An Additional Mechanism of Action of Abciximab: Dispersal of Newly Formed Platelet Aggregates

2002 ◽  
Vol 87 (06) ◽  
pp. 1020-1025 ◽  
Author(s):  
Stanley Marciniak ◽  
Mark Furman ◽  
Alan Michelson ◽  
Joseph Jakubowski ◽  
Robert Jordan ◽  
...  
Keyword(s):  
Single Cells ◽  
Dense Granule ◽  
Light Transmittance ◽  
Dependent Manner ◽  
Aggregate Formation ◽  
Fibrinogen Binding ◽  
Platelet Disaggregation ◽  
Platelet Aggregates

SummaryThe ability of abciximab to prevent fibrinogen binding to activated platelets indicates it may also promote dissolution of platelet-rich thrombi. The present study examined the capacity of abciximab to reverse platelet aggregation in vitro. Experiments were performed on blood from healthy non-medicated donors. Platelet aggregate formation and disaggregation were monitored turbidimetrically. Platelet-bound fibrinogen was measured by flow cytometry. For disaggregation studies, platelets were first stimulated with either ADP or the 11-mer thrombin receptor activating peptide (TRAP), then varying amounts of abciximab were added at periodic intervals after agonist addition. Platelet disaggregation was detected by comparing the extent of light transmittance at 4 min after addition of either abciximab or saline to PRP. ATP release was simultaneously monitored by chemi-luminescence. When added 1 min after low concentrations of ADP, abciximab rapidly (<1 min) dispersed platelet aggregates in a dose-dependent manner, with complete disaggregation observed with 6.25 µg/mL of the β3 antagonist. In contrast, equivalent concentrations of abciximab did not induce appreciable disaggregation to platelets stimulated with TRAP (10 µM). Platelet counts of samples that had undergone complete disaggregation, as assessed by aggregometry, were equivalent to baseline, indicating dispersal of aggregates to single cells. Concentrations of abciximab that produced complete disaggregation induced partial displacement of platelet-bound fibrinogen (52 ± 8% inhibition of fibrinogen binding at 12.5 µg/ml abciximab). The disaggregation effectiveness of abciximab decreased as the time between ADP and subsequent abciximab addition widened, and as the amount of both dense granule release and agonist stimulation increased. However, pre-treatment of platelets with acetylsalicylic acid (ASA) did not potentiate platelet disaggregation induced by abciximab.These data indicate that abciximab facilitates the dispersal of newly formed platelet aggregates in vitro, by partially displacing fibrinogen from activated GPIIb/IIIa receptors. In vivo, abciximab may destabilize coronary thrombi by preventing aggregate formation and dispersing mural thrombi.

Akt2 Supports aIIbb3-Dependent Reorganization of the Actin Cytoskeleton in Platelets.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3565-3565
Author(s):  
Shelley August ◽  
Donna S. Woulfe
Keyword(s):  
Platelet Rich Plasma ◽  
Thrombus Formation ◽  
Active Form ◽  
Dense Granule ◽  
Clot Retraction ◽  
Akt Activation ◽  
Cytoskeletal Remodeling ◽  
Fibrinogen Binding

Abstract Akt is a serine-threonine kinase with well-described roles in growth and metabolism. Previous studies from our laboratory have shown that Akt also plays important roles in platelet function in vitro and in thrombus formation in vivo. Two isoforms are present in mouse platelets, Akt1 and Akt2, with Akt2 being the dominant isoform. Our previous studies have shown that platelets from Akt2−/− mice have marked defects in aggregation, a- and dense granule secretion, and fibrinogen binding. Each of these platelet functions depends in part on the function of the major platelet integrin, aIIbb3. However, whether Akt might regulate the function of aIIbb3 is still unknown. To determine whether Akt regulates aIIbb3-dependent signaling, first the rate of thrombin-initiated clot retraction was compared in platelet-rich plasma (PRP) from wild-type versus Akt2−/− mice. Akt2−/− platelets have a delay in the aIIbb3-dependent retraction of the fibrin clot compared to their wildtype counterparts. Akt2−/− platelets also have a delay in aIIbb3-dependent spreading on immobilized fibrinogen. However, adhesion to immobilized fibrinogen was normal in these platelets, suggesting that the spreading defect is due to a defect in outside-in signaling by the integrin, rather than in fibrinogen binding. Furthermore, unstimulated platelets expressing a constitutively active form of Akt spread more rapidly on fibrinogen-coated slides and generate more filopodial extensions than wildtype platelets, suggesting that Akt activation may be sufficient to induce outside-in signaling and /or cytoskeletal remodeling. Interestingly, integrin activation was not sufficient to induce Akt phoshorylation, suggesting that integrin activity is downstream rather than upstream of Akt activation. Taken together, these results suggest that integrin aIIbb3 is not necessary for Akt activation; however, Akt promotes outside-in signaling and cytoskeletal remodeling by aIIbb3.

scholarly journals NPP4 is a procoagulant enzyme on the surface of vascular endothelium

Blood ◽  
2012 ◽  
Vol 120 (22) ◽  
pp. 4432-4440 ◽  
Author(s):  
Ronald A. Albright ◽  
William C. Chang ◽  
Donna Robert ◽  
Deborah L. Ornstein ◽  
Wenxiang Cao ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Vascular Endothelium ◽  
Platelet Rich Plasma ◽  
Dense Granule ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Phosphodiesterase 4 ◽  
Adp Receptors

Abstract Ap3A is a platelet-dense granule component released into the extracellular space during the second wave of platelet aggregation on activation. Here, we identify an uncharacterized enzyme, nucleotide pyrophosphatase/phosphodiesterase-4 (NPP4), as a potent hydrolase of Ap3A capable of stimulating platelet aggregation and secretion. We demonstrate that NPP4 is present on the surface of vascular endothelium, where it hydrolyzes Ap3A into AMP and ADP, and Ap4A into AMP and ATP. Platelet aggregation assays with citrated platelet-rich plasma reveal that the primary and secondary waves of aggregation and dense granule release are strongly induced by nanomolar NPP4 in a concentration-dependent manner in the presence of Ap3A, while Ap3A alone initiates a primary wave of aggregation followed by rapid disaggregation. NPP2 and an active site NPP4 mutant, neither of which appreciably hydrolyzes Ap3A, have no effect on platelet aggregation and secretion. Finally, by using ADP receptor blockade we confirm that NPP4 mediates platelet aggregation via release of ADP from Ap3A and activation of ADP receptors. Collectively, these studies define the biologic and enzymatic basis for NPP4 and Ap3A activity in platelet aggregation in vitro and suggest that NPP4 promotes hemostasis in vivo by augmenting ADP-mediated platelet aggregation at the site of vascular injury.

In Vivo Effect of Suloctidil as an Antiplatelet Agent

1979 ◽  
Vol 41 (03) ◽  
pp. 465-474 ◽  
Author(s):  
Marcia R Stelzer ◽  
Thomas S Burns ◽  
Robert N Saunders
Keyword(s):  
Ex Vivo ◽  
Antiplatelet Agent ◽  
Ratio Method ◽  
Platelet Aggregate ◽  
Permanent Effect ◽  
Platelet Aggregates ◽  
5 Ht Uptake ◽  
High Doses

SummaryThe relationship between the effects of suloctidil in vivo as an antiplatelet agent and in vitro as a modifier of platelet serotonin (5-HT) parameters was investigated. Suloctidil was found to be effective in reducing platelet aggregates formation in the retired breeder rat as determined using the platelet aggregate ratio method (PAR) with an ED50 of 16.1 mg/kg 24 hours post administration. In contrast to the hypothesis that 5-HT depletion is involved in the anti-aggregatory mechanism of suloctidil, no correlation was found between platelet 5- HT content and this antiplatelet activity. Reduction of platelet 5-HT content required multiple injections of high doses (100 mg/kg/day) of suloctidil. Suloctidil administration for 8 days at 100 mg/kg/day, which lowered platelet 5-HT content by 50%, resulted in no permanent effect on ex vivo platelet 5-HT uptake or thrombin-induced release, nor alteration in the plasma 5-HT level. However, these platelets exhibited a short-lived, significant increase in percent leakage of 5-HT after 30 minutes of incubation. Therefore, suloctidil treatment at high doses may with time result in platelet 5-HT depletion, however this effect is probably not related to the primary anti-aggregatory activity of the drug.

Accumulation of Macromolecular Particles in the Reticuloendothelial System (RES) Mediated by Platelet Aggregation and Disaggregation

1969 ◽  
Vol 22 (03) ◽  
pp. 496-507 ◽  
Author(s):  
W.G van Aken ◽  
J Vreeken
Keyword(s):  
Platelet Aggregation ◽  
Degradation Products ◽  
Reticuloendothelial System ◽  
Caproic Acid ◽  
Carbon Particles ◽  
Carbon Clearance ◽  
Aggregation And Disaggregation ◽  
Platelet Aggregates

SummaryCarbon particles cause platelet aggregation in vitro and in vivo. Prior studies established that substances which modify thrombocyte aggregation also influence the rate at which carbon is cleared from the blood.This study was performed in order to elucidate the mechanism by which the carbon-platelet aggregates specifically accumulate in the RES.Activation of fibrinolysis by urokinase or streptokinase reduced the carbon clearance rate, probably due to generated fibrinogen degradation products (FDP). Isolated FDP decreased the carbon clearance and caused disaggregation of platelets and particles in vitro. Inhibition of fibrinolysis by epsilon-amino-caproic acid (EACA), initially accelerated the disappearance of carbon and caused particle accumulation outside the RES, predominantly in the lungs. It is supposed that platelet aggregation and locally activated fibrinolysis act together in the clearance of particles. In the normal situation the RES with its well known low fibrinolytic activity, becomes the receptor of the particles.

Enhanced Increases in Cytosolic Ca2+ in ADP-Stimulated Platelets from Patients with Delta-Storage Pool Deficiency – A Possible Indicator of Interactions between Granule-Bound ADP and the Membrane ADP Receptor

1997 ◽  
Vol 77 (02) ◽  
pp. 376-382 ◽  
Author(s):  
Bruce Lages ◽  
Harvey J Weiss
Keyword(s):  
Platelet Rich Plasma ◽  
Limited Range ◽  
Dense Granule ◽  
Receptor Desensitization ◽  
Fura 2 ◽  
Storage Pool ◽  
Low Levels ◽  
The Right

SummaryThe possible involvement of secreted platelet substances in agonist- induced [Ca2+]i increases was investigated by comparing these increases in aspirin-treated, fura-2-loaded normal platelets and platelets from patients with storage pool deficiencies (SPD). In the presence and absence of extracellular calcium, the [Ca2+]i response induced by 10 µM ADP, but not those induced by 0.1 unit/ml thrombin, 3.3 µM U46619, or 20 µM serotonin, was significantly greater in SPD platelets than in normal platelets, and was increased to the greatest extent in SPD patients with Hermansky-Pudlak syndrome (HPS), in whom the dense granule deficiencies are the most severe. Pre-incubation of SPD-HPS and normal platelets with 0.005-5 µM ADP produced a dose-dependent inhibition of the [Ca2+]i response induced by 10 µ M ADP, but did not alter the [Ca2+]i increases induced by thrombin or U46619. Within a limited range of ADP concentrations, the dose-inhibition curve of the [Ca2+]i response to 10 µM ADP was significantly shifted to the right in SPD-HPS platelets, indicating that pre-incubation with greater amounts of ADP were required to achieve the same extent of inhibition as in normal platelets. These results are consistent with a hypothesis that the smaller ADP-induced [Ca2+]i increases seen in normal platelets may result from prior interactions of dense granule ADP, released via leakage or low levels of activation, with membrane ADP receptors, causing receptor desensitization. Addition of apyrase to platelet-rich plasma prior to fura-2 loading increased the ADP-induced [Ca2+]i response in both normal and SPD-HPS platelets, suggesting that some release of ADP derived from both dense granule and non-granular sources occurs during in vitro fura-2 loading and platelet washing procedures. However, this [Ca2+]i response was also greater in SPD-HPS platelets when blood was collected with minimal manipulation directly into anticoagulant containing apyrase, raising the possibility that release of dense granule ADP resulting in receptor desensitization may also occur in vivo. Thus, in addition to enhancing platelet activation, dense granule ADP could also act to limit the ADP-mediated reactivity of platelets exposed in vivo to low levels of stimulation.

Chickpea (Cicer arietinum L.) Lectin Exhibit Inhibition of ACE-I, α-amylase and α-glucosidase Activity

2019 ◽  
Vol 26 (7) ◽  
pp. 494-501 ◽  
Author(s):  
Sameer Suresh Bhagyawant ◽  
Dakshita Tanaji Narvekar ◽  
Neha Gupta ◽  
Amita Bhadkaria ◽  
Ajay Kumar Gautam ◽  
...  
Keyword(s):  
Biological Effects ◽  
Exchange Chromatography ◽  
Health Concern ◽  
Carbohydrate Binding ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Ic50 Values ◽  
Chickpea Lectin

Background: Diabetes and hypertension are the major health concern and alleged to be of epidemic proportions. This has made it a numero uno subject at various levels of investigation. Glucosidase inhibitor provides the reasonable option in treatment of Diabetes Mellitus (DM) as it specifically targets post prandial hyperglycemia. The Angiotensin Converting Enzyme (ACE) plays an important role in hypertension. Therefore, inhibition of ACE in treatment of elevated blood pressure attracts special interest of the scientific community. Chickpea is a food legume and seeds contain carbohydrate binding protein- a lectin. Some of the biological properties of this lectin hitherto been elucidated. Methods: Purified by ion exchange chromatography, chickpea lectin was tested for its in vitro antioxidant, ACE-I inhibitory and anti-diabetic characteristic. Results: Lectin shows a characteristic improvement over the synthetic drugs like acarbose (oral anti-diabetic drug) and captopril (standard antihypertensive drug) when, their IC50 values are compared. Lectin significantly inhibited α-glucosidase and α-amylase in a concentration dependent manner with IC50 values of 85.41 ± 1.21 ҝg/ml and 65.05 ± 1.2 µg/ml compared to acarbose having IC50 70.20 ± 0.47 value of µg/ml and 50.52 ± 1.01 µg/ml respectively. β-Carotene bleaching assay showed antioxidant activity of lectin (72.3%) to be as active as Butylated Hydroxylanisole (BHA). In addition, lectin demonstrated inhibition against ACE-I with IC50 value of 57.43 ± 1.20 µg/ml compared to captopril. Conclusion: Lectin demonstrated its antioxidant character, ACE-I inhibition and significantly inhibitory for α-glucosidase and α-amylase seems to qualify as an anti-hyperglycemic therapeutic molecule. The biological effects of chickpea lectin display potential for reducing the parameters of medically debilitating conditions. These characteristics however needs to be established under in vivo systems too viz. animals through to humans.

scholarly journals Exploration of in vitro thrombolytic, anthelminthic, cytotoxic and in vivo anxiolytic potentials with phytochemical screening of flowers of Brassica nigra

2020 ◽  
Vol 6 (1) ◽  
Author(s):  
Mohammad Sarowar Uddin ◽  
Md. Shalahuddin Millat ◽  
Mohammad Safiqul Islam ◽  
Md. Saddam Hussain ◽  
Md. Giash Uddin ◽  
...  
Keyword(s):  
Brassica Nigra ◽  
Anxiolytic Activity ◽  
Clot Lysis ◽  
Dependent Manner ◽  
Standard Drug ◽  
Lead Compounds ◽  
Lysis Activity ◽  
Test Models

Abstract Background Brassica nigra is a plant of Brassicaceae family, which possesses numerous medicinal values. Our present study is intended to assess the potential in vitro thrombolytic, anthelminthic, cytotoxic and in vivo anxiolytic properties of MCE of B. nigra flowers. MCE was fractioned for separating the compound on the basis of polarity by using chloroform, n-hexane and ethyl acetate solvent. Thrombolytic and anthelminthic activities were explained by collecting human erythrocytes and earthworms as test models, respectively. Anxiolytic activity was evaluated by elevated plus maze and hole board models while cytotoxic test was conducted through brine shrimp lethality bioassay. Results MCE revealed the presence of alkaloids, flavonoids, tannin, diterpenes, glycosides, carbohydrates, phenols, fixed oils and fat. In case of thrombolytic test, the MCE, CSF, ASF and n-HSF had produced maximum clot lysis activity at 5 and 10 mg/ml dose conditions. Two different concentrations (10 and 20 mg/ml) of MCE and its fractions showed significant (p < 0.05) anthelminthic activities in a dose-dependent manner. Significant anxiolytic activity was observed for all fractions which was comparable to the standard drug diazepam (p < 0.05). Again, the cytotoxic screening also presented good potentials for all fractions. Conclusion From the findings of present study, we can conclude that MCE of B. nigra flowers and its fraction possess significant anxiolytic, anthelmintic, anticancer and thrombolytic properties which may be a good candidate for treating these diseases through the determination of bio-active lead compounds.

scholarly journals Anti-tumor activity of a novel proteasome inhibitor D395 against multiple myeloma and its lower cardiotoxicity compared with carfilzomib

2021 ◽  
Vol 12 (5) ◽  
Author(s):  
Xuxing Shen ◽  
Chao Wu ◽  
Meng Lei ◽  
Qing Yan ◽  
Haoyang Zhang ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Proteasome Inhibitor ◽  
Osteoclast Differentiation ◽  
Dependent Manner ◽  
Serum Enzyme ◽  
Herg Channels ◽  
Anti Tumor Activity ◽  
Dose Dependent

AbstractCarfilzomib, a second-generation proteasome inhibitor, has significantly improved the survival rate of multiple myeloma (MM) patients, but its clinical application is still restricted by drug resistance and cardiotoxicity. Here, we identified a novel proteasome inhibitor, D395, and assessed its efficacy in treating MM as well as its cardiotoxicity at the preclinical level. The activities of purified and intracellular proteasomes were measured to determine the effect of D395 on the proteasome. CCK-8 and flow cytometry experiments were designed to evaluate the effects of D395 on cell growth and apoptosis. The effects of D395 and carfilzomib on serum enzyme activity, echocardiography features, cardiomyocyte morphology, and hERG channels were also compared. In our study, D395 was highly cytotoxic to MM cell lines and primary MM cells but not normal cells, and it was well tolerated in vivo. Similar to carfilzomib, D395 inhibited osteoclast differentiation in a dose-dependent manner. In particular, D395 exhibited lower cardiotoxicity than carfilzomib in all experiments. In conclusion, D395 is a novel irreversible proteasome inhibitor that has remarkable anti-MM activity and mild cardiotoxicity in vitro and in vivo.

scholarly journals Secreted KIAA1199 promotes the progression of rheumatoid arthritis by mediating hyaluronic acid degradation in an ANXA1-dependent manner

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Wei Zhang ◽  
Guoyu Yin ◽  
Heping Zhao ◽  
Hanzhi Ling ◽  
Zhen Xie ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Hyaluronic Acid ◽  
Dependent Manner ◽  
Collagen Induced Arthritis ◽  
Intracellular Degradation ◽  
Main Pathway ◽  
First Time

AbstractIn inflamed joints, enhanced hyaluronic acid (HA) degradation is closely related to the pathogenesis of rheumatoid arthritis (RA). KIAA1199 has been identified as a hyaladherin that mediates the intracellular degradation of HA, but its extracellular function remains unclear. In this study, we found that the serum and synovial levels of secreted KIAA1199 (sKIAA1199) and low-molecular-weight HA (LMW-HA, MW < 100 kDa) in RA patients were significantly increased, and the positive correlation between them was shown for the first time. Of note, treatment with anti-KIAA1199 mAb effectively alleviated the severity of arthritis and reduced serum LMW-HA levels and cytokine secretion in collagen-induced arthritis (CIA) mice. In vitro, sKIAA1199 was shown to mediate exogenous HA degradation by attaching to the cell membrane of RA fibroblast-like synoviosytes (RA FLS). Furthermore, the HA-degrading activity of sKIAA1199 depended largely on its adhesion to the membrane, which was achieved by its G8 domain binding to ANXA1. In vivo, kiaa1199-KO mice exhibited greater resistance to collagen-induced arthritis. Interestingly, this resistance could be partially reversed by intra-articular injection of vectors encoding full-length KIAA1199 instead of G8-deleted KIAA119 mutant, which further confirmed the indispensable role of G8 domain in KIAA1199 involvement in RA pathological processes. Mechanically, the activation of NF-κB by interleukin-6 (IL-6) through PI3K/Akt signaling is suggested to be the main pathway to induce KIAA1199 expression in RA FLS. In conclusion, our study supported the contribution of sKIAA1199 to RA pathogenesis, providing a new therapeutic target for RA by blocking sKIAA1199-mediated HA degradation.

scholarly journals Eap1p, an Adhesin That Mediates Candida albicans Biofilm Formation In Vitro and In Vivo

2007 ◽  
Vol 6 (6) ◽  
pp. 931-939 ◽  
Author(s):  
Fang Li ◽  
Michael J. Svarovsky ◽  
Amy J. Karlsson ◽  
Joel P. Wagner ◽  
Karen Marchillo ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Cell Adhesion ◽  
Biofilm Formation ◽  
Fungal Infections ◽  
Gene Dosage ◽  
Venous Catheter ◽  
Flow Chamber ◽  
Dependent Manner

ABSTRACT Candida albicans is the leading cause of systemic fungal infections in immunocompromised humans. The ability to form biofilms on surfaces in the host or on implanted medical devices enhances C. albicans virulence, leading to antimicrobial resistance and providing a reservoir for infection. Biofilm formation is a complex multicellular process consisting of cell adhesion, cell growth, morphogenic switching between yeast form and filamentous states, and quorum sensing. Here we describe the role of the C. albicans EAP1 gene, which encodes a glycosylphosphatidylinositol-anchored, glucan-cross-linked cell wall protein, in adhesion and biofilm formation in vitro and in vivo. Deleting EAP1 reduced cell adhesion to polystyrene and epithelial cells in a gene dosage-dependent manner. Furthermore, EAP1 expression was required for C. albicans biofilm formation in an in vitro parallel plate flow chamber model and in an in vivo rat central venous catheter model. EAP1 expression was upregulated in biofilm-associated cells in vitro and in vivo. Our results illustrate an association between Eap1p-mediated adhesion and biofilm formation in vitro and in vivo.

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