Late Aggregation Research Articles

Real-Time BODIPY-Binding Assay To Screen Inhibitors of the Early Oligomerization Process of Aβ1-42 Peptide.

Misfolding and aggregation of amyloid β1–42 peptide (Aβ1–42) play a central role in the pathogenesis of Alzheimer's disease (AD). Targeting the highly cytotoxic oligomeric species formed during the early stages of the aggregation process represents a promising therapeutic strategy to reduce the toxicity associated with Aβ1–42. Currently, the thioflavin T (ThT) assay is the only established spectrofluorometric method to screen aggregation inhibitors. The success of the ThT assay is that it can detect Aβ1–42 aggregates with high β‐sheet content, such as protofibrils or fibrils, which appear in the late aggregation steps. Unfortunately, by using the ThT assay, the detection of inhibitors of early soluble oligomers that present a low β‐sheet character is challenging. Herein, a new, facile, and robust boron‐dipyrromethene (BODIPY) real‐time assay suitable for 96‐well plate format, which allows screening of compounds as selective inhibitors of the formation of Aβ1–42 oligomers, is reported. These inhibitors decrease the cellular toxicity of Aβ1–42, although they fail in the ThT assay. The findings have been confirmed and validated by structural analysis and cell viability assays under comparable experimental conditions. It is demonstrated that the BODIPY assay is a convenient method to screen and discover new candidate compounds that slow down or stop the pathological early oligomerization process and are active in the cellular assay. Therefore, it is a suitable complementary screening method of the current ThT assay.

Aggregation of disease-related peptides.

Protein misfolding and aggregation of amyloid proteins is the fundamental cause of more than 20 diseases. Molecular mechanisms of the self-assembly and the formation of the toxic aggregates are still elusive. Computer simulations have been intensively used to study the aggregation of amyloid peptides of various amino acid lengths related to neurodegenerative diseases. We review atomistic and coarse-grained simulations of short amyloid peptides aimed at determining their transient oligomeric structures and the early and late aggregation steps.

Dynamics of elastin in liver fibrosis: Accumulates late during progression and degrades slowly in regression.

Elastin is an amorphous protein highly resistant to elastase degradation and is believed to be the most stable component among the extracellular matrix (ECM) members. Thus the excessive deposition of elastin in advanced liver fibrosis may contribute to the declining reversibility of the disease. Our previous study has found that elastin crosslinking inhibition can effectively arrest liver fibrosis progression. To further understand the roles of elastin involved in liver fibrosis, we systematically investigated the expression, accumulation, and degradation based on dynamic and bidirectional CCl4 -induced liver fibrosis mouse models and visualized the ultrastructure of elastin globules in cultured LX-2 cells. We found that the expression pattern of tropoelastin (soluble elastin) and collagen I was not completely comparable at both the transcriptional and posttranscriptional levels during liver fibrosis progression and regression. Elastin mainly accumulated onto the internodular fibrous septa and enlarged portal areas and intertwined with collagen I at the late stage of liver fibrosis. Three-dimensional analysis of elastin and collagen I by confocal immunofluorescence coupled with biochemical analyses revealed that with respect to collagen, elastin deposition was characterized by late aggregation in progression and slow turnover in regression. In addition, we visualized the dynamic ultrastructure of ECM fibers during liver fibrogenesis and fibrolysis and the ultrastructure of elastin globules self-aggregated by tropoelastin crosslinking. Our current study established new general hallmarks of elastin levels and forms in progressive and regressive liver fibrosis and provided a foundation for further experimental investigation of the growing role of elastin in liver fibrosis regression.

Small Molecules Attenuate the Interplay between Conformational Fluctuations, Early Oligomerization and Amyloidosis of Alpha Synuclein

Aggregation of alpha synuclein has strong implications in Parkinson’s disease. The heterogeneity of folding/aggregation landscape and transient nature of the early intermediates result in difficulty in developing a successful therapeutic intervention. Here we used fluorescence measurements at ensemble and single molecule resolution to study how the late and early events of alpha synuclein aggregation modulate each other. In-vitro aggregation data was complemented using measurements inside live neuroblastoma cells by employing a small molecule labeling technique. An inhibitor molecule (arginine), which delayed the late event of amyloidosis, was found to bind to the protein, shifting the early conformational fluctuations towards a compact state. In contrast, a facilitator of late aggregation (glutamate), was found to be excluded from the protein surface. The presence of glutamate was found to speed up the oligomer formation at the early stage. We found that the effects of the inhibitor and facilitator were additive and as a result they maintained a ratio at which they cancelled each other’s influence on different stages of alpha synuclein aggregation.

Medicinal importance of Darbha- A review

Darbha is an important medicinal plant which has been used in Indian system of medicine since antiquity for the treatment of various ailments. There is enormous controversy between Kusha and Darbha in Ayurveda literature. Though the synonyms, actions and indications of Kusha, Darbha are mentioned together in different texts, the most of the Nighantus accepted both Kusha and Darbha as two different varieties and mentioned as synonyms for each other. Present study aimed to extensive review on the drug from Ayurveda as well as modern points of views. The review shows in spite of great controversy for botanical identity of this plant, most of the authors has considered Darbha as Imperata cylindrica Beauv., belongs to family Graminae. It contains various active principles like Cylindrine, Arundoin, Fermenol, Isoarborinol, Semiarenol, Cylindol A & B Gramenone A & B, Imperanene etc. On The basis of experimental and clinical studies conducted in various research centers, the drug possess various properties like diuretic, tonic, antipyretic, sedative, astringent, antiviral, vasodilator, vascular smooth muscle contraction inhibitory, plate late aggregation inhibitory properties and useful in hypertension, calculi, snake bite, thirst, gonorrhea, jaundice, diseases of blood, bladder and skin. Study may provide the scope of further research in different pharmacological area of this important medicinal plant.

Effects of P2Y1 receptor antagonism on the reactivity of platelets from patients with stable coronary artery disease using aspirin and clopidogrel

P2Y(1) is a purine receptor that triggers platelet aggregation. Its inhibition was studied in patients with stable coronary artery disease (CAD) receiving standard anti-platelet therapy. Blood samples from 10 patients on aspirin therapy (ASA, 80 mg·day(-1) ) were withdrawn before and 24 h after the administration of 450 mg clopidogrel (ASA/C) and were anti-coagulated with citrate or hirudin/PPACK in the presence or absence of the P2Y(1 ) inhibitor MRS2179 (M, 100 µM). Platelet responses to ADP (2.5 µM) and TRAP (2.5 µM), and collagen-induced thrombosis under flow conditions were analysed. Compared with ASA, ASA + M strongly inhibited ADP-induced peak platelet aggregation (88%), late aggregation (84%), P-selectin expression (85%) and α(IIb) β(3) activation (62%) (28%, 65%, 70% and 51% inhibition, respectively, for ASA/C vs. ASA). ASA + M also inhibited platelet/monocyte and platelet/neutrophil conjugate formation by 69% and 71% (57% and 59% for ASA/C vs. ASA). In TRAP-activated blood, ASA + M unexpectedly inhibited α(IIb) b(3) activation by 30%. In blood perfused in collagen-coated glass capillaries (shear rate of 1500 s(-1) ), ASA/C prevented thrombus growth beyond 5 min in relation to thrombus fragments embolization. ASA + M with or without clopidogrel completely prevented thrombus formation. Finally, ex vivo addition of MRS2179 and ASA to the blood of healthy donors markedly blocked thrombus formation on collagen in flow conditions, in contrast to ASA plus the P2Y(12) inhibitor 2-MeSAMP. Through particularly efficient complementarities with ASA to inhibit platelet activation and thrombus formation, the inhibition of P2Y(1) in the blood of patients with CAD appears to play a more important role than previously anticipated.

Both peak and late aggregation are capable of identifying patients at risk for atherothrombotic events

Both peak and late aggregation are capable of identifying patients at risk for atherothrombotic events -

Comparison of conventional aggregomerty with VASP for monitoring P2Y12-specifc platelet inhibition

No consensus exists regarding the optimal estimate of light transmission aggregometry (LTA) to reflect P2Y12 ADP receptor inhibition in patients receiving thienopyridine therapy. Currently, the only completely P2Y12-receptor specific method is the measurement of vasodilator stimulated phosphoprotein (VASP) phosphorylation (PRI) with flow cytometry. In the current analysis, we aimed to test the superiority of the late platelet aggregation value over other estimates of light transmission aggregometry in determining P2Y12-receptor inhibition by comparing them to VASP-PRI. On-clopidogrel platelet reactivity was measured in 121 clopidogrel-naïve patients who underwent elective coronary stent implantation. Samples for LTA and VASP assessments were drawn at 12–18 hours after a 600-mg loading dose of clopidogrel and 25 days after the intervention. ADP 5 µM-induced maximal aggregation (AGGmax), 6-minute late aggregation (AGGlate), 6-minute disaggregation (disAGG) and area under the aggregation curve (AUC) were compared to VASP-PRI. Categorical agreement with VASP-defined normal and high platelet reactivity (HPR: PRI≥50%) was calculated according to the optimal cutoff values obtained with receiver-operating characteristic (ROC) curves. The evaluation of 242 measurements showed significant, moderate-strength correlations between VASP-PRI and LTA estimates without the superiority of AGGlate over other estimates (AGGmax: ρ = 0.47; AGGlate: ρ = 0.45; disAGG: ρ = −0.44; AUC: ρ = 0.50). Notably, there were considerable intra-individual differences between VASP and LTA testing. LTA estimates were similar in classifying patients to VASP-defined normal or HPR categories (AGGmax: κ = 0.41; AGGlate: κ = 0.45; disAGG: κ = 0.44; AUC: κ = 0.44). When all estimates of LTA were compared in multivariable models, AUC proved to be the independent linear determinant of VASP-PRI and the best predictor of HPR. Estimates of LTA seem equal in determining the degree of P2Y12-receptor inhibition or in predicting VASP-defined HPR without the superiority of AGGlate over others. These results reject a commonly used hypothesis and might contribute to the standardization of the LTA assay.

Adding Cilostazol to Dual Antiplatelet Therapy Achieves Greater Platelet Inhibition than High Maintenance Dose Clopidogrel in Patients With Acute Myocardial Infarction

Background— Optimal platelet inhibition is an important therapeutic adjunct in patients acute myocardial infarction (AMI) undergoing coronary stenting. Whether adjunctive cilostazol to dual antiplatelet therapy (triple antiplatelet therapy) can inhibit enhanced platelet reactivity in patients with AMI yet has not been determined. The aim of this study was to assess the degree of platelet inhibition by triple antiplatelet therapy in patients with AMI. Methods and Results— Immediately after emergency room arrival, patients with AMI received clopidogrel (600-mg loading dose, followed by 75 mg daily) and aspirin (300-mg loading dose and 200 mg daily throughout the study period). After patients underwent coronary stenting (n=90), they were randomly assigned to 1 of 3 groups before discharge: standard group, clopidogrel of 75 mg daily (n=30); high maintenance dose (MD) group, clopidogrel of 150 mg daily (n=30); and triple group, adjunctive cilostazol of 100 mg twice daily to clopidogrel of 75 mg daily (n=30). Platelet reactivity was assessed at predischarge and 30-day follow-up by conventional aggregometry and the VerifyNow P2Y12 assay. Predischarge platelet reactivities were similar in the 3 groups. At 30-day follow-up, inhibition of maximal aggregation with 20 μM ADP stimuli was 6.0% in the standard group, 19.1% in the high-MD group, and 42.4% in the triple group ( P <0.001), whereas inhibition of late aggregation with 20μM ADP stimuli was 10.8%, 38.1%, and 66.4%, respectively ( P <0.001). Similar results were demonstrated when 5 μM ADP was used. Furthermore, percent changes of P2Y12 reaction unit were significantly different among regimens (10.6% in the standard group, 30.7% in the high-MD group, and 43.0% in the triple group; P <0.001). With respect to high-postclopidogrel platelet reactivity (prespecified as 20 μM ADP-induced maximal aggregation >50% of light transmission), fewer patients in the triple group (13.3%) met the criteria as compared with those in the standard (76.7%) and high-MD groups (56.7%) at 30-day follow-up ( P <0.001). In the triple group, there were more potent and consistent platelet inhibitions by all parameters as compared with the high-MD group except for percent changes of P2Y12 reaction unit ( P =0.071). Conclusions— Among patients with AMI undergoing coronary stenting, triple antiplatelet therapy results in a greater antiplatelet effect at 30 days as compared with a high-MD clopidogrel or standard dual antiplatelet therapy.

Variability of residual platelet function despite clopidogrel treatment in patients with peripheral arterial occlusive disease

Residual platelet function despite treatment with clopidogrel may predict an unfavourable cardiovascular outcome. The majority of studies have investigated the effects of clopidogrel administration in conjunction with aspirin in patients undergoing percutaneous coronary intervention. The primary objective of the present study was to assess the platelet response to clopidogrel in the absence of aspirin in patients with peripheral arterial occlusive disease (PAOD) and to investigate whether non-responsiveness to clopidogrel is reproducible during long-term follow-up. Fifty-four clinically stable PAOD patients on a maintenance dose of 75 mg/d clopidogrel were enrolled in this study. Platelet function was assessed at baseline and after a median follow-up of 18 months using light transmittance aggregometry (LTA) with 2 μM ADP as an agonist. HPLC-coupled mass spectrometry was used to detect clopidogrel and clopidogrel carboxylic acid, the main metabolite of clopidogrel. Residual platelet function, as defined by late aggregation values within the reference range (i.e., >43%), was observed in 35.2% of patients at baseline and 17.6% during follow-up. During the observation period, 26.5% had switched from responder to non-responder status or vice versa. Among non-responders, either clopidogrel or its metabolite was detected in 89.5% and 83.3% of patients at baseline and at follow-up, respectively. We conclude that non-responsiveness to clopidogrel as determined by ADP-induced LTA is not stable over time. This phenomenon cannot be attributed to non-compliance alone.

Low platelet disaggregation predicts poor response to 150 mg clopidogrel in patients with elevated platelet reactivity

Raising the maintenance dose of clopidogrel to 150 mg enhances platelet inhibition (PI) in patients with elevated platelet reactivity (EPR); however, large differences were observed between individuals in the extent of this benefit. We aimed to find clinical and platelet function variables that might predict the response to 150 mg clopidogrel in patients with EPR. ADP 5 µM-stimulated peak aggregation (Aggmax), 6-minute late aggregation (Agglate) and 6-minute disaggregation (disAgg) were measured with light transmission aggregometry (LTA) in a consecutive cohort of 202 patients admitted for emergent or elective percutaneous coronary intervention (PCI) after receiving a 600-mg loading dose of clopidogrel. PI was assessed 12 to 18 hours after loading dose and 30 days after the intervention. EPR was defined as a Aggmax value greater than 34%. From the first day of PCI, 85 patients with EPR received 150 mg clopidogrel for 30 days, while others with normal platelet reactivity (NPR) took 75 mg clopidogrel. Baseline clinical and LTA variables were analyzed in multivariable regression models. Administration of 150 mg clopidogrel enhanced PI and decreased the prevalence of EPR at 30 days compared to the peri-interventional period (Aggmax: 46.9 ± 8.3 vs. 37.3 ± 11.5; EPR: 100% vs. 62.4%; p < 0.001 in both cases); however, the achieved level of platelet reactivity was higher than patients with NPR (Aggmax: 20.6 ± 8.1 vs. 25.3 ± 10.8, p < 0.001). Multivariable logistic regression revealed low platelet disaggregation (odds ratio (OR): 12.49; 95% CI: 2.52–62.00; P = 0.002) and acute coronary syndrome (OR: 4.83; 95% CI: 1.54–15.09; P = 0.008) as independent predictors of EPR. The area under the receiver-operating-characteristic curve was 0.72 ± 0.06 for disaggregation to predict NPR after 150 mg clopidogrel. The optimal disaggregation cut-off was 16.5% with a sensitivity of 94% and a specificity of 43%. Administering 150 mg maintenance dose of clopidogrel enhanced antiplatelet potency; however, more than half of the patients persisted with EPR. Acute coronary syndrome and low (<16.5%) platelet disaggregation are independent predictors of poor benefit from dose shift.

Insights into the interpretation of light transmission aggregometry for evaluation of platelet aggregation inhibition by clopidogrel

Introduction When studying the efficacy of clopidogrel to inhibit platelet aggregation by light transmission aggregometry, technical decisions must be taken prior to assessment or during analysis, including, but not limited to, concentration of agonist to use and timing of the evaluation of the response on the aggregation curve obtained (peak ADP-stimulated platelet aggregation vs. late aggregation). We investigated how some of these technical modalities affected the results of platelet aggregation obtained after clopidogrel administration. Materials and methods One hundred and twenty stable coronary artery disease patients requiring a diagnostic angiography were recruited prior to pre-treatment with clopidogrel. Blood samples were tested before clopidogrel initiation and immediately preceding coronary angiography using light transmission aggregometry with either 5 or 20 µM of ADP. Aggregation was measured at maximal amplitude (peak), and 5 minutes after agonist addition (late). Results While measurements of platelet aggregation as either peak or late aggregation were strongly correlated, peak platelet aggregation was significantly higher than late aggregation, by 10.8% and by 10.3% with ADP 5 and 20 µM, respectively. Moreover, the use of ADP 20 µM resulted in less spontaneous disaggregation than 5 µM in the absence of clopidogrel (11.8% and 4.8% with ADP 5 µM and 20 µM, respectively). Conclusions When assessing platelet aggregation following clopidogrel, measurement of late aggregation after addition of ADP 20 µM should be preferred. Large clinical trials should be conducted to assess which parameter between residual aggregation or inhibition of platelet aggregation by clopidogrel best predicts clinical efficacy of the drug.

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.).

Abstract 2014: Additive Cilostazol to Dual Antiplatelet Therapy Achieves Greater Inhibition of Platelet Aggregation Compared With High Maintenance-Dose Clopidogrel in Patients With Acute Myocardial Infarction

Objectives The aim of study was to compare platelet inhibition by additive cilostazol to dual antiplatelet therapy (triple antiplatelet therapy) and high maintenance-dose (MD) clopidogrel 150mg/d in patients with acute myocardial infarction (AMI). Methods AMI patients undergoing successful stenting (n =90) were randomly assigned to 1 of 3 MD regimens: Standard group, clopidogrel 75 mg/d; High MD group, clopidogrel 150 mg/d; Triple group, additive cilostazol 100 mg twice daily. Platelet functions were evaluated before discharge and 30 days after discharge. Percent inhibition (PI) and a rate of poor responder to clopidogrel were assessed, based on maximal and late platelet aggregation (Agg max and Agg late ). Results Baseline platelet measures were similar in 3 groups. There was more potent and consistent PI by Triple group in all platelet measures. PI of 5 uM ADP-induced Agg max was 3.5% in Standard group, 22.6% in High MD group, and 44.1% in Triple group (p &lt; 0.001). In addition, PI of 5 uM ADP-induced Agg late was 12.3%, 31.5%, and 65.0% in each group, respectively (p &lt; 0.001). Similar results were demonstrated when 20 uM ADP and 6 ug/ml collagen were used. In terms of poor response to clopidogrel (5 and 20 uM ADP-induced Agg max &gt; 50%), fewer patients in triple group (4.3% and 23.3%) met a criteria than those in standard group (23.3% and 73.3%) and high MD group (16.7% and 60.0%) at 30 days (p = 0.028 and &lt; 0.001, respectively). All parameter between Triple and High MD groups were significantly different. Conclusions Triple antiplatelet therapy achieves greater platelet inhibition and lesser rate of poor response to clopidogrel compared with high MD clopidogrel in AMI patients.

Clopidogrel 600-Mg Double Loading Dose Achieves Stronger Platelet Inhibition Than Conventional Regimens: Results From the PREPAIR Randomized Study

The objective of this study was to compare the level of platelet inhibition achieved by 3 different clopidogrel loading regimens in patients undergoing elective angiography and percutaneous coronary intervention when appropriate. Optimal platelet inhibition is a key therapeutic goal for patients undergoing percutaneous coronary intervention. Although 600 mg has been described as the maximum absorbed dose when given as a single bolus, the effects of 2 boluses given 24 h apart have not been described. Patients (n = 148) were randomly assigned to one of 3 regimens: Group A, clopidogrel 300 mg the day before (>or=15 h) + 75 mg the morning of the procedure; Group B, clopidogrel 600 mg the morning of the procedure (>or=2 h); and Group C, clopidogrel 600 mg the day before (>or=15 h) and 600 mg the morning of the procedure (>or=2 h). Blood samples were obtained at baseline and immediately before angiography. Peak and late platelet aggregation were measured in platelet rich plasma, with researchers blinded to treatment allocation. There was a consistent difference favoring Group C in all aggregation parameters. Percent inhibition in Groups A, B, and C was 31.4%, 29.0%, and 49.5%, respectively, for peak aggregation (5 micromol/l adenosine diphosphate; p < 0.0001) and 54.1%, 57.7%, and 81.1%, respectively, for late aggregation (p < 0.0001). Similar striking reductions were observed when 20 micromol/l adenosine diphosphate was used. All comparisons between Group C and the other 2 groups were statistically significant, and those between Groups A and B were not. Clopidogrel 600-mg double bolus achieves greater platelet inhibition than conventional single loading doses.

Standardization of light transmittance aggregometry for monitoring antiplatelet therapy: an adjustment for platelet count is not necessary.

Light transmittance aggregometry (LTA) is considered to be the 'gold standard' of platelet function testing. As LTA has been poorly standardized, we analyzed the results of LTA in healthy subjects and patients with antiplatelet therapy using different concentrations of agonists and performing tests in non-adjusted and platelet count-adjusted platelet-rich plasma (PRP). LTA was performed in 20 healthy subjects and in patients treated with aspirin (n = 30) or clopidogrel (n = 30) monotherapy, as well as in patients on combination therapy (n = 20), using arachidonic acid (ARA 0.25 and 0.5 mg mL(-1)) and adenosine diphosphate (ADP 2 and 5 microm) as agonists and performing platelet function tests in non-adjusted and platelet count (250 nL(-1) +/- 10%)-adjusted PRP. The overall platelet aggregation response is decreased after adjusting the PRP for platelet count compared with measurements in unadjusted PRP. The variability of aggregation results is high in adjusted PRP in the subgroup of healthy subjects, ranging from 9.2-95.3% (5th-95th percentile) relative to 77.6-95.5% in non-adjusted PRP when determining maximum aggregation to ARA 0.5 mg mL(-1). Late aggregation using ADP 2 microm ranges from 3.8-89.9% in adjusted PRP compared with 42.9-92.5% in non-adjusted PRP. Maximum aggregation using ARA 0.5 mg mL(-1) in non-adjusted PRP differentiates between aspirin-treated patients and healthy controls well, whereas late aggregation using ADP 2 microm in non-adjusted PRP offers the best discrimination between clopidogrel-treated patients and healthy controls. Adjustment of PRP for platelet count does not provide any advantage and therefore the time-consuming process of platelet count adjustment is not necessary.

Biomarker Optimization to Track the Antithrombotic and Hemostatic Effects of Clopidogrel in Rats

We determined the dose response of the ADP antagonist clopidogrel (0.3-50 mg/kg p.o.) in rat models of thrombosis and provoked bleeding and correlated these activities to ex vivo platelet activation. Carotid artery thrombosis was induced by FeCl(2). Bleeding time was measured by mesenteric vessel puncture and renal cortex or cuticle incision. Platelet biomarkers included standard ADP-induced aggregation, P2Y(12) receptor occupancy, and phosphorylation of vasodilator-stimulated phosphoprotein. Clopidogrel decreased thrombus weight up to 78%, caused maximal prolongation of cuticle and mesenteric bleeding, but had little effect on renal bleeds. Due to the steep mesenteric dose response, further comparisons concentrated on cuticle bleeding. The half-maximal inhibitory dose (ED(50)) for thrombus reduction was 2.4 +/- 0.4 mg/kg, with 10 mg/kg providing optimal blood flow preservation and thrombus reduction. The ED(50) for bleeding was 10.5 +/- 3.4 mg/kg. Increased bleeding was intermediate (3-fold) at 10 mg/kg and maximal (6-fold) at 30 mg/kg. All biomarkers were affected, but with differing sensitivity. ED(50)s for peak platelet aggregation to 10 microM ADP (11.9 +/- 0.4 mg/kg) and the vasodilator-stimulated phosphoprotein index (16.4 +/- 1.3 mg/kg) approximated the higher ED(50) for bleeding. ED(50)s for ligand binding (3.0 +/- 0.3 mg/kg) and late aggregation (5.1 +/- 0.4 mg/kg) better matched the lower ED(50) for antithrombotic activity. Aspirin exerted lesser effects on bleeding (42-70% increase in all models) and thrombosis (24% inhibition). In summary, antithrombotic doses of clopidogrel have limited effects on bleeding and standard measures of platelet aggregation. Other biomarkers may be more sensitive for tracking antithrombotic efficacy.

Matching the Evaluation of the Clinical Efficacy of Clopidogrel to Platelet Function Tests Relevant to the Biological Properties of the Drug

This study aimed to explore platelet function tests relevant to the biological effects of clopidogrel that could help the clinical monitoring of drug efficacy. Clopidogrel selectively inhibits the P2Y12 receptor, the major role of which is stabilization of aggregation, whereas initiation of aggregation depends on activity of both P2Y1 and P2Y12 receptors. Tests used were peak aggregation (Agg(max)) and late aggregation (Agg(6min)), and disaggregation, relating to P2Y1 and P2Y12 activity, respectively; and monoclonal antibody binding activated glycoprotein (GP) IIb/IIIa receptors (PAC-1) and P-selectin, measuring activation and secretion. A first study compared hirudin/PPACK (r-hirudin and D-phenylalanyl-prolyl-arginine chloromethyl ketone) with citrate as blood anticoagulant (16 patients), and a second control study compared the effects of clopidogrel, aspirin, or both (20 normal controls). Clopidogrel similarly inhibited adenosine 5'-diphosphate (ADP)-induced Agg(max) with either anticoagulant, but significantly more Agg(6min) (75% vs. 31%), P-selectin (72% vs. 53%), and PAC-1 (62% vs. 24%) in hirudin/PPACK. In the control study, it inhibited Agg(max) by 22%, and Agg(6min), P-selectin, and PAC-1, by 69%, 66%, and 55%, respectively (all p < 0.05). Disaggregation at six min reached 62% with clopidogrel, but was virtually absent with placebo and aspirin. Non-responsiveness as evaluated by inhibition of Agg(max) in citrate was diagnosed in 35% of patients; in half this rate by Agg(6min), P-selectin, and PAC-1; and in 6% to 12% with the latter tests performed in hirudin/PPACK. The evaluation of clopidogrel responsiveness by platelet function tests is largely influenced by the choice of blood preservative and functional tests. Measures of aggregation stabilization, and of consequent secretion and activation, identified most patients as responders, contrasting with measures of peak aggregation, by likely reflecting better the interactions clopidogrel and the P2Y12 receptor.

Coupling of multicellular morphogenesis and cellular differentiation by an unusual hybrid histidine protein kinase in Myxococcus xanthus

We describe an unusual hybrid histidine protein kinase, which is important for spatially coupling cell aggregation and sporulation during fruiting body formation in Myxococcus xanthus. A rodK mutant makes abnormal fruiting bodies and spores develop outside the fruiting bodies. RodK is a soluble, cytoplasmic protein, which contains an N-terminal sensor domain, a histidine protein kinase domain and three receiver domains. In vitro phosphorylation assays showed that RodK possesses kinase activity. Kinase activity is essential for RodK function in vivo. RodK is present in vegetative cells and remains present until the late aggregation stage, after which the level decreases in a manner that depends on the intercellular A-signal. Genetic evidence suggests that RodK may regulate multiple temporally separated events during fruiting body formation including stimulation of early developmental gene expression, inhibition of A-signal production and inhibition of the intercellular C-signal transduction pathway. We speculate that RodK undergoes a change in activity during development, which is reflected in changes in phosphotransfer to the receiver domains.

Increased rate of chondrocyte aggregation in a wavy‐walled bioreactor

A novel wavy-walled bioreactor designed to enhance mixing at controlled shear stress levels was used to culture chondrocytes in suspension. Chondrocyte aggregation in suspensions mixed at 30, 50, and 80 rpm was characterized in the wavy-walled bioreactor and compared with that in conventional smooth-walled and baffled-walled spinner flask bioreactors. Aggregation was characterized in terms of the percentage of cells that aggregated over time, and aggregate size changes over time. The kinetics of chondrocyte aggregation observed in the bioreactors was composed of two phases: early aggregation between 0 and 2 h of culture, and late aggregation between 3 and 24 h of culture. At 50 rpm, the kinetics of early aggregation in the wavy-walled bioreactor was approximately 25% and 65% faster, respectively, than those in the smooth-walled and baffled-walled spinner flask bioreactors. During the late aggregation phase, the kinetics of aggregation in the wavy-walled bioreactor were approximately 45% and 65% faster, respectively, than in the smooth-walled and baffled-walled spinner flasks. The observed improved kinetics of chondrocyte aggregation was obtained at no cost to the cell survival rate. Results of computerized image analysis suggest that chondrocyte aggregation occurred initially by the formation of new aggregates via cell-cell interactions and later by the joining of small aggregates into larger cell clumps. Aggregates appeared to grow for only a couple of hours in culture before reaching a steady size, possibly determined by limitations imposed by the hydrodynamic environment. These results suggest that the novel geometry of the wavy-walled bioreactor generates a hydrodynamic environment distinct from those traditionally used to culture engineered cartilage. Such differences may be useful in studies aimed at distinguishing the effects of the hydrodynamic environment on tissue-engineered cartilage. Characterizing the wavy-walled bioreactor's hydrodynamic environment and its effects on cartilage cell/tissue culture can help establish direct relationships between hydrodynamic forces and engineered tissue properties.