Platelet Aggregation Research Articles

Attenuation of Atherosclerosis with PAR4 Deficiency: Differential Platelet Outcomes in apoE -/- vs. Ldlr -/- Mice.

Cardiovascular disease (CVD) is a significant burden globally and, despite current therapeutics, remains the leading cause of death. Platelet inhibitors are of interest in CVD treatment to reduce thrombus formation post-plaque rupture as well their contribution to inflammation throughout the progression of atherosclerosis. Protease activated receptor 4 (PAR4) is a receptor highly expressed by platelets, strongly activated by thrombin, and plays a vital role in platelet activation and aggregation. However, the role of PAR4. Mice on a low-density lipoprotein receptor-deficient ( Ldlr -/- ) background were bred with Par4 deficient ( Par4 -/- ) mice to create Ldlr -/- /Par4 +/+ and Ldlr -/- /Par4 -/- cousin lines. Mice were fed high fat (42%) and cholesterol (0.2%) 'Western' diet for 12 weeks for all studies. Bone marrow transplant (BMT) studies were conducted by irradiating Ldlr -/- /Par4 +/+ and Ldlr -/- /Par4 -/- mice with 550 rads (2x, 4 hours apart) and then repopulated with Par4 +/+ or Par4 -/- bone marrow. To determine if the effects of thrombin were mediated solely by PAR4, the thrombin inhibitor dabigatran was added to the 'Western' diet. Ldlr -/- /Par4 -/- given dabigatran did not further decrease their atherosclerotic burden. Differences between apolipoprotein E deficient ( apoE -/- ) and Ldlr -/- platelets were assessed for changes in reactivity. We observed higher PAR4 abundance in arteries with atherosclerosis in human and mice versus healthy controls. PAR4 deficiency attenuated atherosclerosis in the aortic sinus and root versus proficient controls. BMT studies demonstrated this effect was due to hematopoietic cells, most likely platelets. PAR4 appeared to be acting independent of PAR1, as there werer no changes with addition of dabigatran to PAR4 deficient mice. apoE -/- platelets are hyperreactive compared to Ldlr -/- platelets. Hematopoietic-derived PAR4, most likely platelets, plays a vital role in the development and progression of atherosclerosis. Specific targeting of PAR4 may be a potential therapeutic target for CVD. Deficiency of protease-activated receptor 4 attenuates the development of diet-induced atherosclerosis in a Ldlr -/- mouse model. PAR4 deficiency in hematopoietic cells is atheroprotective. PAR4 deficiency accounts for the majority of thrombin-induced atherosclerosis in a Ldlr -/- mouse model. The examination of platelet-specific proteins and platelet activation should be carefully considered before using the apoE -/- or Ldlr -/- mouse models of atherosclerosis.

Acute Hyperglycemia-Induced Injury in Myocardial Infarction.

Acute hyperglycemia is a transient increase in plasma glucose level (PGL) frequently observed in patients with ST-elevation myocardial infarction (STEMI). The aim of this review is to clarify the molecular mechanisms whereby acute hyperglycemia impacts coronary flow and myocardial perfusion in patients with acute myocardial infarction (AMI) and to discuss the consequent clinical and prognostic implications. We conducted a comprehensive literature review on the molecular causes of myocardial damage driven by acute hyperglycemia in the context of AMI. The negative impact of high PGL on admission recognizes a multifactorial etiology involving endothelial function, oxidative stress, production of leukocyte adhesion molecules, platelet aggregation, and activation of the coagulation cascade. The current evidence suggests that all these pathophysiological mechanisms compromise myocardial perfusion as a whole and not only in the culprit coronary artery. Acute hyperglycemia on admission, regardless of whether or not in the context of a diabetes mellitus history, could be, thus, identified as a predictor of worse myocardial reperfusion and poorer prognosis in patients with AMI. In order to reduce hyperglycemia-related complications, it seems rational to pursue in these patients an adequate and quick control of PGL, despite the best pharmacological treatment for acute hyperglycemia still remaining a matter of debate.

Abstract Tu032: LRRC8 complex regulates platelet activation and thrombosis

During activation, platelets undergo both shape and volumetric changes which are mechanical events essential for thrombosis. Volume-Regulated Anion Channels (VRACs) are ubiquitously expressed mechanoresponsive, heterohexameric anion channels comprised of leucine-rich repeat containing protein 8 (LRRC8) A (essential for channel activity) in combination with B, C, D and/or E subunits. In this study, we identify single-nucleotide polymorphisms (SNPs) in three LRRC8 subunits (LRRC8A, C, and D) which are expressed in human platelets and are independently associated with altered mean platelet volume in human genetic studies – implicating the volume-sensing LRRC8 channel complex as a regulator of platelet function in humans. We show that LRRC8A is required for functional LRRC8/VRAC in megakaryocytes (MKs). MK-specific LRRC8A conditional knockout (cKO) mice give rise to LRRC8A-null platelets with larger mean volumes. LRRC8A-null platelets and CRISPR-edited human LRRC8A KO MKs (hiPSC MKs) have reduced agonist-stimulated P-selectin exposure and aIIbb3 integrin activation. LRRC8A-null platelets exhibit impaired adhesion to collagen-coated surfaces and reduced agonist-stimulated aggregation and ATP secretion. Additionally, VRAC in MKs mediate ATP currents which are abolished in LRRC8A-null MKs. In vivo, MK-specific LRRC8A cKO mice have reduced platelet thrombus formation in laser-induced cremaster arteriolar thrombosis, and prolonged occlusion times in FeCl 3 -induced carotid arterial thrombosis, compared to control mice, without affecting tail bleeding times. Mechanistically, platelet LRRC8A mediates swell-induced ATP release to amplify agonist-stimulated calcium influx and platelet aggregation. Small-molecule LRRC8 channel inhibitors largely recapitulate the defects observed in LRRC8A-null platelets. These studies identify the mechanoresponsive LRRC8 channel complex as an ATP release channel in platelets which regulates platelet function and arterial thrombosis, providing a proof-of-concept for a novel anti-thrombotic drug target.

Inhibitory effects of resveratrol on platelet activation and thrombosis in colon cancer through regulation of the MAPK and cGMP/VASP pathways

Thrombosis is the primary cause of death in patients with cancer. Resveratrol inhibits platelet activation, a crucial pathophysiological basis of thrombosis, in healthy individuals. However, its effects and mechanisms of action in patients with colon cancer remain unknown. Here, we investigated the effect of resveratrol on platelet adhesion and aggregation in patients with colon cancer. Through numerous in vitro and in vivo analyses, including flow cytometry, western blotting, ELISA, and immunofluorescence and colon cancer rat models, we demonstrated that resveratrol reduced thrombosis in patients with colon cancer by inhibiting the phosphorylation of the MAPK and activating the cyclic-GMP/vasodilator-stimulated phosphoprotein pathway. These findings demonstrate the potential of resveratrol in reducing thrombosis in patients with colon cancer and could be used to develop novel therapeutic strategies for this condition.

Novel GPIb-independent platelet aggregation induced by botrocetin: implications for diagnosis and antithrombotic therapy

BackgroundSnake venom botrocetin facilitates von Willebrand factor (VWF) binding to platelet GPIbα and has been widely used for the diagnosis of von Willebrand disease and GPIb-related disorders. Botrocetin is also commonly employed for the development/characterization of antithrombotics targeting the GPIb-VWF axis. ObjectivesTo explore the alternative receptor(s)/mechanisms that participate in botrocetin-induced platelet aggregation. MethodsThe effects of botrocetin on platelet aggregation were examined using platelets from wild-type, VWF- and fibrinogen-deficient, GPIbα-deficient, IL4Rα/GPIbα-transgenic and αIIbβ3-deficient mice and Bernard–Soulier syndrome and healthy human samples. Platelet-fibrinogen and platelet-VWF interaction were measured using flow cytometry. GPIbα-VWF binding was evaluated utilizing enzyme-linked immunosorbent assay. Botrocetin-αIIbβ3 and botrocetin-GPIbα interactions were measured using enzyme-linked immunosorbent assay and fluorescence anisotropy assays. Heparinized whole blood from healthy donors was examined for thrombus formation and growth in a perfusion chamber. ResultsBotrocetin could induce aggregation of platelets from a Bernard–Soulier syndrome patient and GPIbα-deficient mice as well as platelets lacking the N-terminal extracellular domain of GPIbα. Botrocetin could interact with αIIbβ3 and facilitated αIIbβ3-VWF interaction independent of GPIb. Botrocetin competitively bound to the ligand-binding domain of activated rather than resting αIIbβ3. Although botrocetin-induced platelet aggregation requires VWF, strikingly, in the absence of VWF, botrocetin blocked fibrinogen and other ligand binding to αIIbβ3 and inhibited platelet aggregation and thrombus formation. Consistently, recombinant botrocetin defective in VWF binding inhibited αIIbβ3- and GPIb-mediated platelet aggregation, spreading, and thrombus formation. ConclusionOur study provides insights into avoiding the misdiagnosis of GPIb-related disorders and developing botrocetin mutants as potential new antithrombotics that may simultaneously target both αIIbβ3 and GPIbα.

The Causes of Platelet Aggregation in Version 6.4 Trima Accel Automated Blood Collection System and the Comparison of Two Intervention Measures

To explore the causes of platelet aggregation in version 6.4 Trima Accel automated blood collection system and the effect of 2 intervention measures. The data on platelet aggregation (n=61) and non-aggregation (n=323) of 61 donors in 2020 were collected and the causes of aggregation were analyzed. Then the 72 donors with platelet aggregation in 2021 were randomized into intervention group A (increasing the anticoagulant-to-blood ratio) and intervention group B (wrapping the donor's arm with an electric blanket to keep warm and improve the blood flow speed). The collection time, average blood flow speed, number of machine alarms, anticoagulant usage, deaggregation and citrate reaction of the two groups were compared. Platelet aggregation was negatively correlated with the average blood flow speed (r =-0.394) and positively correlated with the collection time (r =0.458). The equations for predicting aggregation and non-aggregation were constructed based on Bayesian and Fisher discriminant analysis, and the predicted accuracy was 77.1%. The comparison of the effects of two intervention measures showed that the average blood flow speed in group B was higher than that in group A; the collection time, number of machine alarms, anticoagulant usage and proportion of citrate reaction in blood donors in group B were all lower than those in Group A, all these differences were significant (P < 0.05). In the entire cohort in 2021, 90.28% of the products were immediately deaggregated after collection, and 9.72% of the products were deaggregated within 4 hours. There was no statistically significant difference in deaggregation between the two intervention groups (P >0.05). During apheresis platelet collection, the predictive equations for aggregation and non-aggregation can be used to predict the occurrence probability of aggregation, and the intervention can be made in advance. Both intervention measures are effective in reducing platelet aggregation, however, measure B has the advantages of improving the speed of blood collection, shortening the collection time, reducing the alarm frequency and the anticoagulant usage, and reducing the incidence of citrate reaction in blood donors.

Brain-derived gangliosides prime human platelet aggregation and induce platelet-leukocyte aggregate formation

BackgroundPlatelet activation and interaction with leukocytes are crucial in inflammation. Gangliosides, sialic acid–containing glycosphingolipids, have been linked to different inflammatory conditions related to cardio- and neurodegenerative disorders. The role of gangliosides in platelet and leukocyte function, although reported, still needs further investigation. ObjectivesWe aimed to study the role of gangliosides in platelet activation and platelet-leukocyte interaction in vitro. MethodsPlatelet activation was studied through aggregometry in platelet-rich plasma from apparently healthy human volunteers. Signaling protein phosphorylation was analyzed by immunoblotting. Platelet P-selectin expression and platelet-leukocyte aggregate formation were measured by flow cytometry. ResultsThe gangliosides monosialoganglioside GM1, disialoganglioside GD1a, and trisialoganglioside GT1b did not induce by themselves any platelet aggregation. Conversely, when preincubated with platelets, they potentiate platelet aggregation induced by submaximal adenosine diphosphate and collagen concentrations and increased P-selectin expression. Incubation of platelets with free sialic acid and the soluble part of monosialoganglioside GM1 induced a similar potentiating effect on platelet aggregation but not on platelet P-selectin expression. Consistently, analyzing the signaling protein phosphorylation, only the entire gangliosides activated extracellular stimuli–responsive kinase 1/2 suggesting that a complete ganglioside is crucial for its action on platelets. Both the priming effect on platelet aggregation and ERK1/2 activation were prevented by aspirin. Moreover, incubation of citrated whole blood with gangliosides induced platelet-leukocyte aggregate formation accompanied by increased expression of granulocyte and monocyte CD11b compared with untreated blood, suggesting a primary leukocyte activation. ConclusionGangliosides may act in vitro both on platelet and leukocyte activation and on their interaction. The observed effects might contribute to inflammatory processes in clinical conditions.

Platelet aggregation in hypercholesterolemia: Effect of approved antiplatelet compounds and 4-methylcatechol

Platelet aggregation in hypercholesterolemia: Effect of approved antiplatelet compounds and 4-methylcatechol

Comparison of vortex formation in expanded and curved aortic sinuses: The effect of sinus curvature

Aortic valve disease is a significant health issue. Research on the effects of aortic sinus geometry and aortic valve motion during a heartbeat is imperative, as numerous valvular diseases are associated with blood flow near the heart valve. Changes in the internal space of the sinus and the formation of blood clots have been reported after the implantation of a transcatheter aortic valve, an artificial heart valve. Although the sinus shape and leaflet motion significantly affect the hemodynamic characteristics and platelet aggregation, the blood flow behaviors near the heart valve associated with clot formation have not been fully elucidated. This study conducted in vitro experiments to investigate the flow behavior near an aortic sinus model based on the sinus shape. Additionally, hemodynamic changes associated with variations in the aortic sinus geometry and leaflet length within an aortic sinus model under pulsatile-flow conditions were elucidated. In a curved sinus model, a large single vortex formed near the center of the sinus region. Meanwhile, the center of the vortex in the sinus region shifted during a cycle in an expanded sinus model. Furthermore, a small vortex remained where the leaflet was initiated, thus increasing the likelihood of thrombus formation. Thus, the curved sinus model is advantageous for preventing material accumulation by maintaining a large vortex structure. The experimental results confirmed that the aortic sinus shape and leaflet length affect the likelihood of thrombus formation inside the aortic sinus.

Real-time platelet P2Y12 receptor occupancy as a promising pharmacodynamics biomarker for bridging the gap between PK/PD of clopidogrel therapy

Clopidogrel effectively inhibits platelet aggregation in response to ADP by irreversibly binding to the platelet P2Y12 receptor through its active metabolite. However, the observed discrepancies between the pharmacokinetics (PK) and pharmacodynamics (PD) of clopidogrel present substantial challenges in individualizing of antiplatelet therapy. To address these challenges, a robust liquid chromatography–tandem mass spectrometry method has been developed to facilitate the real-time assessment of platelet P2Y12 receptor occupancy. This method has been validated in animal models, providing a reliable link between individual PK profiles and PD effects. Target receptor occupancy offers a comprehensive overview of interindividual variations in clopidogrel metabolism, regulation of P2Y12 receptor expression, and platelet turnover. Moreover, it directly correlates with the inhibitory effect on platelet aggregation. The levels of platelet P2Y12 occupancy accurately reflect the extent of clinical factors influencing the PD of clopidogrel, including dosage, drug–drug interactions (DDI), and type 2 diabetes mellitus (T2DM). As a normalized metric, platelet P2Y12 occupancy not only serves potential as a diagnostic tool for personalized clopidogrel therapy but also aids in elucidating the role of the P2Y12 signaling pathway in cases of abnormal on-treatment platelet reactivity.

Natural Phenolic Compounds with Antithrombotic and Antiplatelet Effects: A Drug-likeness Approach.

Thrombosis is one of the major causes of morbidity and mortality in a wide range of vessel diseases. Several studies have been conducted to identify antithrombotic agents from medicinal plants, and phenolic compounds (PCs) have been shown to effectively inhibit plasma coagulation and platelet aggregation. This study aimed to conduct a survey of the natural PCs with proven antithrombotic and antiplatelet activities, as well as to evaluate by computational modeling the physicochemical and toxicological properties of these compounds using drug-likeness approaches. The data were collected from the scientific database: 'Web of Science', 'Scifinder', 'Pubmed', 'ScienceDirect' and 'Google Scholar', the different classes of PCs with antithrombotic or antiplatelet effects were used as keywords. These molecules were also evaluated for their Drug-Likeness properties and toxicity to verify their profile for being candidates for new antithrombotic drugs. In this review, it was possible to register 85 lignans, 73 flavonoids, 28 coumarins, 21 quinones, 23 phenolic acids, 8 xanthones and 8 simple phenols. Activity records for tannins were not found in the researched databases. Of these 246 compounds, 213 did not violate any of Lipinski's rules of five, of which 125 (59%) showed non-toxicity, being promising candidates for new potential antithrombotic drugs. This review arouses interest in the isolation of phenolic compounds that may allow a new approach for the prevention of both arterial and venous thrombosis, with the potential to become alternatives in the prevention and treatment of cardiovascular diseases.

Waste-derived Ca and Zn-based bimetallic (Ca/Zn) nanorods encapsulated chitosan-based haemostatic dressing bandage: A step towards waste to bandages

Controlling loss of blood during militant war, traumatic injury, and surgical procedures is the greatest challenge nowadays, which leads to death, thereby the necessity of hemostatic bandages. The present work focuses on the synthesis of waste-derived (WD)-Ca incorporated Zn nanorods encapsulated with chitosan polymers (CZC) to control the loss of blood as well as bacterial infection. The as-prepared CZC bandage was tested against various biochemical tests such as PBS absorption, hemolysis, adsorption of protein, platelets aggregation/adhesion, blood clotting ability, and antibacterial test assay. The data suggested that the CZC bandage has high biocompatibility, exceptional PBS absorption ability (∼1402%), platelets aggregation (∼65%) within 10 min of exposure, protein a loading ability (∼37 mg/cm2), and ∼84% blood clotting within a 1 min of exposure. Moreover, the CZC bandage effectively kills/inhibits both E. Coli and S. aureus bacteria. Therefore, the prepared CZC bandage in this study is simple, economically viable, and sustainable development.

Pharmacological inhibition of Septins with Forchlorfenuron attenuates thrombus formation in experimental thrombotic mice models with modulating multiple signaling pathways in platelets

IntroductionThe Septin family of cytoskeletal proteins is abundant in platelets. When these proteins are functionally blocked using the compound forchlorfenuron (FCF), it hampers the normal activation processes of purified human platelets. ObjectivesTo evaluate the in vivo effects of FCF on physiological haemostasis and pathological thrombosis in mice and to investigate possible molecular mechanisms. MethodsThe impact of FCF on haemorrhage risk in the brain, liver, and tail of mice was investigated. Using several experimental models, thrombus development in the lung, mesenteric arteries, and postcava was studied. Functional assays were performed on mice and human platelets, both with and without FCF pretreatment. These tests included aggregation, granule release, ROS production, integrin αIIbβ3 activation, cytoskeletal remodeling imaging, and clot retraction. ResultsNeither oral nor intravenous administration of FCF showed any apparent impairment of haemostasis in the tissues studied, but only later administration resulted in a significant reduction in thrombus formation in different mice vessel types. FCF generally inhibited agonist-induced platelet aggregation, degranulation, ROS burst, morphological expansion on the fibrinogen matrix with completely disordered dynamic organizations of the cytoskeleton for septin, tubulin and actin. In addition, FCF was found to antagonise agonist-induced dephosphorylation of VASP (Ser239) and PI3K/AKT and ERK1/2 phosphorylation. ConclusionFCF showed preferences in attenuating pathological thrombus formation, apart from physiological haemostasis, with possible mechanisms to prevent cytoskeletal remodelling and signal transduction of AKT, ERK1/2 and VASP signalling pathways, suggesting that Septin may serve as a promising target for the prevention and treatment of thrombotic diseases.

Effects of batroxobin on the antithrombotic system in patients with cerebral venous thrombosis: Clues to mechanisms.

More evidence supports the benefits of batroxobin combined with anticoagulation in correcting acute cerebral venous thrombosis (CVT). The dynamic fluctuations of peripheral blood platelets, fibrinolysis, and coagulation biomarkers during this therapy were analyzed. We investigated batroxobin's effects on the antithrombotic system under two regimens. The pretreatment group included patients on anticoagulants for at least 1 week before starting batroxobin. The simultaneous treatment group began both treatments upon admission. The control group received only anticoagulation. Batroxobin was given on alternate days at doses of 10BU, 5BU, and 5BU, totaling three doses. Anticoagulation was continuous. Baseline data were T0; the next day after each batroxobin dose was T1, T2, and T3. Data from these four time points was analyzed. The time-point paired sample T-test results of the pretreatment group [n = 60; mean age (SD), 43.3(16.5); 38 (63.35%) women] showed that batroxobin significantly inhibited ADP-induced platelet aggregation rate (T1-T0: p = 0.015; T2-T0: p = 0.025; T3-T0: p = 0.013), decreased fibrinogen level (T1-T0: p < 0.001; T2-T0: p < 0.001; T3-T0: p < 0.001), and increased D-dimer (T1-T0:p < 0.001; T2-T0: p < 0.001; T3-T0: p < 0.001), TT (T1-T0:p = 0.046; T2-T0: p = 0.003; T3-T0: p < 0.001), and APTT (T1-T0:p = 0.021; T2-T0: p = 0.012; T3-T0: p = 0.026). Compared to the control group, the simultaneous treatment group showed significantly higher TT (T2: p = 0.002; T3: p = 0.004) and D-dimer (T1: p < 0.001; T2: p < 0.001; T3: p < 0.001) values, while fibrinogen (T2: p < 0.001; T3: p < 0.001) levels were significantly lower. Using batroxobin can alleviate the amplitude of changes in coagulation indicators other than TT caused by anticoagulants. The above conclusions are consistent with the results of repeated measurement data analysis. Batroxobin can significantly inhibit ADP-induced platelet aggregation rate, increase D-dimer, decrease fibrinogen, and prolong TT and APTT in the presence of anticoagulant agents. Using batroxobin can reduce the amplitude of changes in coagulation indicators caused by anticoagulants. These results reveal the potential mechanism of batroxobin combined with anticoagulation in the safe and effective treatment of CVT.

Platelet aggregation response to cyclooxygenase inhibition and thromboxane receptor antagonism using impedance aggregometry: A pilot study.

Impedance aggregometry is an alternative to light transmission aggregometry that allows analysis of platelet function in whole blood samples. We hypothesized (1) impedance aggregometry would produce repeatable results, (2) inhibition of cyclooxygenase with aspirin would attenuate aggregation responses to collagen and abolish the aggregation response to arachidonic acid (AA), and (3) thromboxane receptor antagonism (terutroban) would attenuate the aggregation response to AA. Venous blood was obtained from 11 participants three times separated by at least 2 weeks. One sample followed 7-day-aspirin intervention (81 mg once daily; ASA), the others no intervention (control). Aggregation was induced using 1 μg/mL collagen ([col 1]), 5 μg/mL collagen ([col 5]), and 50 mM AA via impedance aggregometry to determine total aggregation (AUC) analyzed for intra-test repeatability, inter-test repeatability, intervention (ASA or control), and incubation (saline or terutroban). [col 1] showed high intra-test (p ≤ 0.03 visit 1 and 2) and inter-test repeatability (p < 0.01). [col 5] and AA showed intra- ([col 5] p < 0.01 visit 1 and 2; AA p < 0.001 visit 1 and 2) but not inter-test repeatability ([col 5] p = 0.48; AA p = 0.06). ASA attenuated AUC responses to [col 1] (p < 0.01), [col 5] (p = 0.03), and AA (p < 0.01). Terutroban attenuated AUC in response to AA (p < 0.01). [col 1] shows sufficient repeatability for longitudinal investigations of platelet function. [col 5] and AA may be used to investigate mechanisms of platelet function and metabolism at a single time point.

Autophagy-enabled protein degradation: Key to platelet activation and ANGII production in patients with type 2 diabetes mellitus

BackgroundType 2 diabetes mellitus (T2DM) presents a thrombotic environment, contributing to diabetic macroangiopathy and microangiopathy. In this study, the regulation of microthrombosis in T2DM was assessed. MethodsPlatelets from T2DM patients and healthy controls were analyzed using 4D label-free proteomics and bioinformatics. The role of autophagy in T2DM platelet activation and conversion of platelet-derived angiotensinogen (AGT) was investigated. ResultsThe results showed that complement and coagulation cascades, platelet activation, metabolic pathways, endocytosis, autophagy, and other protein digestion-related pathways were enriched. The levels of the key protein AGT were increased in T2DM platelets. Chloroquine (CQ) inhibited ADP- or arachidonic acid (AA)-stimulated platelet aggregation and granule release in a dose-dependent manner, while the effects were less pronounced or even reversed for the proteasome inhibitor PYR-41 and the endocytosis inhibitor Pitstop 2. This indicated the dependence of platelet activation and the accompanying protein digestion on the autophagy-lysosome pathway. Mitophagy occurred in fresh T2DM platelets and ADP- or storage-stimulated platelets; mitophagy was inhibited by CQ. However, the mitophagy inhibitor Mdivi-1 failed to show effects similar to those of CQ. AGT, which could be transformed into ANGII in vitro by ADP-stimulated platelets, was upregulated in T2DM platelets and in MEG-01 cell-derived platelets cultured in a high-glucose medium. Finally, microthrombosis was alleviated as indicated by a reduction in the levels of red blood cells in the liver, spleen, heart, and kidney tissues of db/db mice treated with CQ or valsartan. ConclusionIn platelets, macroautophagy promotes protein digestion, subsequently facilitating platelet activation, ANGII-mediated vasoconstriction, and microthrombosis. Our results suggested that lysosome is a promising therapeutic target for antithrombotic treatment in T2DM.

Prospective Trial on the Pharmacokinetics of Clopidogrel in Hemodialysis Patients

IntroductionPatients undergoing hemodialysis (HDP) exhibit extensive cardiovascular risk. The widely prescribed anti-platelet agent clopidogrel is metabolically activated by cytochrome enzymes, which may be impaired by uremia and chronic low-grade inflammation, typically present in HDP. We conducted a prospective multicenter study to investigate the pharmacokinetics and pharmacodynamics of clopidogrel in HDP and healthy volunteers (HV). MethodsWe enrolled HDP receiving long-term clopidogrel (75mg) and pantoprazole treatment (40 mg). HV received a loading dose of 300 mg clopidogrel, followed by 75 mg once daily. Pantoprazole, a substrate and probe drug of CYP2C19, was administered intravenously (40 mg). Plasma concentrations were quantified by mass spectrometry. Pharmacokinetics were calculated, and a population pharmacokinetic model was developed. The primary endpoint was the maximum concentration of clopidogrel’s active metabolite (Cmax_CAM). Platelet aggregation was measured using ADP-induced whole-blood aggregometry. ResultsSeventeen HDP and 16 HV were included. Cmax_CAM was significantly lower in HDP compared to HV (median [IQR] 12.2 [4.6-23.4] vs. 24.7 [17.8-36.5] ng/mL, p=0.02). The Cmax ratio of CAM to prodrug was 8.5-fold lower in HDP, and an 82.7% reduced clopidogrel clearance, including CAM formation, was found using population pharmacokinetic modeling. ADP-induced platelet aggregation at 120 min was significantly higher in HDP than in HV (median [IQR]: 26 [14-43] vs. 12 [11-18] U, p=0.004. Pantoprazole terminal half-life was ∼1.7-fold higher in HDPs compared to HVs. ConclusionOur data demonstrate an altered metabolism of clopidogrel in HDP in the context of lower CYP2C19 activity, with potential implications for other substances metabolized by this enzyme.

Evaluation of Haemostatic Potential and HPTLC Fingerprinting of Annona squamosa Linn. Bark Extract

Objectives: The present work focuses on exploring and evaluating the hemostatic property of Annona squamosa Linn. bark. Being a renewable resource, it has the potential to contribute to the hemostatic agent. Hemostatic efficacy of the cold and hot bark extract at different concentrations of 10, 50, 100 &amp; 250 mg/ml of saline were evaluated using whole blood clotting, platelet aggregation test, prothrombin time CAMAG HPTLC system. The hemostatic efficacy. At 250 mg/ml saline, both the extracts showed excellent hemostatic efficacy by clotting blood in 1.2 s and 3 s, respectively. The prothrombin time of cold and hot were 5 s and 5.1 s which were less than that of Liquiplastin partial prothrombin time which was Liquicelin E (13.85 s). Annona squamosa Linn. to be used as a topical hemostatic agent. Saponins, which are known to be antihemorrhagic, were detected by HPTLC. hemostatic efficacy of Annona squamosa Linn. bark was evaluated, which can contribute to the development of a fast-acting, cost-effective, easy-to-use, and sustainable topical hemostat. Keywords: Annona squamosa Linn, Hemostatic activity, Prothrombin time, Activated partial thromboplastin time, HPTLC fingerprint

Diet and Nutraceuticals for treatment and prevention of primary and secondary stroke: Emphasis on nutritional antiplatelet and antithrombotic agents

Ischemic stroke is a devastating disease that causes morbidity and mortality. Malnutrition following ischemic stroke is common in stroke patients. During the rehabilitation, the death rates of stroke patients are significantly increased due to malnutrition. Nutritional supplements such as protein, vitamins, fish, fish oils, moderate wine or alcohol consumption, nuts, minerals, herbal products, food colorants, marine products, fiber, probiotics and Mediterranean diets have improved neurological functions in stroke patients as well as their quality of life. Platelets and their mediators contribute to the development of clots leading to stroke. Ischemic stroke patients are treated with thrombolytics, antiplatelets, and antithrombotic agents. Several systematic reviews, meta-analyses, and clinical trials recommended that consumption of these nutrients and diets mitigated the vascular, peripheral, and central complications associated with ischemic stroke (Fig. 2). Particularly, these nutraceuticals mitigated the platelet adhesion, activation, and aggregation that intended to reduce the risks of primary and secondary stroke. Although these nutraceuticals mitigate platelet dysfunction, there is a greater risk of bleeding if consumed excessively. Moreover, malnutrition must be evaluated and adequate amounts of nutrients must be provided to stroke patients during intensive care units and rehabilitation periods. In this review, we have summarized the importance of diet and nutraceuticals in ameliorating neurological complications and platelet dysfunction with an emphasis on primary and secondary prevention of ischemic stroke.

Glanzmann's thrombasthenia: A nightmare for hernia surgeons.

Glanzmann's thrombasthenia is a rare inherited disorder affecting one in one million. It is characterised by a lack of platelet aggregation due to a defect in the platelet membrane receptor complex (αIIb/βIIIa), which mediates the aggregation of platelets at the site of vessel injury. We report here the first case of successful perioperative haemostatic management of a male patient with Glanzmann's thrombasthenia, who underwent an elective laparoscopic hernia repair. The patient was posted for elective surgery considering the availability of expertise in minimally invasive surgery and accessibility to recombinant activated factor VII. The patient was operated using the extended-view totally extraperitoneal technique for inguinal hernia repair. The patient recovered successfully and reported no complication during follow-up. We conclude that with proper perioperative haematological consultation, and careful coordination between anaesthetists and surgeons, elective laparoscopic procedure can be done in patients with Glanzmann's thrombasthenia, with only recombinant factor VIIa support.