Collagen-induced Platelet Aggregation Research Articles

498 Bruton’s tyrosine kinase (BTK) inhibitors impede platelet aggregation but not adhesion to collagen.

OBJECTIVES/GOALS: The research objectives of this project are to elucidate the effects of Bruton’s tyrosine kinase inhibitors (BTKi) of varying target specificity on platelet function with regard to platelet aggregation, adhesion, spreading, and intracellular signaling as measured by kinase phosphorylation. METHODS/STUDY POPULATION: Blood from healthy volunteers was obtained and processed to obtain both washed platelets and platelet-rich plasma. The samples were then treated with one of the BTKi drugs or with vehicle (DMSO) at concentrations matching patient blood concentrations derived from clinical trials and pharmacokinetic studies. The incubated samples were then analyzed in an aggregometer using one of several agonists. Aggregation was stopped after five minutes with a perchloric acid-based lysis buffer. The samples were then analyzed by SDS-PAGE and immunoblotting to quantify BTK protein and BTK phosphorylation. Adhesion was assessed by incubating washed platelets treated with BTKi on microtiter wells coated with fibrinogen or collagen and quantifying adherent platelets by their endogenous acid phosphatase activity. RESULTS/ANTICIPATED RESULTS: We found that ibrutinib, zanubrutinib, and pirtobrutinib all completely inhibited collagen-induced platelet aggregation, whereas they did not inhibit aggregation induced by thrombin, ristocetin, arachidonic acid, or the PAR1 activator peptide SFLLRN (T6). Acalabrutinib inhibited collagen-induced platelet aggregation only at high concentrations (1-2 micromolar). At the lower concentration of 200 nanomolar, comparable to the concentration required for the other BTK inhibitors to completely inhibit platelet aggregation, acalabrutinib failed to inhibit aggregation but did inhibit auto-phosphorylation, indicating an impact on signaling. None of the BTKi drugs inhibited adhesion of platelets to collagen-coated surfaces. DISCUSSION/SIGNIFICANCE: Our data show the inhibitory effect of BTKi on collagen-induced platelet aggregation and signaling. However, it remains unclear whether the inhibition is due to an effect on BTK itself or other related kinases. Better insight into the mechanisms of platelet inhibition by BTKi may help guide the development of BTKi with a lower risk of hemorrhage.

Mudanpioside C Discovered from Paeonia suffruticosa Andr. Acts as a Protein Disulfide Isomerase Inhibitor with Antithrombotic Activities.

Paeonia suffruticosa Andr. is a well-known landscape plant worldwide and also holds significant importance in China due to its medicinal and dietary properties. Previous studies have found that Cortex Moutan (CM), the dried root bark of P. suffruticosa, showed antiplatelet and cardioprotective effects, although the underlying mechanism and active compounds remain to be revealed. In this study, protein disulfide isomerase (PDI) inhibitors in CM were identified using a ligand-fishing method combined with the UHPLC-Q-TOF-MS assay. Further, their binding sites and inhibitory activities toward PDI were validated. The antiplatelet aggregation and antithrombotic activity were investigated. The results showed that two structurally similar compounds in CM were identified as the inhibitor for PDI with IC50 at 3.22 μM and 16.73 μM; among them Mudanpioside C (MC) is the most effective PDI inhibitor. Molecular docking, site-directed mutagenesis, and MST assay unequivocally demonstrated the specific binding of MC to the b'-x domain of PDI (Kd = 3.9 μM), acting as a potent PDI inhibitor by interacting with key amino acids K263, D292, and N298 within the b'-x domain. Meanwhile, MC could dose-dependently suppress collagen-induced platelet aggregation and interfere with platelet activation, adhesion, and spreading. Administration of MC can significantly inhibit thrombosis formation without disturbing hemostasis in mice. These findings present a promising perspective on the antithrombotic properties of CM and highlight the potential application of MC as lead compounds for targeting PDI in thrombosis therapy.

Discovery of 5-Hydroxy-1,4-naphthoquinone (Juglone) Derivatives as Dual Effective Agents Targeting Platelet-Cancer Interplay through Protein Disulfide Isomerase Inhibition.

In this study, a series of 2- and/or 3-substituted juglone derivatives were designed and synthesized. Among them, 9, 18, 22, 30, and 31 showed stronger inhibition activity against cell surface PDI or recombinant PDI and higher inhibitory effects on U46619- and/or collagen-induced platelet aggregation than juglone. The glycosylated derivatives 18 and 22 showed improved selectivity for inhibiting the proliferation of multiple myeloma RPMI 8226 cells, and the IC50 values reached 61 and 48 nM, respectively, in a 72 h cell viability test. In addition, 18 and 22 were able to prevent tumor cell-induced platelet aggregation and platelet-enhanced tumor cell proliferation. The molecular docking showed the amino acid residues Gln243, Phe440, and Leu443 are important for the compound-protein interaction. Our results reveal the potential of juglone derivatives to serve as novel antiplatelet and anticancer dual agents, which are available to interrupt platelet-cancer interplay through covalent binding to PDI catalytic active site.

Antiplatelet effects of the CEACAM1-derived peptide QDTT

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) restricts platelet activation via platelet collagen receptor GPVI/FcRγ-chain. In this study, screening against collagen-induced platelet aggregation was performed to identify functional CEACAM1 extracellular domain fragments. CEACAM1 fragments, including Ala-substituted peptides, were synthesized. Platelet assays were conducted on healthy donor samples for aggregation, cytotoxicity, adhesion, spreading, and secretion. Mice were used for tail bleeding and FeCl3-induced thrombosis experiments. Clot retraction was assessed using platelet-rich plasma. Extracellular segments of CEACAM1 and A1 domain-derived peptide QDTT were identified, while N, A2, and B domains showed no involvement. QDTT inhibited platelet aggregation. Ala substitution for essential amino acids (Asp139, Thr141, Tyr142, Trp144, and Trp145) in the QDTT sequence abrogated collagen-induced aggregation inhibition. QDTT also suppressed platelet secretion and “inside-out” GP IIb/IIIa activation by convulxin, along with inhibiting PI3K/Akt pathways. QDTT curtailed FeCl3-induced mesenteric thrombosis without significantly prolonging bleeding time, implying the potential of CEACAM1 A1 domain against platelet activation without raising bleeding risk, thus paving the way for novel antiplatelet drugs.

Daboialipase, a phospholipase A2 from Vipera russelli russelli venom posesses anti-platelet, anti-thrombin and anti-cancer properties.

Snake venoms are known to contain toxins capable of interfering with normal physiological processes of victims. Specificity of toxins from snake venoms give scope to identify new molecules with therapeutic action and/or help to understand different cellular mechanisms. Russell's viper venom (RVV) is a mixture of many bioactive molecules with enzymatic and non-enzymatic proteins. The present article describes Daboialipase (DLP), an enzymatic phospholipase A2 with molecular mass of 14.3kDa isolated from RVV. DLP was obtained after cation exchange chromatography followed by size-exclusion high performance liquid chromatography (SE-HPLC). The isolated DLP presented strong inhibition of adenosine di-phosphate (ADP) and collagen induced platelet aggregation. It also showed anti-thrombin properties by significantly extending thrombin time in human blood samples. Trypan blue and resazurin cell viability assays confirmed time-dependent cytotoxic and cytostatic activities of DLP on MCF7 breast cancer cells, in vitro. DLP caused morphological changes and nuclear damage in MCF7 cells. However, DLP did not cause cytotoxic effects on non-cancer HaCaT cells. Peptide sequences of DLP obtained by O-HRLCMS analysis showed similarity with a previously reported PLA2 (Uniprot ID: PA2B_DABRR/PDB ID: 1VIP_A). An active Asp at 49th position, calcium ion binding site and anticoagulant activity sites were identified in 1 VIP_A. These findings are expected to contribute to designing new anti-platelet, anticoagulant and anti-cancer molecules.

High factor VIII concentrations interfere with glycoprotein VI-mediated platelet activation in vitro

BackgroundThe recruitment of activated factor VIII (FVIII) at the surface of activated platelets is a key step toward the burst of thrombin and fibrin generation during thrombus formation at the site of vascular injury. It involves binding to phosphatidylserine and, possibly, to fibrin-bound αIIbβ3. Seminal work had shown the binding of FVIII to resting platelets, yet without a clear understanding of a putative physiological relevance. ObjectivesTo characterize the effects of FVIII-platelet interaction and its potential modulation of platelet function. MethodsFVIII was incubated with washed platelets. The effects on platelet activation (spontaneously or triggered by collagen and thrombin) were studied by flow cytometry and light transmission aggregometry. We explored the involvement of downstream pathways by studying phosphorylation profiles (Western blot). The FVIII-glycoprotein (GP) VI interaction was investigated by ELISA, confocal microscopy, and proximity ligation assay. ResultsFVIII bound to the surface of resting and activated platelets in a dose-dependent manner. FVIII at supraphysiological concentrations did not induce platelet activation but rather specifically inhibited collagen-induced platelet aggregation and altered glycoprotein VI (GPVI)-dependent phosphorylation. FVIII, freed of its chaperone protein von Willebrand factor (VWF), interacted in close proximity with GPVI at the platelet surface. ConclusionWe showed that VWF-free FVIII binding to, or close to, GPVI modulates platelet activation in vitro. This may represent an uncharacterized negative feedback loop to control overt platelet activation. Whether locally activated FVIII concentrations achieved during platelet accumulation and thrombus formation at the site of vascular injury in vivo are compatible with such a function remains to be determined.

Role of <i>ITGB3</i>, <i>GP1B1</i>, and <i>ITGA2</i> gene polymorphisms in platelet dysfunction in patients with COVID-19-associated lung damage

The aim of the work. To investigate platelet aggregation, polymorphism in the genes that ensure its implementation, and the association between these indicators in patients with COVID-19-associated lung damage, depending on the severity of the clinical course. Methodology. The study involved 75  patients with COVID-19, which, depending on  the severity of lung involvement, were divided into two groups: patients with  damage of up to 50  % of the lung parenchyma (n  =  48) and with damage of more than 50 % (n = 27) respectively. The control group consisted of healthy people (n = 24), comparable in sex and age. In all individuals, the number of platelets, platelet aggregation induced by ADP, collagen and ristomycin were studied; polymorphisms rs6065 in the GP1BA gene, rs1126643 in the ITGA2 gene, and rs5918 in the ITGB3 gene were determined by polymerase chain reaction. The analysis of the obtained data was executed using the IBM SPSS Statistics v. 23 (IMB Corp., USA). Results and discussion. In patients with moderate and severe COVID-19-associated lung damage, platelet aggregation induced by ADP, collagen, and ristomycin accelerated; in severe cases, the number of  platelets decreased. The frequency of variants of the rs6065 polymorphism did not change, the frequency of occurrence of the T/C genotype of the rs5918 polymorphism increased; with moderate severity, the frequency of occurrence of the C/T and T/T genotypes of the rs1126643 polymorphism increased; with severe lung damage, the frequency of occurrence of the mutant C/C genotype polymorphism rs5918 increased. In moderate lung damage, the presence of the  mutant T/T polymorphism rs1126643 accelerated collagen-induced platelet aggregation; in severe cases, the presence of mutant C/C and heterozygous variant C/T polymorphism rs5918 accelerated ADP-induced platelet aggregation. There was no effect of the rs6065 polymorphism on platelet aggregation. The data obtained indicate the possible role of genetic predisposition in the activation of platelet aggregation in patients with COVID-19-associated lung damage.

Functionalization of graphene oxide as a way to increase biocompatibility

Introduction. Now, one of the most promising areas for the use of graphene-based materials, in particular graphene oxide, is biomedicine. Due to the wide variety of functional groups and the possibility of chemical modification of graphene oxide, the creation of composite materials for biomedical use is promising. These nanomaterials have a unique structure and properties, which determines their use for creating targeted drug delivery systems, in tissue engineering, bioimaging, as well as for creating new materials with antimicrobial and antiviral properties.The objective was to perform synthesis and identification of graphene oxide and its conjugate with glycine, and to study the biocompatibility of the obtained nanomaterials: the effect on haemolysis and platelet aggregation, genotoxicity and cytotoxicity.Methods and materials. Graphene oxide was synthesized from graphite using the modified Hummers and Offeman method, after which the graphene oxide-glycine conjugate was also obtained. Identification was carried out using nuclear magnetic resonance spectroscopy. Estimation of biocompatibility of the obtained nanomaterials included the study of their hemolytic activity, effect on collagen-induced platelet aggregation, cyto- and genotoxicity.Results. Graphene oxide and its conjugate with glycine were synthesized. Identification with using nuclear magnetic resonance spectroscopy confirmed the structure and composition of the substances. The study of the biocompatibility of the obtained nanomaterials showed the absence of hemolytic activity (the degree of hemolysis did not exceed 2.5% at the studied concentration range); the presence of antiplatelet properties (at C=10–100 mg·ml–1); the absence of geno- and cytotoxicity (graphene oxide at C=0.25–25 mg·L–1 does not affect the viability of HEK293 cells; in turn, the conjugate with glycine at C=100–200 mg·L–1 causes a dose-dependent increase proliferation of HEK293 cells).Conclusion. The study demonstrates that functionalization of the graphene surface with oxygen-containing groups and amino acids leads to increased hemocompatibility, as well as to the production of nanomaterials that do not exhibit genoand cytotoxicity.

АНТИКОАГУЛЯЦИОННАЯ И АНТИАГРЕГАЦИОННАЯ АКТИВНОСТЬ ФИТОЭКСТРАКТОВ ЛЕКАРСТВЕННЫХ РАСТЕНИЙ: IN VITRO СКРИНИНГ-ИССЛЕДОВАНИЕ

Objective: In vitro evaluate the antiaggregation and anticoagulation properties of aqueous plant extracts, including Viburnum opulus flowers and fruits, Urtica dioica leaves, Coffea arabica leaves, and shepherd's purse (Capsella bursa-pastoris) herb. Methods: In vitro assessment of anticoagulation activity was conducted by examining the impact on activated partial thromboplastin time (APTT), prothrombin time (PT), and fibrinogen levels. Anti-aggregation activity was evaluated using the Born turbidimetric method. Results: The research found that the phytoextracts examined did not impact the concentration of fibrinogen and PT. However, they did slightly prolong the APTT, and this effect was statistically significant compared to the control. Coffea arabica leaves, and Viburnum opulus fruit extracts showed substantial anticoagulant activity (30% and 29% sodium heparin, respectively; p-value <0.05 for both). Extracts of Viburnum opulus fruits and Coffea arabica leaves significantly prolong the latent period before collagen-induced platelet aggregation by up to 116.7% and 118.8% of the intact platelet index, respectively. Every sample analyzed showed a significant decrease in the maximum platelet aggregation amplitude compared to the control. The most notable impact was with Urtica dioica and Coffea arabica leaf extracts, which reduced the percentage to 85.9% and 89.2% of the control, respectively. These extracts exhibited 77.6% and 59.7% of the effectiveness of acetylsalicylic acid, with Coffea arabica extract showing a significant difference (p<0.05) compared to the reference drug. All plant extracts, except Capsella bursa-pastoris extract, which reduced this indicator, significantly prolonged the time needed to reach maximum aggregation. Coffea arabica extract had the most significant impact, showing 223.8% of the efficacy of acetylsalicylic acid (p<0.05). The extracts (excluding Capsella bursa-pastoris) significantly decreased the platelet aggregation rate. The most notable impact was observed with extracts of Viburnum opulus flowers and Coffea arabica leaves, which yielded an inhibitory effect on the rate of aggregation of acetylsalicylic acid (-10%/min relative to intact platelets, p<0.05), reducing aggregation rate by -5.2% and -6%/min compared to control, respectively. Conclusion: The samples analyzed in our study show weak anticoagulation activity, with the most noticeable effects in extracts of coffee leaves and viburnum fruits. Additionally, we found that the antiaggregation activity of the extracts of coffee leaves, nettles, and viburnum fruits was much more substantial, comparable to or even surpassing the effect of acetylsalicylic acid in some cases. Keywords: Anticoagulation properties, antiaggregation activity, Viburnum opulus, Urtica dioica, Coffea arabica, Capsella bursa-pastoris.

Diphenyl-tetrazol-propanamide Derivatives Act as Dual-Specific Antagonists of Platelet CLEC-2 and Glycoprotein VI.

Platelet C-type lectin-like receptor 2 (CLEC-2) induces platelet activation and aggregation after clustering by its ligand podoplanin (PDPN). PDPN, which is not normally expressed in cells in contact with blood flow, is induced in inflammatory immune cells and some malignant tumor cells, thereby increasing the risk of venous thromboembolism (VTE) and tumor metastasis. Therefore, small-molecule compounds that can interfere with the PDPN-CLEC-2 axis have the potential to become selective antiplatelet agents. Using molecular docking analysis of CLEC-2 and a PDPN-CLEC-2 binding-inhibition assay, we identified a group of diphenyl-tetrazol-propanamide derivatives as novel CLEC-2 inhibitors. A total of 12 hit compounds also inhibited PDPN-induced platelet aggregation in humans and mice. Unexpectedly, these compounds also fit the collagen-binding pocket of the glycoprotein VI molecule, thereby inhibiting collagen interaction. These compounds also inhibited collagen-induced platelet aggregation, and one compound ameliorated collagen-induced thrombocytopenia in mice. For clinical use, these compounds will require a degree of chemical modification to decrease albumin binding. Nonetheless, as dual activation of platelets by collagen and PDPN-positive cells is expected to occur after the rupture of atherosclerotic plaques, these dual antagonists could represent a promising pharmacophore, particularly for arterial thrombosis, in addition to VTE and metastasis.

Loss of Arl6ip5 Gene Function Suppresses Thrombosis in Mice

Background: The genetic contributors to thrombosis have only been partly described. Heterozygous tissue factor pathway inhibitor deficiency ( Tfpi +/-) is a powerful negative genetic modifier of the homozygous FVL ( F5 L/L) phenotype, demonstrating a nearly complete perinatal lethal genetic interaction in F5 L/L Tfpi +/- mice. To identifymutations suppressing lethal thrombosis, we used this F5 L/L Tfpi +/- phenotype for a sensitized genome wide ENU mutagenesis screen. We screened over 12,000 mice and generated a total of 31 thrombosis suppressor variant lines. We were able to identify ENU-induced mutations in 10 of these lines, each potentially representing an independent pathway for reducing thrombosis severity. One of the mutations that suppressed thrombosis in F5 L/L Tfpi +/- mice was a dominant loss of function mutation (i.e. mutation in a single allele) in the ADP Ribosylation Factor Like GTPase 6 Interacting Protein-5 ( Arl6ip5) gene. Interestingly, no direct role(s) of Arl6ip5 gene or protein in hemostasis have been reported. Therefore, the aim of our studies was to investigate the hypothesis that Arl6ip5 deficiency suppresses thrombosis by reducing blood coagulation and platelet reactivity in mice. Methods: We previously generated a mouse model of Arl6ip5 ( Arl6ip5 -) by genome editing, creating a 151 base pair deletion removing an exon 1 splice site. After backcrossing mice carrying the Arl6ip5 - allele to the wildtype C57BL/6J (B6) background, we performed Arl6ip5 +/-intercrosses to determine a potential difference in genotype frequency at weaning. To investigate whether the Arl6ip5 - allele was able to produce a stable mRNA transcript we performed RT-qPCR on whole liver, lung, heart, kidney, and brain samples derived from Arl6ip5 +/+ Arl6ip5 +/- and Arl6ip5 -/- mice. Furthermore, complete blood cell counts were analyzed using the Advia 2120 (N ≥ 10 for each group), and to investigate potential blood coagulation defects, Prothrombin Time (PT) (N ≥ 5 for each group) and activated Partial Thromboplastin Time (aPTT) (N ≥ 8 for each group) assays were performed on plasma from Arl6ip5 +/+ Arl6ip5 +/- and Arl6ip5 -/- mice with the operator blinded to the genotype. Similarly, we performed platelet function analyses on whole blood from Arl6ip5 +/+ Arl6ip5 +/- and Arl6ip5 -/- mice using the Roche Multiplate, with ADP and collagen as agonists (N ≥ 6 for each group). Results: Arl6ip5 +/- x Arl6ip5 +/- crosses resulted in normal Mendelian proportions of progeny observed at weaning (N > 400). No Arl6ip5transcripts were present in any of the Arl6ip5 -/- tissue samples analyzed, whereas heterozygous mice had the expected 50% Arl6ip5 expression levels across all organs as compared to wildtype mice. A significant prolongation was observed in aPTT between Arl6ip5 +/+ and Arl6ip5 -/- animals (p<0.01). Although no differences in platelet counts between genotypes were observed, platelet aggregometry showed significant differences between wildtype Arl6ip5 +/+and Arl6ip5 -/-littermates for platelet aggregation, velocity, and area under the curve in response to ADP agonist (normalized to platelet counts, p<0.01). In addition, collagen-induced aggregometry also yielded significant differences in aggregation velocity. Conclusions: Our results support the hypothesis that Arl6ip5 deficiency contributes to thrombosis suppression by increasing the aPTT and reducing platelet reactivity. Since the PT in Arl6ip5 -/- mice is normal, we speculate that Arl6ip5 -/- is either directly or indirectly affecting one or more of the levels or activity of coagulation factors VIII, IX, XI and XII. Significant differences in ADP- and collagen-induced platelet aggregation between Arl6ip5 +/+wildtype littermates and Arl6ip5 -/- suggests that the lack of Arl6ip5affects an aggregation process common to both pathways. Additional analyses are being performed to refine these mechanisms. Understanding the mechanism by which Arl6ip5 deficiency suppresses thrombosis could provide insights into human thrombosis regulation.

Isoquercetin for thromboinflammation in sickle cell disease: a randomized double-blind placebo-controlled trial

AbstractData from a small trial in patients with cancer suggest that isoquercetin (IQ) treatment lowered thrombosis biomarkers and prevented clinical thrombosis, but, to our knowledge, no studies of IQ have been conducted to target thromboinflammation in adults with sickle cell disease (SCD). We conducted a randomized, double-blind, placebo-controlled trial in adults with steady-state SCD (hemoglobin SS [HbSS], HbSβ0thal, HbSβ+thal, or HbSC). The primary outcome was the change in plasma soluble P-selectin (sP-selectin) after treatment compared with baseline, analyzed in the intention-to-treat population. Between November 2019 and July 2022, 46 patients (aged 40 ± 11 years, 56% female, 75% under hydroxyurea treatment) were randomized to receive IQ (n = 23) or placebo (n = 23). IQ was well tolerated and all the adverse events (AEs; n = 21) or serious AEs (n = 14) recorded were not attributable to the study drug. The mean posttreatment change for sP-selectin showed no significant difference between the treatment groups (IQ, 0.10 ± 6.53 vs placebo, 0.74 ± 4.54; P = .64). In patients treated with IQ, whole-blood coagulation (P = .03) and collagen-induced platelet aggregation (P = .03) were significantly reduced from the baseline. Inducible mononuclear cell tissue factor gene expression and plasma protein disulfide isomerase reductase activity were also significantly inhibited (P = .003 and P = .02, respectively). Short-term fixed-dose IQ in patients with SCD was safe with no off-target bleeding and was associated with changes from the baseline in the appropriate direction for several biomarkers of thromboinflammation. The trial was registered at www.clinicaltrials.gov as #NCT04514510.

Interleukin 37 attenuates platelet activation and thrombosis formation

Abstract Aims Interleukin 37 (IL-37) emerges as a natural suppressor of innate inflammatory and immune responses. Plasma IL-37 is abnormally increased in cardiovascular disease. Pleiotropic effects of IL-37 beyond anti-inflammation have been shown. However, the direct effects of IL-37 on platelet activation and thrombosis remain unclear. This study explored the direct effects of IL-37 on platelet activation and thrombosis. Methods and results A negative correlation between plasma IL-37 concentration and platelet aggregation was observed in patients with myocardial infarction. IL-37 directly inhibited ADP, thrombin and collagen induced platelet aggregation and dense granule release (Figure 1A, B). Platelet spreading in immobilized fibrinogen and clot retraction driven by integrin αIIbβ3 mediated out-side in signaling were inhibited by IL-37 concentration-dependently. In vivo thrombosis formation and stability in a FeCl3-injured mesenteric arteriole thrombosis mouse model was also severely suppressed by IL-37. Using co-immunoprecipitation, confocal microscopy and platelet-specific IL-1R8-deficient mice, we found that IL-37 bound to platelet IL-1R8 and IL-18Rα, and silencing of IL-1R8 impaired inhibitory effects of IL-37 on platelet activation. Mechanistic studies revealed that IL-37 combined with IL-1R8 to enhance PTEN activity and inhibited Akt, mitogen-activated protein kinase pathways. Platelet-specific IL-1R8-deficient mice presented impaired thrombosis and hemostasis but had improved cardiac function, reduced infarct size, decreased inflammatory response, and microthrombus after permanent ligation of the left anterior descending coronary. While control mice treated with IL-37 were protected from myocardial injury, IL-1R8-/- mice were not (Figure 1C, D). Conclusions IL-37 directly attenuated platelet activation and thrombosis formation by binding to platelet IL-18Rα and IL-1R8 and regulating the downstream signaling pathways, suggesting the potential role of exogenous IL-37 protein to prevent platelet hyperactivity thrombotic complications in patients with low plasma IL-37 levels.resultsmode picture

Potential antithrombotic effect of two new phenylpropanoid sucrose esters and other secondary metabolites of Canna indica L. rhizome

Canna indica L. has been traditionally used to treat various diseases. Based on previously reported antithrombotic effect for this plant, two new phenylpropanoid sucrose esters (canindicoside A (1) and canindicoside B (2)) and seven known compounds: nepetoidin B (3), caffeic acid (4), ferulic acid (5), (R)-(+)-rosmarinic acid (6), isorinic acid (7), (S)-(-)-rosmarinic acid (8) and (S)-(-)-rosmarinic acid methyl ester (9) were isolated from the ethyl acetate extract. Compounds were elucidated by NMR and MS spectroscopic methods. The antiplatelet effect was evaluated using turbidimetric method. Anticoagulant activity was examined by measuring activated partial thromboplastine time (APTT), prothrombin time, and thrombine time (TT). It was shown for the first time that both new phenylpropanoid sucrose esters 1 and 2, 7 and 9 displayed dose-dependent antiplatelet effects. 2 and 9 had the highest inhibitory activity on both adenosine diphosphate (ADP)- and collagen-induced platelet aggregation. Moreover, 1, 7 and 9 also exhibited anticoagulant activity. At 0.4 mg/mL, both 1 and 7 prolonged APTT compared to the negative control (p < 0.05), suggesting the possible inhibitory impact on the intrinsic coagulation pathway. Moreover, 9 at 0.4 mg/mL exerted higher TT values than the negative control (p < 0.05). C. indica and its bioactive phytochemicals are potential candidates for development of anti-thrombosis therapy.

Plasma Soluble Glycoprotein VI, Platelet Function, Bleeding, and Ischemic Events in Patients Undergoing Elective Percutaneous Coronary Intervention.

Glycoprotein VI (GPVI) is the major platelet-specific collagen receptor. GPVI shedding with generation of soluble GPVI (sGPVI) is an endogenous feedback mechanism preventing platelet overstimulation. sGPVI has not been investigated in patients with chronic coronary syndrome (CCS) undergoing percutaneous coronary intervention (PCI), especially regarding its potential value as a predictor of ischemic and bleeding risk. Baseline plasma sGPVI levels were available in 318 patients with CCS undergoing PCI. Platelet function was assessed by measuring both adenosine diphosphate (ADP) and collagen-induced platelet aggregation. Co-primary endpoints were a composite of death or myocardial injury at 48 hours after PCI, and Bleeding Academic Research Consortium (BARC) type 1 to 5 bleeding at 30 days. There was no significant correlation between sGPVI and platelet function at baseline or at 48 hours after PCI and loading with antiplatelet drugs. Baseline plasma sGPVI levels were not associated with the ischemic risk: the incidence of the ischemic endpoint was 25.0% in the lower, 22.9% in the middle, and 26.7% in the upper sGPVI tertile (p = 0.82). There was a significant nonlinear relationship between sGPVI and the risk of bleeding: the incidence of the bleeding endpoint was 11.8% in the lower, 12.6% in the middle, and 26.4% in the upper sGPVI tertile (p = 0.006). In patients with CCS undergoing PCI, plasma levels of sGPVI did not correlate with ADP- or collagen-induced platelet aggregation. Patients with higher baseline levels of sGPVI may carry an increased risk of bleeding at 30 days after PCI but no excess risk of ischemic events.

Daidzein Inhibits Human Platelet Activation by Downregulating Thromboxane A2 Production and Granule Release, Regardless of COX-1 Activity.

Platelets play crucial roles in cardiovascular diseases (CVDs) by regulating hemostasis and blood coagulation at sites of blood vessel damage. Accumulating evidence indicates daidzein inhibits platelet activation, but the mechanism involved has not been elucidated. Thus, in this study, we investigated the mechanism responsible for the inhibition of collagen-induced platelet aggregation by daidzein. We found that in collagen-induced platelets, daidzein suppressed the production of thromboxane A2 (TXA2), a molecule involved in platelet activation and aggregation, by inhibiting the cytosolic phospholipase A2 (cPLA2) signaling pathway. However, daidzein did not affect cyclooxygenase-1 (COX-1). Furthermore, daidzein attenuated the PI3K/PDK1/Akt/GSK3αβ and MAPK (p38, ERK) signaling pathways, increased the phosphorylation of inositol trisphosphate receptor1 (IP3R1) and vasodilator-stimulated phosphoprotein (VASP), and increased the level of cyclic adenosine monophosphate (cAMP). These results suggest that daidzein inhibits granule release (ATP, serotonin, P-selectin), integrin αIIbβ3 activation, and clot retraction. Taken together, our study demonstrates that daidzein inhibits collagen-induced platelet aggregation and suggests that daidzein has therapeutic potential for the treatment of platelet aggregation-related diseases such as atherosclerosis and thrombosis.

Daboxin P, a phospholipase A2 of Indian Daboia russelii venom, modulates thrombin-mediated platelet aggregation.

Daboxin P, reported earlier from the venom of Daboia russellii, disturbs the blood coagulation cascade by targeting factor X and factor Xa. The present study exhibits that Daboxin P also inhibits platelet aggregation induced by various agonists. The thrombin-induced platelet aggregation was inhibited maximum whereas inhibition of collagen-induced platelet aggregation was found to be 50% and no inhibition of adenosine diphosphate (ADP)and arachidonic acid-induced aggregation was observed. Daboxin P dose-dependently inhibited the thrombin-induced platelet aggregation with Anti-Aggregation 50 (AD50 ) dose of 55.166 nM and also reduced the thrombin-mediated calcium influx. In-silico interaction studies suggested that Daboxin P binds to thrombin and blocks its interaction with its receptor on the platelet surface. Quenching of thrombin's emission spectrum by Daboxin P and electrophoretic profiles of pull-down assay further reveals the binding between Daboxin P and thrombin. Thus, the present study demonstrates that Daboxin P inhibits thrombin-induced platelet aggregation by binding to thrombin.

The Anti-Aggregative Potential of Resolvin E1 on Human Platelets.

Resolvin E1 is a metabolite of eicosapentaenoic acid (EPA) which is one of the omega-3 polyunsaturated fatty acids (omega-3 PUFAs). The antiplatelet properties of omega-3 PUFAs are well known, but the effect of resolvin E1 on platelets via the collagen receptors is extremely poorly reported. We investigated the effect of resolvin E1 on collagen-induced platelet aggregation, activation, and reactivity, and also platelet membrane fluidity. The ultimate and statistically significant results showed that resolvin E1 may inhibit platelet reactivity due to the reduction of collagen-induced platelet aggregation in platelet-rich plasma and isolated platelets, but not in whole blood. Also, resolvin E1 significantly reduced P-selectin exposure on collagen-stimulated platelets. Moreover, we demonstrated that resolvin E1 can maintain platelet membrane structure (without increasing membrane fluidity). The association between platelet reactivity and membrane fluidity, including resolvin E1 and collagen receptors requires further research. However, the goal of this study was to shed light on the molecular mechanisms behind the anti-aggregative effects of resolvin E1 on platelets, which are still not fully clarified. We also indicate an innovative research direction focused on further analysis and then use of omega-3 PUFAs metabolites as antiplatelet compounds for future applications in the treatment and prevention of cardiovascular diseases.

Agomelatine inhibits platelet aggregation through melatonin receptor-dependent and independent mechanisms

AimsMelatonin is known to inhibit platelet aggregation induced by arachidonic acid (AA). In the present study we investigated whether agomelatine (Ago), an antidepressant with agonist activity at melatonin receptor 1 (MT1) and MT2 could reduce platelets aggregation and adhesion. Main methodsHuman platelets from healthy donors were used to test the in vitro effects of Ago in the presence of different platelet activators. We performed aggregation and adhesion assays, thromboxane B2 (TxB2), cAMP and cGMP measurements, intra-platelet calcium registration and flow cytometry assays. Key findingsOur data revealed that different concentrations of Ago reduced AA- and collagen-induced human platelet aggregation in vitro. Ago also reduced AA-induced increase in thromboxane B2 (TxB2) production, intracellular calcium levels and P-selectin expression at plasma membrane. The effects of Ago in AA-activated platelets were likely dependent on MT1 as they were blocked by luzindole (a MT1/MT2 antagonist) and mimicked by the MT1 agonist UCM871 in a luzindole-sensitive manner. The MT2 agonist UCM924 was also able to inhibit platelet aggregation, but this response was not affected by luzindole. On the other hand, although UCM871 and UCM924 reduced collagen-induced platelet aggregation and adhesion, inhibition of collagen-induced platelet aggregation by Ago was not mediated by melatonin receptors because it was not affected by luzindole. SignificanceThe present data show that Ago suppresses human platelet aggregation and suggest that this antidepressant may have the potential to prevent atherothrombotic ischemic events by reducing thrombus formation and vessel occlusion.

Mouse skin peptidomic analysis of the hemorrhage induced by a snake venom metalloprotease.

Hemorrhage induced by snake venom metalloproteases (SVMPs) results from proteolysis, capillary disruption, and blood extravasation. HF3, a potent SVMP of Bothrops jararaca, induces hemorrhage at pmol doses in the mouse skin. To gain insight into the hemorrhagic process, the main goal of this study was to analyze changes in the skin peptidome generated by injection of HF3, using approaches of mass spectrometry-based untargeted peptidomics. The results revealed that the sets of peptides found in the control and HF3-treated skin samples were distinct and derived from the cleavage of different proteins. Peptide bond cleavage site identification in the HF3-treated skin showed compatibility with trypsin-like serine proteases and cathepsins, suggesting the activation of host proteinases. Acetylated peptides, which originated from the cleavage at positions in the N-terminal region of proteins in both samples, were identified for the first time in the mouse skin peptidome. The number of peptides acetylated at the residue after the first Met residue, mostly Ser and Ala, was higher than that of peptides acetylated at the initial Met. Proteins cleaved in the hemorrhagic skin participate in cholesterol metabolism, PPAR signaling, and in the complement and coagulation cascades, indicating the impairment of these biological processes. The peptidomic analysis also indicated the emergence of peptides with potential biological activities, including pheromone, cell penetrating, quorum sensing, defense, and cell-cell communication in the mouse skin. Interestingly, peptides generated in the hemorrhagic skin promoted the inhibition of collagen-induced platelet aggregation and could act synergistically in the local tissue damage induced by HF3.