LUMI-AGGREGOMETER STUDIES OF THE INITIAL ATP-SECRETION FROM COLLAGEN-ADHERENT PLATELETS

We have earlier, with the use of a lumi-aggregometer and sub-aggregating doses of collagen (0.2-0.8 ug/ml PRP), been able to detect the initial, aspirin-insensitive secretion of ATP from the collagen-adherent platelets, and to correlate this secretion to the doses of collagen, and onset and degree of subsequent shape change of non-adherent platelets (Malmgren, Thromb Res 4:445, 1986). The present study shows, that 200 ATU of hirudin,which reduced near-maximal aggregation and ATP-secretion induced by high collagen doses (2.5 ug/ml PRP) from 3.35 ± 0.2 uM to 2.85 ± 0.1 uM, did neither reduce the secreted amount of ATP that were 82.5 ± 15 nM in control samples and 90 ± 27.5 nM in hirudin-treated samples, nor reduce platelet shape change when platelets were challenged with 0.31 ug collagen /ml PRP. (200 ATU hirudin completely abolished an equal degree of platelet shape change induced by 0.01 U thrombin). Assuming that 3 % of the platelets in PRP were actually adhering to the collagen fibrils, the secreted amount corresponds to 14.6 ±0.04 pmoles ATP/106adheringplatelets, amounts which closely represented 100 % of their dense granule content. The finding confirms that hirudin does not inhibit platelet adhesion and also indicates, that thrombin-mediated activation of secretory pathways appears not to be involved during the initial phase of platelet-collagen interactions.Dipyridamole (DPA) and dibutyryl cAMP (DBcAMP) inhibited ATP-secretion and platelet aggregation in a dose-dependent manner at high collagen concentrations, but only DBcAMP caused a dose-dependent reduction of ATP secretion (IC50 =10-4 M) induced by sub-aggregating doses of collagen. DPA was devoid of effect in this respect and thus did not inhibit platelet adhesion.Yohimbine, dihydroergotamine and phentolamine reduced ATP-secretion induced by sub-aggregating collagen doses in the mentioned rank order of potency, and with IC50 values in the micromolar range. Ketanserin, ritanserin and propranolol were devoid of effect. The findings suggest that the initial collagen-plate-let interaction involve alfareceptor-mediated mechanisms that may encompass adhesion, while DBcAMP probably interacts with secretory mechanisms connected to phosphatidylinositol turnover.

Platelet Aggregation Caused by Dithiothreitol

Thrombosis and Haemostasis ◽

10.1055/s-0038-1661036 ◽

1984 ◽

Vol 51 (01) ◽

pp. 119-124 ◽

Cited By ~ 59

Author(s):

M B Zucker ◽

N C Masiello

Keyword(s):

Shape Change ◽

Blood Platelets ◽

Dense Granule ◽

Metabolic Inhibitors ◽

Electrophoretic Patterns ◽

Discernible Effect ◽

Variable Extent ◽

Triton X ◽

Induced Aggregation ◽

SummaryMacIntyre et al. showed that over 1 mM dithiothreitol (DTT) aggregates blood platelets in the presence of fibrinogen; aggregation is not inhibited by prostaglandin E1. We confirmed their data and found that 70 mM 2-mercaptoethanol was also active. DTT- induced aggregation was not associated with platelet shape change or secretion of dense granule contents, was not inhibited by tetracaine or metabolic inhibitors, was prevented at pH 6.5, and prevented, reversed, or arrested by EDTA, depending on when the EDTA was added. DTT did not cause aggregation of thrombasthenic, EDTA-treated, or cold (0° C) platelets, which also failed to aggregate with ADP. Platelets stimulated with DTT bound 125I-labeled fibrinogen. Thus DTT appears to “expose” the fibrinogen receptors. SDS gel electrophoresis of platelet fractions prepared by use of Triton X-114 showed that aggregating concentrations of DTT reduced proteins of apparent Mr 69,000 and 52,000 (probably platelet albumin) and, to a variable extent, glycoproteins Ib, IIb and III. Exposure of unlabeled or 125I- labeled platelets to ADP had no discernible effect on the electrophoretic patterns.

Adenosine Diphosphate (ADP)-Induced ⍺-Granules Release from Platelets of Native Whole Blood Is Reduced by Ticlopidine but Not by Aspirin or Dipyridamole

Thrombosis and Haemostasis ◽

10.1055/s-0038-1661629 ◽

1986 ◽

Vol 56 (02) ◽

pp. 147-150 ◽

Cited By ~ 18

Author(s):

V Pengo ◽

M Boschello ◽

A Marzari ◽

M Baca ◽

L Schivazappa ◽

...

Keyword(s):

Whole Blood ◽

Light Transmission ◽

Ex Vivo ◽

Early Stage ◽

Shape Change ◽

Adenosine Diphosphate ◽

Dose Dependent ◽

Plateletderived Growth Factor ◽

SummaryA brief contact between native whole blood and ADP promotes a dose-dependent release of platelet a-granules without a fall in the platelet number. We assessed the “ex vivo” effect of three widely used antiplatelet drugs, aspirin dipyridamole and ticlopidine, on this system. Aspirin (a single 800 mg dose) and dipyridamole (300 mg/die for four days) had no effect, while ticlopidine (500 mg/die for four days) significantly reduced the a-granules release for an ADP stimulation of 0.4 (p <0.02), 1.2 (p <0.01) and 2 pM (p <0.01). No drug, however, completeley inhibits this early stage of platelet activation. The platelet release of α-granules may be related to platelet shape change of the light transmission aggregometer and may be important “in vivo” by enhancing platelet adhesiveness and by liberating the plateletderived growth factor.

Synthesis and Assessment of Novel Anti-chlamydial Benzylidene Acylhydrazides Derivatives

Letters in Drug Design & Discovery ◽

10.2174/1570180814666170526170227 ◽

2018 ◽

Vol 15 (1) ◽

pp. 31-36 ◽

Cited By ~ 5

Author(s):

Xiaofeng Bao ◽

Ying Xue ◽

Chao Xia ◽

Yin Lu ◽

Ningjing Yang ◽

...

Keyword(s):

Inhibitory Effect ◽

Dependent Manner ◽

Iron Starvation ◽

Hydrochloride Salt ◽

Obligate Intracellular ◽

Biphasic Life Cycle ◽

Ic50 Value ◽

Ic50 Values ◽

Dose Dependent ◽

Better Than

Background: Chlamydiae, characterized by a unique biphasic life cycle, are a group of Gram-negative obligate intracellular bacterial pathogens responsible for diseases in a range of hosts including humans. Benzylidene acylhydrazide CF0001 could inhibit chlamydiae independent of iron starvation and T3SS inhibition. This finding promoted us to design and synthesize more benzylidene acylhydrazides to find novel anti-chlamydial agents. Methods: The carboxylic acids 1a-1d were coupled with Boc-hydrazide inpresence of EDCI and DMAP to obtain the intermediate 2a-2d in 60-62% yields. N-Boc deprotections were performed to obtain hydrazide hydrochloride salt 3a-3d. Nextly, the hydrazides were subjected to condensation with aldehydes to obtain benzylidene acylhydrazides 4a-4g in 30-52% yields in two steps. Results: Compound 4d exhibited best inhibitory effect on the formation and growth of chlamydial inclusions. The IC50 value of compound 4d for infectious progenies was 3.55 µM, better than 7.30 µM of CF0001. Conclusion: To find novel anti-chlamydial agents, we have designed and synthesized benzylidene acylhydrazides 4a-4g. Compounds 4a, 4d, 4g showed inhibitory activity on C. muridarum with the IC50 values from 3.55-12 µM. The 3,5-dibromo-4-hydroxyl substitutes on ring B are critical to keep their anti-chlamydial activity. Compound 4d inhibited C. muridarum in a dose-dependent manner without apparent cytotoxicity.

Targeting BMI-1 with PLGA–PEG nanoparticle-containing PTC209 modulates the behavior of human glioblastoma stem cells and cancer cells

Cancer Nanotechnology ◽

10.1186/s12645-021-00078-8 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Elham Poonaki ◽

Fatemeh Ariakia ◽

Mohammad Jalili-Nik ◽

Mehdi Shafiee Ardestani ◽

Gholamhossein Tondro ◽

...

Keyword(s):

Drug Loading ◽

Polycomb Group ◽

Dependent Manner ◽

Migration Ability ◽

Jnk Signaling ◽

Human Glioblastoma ◽

Core Member ◽

Ic50 Values ◽

Human Gbm ◽

AbstractDespite advances in glioblastoma (GBM) treatments, current approaches have failed to improve the overall survival of patients. The oncogene BMI-1, a core member of the polycomb group proteins, is a potential novel therapeutic target for GBM. To enhance the efficacy and reduce the toxicity, PTC209, a BMI-1 inhibitor, was loaded into a PLGA–PEG nanoparticle conjugated with CD133 antibody (Nano-PTC209) and its effect on the behavior of human GBM stem-like cells (GSCs) and the human glioblastoma cell line (U87MG) was assessed. Nano-PTC209 has a diameter of ~ 75 nm with efficient drug loading and controlled release. The IC50 values of Nano-PTC209 for GSCs and U87MG cells were considerably lower than PTC209. Nano-PTC209 significantly decreased the viability of both GSCs and U87MG cells in a dose-dependent manner and caused a significant enhancement of apoptosis and p53 levels as well as inhibition of AKT and JNK signaling pathways. Furthermore, Nano-PTC209 significantly inhibited the migration ability, decreased the activity of metalloproteinase-2 and -9, and increased the generation of reactive oxygen species in both GSCs and U87MG cells. Our data indicate that PLGA–PEG nanoparticle conjugated with CD133 antibody could be an ideal nanocarrier to deliver PTC209 and effectively target BMI-1 for potential approaches in the treatment of GBM.

High molecular weight kininogen inhibits thrombin-induced platelet aggregation and cleavage of aggregin by inhibiting binding of thrombin to platelets

Blood ◽

10.1182/blood.v77.3.500.bloodjournal773500 ◽

1991 ◽

Vol 77 (3) ◽

pp. 500-507 ◽

Cited By ~ 2

Author(s):

RN Puri ◽

F Zhou ◽

CJ Hu ◽

RF Colman ◽

RW Colman

Keyword(s):

Molecular Weight ◽

Platelet Aggregation ◽

Plasma Concentration ◽

Human Plasma ◽

Shape Change ◽

Dependent Manner ◽

Normal Human Plasma ◽

High Molecular Weight Kininogen ◽

Normal Human ◽

In this study we show that high molecular weight kininogen (HK) inhibited alpha-thrombin-induced aggregation of human platelets in a dose-dependent manner with complete inhibition occurring at plasma concentration (0.67 mumol/L) of HK. HK (0.67 mumol/L) also completely inhibited thrombin-induced cleavage of aggregin (Mr = 100 Kd), a surface membrane protein that mediates adenosine diphosphate (ADP)- induced shape change, aggregation, and fibrinogen binding. The inhibition of HK was specific for alpha- and gamma-thrombin-induced platelet aggregation, because HK did not inhibit platelet aggregation induced by ADP, collagen, calcium ionophore (A23187), phorbol myristate acetate (PMA), PMA + A23187, or 9,11-methano derivative of prostaglandin H2 (U46619). These effects were explained by the ability of HK, at physiologic concentration, to completely inhibit binding of 125I-alpha-thrombin to washed platelets. As a result of this action of HK, this plasma protein also completely inhibited thrombin-induced secretion of adenosine triphosphate, blocked intracellular rise in Ca2+ in platelets exposed to alpha- and gamma-thrombin, inhibited thrombin- induced platelet shape change, and blocked the ability of thrombin to antagonize the increase in intracellular cyclic adenosine monophosphate (cAMP) levels induced by iloprost. Because elevation of cAMP is known to inhibit binding of thrombin to platelets, we established that HK did not increase the intracellular concentration of platelet cAMP. Finally, HK did not inhibit enzymatic activity of thrombin. To study the role of HK in the plasma environment, we used gamma-thrombin to avoid fibrin formation by alpha-thrombin. Platelet aggregation induced by gamma- thrombin was also inhibited by HK in a dose-dependent manner. The EC50 (concentration to produce 50% of the maximum rate of aggregation) of gamma-thrombin for washed platelets was 7 nmol/L and increased to 102 nmol/L when platelets were suspended in normal human plasma. The EC50 for platelet aggregation induced by alpha-thrombin in plasma deficient in total kininogen was 40 nmol/L. When supplemented with HK at plasma concentration (0.67 mumol/L), the EC50 increased to 90 nmol/L, a value similar to that for normal human plasma. These results indicate that (1) HK inhibits thrombin-induced platelet aggregation and cleavage of aggregin by inhibiting binding of thrombin to platelets; (2) HK is a specific inhibitor of platelet aggregation induced by alpha- and gamma- thrombin; and (3) HK plays a role in modulating platelet aggregation stimulated by alpha-thrombin in plasma.

ROLE OF PLATELET GLYCOPROTEINS lb AND llb/llla IN TUMOR CELL INDUCED PLATELET AGGREGATION AND TUMOR CELL ADHESION TO EXTRACELLULAR MATRIX. I. Grossi

10.1055/s-0038-1644670 ◽

1987 ◽

Author(s):

L Grossi ◽

K V Honn ◽

B F Sloane ◽

J Thomopson ◽

D Ohannesian ◽

...

Keyword(s):

Extracellular Matrix ◽

Cell Adhesion ◽

Platelet Aggregation ◽

Tumor Cell ◽

Platelet Adhesion ◽

Eicosatetraenoic Acid ◽

Platelet Glycoprotein ◽

Dependent Manner ◽

Tumor Cell Adhesion ◽

Platelet glycoproteins are known to play a role in platelet platelet interactions, platelet activation, and platelet adhesion to extracellular matrix (ECM). Monoclonal antibody to human platelet glycoprotein lb (mAblb) and polyclonal antibodies to the llb/llla complex (pAbllb/llla) were used to evaluate the involvement of these glycoproteins in tumor cellinduced platelet aggregation (TCIPA and tumor cell adhesion to the ECM. We have demonstrated that human cervical carcinoma (MS5I7), human colon carcinoma (Clone A), and rat Walker 256 carcinosarcoma (W256) cells induce aggregation of homologous platelets via thrombin generation. MAblb and pAbllb/llla were shown to inhibit TCIPA by MS517, Clone A, and W256 in a dose dependent manner. MAblb was also shown to inhibit platelet thromboxane B2 production in response to tumor cells in a dose dependent manner. Neither mAblb nor pAbllb/llla had any effect on ADP stimulated platelet aggregation. Concentrations of mAblb and pAbllb/llla which produced half maximal inhibition alone were combined resulting in complete inhibition of TCIPA. Preincubation of MS5I7 and W256 with mAblb also resulted in inhibition of TCIPA, while preincubation of Clone A with mAblb did not, suggesting the presence of this glycoprotein on the cell membranes of MS5I7 and W256, but not on Clone A. Immunofluorescence studies confirmed the presence of this glycoprotein on the cell plasma membrane of the MS5I7 and W256, but not on Clone A. Preincubation of MS5I7 and W256 with both mAblb and pAbllb/llla alone or in combination, also resulted in decreased (12S)-12 -hydroxy -5, 8,10, 14 -eicosatetraenoic acid (12-HETE) production, while platelets preincubated with these antibodies had no effect on the concentration of 12-HETE produced. Isolation of platelet membranes and released platelet contentswere tested separately and in combination on platelet adhesion to ECM. Platelet release factors were ineffective, while isolated platelet membrane ghosts enhanced adhesion. Disruption of the platelet cytoskeleton andinhibition of the formation of the llb/llla complex decreased platelet enhanced tumor cell adhesion. These findings suggest a role for these platelet glycoproteins in TCIPA, platelet enhanced tumor cell adhesion to ECM and subsequent tumor metastasis.

Adenosine Inhibits Tissue Factor Expression by LPS-stimulated Human Monocytes: Involvement of the A3 Adenosine Receptor

Thrombosis and Haemostasis ◽

10.1055/s-0037-1613164 ◽

2002 ◽

Vol 88 (07) ◽

pp. 123-130 ◽

Cited By ~ 32

Author(s):

Matthieu Broussas ◽

Pascale Cornillet-Lefèbvre ◽

Gérard Potron ◽

Philippe Nguyên

Keyword(s):

Tissue Factor ◽

Rank Order ◽

Mrna Level ◽

Dependent Manner ◽

Human Monocytes ◽

Extrinsic Pathway ◽

A3 Adenosine Receptor ◽

Factor Expression ◽

Cardioprotective Effects ◽

SummaryTissue Factor (TF), an integral membrane glycoprotein that initiates the extrinsic pathway of blood coagulation, is thought to play a major part in coronary acute events. Adenosine, an endogenous nucleoside produced by the degradation of intracellular adenosine triphosphate, has been shown to exert many cardioprotective effects via an inhibition of platelets and neutrophils. This study was conducted to determine whether adenosine (ADO) could modulate the expression of TF by human monocytes. We found that ADO inhibited TF antigen and activity on endotoxin-stimulated monocytes in a dose-dependent manner. The mechanism was at least pre-translational since ADO caused a change in the TF mRNA level. Using ADO receptor-specific analogs, we showed that highly selective A3 agonist N6-(3-iodobenzyl)-adenosine-5’-N’-methyluronamide (IB-MECA) inhibited LPSinduced TF activity expression more potently than A1 agonist R-phenylisopropyladenosine (R-PIA) and A2 agonist CGS 2180. Furthermore, A1/A3 antagonist, xanthine amine congener (XAC) blocked the effect of ADO whereas A2a, A2b and A1 antagonists were ineffective. In addition, we observed that ADO agonists inhibited monocyte TF expression in LPS-stimulated whole blood. The rank order of agonist potency suggested that A2 and A3 receptors might be involved (2-Cado > CGS = IB-MECA > R-PIA). This was supported by the fact that A2 and A3 antagonists reversed the action of 2-Cado. We conclude that TF inhibition by ADO on human purified monocytes involved A3 receptors.

GPR56/ADGRG1 is a platelet collagen-responsive GPCR and hemostatic sensor of shear force

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2008921117 ◽

2020 ◽

Vol 117 (45) ◽

pp. 28275-28286 ◽

Cited By ~ 1

Author(s):

Jennifer Yeung ◽

Reheman Adili ◽

Emily N. Stringham ◽

Rong Luo ◽

Alexander Vizurraga ◽

...

Keyword(s):

Blood Flow ◽

G Protein ◽

Shear Force ◽

Platelet Adhesion ◽

Shape Change ◽

Blood Perfusion ◽

Perfusion Studies ◽

Platelet Receptor ◽

Plug Formation ◽

Circulating platelets roll along exposed collagen at vessel injury sites and respond with filipodia protrusion, shape change, and surface area expansion to facilitate platelet adhesion and plug formation. Various glycoproteins were considered to be both collagen responders and mediators of platelet adhesion, yet the signaling kinetics emanating from these receptors do not fully account for the rapid platelet cytoskeletal changes that occur in blood flow. We found the free N-terminal fragment of the adhesion G protein-coupled receptor (GPCR) GPR56 in human plasma and report that GPR56 is the platelet receptor that transduces signals from collagen and blood flow-induced shear force to activate G protein 13 signaling for platelet shape change.Gpr56−/−mice have prolonged bleeding, defective platelet plug formation, and delayed thrombotic occlusion. Human and mouse blood perfusion studies demonstrated GPR56 and shear-force dependence of platelet adhesion to immobilized collagen. Our work places GPR56 as an initial collagen responder and shear-force transducer that is essential for platelet shape change during hemostasis.

Cytotoxic, Anti-Migration, and Anti-Invasion Activities on Breast Cancer Cells of Angucycline Glycosides Isolated from a Marine-Derived Streptomyces sp.

Marine Drugs ◽

10.3390/md17050277 ◽

2019 ◽

Vol 17 (5) ◽

pp. 277 ◽

Cited By ~ 5

Author(s):

Xin-Ying Qu ◽

Jin-Wei Ren ◽

Ai-Hong Peng ◽

Shi-Qi Lin ◽

Dan-Dan Lu ◽

...

Keyword(s):

Breast Cancer ◽

Cancer Cells ◽

Breast Cancer Cells ◽

Ring Cleavage ◽

Migration And Invasion ◽

Dependent Manner ◽

Streptomyces Sp ◽

Deoxy Sugar ◽

Ic50 Values ◽

Four angucycline glycosides were previously characterized from marine-derived Streptomyces sp. OC1610.4. Further investigation of this strain cultured on different fermentation media from that used previously resulted in the isolation of two new angucycline glycosides, vineomycins E and F (1–2), and five known homologues, grincamycin L (3), vineomycinone B2 (4), fridamycin D (5), moromycin B (7), and saquayamycin B1 (8). Vineomycin F (2) contains an unusual ring-cleavage deoxy sugar. All the angucycline glycosides isolated from Streptomyces sp. OC1610.4 were evaluated for their cytotoxic activity against breast cancer cells MCF-7, MDA-MB-231, and BT-474. Moromycin B (7), saquayamycin B1 (8), and saquayamycin B (9) displayed potent anti-proliferation against the tested cell lines, with IC50 values ranging from 0.16 to 0.67 μM. Saquayamycin B (9) inhibited the migration and invasion of MDA-MB-231 cells in a dose-dependent manner, as detected by Transwell and wound-healing assays.

Bersavine: A Novel Bisbenzylisoquinoline Alkaloid with Cytotoxic, Antiproliferative and Apoptosis-Inducing Effects on Human Leukemic Cells

Molecules ◽

10.3390/molecules25040964 ◽

2020 ◽

Vol 25 (4) ◽

pp. 964 ◽

Cited By ~ 2

Author(s):

Darja Koutova ◽

Monika Kulhava ◽

Radim Havelek ◽

Martina Majorosova ◽

Karel Královec ◽

...

Keyword(s):

Leukemic Cells ◽

Dependent Manner ◽

Carcinoma Cell Lines ◽

Ic50 Values ◽

Bisbenzylisoquinoline Alkaloid ◽

Human Leukemic Cells ◽

Induced Apoptosis ◽

Dose Dependent ◽

Increased Activity ◽

Mcf 7

Bersavine is the new bisbenzylisoquinoline alkaloid isolated from the Berberis vulgaris L. (Berberidaceae) plant. The results of cytotoxicity screening 48 h post-treatment showed that bersavine considerably inhibits the proliferation and viability of leukemic (Jurkat, MOLT-4), colon (HT-29), cervix (HeLa) and breast (MCF-7) cancer cells with IC50 values ranging from 8.1 to 11 µM. The viability and proliferation of leukemic Jurkat and MOLT-4 cells were decreased after bersavine treatment in a time- and dose-dependent manner. Bersavine manifested concentration-dependent antiproliferative activity in human lung, breast, ovarian and hepatocellular carcinoma cell lines using a xCELLigence assay. Significantly higher percentages of MOLT-4 cells exposed to bersavine at 20 µM for 24 h were arrested in the G1 phase of the cell cycle using the flow cytometry method. The higher percentage of apoptotic cells was measured after 24 h of bersavine treatment. The upregulation of p53 phosphorylated on Ser392 was detected during the progression of MOLT-4 cell apoptosis. Mechanistically, bersavine-induced apoptosis is an effect of increased activity of caspases, while reduced proliferation seems dependent on increased Chk1 Ser345 phosphorylation and decreased Rb Ser807/811 phosphorylation in human leukemic cells.