECs). It was observed that F2RL3 enhanced the activity, metastasis, invasion, and angiogenesis of GC cells; promoted the epithelial-mesenchymal transition (EMT) of GC cells; and impacted the Ras-associated protein 1 (Rap1)/mitogen-activated protein kinase (MAPK) pathway. To further explore the involvement of the Rap1/MAPK pathway in GC development, a pathway activator was added to GC cells with knockdown of F2RL3 expression. This pathway activator not only enhanced the activity, invasion, and migration of GC cells but also promoted the EMT and blood vessel formation. F2RL3 regulates the angiogenesis and EMT of GC cells through the Rap1/MAPK pathway, thus influencing the onset and progression of GC.

The thrombin receptor PAR4 supports visceral adipose tissue inflammation.

Thrombin inhibition suppresses adiposity, WAT inflammation and metabolic dysfunction in mice. Protease-activated receptor (PAR)1 does not account for thrombin-driven obesity, so we explored the culprit role of PAR4 in this context. Male WT and PAR-4-/- mice received a high fat diet (HFD) for 8 weeks, WT controls received standard chow. Body fat was quantified by NMR. Epididymal WAT was assessed by histology, immunohistochemistry, qPCR and lipase activity assay. 3T3-L1 preadipocytes were differentiated ± thrombin, acutely stimulated ± PAR4 activating peptide (AP) and assessed by immunoblot, qPCR and U937 monocyte adhesion. Epicardial adipose tissue (EAT) from obese and lean patients was assessed by immunoblot. PAR4 was upregulated in mouse WAT under HFD. PAR4-/- mice developed less visceral adiposity and glucose intolerance under HFD, featuring smaller adipocytes, fewer macrophages and lower expression of adipogenic (leptin, PPARγ) and pro-inflammatory genes (CCL2, IL-1β) in WAT. HFD-modified activity and expression of lipases or perilipin were unaffected by PAR4 deletion. 3T3-L1 adipocytes differentiated with thrombin retained Ki67 expression, further upregulated IL-1β and CCL2 and were more adhesive for monocytes. In mature adipocytes, PAR4-AP increased phosphorylated ERK1/2 and AKT, upregulated Ki67, CCl2, IL-β and hyaluronan synthase 1 but not TNF-α mRNA, and augmented hyaluronidase-sensitive monocyte adhesion. Obese human EAT expressed more PAR4, CD68 and CD54 than lean EAT. PAR4 upregulated in obesity supports adipocyte hypertrophy, WAT expansion and thrombo-inflammation. The emerging PAR4 antagonists provide a therapeutic perspective in this context beyond their canonical antiplatelet action.