Reduction In ADP-induced Platelet Aggregation Research Articles

The antiplatelet and anticoagulant activity of 7-O-gentiobiozide formononetin in vitro and in vivo

Aim. In experiments in vitro and in vivo it was investigated the effect of isoflavone 7-O-gentiobiozide formononetin (GBF) isolated from the roots of the plant Maackia аmurensis (Maackia amurensis Rupr. et Maxim.), on the processes of vascular-platelet and coagulation hemostasis. Materials and methods. In experiments using blood of healthy human plasma in concentrations of GBF 1,0–50,0 mM promoted dose-dependently ADP-induced weakening of platelet aggregation. Since the concentration of 10,0 mM GBF induced hypocoagulative changes in blood plasma, comparable with the effect of 0,2–0,5 IU/ml heparin. Revealed hypocoagulative effect was confirmed in the application thromboelastometry, showing pronounced hypocoagulation and a significant reduction in fibrin formation dynamics. Results. In chronic GBF oral administration to rats at a dose of 25 mg/kg was fixed almost a 10-fold reduction in ADP-induced platelet aggregation, with increased content of these cells in the peripheral blood. Furthermore, in these conditions, there was a pronounced effect of GBF’s hypocoagulation which was implemented in inhibiting reactions inner and outer tracks of blood clotting, reducing the rate of formation of fibrin and its mechanical density. Conclusion. Thus, in experiments in vitro and in vivo for the first time revealed the ability of isoflavone 7-O-gentiobiozideformononetin extracted from the roots Maackia amurensis, inhibit the processes of vascularplatelet and coagulation hemostasis. This fact is of great practical importance, because it opens the prospective of the development of a new drug that can reduce the risk of thrombosis in various cardiovascular diseases.

PKC and AKT Modulate cGMP/PKG Signaling Pathway on Platelet Aggregation in Experimental Sepsis

Sepsis severity has been positively correlated with platelet dysfunction, which may be due to elevations in nitric oxide (NO) and cGMP levels. Protein kinase C, Src kinases, PI3K and AKT modulate platelet activity in physiological conditions, but no studies evaluated the role of these enzymes in platelet aggregation in sepsis. In the present study we tested the hypothesis that in sepsis these enzymes positively modulate upstream the NO-cGMP pathway resulting in platelet inhibition. Rats were injected with lipopolysaccharide (LPS, 1 mg/kg, i.p.) and blood was collected after 6 h. Platelet aggregation was induced by ADP (10 μM). Western blotting assays were carried out to analyze c-Src and AKT activation in platelets. Intraplatelet cGMP levels were determined by enzyme immunoassay kit. Phosphorylation of c-SRC at Tyr416 was the same magnitude in platelets of control and LPS group. Incubation of the non-selective Src inhibitor PP2 (10 μM) had no effect on platelet aggregation of LPS-treated rats. LPS increased intraplatelet cGMP levels by 5-fold compared with control group, which was accompanied by 76% of reduction in ADP-induced platelet aggregation. The guanylyl cyclase inhibitor ODQ (25 μM) and the PKG inhibitor Rp-8-Br-PET-cGMPS (25 μM) fully reversed the inhibitory effect of LPS on platelet aggregation. Likewise, the PKC inhibitor GF109203X (10 μM) reversed the inhibition by LPS of platelet aggregation and decreased cGMP levels in platelets. AKT phosphorylation at Thr308 was significantly higher in platelets of LPS compared with control group, which was not reduced by PI3K inhibition. The AKT inhibitor API-1 (20 μM) significantly increased aggregation and reduced cGMP levels in platelets of LPS group. However, the PI3K inhibitor wortmannin and LY29004 had no effect on platelet aggregation of LPS-treated rats. Therefore, inhibition of ADP-induced platelet aggregation after LPS injection is mediated by cGMP/PKG-dependent mechanisms, and PKC and AKT act upstream upregulating this pathway.

Inhibitory effects of bortezomib on platelet aggregation in patients with multiple myeloma

IntroductionMultiple myeloma (MM) therapy affects prothrombotic and anticoagulant processes. Patients receiving thalidomide, especially in combination with steroids, are at increased risk of venous thromboembolism (VTE), while the incidence of VTE on bortezomib is low. In vitro studies indicate that bortezomib causes a reduction in ADP-induced platelet aggregation. ObjectivesTo analyse the influence of bortezomib on platelet aggregation induced by various agonists in patients with MM. Patients and MethodsA total of 30 patients (median age 57.5years) with relapsed/refractory MM receiving bortezomib-based regimens were analysed. Optical platelet aggregometry was performed with the agonists collagen, ADP and ristocetin and measured over two 21-day cycles. The results from two groups: those treated with bortezomib and thalidomide (BT group, n=11) and those without thalidomide (B group, n=19) were analysed. ResultsDuring the second cycle, significantly decreased platelet aggregation was observed in the B group: 5μM ADP (p=0.0285, day 1 versus 8); 3.5μM ADP (p=0.0005, day 1 versus 8 and day 1 versus 11), collagen (p=0.0014, day 4 versus 8, day 4 versus 11), 1.25mg/ml ristocetin (p=0.0017, day 1 versus 8 and day 1 versus 11). Agonist-induced platelet aggregation tended to be reduced over time during the 1st cycle in group B. In the thalidomide group, significant platelet aggregation inhibition by collagen only was found. Transient reduction in platelet count was observed in all patients, but more prominently in group B. ConclusionThe inhibitory effects of prolonged exposure of bortezomib on platelet aggregation were demonstrated in relapsed/refractory MM patients, but antithrombotic activity of bortezomib should be clarified in further prospective studies.

Aspirin effectiveness in patients with rheumatoid arthritis and coronary heart disease who receive non-steroidal anti-inflammatory drugs

Aim. To study coagulation and vascular-platelet haemostasis in patients with rheumatoid arthritis (RA) and coronary heart disease (CHD), who receive various non-steroidal anti-inflammatory drugs (NSAID) in combination with low doses of aspirin. Material and methods. The study included 79 patients (59 women and 20 men; mean age 61,0 years; mean disease duration 8,5 years) with confirmed RA diagnosis. All participants received disease-modifying anti-inflammatory therapy and NSAID, as well as standard pharmacological CHD therapy. The parameters of coagulation and vascular-platelet haemostasis were compared by the type of administered NSAID (diclofenac, tenoxicam, nimesulide, or meloxicam). In total, 40 patients with increased platelet aggregation but no previous antiaggregant therapy were administered aspirin (100 mg/day). Platelet aggregation was reassessed at Day 7–8 of aspirin therapy. The control group included 25 untreated healthy men (mean age 55 years). Results. Activated coagulation haemostasis was observed in 58,2% of patients with RA and CHD, as manifested in increased levels of fibrinogen, soluble fibrin monomer complexes (SFMC), factor XII-dependent fibrinolysis, and von Willebrand factor, compared to controls. The therapy with most NSAID was linked to similar changes in coagulation haemostasis. The patients receiving diclofenac, nimesulide, and meloxicam demonstrated an activation of vascular-platelet haemostasis, as manifested in a significant increase of spontaneous platelet aggregation and ADPinduced platelet aggregation, compared to controls. Among patients receiving tenoxicam, there was a tendency towards a reduction in ADP-induced platelet aggregation (aspirin-like effect). Among patients already receiving diclofenac, nimesulide, or meloxicam, aspirin administration typically resulted in reduced platelet aggregation. In total, 42,4% of the patients did not respond to aspirin therapy. Conclusion. Patients with RA and CHD who receive NSAID are also in need of antiaggregant therapy. The latter should be administered under control of vascularplatelet haemostasis, as in a substantial proportion of these patients (42,4%), aspirin effectiveness is not adequate.

Doubling the clopidogrel dose in patients with reduced responsiveness to the standard dose is associated with a limited effectiveness as evaluated by impedance aggregometry

Different methods are available for quantifying platelet function inhibition. Measuring vasodilator-stimulated phosphoprotein (VASP) phosphorylation is currently the most specific method for assessing the clopidogrel effect. The aim of our study was to compare different tests in view of a clinically applicable bedside test. Further, we examined whether doubling the clopidogrel dose to 150mg/d in clopidogrel low-responder would lead to a reduction in platelet reactivity. ADP-, ADP Hs-, and TRAP-induced platelet aggregation were measured by impedance aggregometry in 100 patients with CAD and 18 healthy controls. Moreover, platelet aggregation was assessed by flow cytometrical detection of VASP-phosphorylation and surface P-selectin in a subgroup of 34 patients and in healthy controls. Another 10 patients with CAD, identified as low-responder, were treated with a clopidogrel dose of 150mg/d. Thereafter, ADP-induced platelet aggregation was assessed by impedance aggregometry. Significant correlations were observed between ADP-induced platelet aggregation assessed by VASP-phosphorylation and by impedance aggregometry. Doubling the dose of clopidogrel to 150mg/d was associated with a reduction of ADP-induced platelet aggregation in only 60% of the patients. Impedance aggregometry is a valuable bedside test to assess platelet function inhibition. Doubling the clopidogrel dose is not effective to reduce high on-treatment platelet reactivity in almost half of these patients, pointing to the need of a more powerful platelet inhibitor.

Can we extrapolate the outcomes of in vitro studies on murine endothelium to studies of human platelet-endothelium interactions? A technical note

IntroductionInteractions between vascular endothelium and blood platelets play a crucial role in cardiovascular diseases. Ex vitro models which use endothelial cells and platelets were the essential tools to investigate these interactions and their impact on haemostasis. The impaired interplay between vascular endothelium, blood platelets and leukocytes is believed to contribute to the development of cardiovascular disease. In this study we compared the ability of human (HUVECs) and murine (HECa10) endothelial cells to inhibit human platelet function and reactivity under in vitro conditions.Material and methodsThe aliquots of platelet-rich plasma obtained from 20 healthy donors were incubated with murine endothelial cell line HECa10 or human umbilical vein endothelial cells (HUVECs) (10 min, 37°C) prior to agonizing platelets with 5 µM ADP and monitoring platelet reactivity for 10 min using optical aggregation.ResultsSignificant reduction in ADP-induced platelet aggregation in the presence of endothelial cell cultures remained independent of cell count. HUVECs appeared much more effective in the inhibition of platelet aggregation compared to HECa10 (35.2 ±2.3 AU vs. 43.7 ±2.0 AU, p= 0.025).ConclusionsHECa10 cells have much lower potential to inhibit platelet aggregation than HUVECs. This implies that these two cell lines may not be freely used interchangeably in in vitro experiments. These findings clearly indicate that the outcomes of in vitro studies performed with murine EC lines cannot be unreservedly extrapolated to human platelet-endothelium interactions.

Cangrelor increases the magnitude of platelet inhibition and reduces interindividual variability in clopidogrel-pretreated subjects.

Inadequate platelet inhibition despite aspirin and clopidogrel therapy during and after a percutaneous coronary intervention is associated with an impaired clinical outcome. Cangrelor, a direct and reversible P2Y(12) inhibitor that is currently in development, has the potential to achieve higher levels of inhibition of ADP-induced platelet aggregation than clopidogrel. The aim of the present study was to compare the magnitude of platelet inhibition in clopidogrel-pretreated patients before and after the in vitro addition of a subtherapeutic dose of cangrelor. Blood samples were drawn from patients pretreated with clopidogrel and aspirin who were undergoing elective percutaneous coronary intervention (n=39). Platelet function analysis with 'classical' light transmittance aggregometry (both peak and late aggregation [at 6 min]) was performed before and after the in vitro addition of cangrelor (0.25 mumol/l) to platelet-rich plasma (PRP). After an incubation period of five minutes, platelet aggregation was induced by 5 and 20 mumol/l ADP. The in vitro addition of 0.25mumol/l cangrelor to the PRP from clopidogrel-treated subjects resulted in an additional reduction in ADP-induced platelet aggregation. For ADP concentrations of 5 and 20 mumol/l, peak aggregation showed a decrease of 75 and 85%, respectively (p<0.001 for both), while late aggregation was almost completely diminished (p=0.003 and p<0.001, respectively). Furthermore, the interindividual variation in inhibition of ADP-induced platelet aggregation by clopidogrel was greatly reduced after the addition of cangrelor. We demonstrate that the in vitro addition of even a subtherapeutic dose of cangrelor to the PRP of clopidogrel-pretreated patients results in an additional reduction of ADP-induced platelet aggregation. Moreover, cangrelor was able to diminish the interindividual variation observed in clopidogrel-inhibited platelet aggregation. (Neth Heart J 2009;17:195-8.).

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)

Evidence for the involvement of 5-HT 2A receptors in mild mesenteric ischemia/reperfusion dysfunctions in mice

In this study, the involvement of 5-HT 2A receptors on mesenteric ischemia–reperfusion injury was examined in mice. Intestinal ischemia produced by 45 min occlusion of superior mesenteric artery was followed by 24 h reperfusion (I/R). The 5-HT 2A selective antagonist, ketanserin (0.5 mg kg −1) or the 5-HT 2A agonist DOI (0.25 mg kg −1) was intravenously administered before ischemia and 8 h after the beginning of reperfusion. The effects were compared with those obtained in sham operated animals (S). Ketanserin prevented the upper gastrointestinal transit delay induced by I/R ( P < 0.01), protected intestine from leukocyte recruitment as indicated by jejunal myeloperoxidase activity ( P < 0.05) and reverted Evans Blue extravasation elicited by I/R in lung, colon and jejunum ( P < 0.05). On the other hand, 5-HT 2A activation by DOI mimicked the effects of I/R in S mice prolonging small intestine transit ( P < 0.05) and enhancing neutrophil accumulation in jejunal tissues ( P < 0.05). Furthermore, the reduction of ADP-induced platelet aggregation in plasma of I/R mice was prevented by ketanserin treatment. All together, these findings support the critical involvement of 5-HT 2A receptor subtype in mediating the damage induced by mesenteric I/R in mice.

Comparison of turbidimetric aggregation and in vitro bleeding time (PFA-100) for monitoring the platelet inhibitory profile of antiplatelet agents in patients undergoing stent implantation

The present study compared classical ADP-induced platelet aggregation vs. PFA-100 closure times using collagen/ADP membrane cartridges to monitor the degree of platelet-inhibiting effect of three drug regimens: ticlopidin, abciximab/ticlopidin and loading dose clopidogrel, each on top of aspirin (acetylsalicylic acid, ASA) during and after elective stent placement (intervention) in a total of 31 patients with acute coronary syndrome. Ticlopidin was started directly after stent implantation, abciximab was started before coronary intervention and given intravenously for 12 h, and a clopidogrel loading dose was given before intervention. The 10 patients treated with ticlopidin (500 mg daily) showed no significant prolongation of PFA closure times and a slight increase of ADP-induced platelet aggregation shortly after intervention. In 11 patients treated with abciximab/ticlopidin, the PFA closure times were significantly prolonged, and ADP-induced platelet aggregation was reduced by more than 80% during the 12-h abciximab infusion after intervention. The 10 patients pretreated with loading dose clopidogrel (450 mg followed by 75 mg daily) showed an intermediate but significant prolongation of PFA closure times and reduction of ADP-induced platelet aggregation at levels between the ticlopidin/aspirin- and the abciximab/ticlopidin/aspirin-treated groups. At 20 h after intervention, a similar degree of PFA closure time prolongation and inhibition of ADP-induced aggregation was observed in the abciximab/ticlopidin/aspirin- and the clopidogrel/aspirin-treated patient groups. Both measurement of PFA-100 closure times and inhibition of ADP-induced platelet aggregation showed a similar degree of platelet inhibition, but had rather broad SD ranges, which limit their precision for the follow-up of individual patients. In conclusion, abciximab on top of ticlopidin/aspirin showed a stronger antiplatelet effect for only less than 20 h, as compared to loading dose clopidogrel/aspirin in acute coronary syndrome patients undergoing stent implantation. Whether such a short-term superiority of abciximab, as compared to loading dose clopidogrel, translates into an overall clinical benefit of thombotic and bleeding complications remains to be established in a randomized clinical trial.

Fosinopril Reduces ADP-Induced Platelet Aggregation in Hypertensive Patients

Platelets are intimately involved in atherosclerosis, and hypertension is a known risk factor for coronary artery disease. The angiotensin-converting enzyme (ACE) inhibitors were demonstrated to reduce hypertension and attenuate atherosclerosis. Because increased platelet aggregation was shown in hypertensive patients, the effect of a new ACE inhibitor, fosinopril, on platelet aggregation was studied. Fosinopril therapy (10 mg/day for 4 weeks) in 18 male hypertensive patients showed > or = 31% reduction in ADP-induced platelet aggregation. In vitro studies showed that fosinopril had similar inhibitory effect on ADP-induced platelet aggregation. No inhibitory effect could be detected with collagen as the aggregating agent. Finally, inhibition of platelet aggregation by fosinopril was less effective in platelets derived from hypertensive patients as compared with platelets derived from normal subjects. We conclude that fosinopril possesses a significant inhibitory activity on ADP-induced platelet aggregation both in vitro and in vivo.

Fibrinogen degradation products generation is the major determinant of platelet inhibition induced by plasminogen activators in platelet-rich plasma

The platelet function defect induced by thrombolytic agents has been referred either to the degradation of platelet surface receptors or to the anti-aggregatory effect of fibrinogen degradation products (FgDPs). In the present study we have evaluated platelet aggregation induced by ADP, collagen and ristocetin after incubation of washed platelets or platelet-rich plasma (PRP) with plasmin (1.1–3.4IU/ml), plasminogen activators (PAs) (streptokinase 250–1000 IU/ml; urokinase, 10–1000 IU/ml; t-PA 0.5–10 μg/ml) or FgDPs (0.062–2 mg/ml). In parallel the surface levels of platelet GP lb and IIb/IIIa complex were determined by fluorescence flow cytometry using specific monoclonal antibody. Washed platelets treated with plasmin (1.1IU/ml) for 10 to 90 min showed a progressive reduction of ristocetin-induced platelet agglutination and a progressive reduction of surface GP Ib. Surface expression of GP IIb/IIIa complex was significantly increased after plasmin exposure. The addition of PAs to PRP resulted in a marked reduction of ADP-induced platelet aggregation. Collagen-induced platelet aggregation was only slightly affected. Similar changes were observed when PRP was preincubated with high concentrations of FgDPs. In PRP treated with PAs platelet surface levels of GP Ib and GP IIb/IIIa complex did not show any significant changes. In conclusion our results show that in plasma no proteolysis of platelet adhesive receptors occurs after plasminogen activation. The platelet inhibition observed after incubation of PRP with PAs is likely to be caused by FgDPs generation.

The effect of phospholipase C on rat blood platelets in vivo.

Rat blood platelets were treated with phospholipase C in vitro or phospholipase C was injected i.v. to rats. In both cases its effect on the functions of the platelets in vivo has been studied. No change was found in primary bleeding time or in platelet survival. Treatment with phospholipase C gave a moderate reduction of ADP-induced platelet aggregation in the pulmonary circulation whereas the aggregation induced by thrombin was unchanged. Iv. injection of phospholipase C caused a rapid, very moderate and transient increase of 51Cr-activity in the lungs without concomitant overt respiratory distress. A moderate increase in 51Cr-activity was noted in liver and kidney 24 and 48 h after injection of phospholipase C. This may be caused by a slightly increased leakage of 51Cr-labelled material from the platelets during exposure to phospholipase C.