Thiosulfinates Inhibit Platelet Aggregation and Microparticle Shedding at a Calpain-dependent Step

SummaryThiosulfinates (TSs) are sulfur compounds generated through the processing of different Allium species with antiplatelet property. To further define this platelet inhibitory effect we studied diallyl-TS (Al2TS), dipropyl-TS (Pr2TS), and dimethyl-TS (Me2TS) on platelet responses. The three TSs inhibited dose-dependent platelet aggregation, with IC50 values of 15 ± 2, 19 ± 2, and 9 ± 1 μM for Al2TS, Pr2TS and Me2TS, respectively. TSs had no effect on the expression of a platelet procoagulant surface, measured by flow cytometry as the binding of annexin V-FITC. They inhibited the microparticle shedding and clot retraction. Since the microparticle shedding is a calpain-activation dependent step, we assessed calpain activation by analysis of autoproteolysis in shorter active forms and by talin proteolysis in the presence of TSs. Calpain activation was inhibited by TSs independently of fibrinogen binding. Thus, TSs represent a new category of platelet inhibitors, acting on cal-pain-induced events.

Download Full-text

Ticlopidine Selectively Inhibits Human Platelet Responses to Adenosine Diphosphate

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646487 ◽

1991 ◽

Vol 66 (06) ◽

pp. 694-699 ◽

Cited By ~ 49

Author(s):

Marco Cattaneo ◽

Benjaporn Akkawat ◽

Anna Lecchi ◽

Claudio Cimminiello ◽

Anna M Capitanio ◽

...

Keyword(s):

Platelet Aggregation ◽

Adenosine Diphosphate ◽

Inhibitory Effect ◽

Clot Retraction ◽

Fibrinogen Binding ◽

Low Concentrations ◽

Binding Inhibition ◽

High Concentrations ◽

Formalin Fixed

SummaryPlatelet aggregation and fibrinogen binding were studied in 15 individuals before and 7 days after the oral administration of ticlopidine (250 mg b.i.d.). Ticlopidine significantly inhibited platelet aggregation induced by adenosine diphosphate (ADP), the endoperoxide analogue U46619, collagen or low concentrations of thrombin, but did not inhibit platelet aggregation induced by epinephrine or high concentrations of thrombin. Ticlopidine inhibited 125I-fibrinogen binding induced by ADP, U46619 or thrombin (1 U/ml). The ADP scavengers apyrase or CP/CPK, added in vitro to platelet suspensions obtained before ticlopidine, caused the same pattern of aggregation and 125I-fibrihogen binding inhibition as did ticlopidine. Ticlopidine did not inhibit further platelet aggregation and 125I-fibrinogen binding induced in the presence of ADP scavengers. After ticlopidine administration, thrombin or U46619, but not ADP, increased the binding rate of the anti-GPIIb/IIIa monoclonal antibody 7E3 to platelets. Ticlopidine inhibited clot retraction induced by reptilase plus ADP, but not that induced by thrombin or by reptilase plus epinephrine, and prevented the inhibitory effect of ADP, but not that of epinephrine, on the PGE1-induced increase in platelet cyclic AMP. The number of high- and low-affinity binding sites for 3H-ADP on formalin-fixed platelets and their K d were not modified by ticlopidine. These findings indicate that ticlopidine selectively inhibits platelet responses to ADP.

Download Full-text

Platelet binding to polymerizing fibrin is avidity driven and requires activated αIIbβ3 but not fibrin cross-linking

Blood Advances ◽

10.1182/bloodadvances.2021005142 ◽

2021 ◽

Vol 5 (20) ◽

pp. 3986-4002

Author(s):

Lorena Buitrago ◽

Samuel Lefkowitz ◽

Ohad Bentur ◽

Julio Padovan ◽

Barry Coller

Keyword(s):

Platelet Aggregation ◽

Light Transmission ◽

Potent Inhibitor ◽

Thrombin Receptor ◽

Cross Linking ◽

Inhibit Platelet Aggregation ◽

Clot Retraction ◽

Fibrinogen Binding ◽

Platelet Binding ◽

Binding Inhibition

Abstract The molecular basis of platelet-fibrin interactions remains poorly understood despite the predominance of fibrin in thrombi. We have studied the interaction of platelets with polymerizing fibrin by adding thrombin to washed platelets in the presence of the peptide RGDW, which inhibits the initial platelet aggregation mediated by fibrinogen binding to αIIbβ3 but leaves intact a delayed increase in light transmission (delayed wave; DW) as platelets interact with the polymerizing fibrin. The DW was absent in platelets from a patient with Glanzmann thrombasthenia, indicating a requirement for αIIbβ3. The DW required αIIbb3 activation and it was inhibited by the αIIbβ3 antagonists eptifibatide and the monoclonal antibody (mAb) 7E3, but only at much higher concentrations than needed to inhibit platelet aggregation initiated by a thrombin receptor activating peptide (T6). Surface plasmon resonance and scanning electron microscopy studies both supported fibrin having greater avidity for αIIbβ3 than fibrinogen rather than greater affinity, consistent with fibrin’s multivalency. mAb 10E5, a potent inhibitor of T6-induced platelet aggregation, did not inhibit the DW, suggesting that fibrin differs from fibrinogen in its mechanism of binding. Inhibition of factor XIII–mediated fibrin cross-linking by >95% reduced the DW by only 32%. Clot retraction showed a pattern of inhibition similar to that of the DW. We conclude that activated αIIbβ3 is the primary mediator of platelet-fibrin interactions leading to clot retraction, and that the interaction is avidity driven, does not require fibrin cross-linking, and is mediated by a mechanism that differs subtly from that of the interaction of αIIbβ3 with fibrinogen.

Download Full-text

Inhibition of Integrin-Mediated Platelet Aggregation, Fibrinogen-Binding, and Interactions with Extracellular Matrix by Nonpeptidic Mimetics of Arg-Gly-Asp

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649720 ◽

1993 ◽

Vol 70 (06) ◽

pp. 1030-1036 ◽

Cited By ~ 8

Author(s):

David Varon ◽

Ofer Lider ◽

Rima Dardik ◽

Boris Shenkman ◽

Ronen Alon ◽

...

Keyword(s):

Extracellular Matrix ◽

Platelet Aggregation ◽

Specific Interaction ◽

Adenosine Diphosphate ◽

Inhibitory Effect ◽

Dependent Manner ◽

Rgd Sequence ◽

Fibrinogen Binding ◽

Inhibitory Potential ◽

Dose Dependent

SummaryThe interaction of the activated platelet integrin, glycoprotein IIb-IIIa (GPIIb-IIIa) with fibrinogen and von-Wille-brand factor (vWF) is essential for platelet aggregation. The minimal structure required for this integrin’s binding to fibrinogen is the Arg-Gly-Asp (RGD) sequence. Inasmuch as normal level of GPIIb-IIIa-RGD interactions are required for maintaining hemostasis, elevated platelet aggregation can cause adverse pathological effects. We have previously reported that nonpeptidic mimetics of RGD, consisting of carboxylate and guanidinium groups of Asp and Arg divided by a linear 11-atom spacer, acquired a significant affinity for the GPIIb-IIIa integrin and inhibited platelet aggregation. The structural requirements for the interactions of the RGD sequence with GPIIb-IIIa and the inhibitory potential of a newly designed series of mimetics on platelet aggregation and interactions with extracellular matrix (ECM) were assayed herein. Adenosine-diphosphate (ADP)-induced platelet aggregation was inhibited in a dose-dependent manner by various RGD mimetics, with a maximal inhibition of 80-100% with an IC50 of 3 μM for the most potent inhibitor, NS-11 which a six-membered ring was introduced into the spacer chain, which exceeded the IC50 attained with the original RGDS peptide. The inhibitory effect of the RGD mimetics was attributed to their specific interaction with the GPIIb-IIIa integrin, since these mimetics inhibited the binding of the PAC-1 mAb to GPIIb-IIIA. Furthermore, the binding of 125I-labeled fibrinogen to platelets was inhibited by the RGD surrogates in a dose-dependent and saturable manner. The RGD-mimetics also inhibited up to 70% the adhesion, aggregation, and deposition of platelets onto ECM. Thus, we suggest that the novel nonpeptidic mimetics of RGD described herein, which were shown to be resistant to proteolytic digestion, would be valuable in novel therapeutic approaches to treat in RGD-dependent pathological disorders involving platelet-ECM interactions.

Download Full-text

Inhibitory Effect of Staphylokinase on Platelet Aggregation

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649679 ◽

1993 ◽

Vol 70 (05) ◽

pp. 834-837 ◽

Cited By ~ 6

Author(s):

Akira Suehiro ◽

Yoshio Oura ◽

Motoo Ueda ◽

Eizo Kakishita

Keyword(s):

Platelet Aggregation ◽

Human Platelet ◽

Degradation Products ◽

Platelet Rich Plasma ◽

Inhibitory Effect ◽

Effective Concentration ◽

Inhibit Platelet Aggregation ◽

Platelet Suspension ◽

Washed Platelet ◽

Human Platelet Aggregation

SummaryWe investigated the effect of staphylokinase (SAK), which has specific thrombolytic properties, on human platelet aggregation. Platelet aggregation induced with collagen was observed following preincubation of platelets in platelet-rich plasma (PRP) or washed platelet suspension (WP) with SAK at 37° C for 30 min. SAK inhibited platelet aggregation in PRP only at the highest examined concentration (1 x 10-4 g/ml). Although SAK did not inhibit platelet aggregation in WP which contained fibrinogen, it did when the platelets had been preincubated with SAK and plasminogen. The most effective concentration in WP was 1 x 10-6 g/ml. The effect could be inhibited by adding aprotinin or α2-antiplasmin. The highest generation of plasmin in the same preincubation fluid was detected at 1 x 10-6 g/ml SAK. We concluded that SAK can inhibit platelet aggregation in WP by generating plasmin and/or fibrinogen degradation products, but is only partially effective in PRP because of the existence of α2-antiplasmin.

Download Full-text

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.

Download Full-text

Extracellular fibrinogen binding protein, Efb, from Staphylococcus aureus binds to platelets and inhibits platelet aggregation

Thrombosis and Haemostasis ◽

10.1160/th03-05-0287 ◽

2004 ◽

Vol 91 (04) ◽

pp. 779-789 ◽

Cited By ~ 24

Author(s):

Oonagh Shannon ◽

Jan-Ingmar Flock

Keyword(s):

Platelet Aggregation ◽

Potent Inhibitor ◽

Specific Binding ◽

Binding Protein ◽

Dependent Manner ◽

Platelet Surface ◽

Putative Receptor ◽

Activated Platelets ◽

Fibrinogen Binding ◽

Dose Dependent

Summary S. aureus produces and secretes a protein, extracellular fibrinogen binding protein (Efb), which contributes to virulence in wound infection. We have shown here that Efb is a potent inhibitor of platelet aggregation. Efb can bind specifically to platelets by two mechanisms; 1) to fibrinogen naturally bound to the surface of activated platelets and 2) also directly to a surface localized component on the platelets. This latter binding of Efb is independent of fibrinogen. The specific binding of Efb to the putative receptor on the platelet surface results in a stimulated, non-functional binding of fibrinogen in a dose dependent manner, distinct from natural binding of fibrinogen to platelets. The natural binding of fibrinogen to GPIIb/IIIa on activated platelets could be blocked by a monoclonal antibody against this integrin, whereas the Efb-mediated fibrinogen binding could not be blocked. The enhanced Efb-dependent fibrinogen binding to platelets is of a nature that does not promote aggregation of the platelets; instead it inhibits aggregation. The anti-thrombotic action of Efb may explain the effect of Efb on wound healing, which is delayed in the presence of Efb.

Download Full-text

Bile Acids Induce Platelet Activation Leading to Degranulation and a Prothrombotic Phenotype

Blood ◽

10.1182/blood-2019-126095 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4887-4887

Author(s):

Joachim Zobel ◽

Tanja Strini ◽

Martin Tischitz ◽

Sina Pohl ◽

Theresa Greimel ◽

...

Keyword(s):

Liver Fibrosis ◽

Bile Acids ◽

Conflicts Of Interest ◽

Annexin V ◽

Farnesoid X Receptor ◽

Fibrinogen Binding ◽

Model Study ◽

Serine Protease Activity ◽

Adult Volunteers ◽

Dose Dependent

Background: Previous articles have identified the farnesoid X receptor (FXR) as an integral part in the formation of coated platelets. Coated platelets are preactivated platelets featuring degranulation, increased fibrinogen binding, and increased serine protease activity leading to fibrin generation. Furthermore, phosphatidylserine exposure is increased and integrin α2bβIII is inhibited - leading to a prothrombotic phenotype despite decreased platelet aggregation. We hypothesize that bile acids, as natural ligands of FXR, lead to a change of platelet phenotype and therefore play a pivotal role in the formation of coated platelets, especially in presence of cholestasis. Methods: Based on previous findings, we incubated human washed platelets of healthy adult volunteers with the synthetic FXR ligand GW4064 in various concentrations (0, 10, 20, 50, 100µM) and used flow cytometry to detect a shift in p-selectin expression, PAC-1 binding and annexin-V-binding. Moreover, we used different concentrations (0, 100, 200, 400, 600µM) of three bile acids (ursodeoxycholic acid, UDCA; chenodeoxycholic acid, CDCA; glycochenodeoxycholic acid, GCDCA) to see if natural FXR ligands induce an effect on the platelet phenotype. Results: We observed a dose dependent shift in annexin-V-binding when treating washed platelets with GW4064 as well as CDCA and GCDCA. Similarly, GW4064 led to increased p-selectin expression while increased PAC-1-binding was only detected at the highest concentration. In contrast, CDCA and GCDCA showed merely slight changes in p-selectin expression whereas PAC-1-binding seemed to be unaffected. However, none of these effects were seen when using UDCA. Conclusion: We conclude that pretreatment of washed platelets with CDCA and GCDCA initiate a dose-dependent shift towards a prothrombotic platelet phenotype. Therefore, we assume that increased levels of certain bile acids drive thrombosis in patients with cholestatic liver injury. Furthermore, a recent mouse model study suggested that platelet derived growth factor β (PDGFβ), a component of α-granula, drives liver fibrosis. Hence, in addition to their prothrombotic effects, coated platelets might exacerbate liver fibrosis. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Randomized, Placebo-controlled, Blinded and Cross-matched Study on the Antiplatelet Effect of Inhaled Nitric Oxide in Healthy Volunteers

Thrombosis and Haemostasis ◽

10.1055/s-0037-1613804 ◽

2000 ◽

Vol 83 (02) ◽

pp. 309-315 ◽

Cited By ~ 27

Author(s):

Axel Herr ◽

Johann Motsch ◽

Alexandra Holzmann ◽

Jörg Weimann ◽

Friedemann Taut ◽

...

Keyword(s):

Nitric Oxide ◽

Platelet Aggregation ◽

Healthy Volunteers ◽

Bleeding Time ◽

Inhibitory Effect ◽

Inhaled Nitric Oxide ◽

Men And Women ◽

Fibrinogen Binding ◽

Matched Study

SummaryThe platelet inhibitory effect of 0-40 ppm inhaled nitric oxide (NO) was investigated in healthy men and women. In both groups, ADPand collagen-induced platelet aggregation was significantly inhibited 20 (T20) and 40 min (T40) after the beginning of inhalation of 5, 10, and 40 ppm. Moreover, in both men and women, the in vitro bleeding time was significantly prolonged at T20 and T40 during inhalation of 40 ppm. Inhalation of NO also inhibited P-selectin expression at 5, 10, and 40 ppm and fibrinogen binding to the GPIIb/IIIa-receptor at 40 ppm. In conclusion, in healthy volunteers, the platelet inhibitory effect of inhaled NO was not dose-related, since it was significant at 5 and 10 ppm but did not increase during the administration of higher NO concentrations. In addition, gender-related differences were only observed in ADP-induced platelet aggregation at 10 ppm and in bleeding time prolongation at 40 ppm.

Download Full-text

Inhibition of platelet aggregation by olive oil phenols via cAMP-phosphodiesterase

British Journal Of Nutrition ◽

10.1017/s0007114507837470 ◽

2008 ◽

Vol 99 (5) ◽

pp. 945-951 ◽

Cited By ~ 62

Author(s):

Mario Dell'Agli ◽

Omar Maschi ◽

Germana V. Galli ◽

Rossana Fagnani ◽

Esther Dal Cero ◽

...

Keyword(s):

Platelet Aggregation ◽

Olive Oil ◽

Human Platelet ◽

Inhibitory Effect ◽

Significant Inhibition ◽

Inhibit Platelet Aggregation ◽

Phenol Content ◽

Camp Phosphodiesterase ◽

Aggregation Assay ◽

Phenolic Constituents

The aim of the present study was to confirm that olive oil phenols reduce human platelet aggregability and to verify the hypothesis that cAMP- and cGMP- phosphodiesterases (PDE) could be one of the targets of the biological effect. Four extracts from oils characterized by a high phenol content (HPE), and low phenol levels (LPE) were prepared and analyzed quali- and quantitatively by HPLC-UV and electrospray ionization–MS/MS. Human washed platelets stimulated with thrombin were used for the aggregation assay. Human platelet cAMP-PDE and recombinant PDE5A1 were used as enzyme source. Platelet aggregation and enzyme activity were assayed in the presence of HPE, LPE and individual phenols. The phenol content of HPE ranged between 250 and 500 mg/kg, whereas the LPE content was 46 mg/kg. The compounds identified were hydroxytyrosol (HT), tyrosol (TY), oleuropein aglycone (OleA) and the flavonoids quercetin (QU), luteolin (LU) and apigenin (AP). OleA was the most abundant phenol (range 23·3 to 37·7 %) and LU was the most abundant flavonoid in the extracts. Oil extracts inhibited platelet aggregation with an 50% inhibitory concentration interval of 1·23–11·2 μg/ml. The inhibitory effect of individual compounds (10 μm) including homovanillyl alcohol (HVA) followed this order: OleA>LU>HT = TY = QU = HVA, while AP was inactive. All the extracts inhibited cAMP-PDE, while no significant inhibition of PDE5A1 (50μg/ml) was observed. All the flavonoids and OleA inhibited cAMP-PDE, whereas HT, TY, HVA (100 μm) were inactive. Olive oil extracts and part of its phenolic constituents inhibit platelet aggregation; cAMP-PDE inhibition is one mechanism through which olive oil phenols inhibit platelet aggregation.

Download Full-text

Hemolytic activity and platelet aggregation inhibitory effect of vipoxin’s basic sPLA2 subunit

Interdisciplinary Toxicology ◽

10.2478/intox-2013-0021 ◽

2013 ◽

Vol 6 (3) ◽

pp. 136-140 ◽

Cited By ~ 3

Author(s):

Silviya Stoykova ◽

Yana Goranova ◽

Ivayla Pantcheva ◽

Vasil Atanasov ◽

Dobri Danchev ◽

...

Keyword(s):

Fatty Acids ◽

Platelet Aggregation ◽

Hemolytic Activity ◽

Blood Cells ◽

Inhibitory Effect ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Erythrocyte Morphology ◽

Dose Dependent ◽

Vipera Ammodytes

ABSTRACT In the present study we evaluated the effect of secreted phospholipase A2 (sPLA2) (the toxic subunit of the heterodimeric neurotoxin vipoxin, isolated from the Bulgarian long-nosed viper Vipera ammodytes meridionalis) on hemolysis, erythrocyte morphology and platelet aggregation. Hemolytic activity of sPLA2 was examined in the presence of saturated (palmitic) and unsaturated (oleic) fatty acids and it was found that oleic acid increased the hemolytic activity of sPLA2 in a concentration-dependent manner, compared to the effect of palmitic acid and controls. The addition of heparin to red blood cells (RBC) suspension containing sPLA2 or mixture of sPLA2 and the corresponding fatty acid led to an inhibition of hemolytic activity. The effect of sPLA2 on RBC morphology resulted in formation of echinocytes (spherocyte subtype), suggesting that RBC could be the possible targets attacked by sPLA2. Vipoxin sPLA2 inhibited (in a dose-dependent manner) platelet aggregation when arachidonic acid and collagen were used as inducers, while in the case of ADP its inhibitory effect was inappreciable.

Download Full-text