Agonists Of Platelet Aggregation Research Articles

Synergy by Ristocetin and CXCL12 in Human Platelet Activation: Divergent Regulation by Rho/Rho-Kinase and Rac.

CXCL12, belonging to the CXC chemokine family, is a weak agonist of platelet aggregation. We previously reported that the combination of CXCL12 and collagen at low doses synergistically activates platelets via not CXCR7 but CXCR4, a specific receptor for CXCL12 on the plasma membrane. Recently, we reported that not Rho/Rho kinase, but Rac is involved in the platelet aggregation induced by this combination. Ristocetin is an activator of the von Willebrand factor that interacts with glycoprotein (GP) Ib/IX/V, which generates thromboxane A2 via phospholipase A2 activation, resulting in the release of the soluble CD40 ligand (sCD40L) from human platelets. In the present study, we investigated the effects of a combination of ristocetin and CXCL12 at low doses on human platelet activation and its underlying mechanisms. Simultaneous stimulation with ristocetin and CXCL12 at subthreshold doses synergistically induce platelet aggregation. A monoclonal antibody against not CXCR7 but CXCR4 suppressed platelet aggregation induced by the combination of ristocetin and CXCL12 at low doses. This combination induces a transient increase in the levels of both GTP-binding Rho and Rac, followed by an increase in phosphorylated cofilin. The ristocetin and CXCL12-induced platelet aggregation as well as the sCD40L release were remarkably enhanced by Y27362, an inhibitor of Rho-kinase, but reduced by NSC23766, an inhibitor of the Rac-guanine nucleotide exchange factor interaction. These results strongly suggest that the combination of ristocetin and CXCL12 at low doses synergistically induces human platelet activation via Rac and that this activation is negatively regulated by the simultaneous activation of Rho/Rho-kinase.

The role of hyperbaric oxygen to platelet aggregation in noninsulin-dependent diabetes mellitus (NIDDM)

Hyperglycemia in diabetes mellitus (DM) could cause rheological disorder, such as platelet aggregation and blood hyperviscosity. Hyperbaric oxygen (HBO) could decrease collagen as platelet aggregation agonist. This study aimed to explore the effect of HBO treatment to platelet aggregation parameters (latency time(LT), aggregation speed, aggregation index, and aggregation percentage) with the collagen aggregator in the noninsulin dependent diabetes mellitus (NIDDM). The number of subjects in this study were 16 for each group normoxia normobaric (NONB) and HBO. NIDDM patients from DM polyclinic in Rumah Sakit AngkatanLaut (RSAL) Dr Ramelan Surabaya which was fulfilled inclusion criteria would receive HBO Therapy. Control Group/NONB were treated with NONB condition (20% O2 1 ATA) for 90min and treatment group/HBO were treated with hyperoxia hyperbaric condition (100% O2 2.4 ATA) for 3×30min with interval of 2×5min for inhaling fresh air. Subject has been blood taken for platelet aggregation test before and after HBO Therapy. The length of treatment was 5days for both condition (NONB and HBO). The data from both groups, NONB and HBO were tested first by normality test, homogenity test, correlation test, analysis of covariance, and paired t-test. Based on paired t-test, the decrease on platelet aggregation speed, aggregation index, and aggregation percentage after HBO treatment was showed significant difference on the LT and aggregation index while in aggregation speed and aggregation percentage was not significant. NONB group after 5days was showed a significant difference on the aggregation speed and aggregation index while in LT and aggregation percentage was not significant. The utilization of HBO 2.4 ATA 100% O2 3×30min, once a day, for 5days could decrease the platelet aggregation parameters (LT, aggregation speed, aggregation index, and aggregation percentage) in patients with NIDDM.

Transcatheter Aortic Valve Implantation Induces Transient Drop in Platelet Reactivity

Although recent studies described platelet reactivity (PR) changes in days after transcatheter aortic valve implantation (TAVI), precise time course and duration of these changes have not been fully investigated. The aim of this study was to investigate PR pattern during and after TAVI in multiple time points. Study included 40 consecutive patients undergoing TAVI. All patients underwent the procedure on dual antiplatelet therapy. PR was measured in 7 time points: before induction of anesthesia (T1), after heparin administration (T2), 10 minutes after initial valve implantation (T3), at the end of procedure (T4), and on 3rd, 6th, and 30th postoperative day (T5-T7). PR was measured using impedance aggregometer using 3 different platelet aggregation agonists (arachidonic acid in ASPItest, adenosine diphosphate in ADPtest and thrombin receptor activating peptide 6 in TRAPtest). All patients underwent successful TAVI procedure. Mean PR on T1 was 22.9 ± 23.0 U for ASPItest, 40.5 ± 23.7 U for ADPtest and 91.7 ± 32.5 U for TRAPtest. There was no significant difference in PR on T2. On T3, significant reduction of PR in all 3 tests was observed [ASPI 10.4 ± 11.6 U (P = 0.001), ADP 24.2 ± 14.1 U (P < 0.001) and TRAP 69.3 ± 26.6 U (P < 0.001)]. PR nadir for all tests was reached on T5, with subsequent PR incline. PR values in all tests returned to baseline levels on T7. Our results show that successful TAVI procedure induces transient decrease in PR regardless of the platelet activation pathway.

P3722Platelet reactivity changes following transcatheter aortic valve implantation

Abstract Introduction Although recent studies described changes in platelet reactivity (PR) in days following transcatheter aortic valve implantation (TAVI), precise time course and duration of these changes have not been fully investigated. Purpose To investigate PR changes during and after TAVI. Methods Study included 42 consecutive patients with severe and symptomatic aortic stenosis undergoing TAVI procedure in our institution. Patients' clinical characteristics were collected from medical records. All patients who did not have chronic dual antiplatelet therapy received loading dose of aspirin and clopidogrel (300 mg) one day before the procedure followed by their standard maintenance doses for next three months. PR was measured in six time points: just before induction of anaesthesia (T1), after heparin administration (T2), at the end of procedure (T3), and on 3rd, 6th and 30th postoperative day (T4–6). PR was measured using impedance aggregometer (Multiplate® analyzer, Roche, Munich, Germany) in response to three platelet aggregation agonists: arachidonic acid (ASPItest), ADP (ADPtest) and thrombin receptor activating peptide-6 (TRAPtest). Results Mean patient age was 82.1 years with majority of patients being male 57.1% (N=24). Mean valve area and mean transvalvular gradient prior to procedure were 0.71±0.21 cm2 and 49.1±18.7 mmHg, respectively. All patients underwent successful TAVI procedure using either self-expandable (N=25, 59.5%) or balloon-expandable valve. Two patients (4.7%) underwent transapical TAVI, while transfemoral approach was used in all other patients. Mean postimplantation gradient was 10.2±7.1 mmHg. Mean PR on T1 was 24.3±23.1 U for ASPItest, 41.6±26.5 U for ADPtest and 90.1±33.3 U for TRAPtest. There was no significant difference in PR on T2. However, on T3, ignificant reduction of PR in all 3 tests was observed (ASPI 9.4±10.1 U (p=0.001), ADP 23.1±15.0 U (p&lt;0.001) and TRAP 64.5±27.3 U (p&lt;0.001)). Lowest PR values for all tests were reached on T4, after which incline in PR has been observed. On T5, value of ASPItest reached levels not significantly different to those on T1 (15.1±17.2 U, p=0.13), while ADPtest and TRAPtest remained significantly lower (27.3±18.5 U, p=0.007 and 68.6±33.2 U, p=0.003, respectively). All PR values returned to initial levels on T6 (figure 1). Conclusions Our results show that successful TAVI procedure induces transient decrease in PR regardless of the platelet activation pathway. These findings add new knowledge in understanding complexed relations in intravascular milieu following TAVI. Further research on a larger number of patients is needed to confirm and asses clinical significance of these results. Acknowledgement/Funding Croatian Science Foundation

In Vitro Antithrombotic Properties of Salmon (Salmo salar) Phospholipids in a Novel Food-Grade Extract

Marine and salmon polar lipids (PLs) extracted by conventional extractions with non-food-grade solvents (CE-salmon-PLs) possess antithrombotic bioactivities against platelet-activating factor (PAF) and thrombin. Similar effects of food-grade-extracted (FGE) marine PLs have not yet been reported. In this study, food-grade solvents were used to extract PLs from Irish organic farmed salmon (Salmo salar) fillets (FGE-salmon-PLs), while their antithrombotic bioactivities were assessed in human platelets induced by platelet aggregation agonists (PAF/thrombin). FGE-salmon-PLs were further separated by thin layer chromatography (TLC) into lipid subclasses, and the antithrombotic bioactivities of each subclass were also assessed. LC-MS was utilized to elucidate the structure-activity relationships. FGE-salmon-PLs strongly inhibited PAF-induced platelet aggregation, while their relevant anti-thrombin effects were at least three times more potent than the previously reported activities of CE-salmon-PLs. TLC-derived lipid fractions corresponding to phosphatidylcholines (PC) and phosphatidylethanolamines (PE) were the most bioactive lipid subclasses obtained, especially against thrombin. Their LC-MS analysis elucidated that they are diacyl- or alkyl-acyl- PC and PE moieties baring ω3 polyunsaturated fatty acids (PUFA) at their sn-2 position, such as eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA). Our results concerning the potent antithrombotic effects of FGE-salmon-PLs against both PAF and thrombin pathways strongly suggest that such food-grade extracts are putative candidates for the development of novel cardioprotective supplements and nutraceuticals.

Platelet Function Changes in a Time-Dependent Manner Following Traumatic Brain Injury in a Murine Model.

Traumatic brain injury (TBI) results in systemic changes in coagulation and inflammation that contribute to post-traumatic morbidity and mortality. The potential interaction of platelets and pro-inflammatory cytokines in the modulation of coagulation, microthrombosis, and venous thromboembolic events after moderate TBI has not been determined. Using a murine model, we hypothesized that the degree of platelet-induced coagulation varies depending on the platelet aggregation agonist platelet-induced coagulation changes in a time-dependent manner following TBI, and changes in platelet-induced coagulation are mirrored by changes in the levels of circulating pro-inflammatory cytokines. An established weight-drop model was used to induce TBI in anesthetized mice. Blood samples were collected at intervals after injury for measurements of platelet count, serum fibrinogen, pro-inflammatory cytokines, and determination of soluble P-selectin levels. Thromboelastometry was used to evaluate changes in hemostasis. Platelet function was determined using whole blood impedance aggregometry. Ten minutes following TBI, adenosine diphosphate-induced platelet aggregation decreased as measured by platelet aggregometry. Despite no changes in platelet counts and serum fibrinogen, platelet aggregation, pro-inflammatory cytokines, and soluble P-selectin were increased at 6 h after TBI. Rotation thromboelastometry demonstrated increased maximal clot firmness at 6 h. Platelet function and coagulability returned to baseline levels 24 h following head injury. Our data demonstrate that after TBI, acute platelet dysfunction occurs followed by rebound platelet hyperaggregation. Alterations in post-TBI platelet aggregation are reflected in whole blood thromboelastometry and are temporally associated with the systemic pro-inflammatory response.

On-Treatment Platelet Reactivity is a Predictor of Adverse Events in Peripheral Artery Disease Patients Undergoing Percutaneous Angioplasty

Few data are available on the association between a different entity of platelet inhibition on antiplatelet treatment and clinical outcomes in patients with peripheral artery disease (PAD). The aim of this study was to evaluate the degree of on-treatment platelet reactivity, and its association with ischaemic and haemorrhagic adverse events at follow up in PAD patients undergoing percutaneous transluminal angioplasty (PTA). In this observational, prospective, single centre study, 177 consecutive patients with PAD undergoing PTA were enrolled, and treated with dual antiplatelet therapy with aspirin and a P2Y12 inhibitor. Platelet function was assessed on blood samples obtained within 24 h from PTA by light transmission aggregometry (LTA) using arachidonic acid (AA) and adenosine diphosphate (ADP) as agonists of platelet aggregation. High on-treatment platelet reactivity (HPR) was defined by LTA ≥ 20% if induced by AA, and LTA ≥ 70% if induced by ADP. Follow up was performed to record outcomes (death, major amputation, target vessel re-intervention, acute myocardial infarction and/or myocardial revascularisation, stroke/TIA, and bleeding). HPR by AA and HPR by ADP were found in 45% and 32% of patients, respectively. During follow up (median duration 23 months) 23 deaths (13%) were recorded; 27 patients (17.5%) underwent target limb revascularisation (TLR), two (1.3%) amputation, and six (3.9%) myocardial revascularisation. Twenty-four patients (15.6%) experienced minor bleeding. On multivariable analysis, HPR by AA and HPR by ADP were independent predictors of death [HR 3.8 (1.2–11.7), p = .023 and HR 4.8 (1.6–14.5), p = .006, respectively]. The median value of LTA by ADP was significantly lower in patients with bleeding complications than in those without [26.5% (22–39.2) vs 62% (44.5–74), p < .001). LTA by ADP ≤ 41% was independently associated with bleeding HR 14.6 (2.6–24.0), p = .001] on multivariable analysis. In this study a high prevalence of on-clopidogrel and aspirin high platelet reactivity was found, which was significantly associated with the risk of death. Conversely, a low on-clopidogrel platelet reactivity was associated with a higher risk of bleeding. These results document that the entity of platelet inhibition is associated with both thrombotic and bleeding complications in PAD patients.

On-Treatment Platelet Reactivity is a Predictor of Adverse Events in Peripheral Artery Disease Patients Undergoing Percutaneous Angioplasty

Few data are available on the association between a different entity of platelet inhibition on antiplatelet treatment and clinical outcomes in patients with peripheral artery disease (PAD). The aim of this study was to evaluate the degree of on-treatment platelet reactivity, and its association with ischaemic and haemorrhagic adverse events at follow up in PAD patients undergoing percutaneous transluminal angioplasty (PTA). In this observational, prospective, single centre study, 177 consecutive patients with PAD undergoing PTA were enrolled, and treated with dual antiplatelet therapy with aspirin and a P2Y12 inhibitor. Platelet function was assessed on blood samples obtained within 24h from PTA by light transmission aggregometry (LTA) using arachidonic acid (AA) and adenosine diphosphate (ADP) as agonists of platelet aggregation. High on-treatment platelet reactivity (HPR) was defined by LTA≥20% if induced by AA, and LTA≥70% if induced by ADP. Follow up was performed to record outcomes (death, major amputation, target vessel re-intervention, acute myocardial infarction and/or myocardial revascularisation, stroke/TIA, and bleeding). HPR by AA and HPR by ADP were found in 45% and 32% of patients, respectively. During follow up (median duration 23 months) 23 deaths (13%) were recorded; 27 patients (17.5%) underwent target limb revascularisation (TLR), two (1.3%) amputation, and six (3.9%) myocardial revascularisation. Twenty-four patients (15.6%) experienced minor bleeding. On multivariable analysis, HPR by AA and HPR by ADP were independent predictors of death [HR 3.8 (1.2-11.7), p=.023 and HR 4.8 (1.6-14.5), p=.006, respectively]. The median value of LTA by ADP was significantly lower in patients with bleeding complications than in those without [26.5% (22-39.2) vs. 62% (44.5-74), p<.001). LTA by ADP ≤ 41% was independently associated with bleeding HR 14.6 (2.6-24.0), p=.001] on multivariable analysis. In this study a high prevalence of on-clopidogrel and aspirin high platelet reactivity was found, which was significantly associated with the risk of death. Conversely, a low on-clopidogrel platelet reactivity was associated with a higher risk of bleeding. These results document that the entity of platelet inhibition is associated with both thrombotic and bleeding complications in PAD patients.

Transcriptomic landscape of acute promyelocytic leukemia reveals aberrant surface expression of the platelet aggregation agonist Podoplanin.

Acute promyelocytic leukemia (APL) is a medical emergency because of associated lethal early bleeding, a condition preventable by prompt diagnosis and therapeutic intervention. The mechanisms underlying the hemostatic anomalies of APL are not completely elucidated. RNA-sequencing-based characterization of APL (n = 30) was performed and compared to that of other acute myeloid leukemia (n = 400) samples and normal promyelocytes. Perturbations in the transcriptome of coagulation and fibrinolysis-related genes in APL extend beyond known culprits and now include Thrombin, Factor X and Urokinase Receptor. Most intriguingly, the Podoplanin (PDPN) gene, involved in platelet aggregation, is aberrantly expressed in APL promyelocytes and is the most distinctive transcript for this disease. Using an antibody panel optimized for AML diagnosis by flow cytometry, we also found that PDPN was the most specific surface marker for APL, and that all-trans retinoic acid therapy rapidly decreases its expression. Functional studies showed that engineered overexpression of this gene in human leukemic cells causes aberrant platelet binding, activation and aggregation. PDPN-expressing primary APL cells, but not PDPN-negative primary leukemias, specifically induce platelet binding, activation and aggregation. Finally, PDPN expression on leukemia cells in a xenograft model was associated with thrombocytopenia and prolonged bleeding time in vivo. Together our results suggest that PDPN may contribute to the hemostatic perturbations found in APL.

Pannexin1 Single Nucleotide Polymorphism and Platelet Reactivity in a Cohort of Cardiovascular Patients.

Pannexin1 (Panx1), a membrane channel-forming protein permitting the passage of small-sized molecules, such as ATP, is expressed in human platelets. Recently, we showed that inhibiting Panx1 affects collagen-induced platelet aggregation but not aggregation triggered by other agonists. We also found that a single nucleotide polymorphism (SNP; rs1138800) in the Panx1 gene encoded for a gain-of-function channel (Panx1-400C) and was associated with enhanced collagen-induced platelet reactivity. Here, we assessed the association of this SNP with platelet reactivity in a cohort of 758 stable cardiovascular patients from the ADRIE study treated with aspirin and/or clopidogrel. We found that presence of the Panx1-400C allele was not associated with platelet reactivity in stable cardiovascular patients, irrespective of the platelet aggregation agonist used (collagen, ADP or arachidonic acid) or the anti-platelet drug regimen. Moreover, the Panx1-400A > C SNP did also not affect the re-occurrence of cardiac ischemic events in the same stable cardiovascular patient cohort.

&amp;lt;i&amp;gt;In Vitro&amp;lt;/i&amp;gt; Effect of Soy Isoflavone and Equol on Soluble CD40L Release Stimulated by Ristocetin in Platelets from Postmenopause Women

The inhibition of specific flavonoid on the in vitro platelet aggregation induced by collagen, arachidonic acid and thromboxane A2 (TxA2) agonist, seems to be related mostly to their ability to compete for binding to the TxA2 receptor (TP). The aim of this study was to analyze the effect of soy isoflavone and equol in terms of inhibiting the platelet aggregation and sCD40L release stimulated by ristocetin, an in vitro-activator of glycoprotein Ib/IX/V, in platelets from postmenopausal women. When platelets were stimulated by 0.75 mg/ml ristocetin, equol (10 μM) exhibited a greater inhibitory activity on platelet aggregation (~68%) than genistein or daidzein. The effect of equol was dependent on the concentration of platelet aggregation agonist. In the presence of ristocetin (0.75 mg/ml, 1.125 mg/ml and 1.5 mg/ml), the inhibitory effect of 10 μM equol was 68% ± 5%, 54% ± 4% and 31% ± 5%, respectively. Equol (10 μM) was a potent inhibitor (~35%) of sCD40L release when stimulated with 1.5 mg/ml ristocetin. However, no significant differences were noted in platelets incubated in the presence of genistein or daidzein and stimulated by ristocetin. On the other hand, SQ29548, a high TP antagonist, also inhibited the sCD40L release stimulated by ristocetin. Finally, 10 μM of genistein, daidzein or equol did not significantly affect the thromboxane B2 production when platelets were incubated with 1.5 mg/ml ristocetin. The relevance of this study was to find that equol exhibits a potent activity by inhibiting ristocetin-induced sCD40L release, suggesting that soy isoflavone has important biological effects on the hemostatic system. However, clinical trials will be necessary to assess the effect of equol on platelet and their impact on inflammatory markers.

The evaluation method for antiplatelet effect of acetylsalicylic acid

Reduced platelet aggregation by acetylsalicylic acid administration has been associated with adverse outcomes in patients with thrombotic diseases, thus it is important to determine aspirin resistance in those cases. The antiplatelet effect of acetylsalicylic acid is rarely measured, but it has many problems. The aim of this study was to find the evaluation method for antiplatelet effect after administration of acetylsalicylic acid. We developed a particle counting method based upon laser light scattering, and utilized the platelet aggregation agonists, collagen, at 0.25, 0.5 and 1.0μg/mL, and adenosine diphosphate (ADP), at 0.5, 1.0 and 2.0μM, to determine their effective concentrations. Seventeen healthy volunteers were administered acetylsalicylic acid at 162mg/day, with platelet aggregation determined before and 20min after administration. In all subjects, the rate of platelet aggregation induced by 1.0μg/mL of collagen before taking acetylsalicylic acid was the highest value obtained, while 20min after acetylsalicylic acid administration, aggregation induced by collagen at 1.0μg/mL was significantly decreased as compared to before administration. As for the other concentrations of collagen and all those of ADP tested, platelet aggregation was either not significantly induced before taking acetylsalicylic acid or the rate of aggregation was not significantly decreased after taking acetylsalicylic acid. Our results indicate that collagen at 1.0μg/mL is appropriate as a platelet aggregation agonist for evaluating the antiplatelet effect of acetylsalicylic acid. Thus, it is useful that the measurement is performed only once.

Abstract P065: Changes in Lysophosphatidic Acids and Lysophosphatidylcholines with Fish Oil Consumption wnd their Correlates with Platelet Aggregation

Background: Lysophosphatidic acids (LPA) and lysophosphatidylcholines (LPC) are phospholipids, each containing one fatty acid, and have effects on atherogenic mechanisms. No data currently exist regarding the effects of aspirin and fish oil (FO) ingestion on these potent lipid mediators in humans with diabetes mellitus (DM). We hypothesized that ingestion of these agents alter LPA and LPC concentrations and that these effects correlate with platelet function. Methods: Using a sequential clinical trial approach, 30 adults with type 2 DM were treated with 81 mg/d of aspirin for 7 days, then with a 28-day regimen of FO (4g/day containing 1600 mg EPA and 800 mg DHA) after which they restarted 81 mg/d of aspirin for another 7 days. After the ingestion of a standard low-fat meal, phlebotomies were performed at baseline, 4 hours and 7 days after the ingestion of aspirin alone, 28 days after FO alone, and 4 hours and 7 days after aspirin and FO ingestion together. Eleven LPCs and 11 LPAs were analyzed using a modified Bligh and Dyer extract of plasma and infusion into an ABI QTrap 2000 via an Advion Nanomate robotic device. Platelet function was measured using whole blood aggregometry and via assays. Changes in concentrations of LPAs and LPCs compared to baseline were assessed using mixed models accounting for statin use, blood draw, BMI, presence of HTN, gender, race, smoking status, alcohol use, fish consumption, and education. Pearson’s correlations (r) were calculated for comparing LPAs and LPCs with platelet function. P-values less than 0.05 were considered statistically significant. Results: FO ingestion reduced concentrations of all 11 LPAs, including EPA (20:5n3) and DHA (22:6n3) species. EPA and DHA LPC concentrations significantly increased by FO alone and in combination with aspirin. Stearic acid (18:0) and alpha-linolenic acid (18:3n3) LPCs were significantly reduced by FO combined with aspirin after 4 hours and 7 days. Changes in 18:0 and oleic acid (18:1) LPAs after FO moderately correlated with the platelet aggregation agonist collagen (r=0.41, p=0.02 and r=0.39, p=0.03, respectively) whereas changes in EPA and DHA LPCs did not correlate with platelet aggregation or function. However, EPA changes with FO and aspirin moderately correlated with the platelet aggregation agonist ADP after 4 hours (r=0.46, p=0.01) but not 7 days (r=0.04, p=0.83). Conclusions: The ingestion of FO with and without aspirin has significant effects on plasma lysophospholipid levels in subjects with DM. Given the pleiotropic roles of lysophospholipids in platelet function and atherogenesis, these results suggest that the formation of potent lipid mediators- derived from ingested fats- may be more important than the simple ingestion of the fats. Our results should be used to generate new hypotheses applying different phospholipids in the evaluation of atherogenic mechanisms.

Synthesis, in vitro antiplatelet activity and molecular modelling studies of 10-substituted 2-(1-piperazinyl)pyrimido[1,2-a]benzimidazol-4(10H)-ones

The multistep preparation of the new 10-substituted 2-(1-piperazinyl)pyrimido[1,2-a]benzimidazol-4(10H)-ones 6a–o, and of the two isomers 10-ethyl-2-(diethylamino)pyrimido[1,2-a]benzimidazol-4(10H)-one 6p and 10-ethyl-4-(diethylamino)pyrimido[1,2-a]benzimidazol-2(10H)-one 13, as well as the in vitro evaluation of their inhibitory activity on human platelet aggregation induced in platelet-rich plasma by ADP, collagen or the Ca2+ ionophore A23187 were here described. Nine out of fifteen 2-(1-piperazinyl)derivatives (6g–o) showed good inhibitory properties towards all the platelet aggregation agonists used. Moreover, a molecular modelling study has been performed on two of the best compounds of this series (6i and 6o) to confirm in silico their interactions with the catalytic site of human platelet PDE3, using the X-ray data of the PDE3B isoform in complex with an inhibitor.

A lipid peroxidation product 9-oxononanoic acid induces phospholipase A2 activity and thromboxane A2 production in human blood

Lipid peroxidation products are known to cause toxicity by reacting with biologically significant proteins, but the inducing role of peroxidation products has been not noted to produce degenerative disease-related eicosanoids. Here, 9-oxononanoic acid (9-ONA), one of the major products of peroxidized fatty acids, was found to stimulate the activity of phospholipase A2 (PLA2), the key enzyme to initiate arachidonate cascade and eicosanoid production. An exposure of fresh human blood to the atmosphere at 37°C accumulated 9-ONA, increasing peroxide value and thiobarbituric acid reactive substances in the blood. The lipid peroxidation was accompanied by significant increases of PLA2 activity and thromboxane B2 (TxB2) production, which is a stable metabolite of thromboxane A2 (TxA2) and a potent agonist of platelet aggregation. These events were abolished by standing the blood under nitrogen. The addition of organically synthesized 9-ONA resumed the activity of PLA2 and the production of TxB2. Also, 9-ONA induced platelet aggregation dose-dependently. These results indicated that 9-ONA is the primary inducer of PLA2 activity and TxA2 production, and is probably followed by the development of diseases such as thrombus formation. This is the first report to find that a lipid peroxidation product, 9-ONA, stimulates the activity of PLA2.

Phytochemicalassay and Antiplatelet Activity of Fractions of Velvet Bean Seeds (Mucuna pruriens L.)

Platelet aggregation is an important factor contributing to the formation of thrombus due to an uncontrolled blood clotting. An antiplatelet agent is a compound which decreases platelet aggregation and inhibits thrombus formation. The objectives of this study were to determine the class of compound employing phytochemical assay and to determine the in vitro antiplatelet activity of four fraction, namely hexane, ethyl acetate, butanol, and water fractions of velvet bean seeds ( Mucuna pruriens L.) using epinephrine (EPN) as agonist of platelet aggregation. The antiplatelet activities were tested in human platelet rich plasma with hyperaggregation. To determine the activities, EPN was arranged at 4 level of concentrations (300, 150, 75, and 30 ì M), and antiplatelet agents were at 500 µg/ml. The results indicated that ethyl acetate, butanol and water fraction contained high flavonoids and moderate phenols. The water, butanol and ethyl acetate fractions of velvet bean seeds exhibited potential inhibition of EPN-induced platelet aggregation at all concentrations. The strongest antiplatelet agent was water fraction and had the same antiplatelet activity as aspirin at level 150, 75, and 30 ì M of EPN. Butanol fraction had the same antiplatelet activity as aspirin at the lowest EPN (30 ì M).

Immunomodulatory derivatives induce PU.1 down-regulation, myeloid maturation arrest, and neutropenia

AbstractThe immunomodulatory drugs (IMiDs) lenalidomide and pomalidomide yield high response rates in patients with multiple myeloma, but the use of IMiDs in multiple myeloma is associated with neutropenia and increased risk for venous thromboembolism (VTE) by mechanisms that are unknown. We show that IMiDs down-regulate PU.1, a key transcription factor involved in granulocyte differentiation in vitro and in patients treated with lenalidomide. Loss of PU.1 results in transient maturation arrest with medullary accumulation of immature myeloid precursors and subsequent neutropenia. Accumulation of promyelocytes leads to high levels of the platelet aggregation agonist, cathepsin G stored in the azurophilic granules of promyelocytes. High levels of cathepsin G subsequently may increase the risk of VTE. To our knowledge, this is the first report investigating the underlying mechanism of IMiD-induced neutropenia and increased risk of VTE in multiple myeloma.

Genetic Disruption of □pdl and □pdlr Phospholipases in Mice Leads to Impaired Platelet Adhesion and Aggregation

Platelet adhesion and aggregation are critical events in thrombosis. Bioactive phospholipid LPA (lysophosphatidic acid) has been identified as an important agonist for platelet aggregation. Plasma LPA can be generated from phospholipid substrates by phospholipase A1 (PLA1) and phospholipase D (PLD). We previously identified two novel PLA1 enzymes, designated lpdl (lpd lipase) and lpdlr (lpdl related lipase). Together with phosphatidylserine phospholipase A1 (PS-PLA1), these three phospholipases form a unique PLA1 lipase subfamily. Phospholipids are important structural components of cellular membranes. Recent studies demonstrate that lipid raft microdomains on the platelet membrane contribute to platelet activation. The gathering of membrane lipid rafts is necessary for ADP-mediated platelet activation. Disruption of lipid raft results in a reduction of ADP-induced platelet aggregation. In addition, it is reported that alteration of membrane lipid composition also affects the function of platelet β3 integrin (GPIIbIIIa). Thus, phospholipases may play an important role in platelet function. However, the roles of lpdl and lpdlr in platelet activation and thrombosis are unknown. To study the function of lpdl and lpdlr, we have recently knocked out both lpdl and lpdlr genes in mice by deleting their exons I and II, including their translation start codon ATG. RT-PCR confirmed that neither lpdl nor lpdlr gene is expressed in homozygous lpdl−/− or lpdlr−/− mice but expressed in wild-type (WT) tissues, indicating successful knockout of these phospholipases. Next, optical platelet aggregometry was used to assess platelet aggregation in platelet rich plasma (PRP). Platelet aggregation was induced with 5, 10 and 20 μM ADP in pooled PRP (3×108 platelets/mL) from age- and sex-matched WT, heterozygous and homozygous mice. Our results demonstrated that both the lpdl−/− and lpdlr−/− mice have decreased platelet aggregation after ADP stimulation. We further studied thrombus formation in lpdl−/− mice using an ex vivo perfusion chamber model on collagen type I-coated rectangular glass microcapillary tubes. Our preliminary data showed that both platelet adhesion and aggregation were impaired in the lpdl−/− mice. We are repeating these experiments and are examining lpdlr−/− mice using the same perfusion chamber model. In vivo thrombosis models with intravital microscopy and the mechanisms of how lpdl and lpdlr enzymes regulate platelet activation will also be investigated. These studies could potentially identify a novel pathway for regulation of platelet function, which may lead to development of new diagnostic and/or therapeutic methods for atherothrombosis. (Drs. D. Wang and A. Reheman contribute equally to this work)

In Vitro Antiplatelet Activity of Flavonoids from Leuzea Carthamoides

Plants and their secondary metabolites, including flavonoids, exhibit a wide range of biological effects. Consequently, natural substances are receiving an increased attention in medicinal research. Owing to these facts, in vitro antiplatelet activity of ethanol summary extract and four flavonoids from Leuzea carthamoides was determined in human platelet-rich plasma. Arachidonic acid (AA), adenosine diphosphate (ADP), collagen (COL), and thrombin were used as agonists of platelet aggregation. The summary extract showed a significant inhibition of the aggregation induced by COL and ADP. Of the tested flavonoids, eriodictyol (1) and patuletin (2) influenced COL- and AA-induced aggregation. Their IC50 values are presented. Flavonoid glycosides eriodictyol-7-β-glucopyranoside (3) and 6-hydroxykaempferol-7-O-(6″-O-acetyl-β-D[small cap]-glucopyranoside) (4) were found to be weak antiplatelet agents. These results confirmed the fact that glucosylation decreases the antiplatelet activity. Quantitative composition of tested flavonoids in L. carthamoides extract was also determined. Though two of the tested flavonoids inhibited platelet aggregation, further evaluation of L. carthamoides, in order to discover other antiplatelet active compounds and possible adverse health effects, is needed.

Cathepsin G, a Potent Platelet Aggregation Activator, Is Upregulated in Multiple Myeloma Patients during Treatment with Lenalidomide (Revlimid).

Lenalidomide induces impressive response rates in patients with multiple myeloma. Unfortunately, one of the major side effects of this therapy is the increased risk of thromboembolic events (TEE) up to 17%. The reason for a higher risk of TEE associated with Lenalidomide is not known, but it has been found that aspirin is very effective in preventing TEEs induced by thalidomide and lenalidomide (Rajkumar, 2005). Cathepsin G is a serine proteinase present in the azurophilic granules of polymorphonuclear neutrophils (PMNs) and is a strong platelet aggregation agonist with potency similar to thrombin. In the current study we investigated the effect of the immunomodulatory derivatives of thalidomide (IMiDs) including Actimid and lenalidomide on cathepsin G in vitro and in vivo. To investigate the effect of IMiDs in vitro CD34+ hematopoietic progenitors were cultured in the presence of IL-3, IL-6 and SCF supporting the development of granulocytes and treated with Actimid or DMSO (control). RNA and protein were extracted for the analysis of cathepsin G. A significant up-regulation of cathepsin G (7.7 fold) was detected in oligonucleotide gene array analysis after 3 days of treatment with Actimid as compared to control group. These results were confirmed by RT-PCR, which showed a 3.7 and 7.6 fold mRNA-increase on day 6 and day 10 of culture compared to control. Next we analyzed the cathepsin G levels of patients (n=10) treated with lenalidomide before treatment, on day 15 of each cycle (1–4) as well as 1, 2, and 3 month after stop of lenalidomide treatment. We observed a continuous significant increase of the mRNA levels of PMNs over the course of treatment (baseline: 1 fold, cycle 2: 1.7 fold, cycle 3: 4.8 fold, cycle 4: 20.7 fold). These data were confirmed by measuring cathepsin G levels in patient serum by ELISA. Cathepsin G significantly increased from a baseline mean of 53 ng/ml to 77.5 ng/ml (cycle 2), 129.2 ng/ml (cycle 3) and 145.5 ng/ml (cycle 4). After stop of the lenalidomide treatment we observed a time dependent normalization to baseline levels of the cathepsin G with 61 ng/mL (1 month post treatment), 42.5 ng/mL (2 months post treatment), and 49.2 ng/mL (3 months post treatment). Our data show that IMiDs up-regulate the potent platelet aggregation activator cathepsin G in hematopoietic cells and thereby might contribute to the development of TEE in patients receiving IMiD treatment. Based on our data TEE prophylaxis should be continued for at least 1 more month after stop of therapy. For the first time our results provide an explanation why aspirin is effective in preventing TEE in patients receiving IMiDs. Further studies are needed to determine whether cathepsin G helps to predict TEE. Inhibition of cathepsin G might be a potential therapeutic target for preventing the hypercoaguable state induced by IMiD treatment.