70 research outputs found
Recommended from our members
P2Y12 Receptor Blockade Augments Glycoprotein IIbāIIIa Antagonist Inhibition of Platelet Activation, Aggregation, and Procoagulant Activity
Background: New antiplatelet agents that provide greater, more consistent inhibition of the platelet ADP receptor P2Y12 may be used in combination with glycoprotein (GP) IIbāIIIa antagonists, but their combined effect on platelet function and procoagulant activity is not well studied. Therefore, the objective of this study was to evaluate the independent and complementary effects of P2Y12 and GPIIbāIIIa inhibition on platelet function and procoagulant activity. Methods and Results: Healthy donor blood was treated with the active metabolite of prasugrel (Rā138727 5 Ī¼mol/L), GPIIbāIIIa antagonists (abciximab 3 Ī¼g/mL or eptifibatide 0.9 Ī¼g/mL), and combinations thereof, exposed to physiologically relevant agonists (collagen and ADP) and then evaluated for markers of platelet activation and procoagulant activity. Significant interactions between Rā138727 and GPIIbāIIIa antagonists were observed. Rā138727 and the GPIIbāIIIa antagonists had additive inhibitory effects on collagenāstimulated platelet aggregation and on the collagen plus ADPāstimulated level of activated platelet surface GPIIbāIIIa. Rā138727 and abciximab each inhibited collagen plus ADPāstimulated platelet phosphatidylserine expression and prothrombin cleavage, and the combination produced greater inhibition than achieved with abciximab alone. In contrast, eptifibatide did not inhibit, but instead enhanced, collagen plus ADPāstimulated prothrombin cleavage. Addition of Rā138727 reduced prothrombin cleavage in eptifibatideātreated samples, suggesting a novel mechanism for potential benefit from combined prasugrel and eptifibatide treatment. Conclusions: The complementary effects of abciximab and Rā138727 on platelet activation, aggregation, and procoagulant activity suggest their combined use may, to a greater degree than with either agent alone, reduce thrombus formation in vivo
Genetic variation of CYP2C19 affects both pharmacokinetic and pharmacodynamic responses to clopidogrel but not prasugrel in aspirin-treated patients with coronary artery disease
The metabolic pathways leading to the formation of prasugrel and clopidogrel active metabolites differ. We hypothesized that decreased CYP2C19 activity affects the pharmacokinetic and pharmacodynamic response to clopidogrel but not prasugrel. Ninety-eight patients with coronary artery disease (CAD) taking either clopidogrel 600 mg loading dose (LD)/75 mg maintenance dose (MD) or prasugrel 60 mg LD/10 mg MD were genotyped for variation in six CYP genes. Based on CYP genotype, patients were segregated into two groups: normal function (extensive) metabolizers (EM) and reduced function metabolizers (RM). Plasma active metabolite concentrations were measured at 30 min, 1, 2, 4, and 6 h post-LD and during the MD period on Day 2, Day 14, and Day 29 at 30 min, 1, 2, and 4 h. Vasodilator-stimulated phosphoprotein (VASP) and VerifyNow (TM) P2Y12 were measured predose, 2, and 24 +/- 4 h post-LD and predose during the MD period on Day 14 +/- 3 and Day 29 +/- 3. For clopidogrel, active metabolite exposure was significantly lower (P = 0.0015) and VASP platelet reactivity index (PRI, %) and VerifyNow (TM) P2Y(12) reaction unit (PRU) values were significantly higher (P < 0.05) in the CYP2C19 RM compared with the EM group. For prasugrel, there was no statistically significant difference in active metabolite exposure or pharmacodynamic response between CYP2C19 EM and RM. Variation in the other five genes demonstrated no statistically significant differences in pharmacokinetic or pharmacodynamic responses. Variation in the gene encoding CYP2C19 in patients with stable CAD contributes to reduced exposure to clopidogrel's active metabolite and a corresponding reduction in P2Y(12) inhibition, but has no significant influence on the response to prasugrel
Evaluation of the INNOVANCE PFA P2Y test cartridge:sensitivity to P2Y(12) blockade and influence of anticoagulant
In vitro effects of recombinant activated factor VII on thrombin generation and coagulation following inhibition of platelet procoagulant activity by prasugrel
Introduction Prasugrel is a thienopyridyl P2Y12 antagonist with potent antiplatelet effects. At present, little is known about its effects on thrombin generation or what strategies may emergently reverse its anticoagulant effects. In the current study we evaluated whether recombinant activated factor VII may reverse prasugrel induced effects and increase thrombin generation in an in vitro model. Methods The effect of prasugrel active metabolite, PAM (R-138727), was evaluated on platelet aggregation, thrombin generation, and rotational thromboelastometry parameters using blood from 20 healthy volunteers. Additionally, we evaluated the effects of adenosine diphosphate (ADP) and recombinant activated factor VII on restoring these parameters towards baseline values. Results PAM reduced maximum platelet aggregation and led to platelet disaggregation. It also decreased peak thrombin, increased lag time, and increased time to peak thrombin. Treatment with recombinant activated factor VII restored all three parameters of thrombin generation towards baseline. ADP decreased lag time and time to peak thrombin, but had no effect on peak thrombin. When recombinant activated factor VII and ADP were combined they had a greater effect on thrombin parameters than either drug alone. PAM also increased thromboelastometric clotting time and clot formation time, but had no effect on maximum clot firmness. Treatment with either recombinant activated factor VII or ADP restored these values towards baseline. Conclusions Recombinant activated factor VII restores thrombin generation in the presence of PAM. In patients taking prasugrel with life-threatening refractory bleeding it has the potential to be a useful therapeutic approach. Additional clinical studies are needed to validate our findings. Ā© 2013 Elsevier Ltd. All rights reserved
Correlation between human platelet cytoplasmic membrane outer leaflet fluidity, Na+/H+ exchanger activity and aging
Abstract A cross-sectional survey was undertaken to study the aging effect on platelet cytoplasmic membrane outer leaflet fluidity and Na+/H+ antiporter activity, and to investigate the relative influence of membrane order and age on the Na+/H+ exchanger. The study population consisted of 19 healthy subjects (age range 22ā83 years, mean age 48.5 Ā± 16.5 years; 12 females and 7 males). Washed platelets were used as neuronal models and as eventually contributing to brain pathology by aberrant secretion of their granule content. A significant positive correlation (r = 0.48,p < 0.05) was found between age and the membrane structural order, according to a second-degree equation. Sewall-Wright path coefficient analysis revealed that the age influence on Na+/H+ exchanger activity was unimportant (0.18), but those of cytoplasmic membrane outer leaflet fluidity and mean platelet volume were strong and positive (0.51 and 0.54, respectively). Women presented a marked standard deviation in the countertransporter results. In conclusion, decreased membrane outer leaflet fluidity and unchanged Na+/H+ antiporter activity may contribute to the process of normal brain aging
A Novel Model of Intravital Platelet Imaging Using CD41-ZsGreen1 Transgenic Rats
<div><p>Platelets play pivotal roles in both hemostasis and thrombosis. Although models of intravital platelet imaging are available for thrombosis studies in mice, few are available for rat studies. The present effort aimed to generate fluorescent platelets in rats and assess their dynamics in a rat model of arterial injury. We generated CD41-ZsGreen1 transgenic rats, in which green fluorescence protein ZsGreen1 was expressed specifically in megakaryocytes and thus platelets. The transgenic rats exhibited normal hematological and biochemical values with the exception of body weight and erythroid parameters, which were slightly lower than those of wild-type rats. Platelet aggregation, induced by 20 Ī¼M ADP and 10 Ī¼g/ml collagen, and blood clotting times were not significantly different between transgenic and wild-type rats. Saphenous arteries of transgenic rats were injured with 10% FeCl<sub>3</sub>, and the formation of fluorescent thrombi was evaluated using confocal microscopy. FeCl<sub>3</sub> caused time-dependent increases in the mean fluorescence intensity of injured arteries of vehicle-treated rats. Prasugrel (3 mg/kg, p.o.), administered 2 h before FeCl<sub>3</sub>, significantly inhibited fluorescence compared with vehicle-treated rats (4.5 Ā± 0.4 vs. 14.9 Ā± 2.4 arbitrary fluorescence units at 30 min, respectively, n = 8, <i>P</i> = 0.0037). These data indicate that CD41-ZsGreen1 transgenic rats represent a useful model for intravital imaging of platelet-mediated thrombus formation and the evaluation of antithrombotic agents.</p></div
Actin polymerization stimulated by contractile activation regulates force development in canine tracheal smooth muscle
The role of actin polymerization in the regulation of smooth muscle contractility was investigated in canine trachealis muscle strips. The effect of contractile activation on the content of monomeric globular (G)-actin was estimated by the method of DNase I inhibition. The G-actin content was 30 % lower in extracts of muscle strips activated with 10ā4 M acetylcholine (ACh) than in extracts from unstimulated muscle strips. The decrease in G-actin in response to contractile stimulation was prevented by latrunculin-A, an agent that prevents actin polymerization by binding to G-actin monomers.The inhibition of actin polymerization by latrunculin-A markedly depressed force development in response to ACh but had no effect on ACh-induced myosin light chain (MLC) phosphorylation. Latrunculin also suppressed the length sensitivity of force during ACh-induced isometric contractions. The actin-capping agent cytochalasin-D also markedly inhibited force and caused only a slight decrease in MLC phosphorylation. Cytochalasin-D also inhibited force in Ī±-toxin-permeabilized muscle strips that were activated either by Ca2+ or by ACh at constant pCa. No disorganization of smooth muscle cell ultrastructure was detected by electron microscopy or by immunofluorescence microscopy of muscles treated with either agent.The results suggest that the polymerization of actin is stimulated by the contractile activation of tracheal smooth muscle and that this actin polymerization contributes directly to force development. In addition, actin filament remodelling contributes to the length sensitivity of tracheal smooth muscle contractility
Representative ADP- and collagen-induced platelet aggregation tracings of CD41-ZsGreen1 transgenic rats.
<p>Citrated blood was collected from the abdominal aorta and centrifuged to obtain platelet-rich plasma. Platelet aggregation induced by ADP (20 Ī¼M) and collagen (10 Ī¼g/mL) was measured using light transmission aggregometry. WT: wild-type Sprague-Dawley rats, TG: CD41-ZsGreen1 transgenic rats, ADP: adenosine 5ā-diphosphate.</p
Specific expression of green fluorescent protein in bone marrow megakaryocytes of CD41-ZsGreen1 transgenic rats.
<p>Bone marrow from the femur was excised, fixed with 10% buffered formalin, and embedded in paraffin. Sections were prepared and stained with DAPI or HE. Arrows in the HE photograph indicate megakaryocytes. WT: wild-type Sprague-Dawley rats, TG: CD41-ZsGreen1 transgenic rats, HE: hematoxylin-eosin, DAPI: 4ā²,6-diamidino-2-phenylindole.</p
- ā¦