Inhibition of platelet function by A02131-1, a novel inhibitor of cGMP- specific phosphodiesterase, in vitro and in vivo

The effect of A02131–1 [3-(5′-hydroxymethyl-2′-furyl)-1-benzyl thieno (3,2-c)pyrazole], a cGMP-specific phosphodiesterase (PDE) inhibitor, on platelet function was investigated. The compound was found to inhibit the aggregation of and adenosine triphosphate (ATP) release from human platelet-rich plasma and washed platelets that were induced by aggregation inducing drugs such as arachidonic acid (AA), collagen, U46619, platelet-activating factor (PAF), adenosine diphosphate (ADP) and A23187, and the inhibitory effect was concentration-dependent. A02131–1 also disaggregated the performed platelet aggregates induced by these inducers. Thromboxane B2 (TXB2) formations caused by collagen, PAF, ADP, and A23187 were inhibited by A02131–1 at concentrations that did not affect the AA-induced formation of TXB2 and prostaglandin D2 (PGD2). A02131–1 suppressed both the generation of inositol 1,4,5- triphosphate (IP3) and the increase of intracellular Ca2+ concentration stimulated by these aggregation inducers. A02131–1 was shown to increase the cAMP and cGMP levels in platelets and the extent was found to be dependent on concentration as well as time. A02131–1 increased the cAMP level much more slowly than the cGMP level. Activities of adenylate cyclase, guanylate cyclase, and PDEs (type I and III) were not altered by A02131–1. However, the activity of cGMP-specific PDE (type V) was inhibited by A02131–1. The antiplatelet aggregation activity and the effect on raising cAMP level of A02131–1 were both potentiated by prostaglandin E1 (PGE1). In the mouse tail bleeding test, A02131–1 was clearly shown to be more effective than dipyridamole in prolonging the tail bleeding time of conscious mice. These data indicate that A02131–1 is a cGMP-specific PDE (type V) inhibitor in human platelets.

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The Effect of Barbituric Acid Derivatives on Platelet Function in Vitro and in Vivo

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649080 ◽

1973 ◽

Vol 30 (02) ◽

pp. 315-326

Author(s):

J. Heinz Joist ◽

Jean-Pierre Cazenave ◽

J. Fraser Mustard

Keyword(s):

Platelet Aggregation ◽

Platelet Function ◽

Barbituric Acid ◽

Platelet Rich Plasma ◽

Inhibitory Effect ◽

Primary Response ◽

Sodium Pentobarbital ◽

Barbituric Acid Derivatives

SummarySodium pentobarbital (SPB) and three other barbituric acid derivatives were found to inhibit platelet function in vitro. SPB had no effect on the primary response to ADP of platelets in platelet-rich plasma (PRP) or washed platelets but inhibited secondary aggregation induced by ADP in human PRP. The drug inhibited both phases of aggregation induced by epinephrine. SPB suppressed aggregation and the release reaction induced by collagen or low concentrations of thrombin, and platelet adherence to collagen-coated glass tubes. The inhibition by SPB of platelet aggregation was readily reversible and isotopically labeled SPB did not become firmly bound to platelets. No inhibitory effect on platelet aggregation induced by ADP, collagen, or thrombin could be detected in PRP obtained from rabbits after induction of SPB-anesthesia.

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The influence of the haematocrit on primary haemostasis in vitro

Thrombosis and Haemostasis ◽

10.1160/th05-06-0424 ◽

2005 ◽

Vol 94 (12) ◽

pp. 1213-1218 ◽

Cited By ~ 70

Author(s):

Marco Eugster ◽

Walter H. Reinhart

Keyword(s):

Platelet Function ◽

Type I Collagen ◽

Inverse Correlation ◽

Type I ◽

Membrane Pore ◽

Closure Time ◽

Function Analyzer ◽

Platelet Function Analyzer

SummaryPrimary haemostasis consists of platelet adhesion to subendothelial collagen, their activation and aggregation and finally the formation of a platelet plug. Erythrocytes are involved in this process because they flow in the center of the vessel and push platelets towards the site of action on the vessel wall and enhance shear forces, which activate platelets. In the platelet function analyzer PFA-100® (Dade Behring, Düdingen, Switzerland), the in vivo situation is simulated in vitro with blood being aspirated at high shear rates (5000s-1) through a capillary into a membrane pore with a diameter of 150 μm coated with type I collagen and either epinephrine or adenosine diphosphate. Aggregating platelets plug the pore and stop the flow, which is measured as the closure time. We analysed the influence of erythrocytes on platelet function analyzer measurements by systematic variation of the haematocrit (20,30,40,and 50%) at constant platelet counts of 289±61 ×103/μl plasma, or 152±30 ×103/μl blood, 96±9 ×103/μl blood and 54±5 ×103/μl blood, respectively. An inverse correlation was found between haematocrit and closure time under all circumstances. A decrease of the platelet count by 50 ×103 /μl could be compensated for by a 10% increase in haematocrit. The haematocrit must, therefore, be taken into consideration for the correct interpretation of PFA-100® measurements. Our data also provide a pathophysiological rationale to reduce the risk of bleeding in patients with thrombocytopenia and anaemia by normalizing the haematocrit with erythrocyte transfusions.

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Are Platelet Aggregation Tests the Best Way to Assess New Drugs for Arterial Disease

General medicine and Clinical Practice ◽

10.31579/2639-4162/003 ◽

2018 ◽

Vol 1 (1) ◽

pp. 01-03

Author(s):

Mark I. M. Noble

Keyword(s):

Platelet Aggregation ◽

Platelet Function ◽

Platelet Rich Plasma ◽

New Drugs ◽

In Vitro Test ◽

In Vivo Efficacy ◽

Experimental Animals ◽

Stenosed Artery

Over many years, laboratory testing of platelet aggregability have been carried out in attempts to develop drugs that would prevent thrombosis in arteries. The problems encountered included the question of methodology. Blood samples have to be anticoagulated in order to study the platelets. Anti-coagulation with citrate and tests on derived platelet rich plasma did not correlate at all well with thrombus growth in the stenosed coronary arteries of experimental animals and citrate removes the calcium ions which are vital for platelet function. Anticoagulation with heparin also interfered with platelet function, so that now, hirudins are the preferred anticoagulant. However it was observed that if, instead of stimulating platelet aggregation with adrenaline or ADP, serotonin was applied to the preparation, very little aggregation took place in spite of serotonin 5HT2A antagonists being the most potent inhibitors of thrombus growth in experimental animals. Another indicator that primary platelet agggregation is not a predictor of in vivo efficacy was the finding that 5HT2A antagonism inhibited aggregate growth. In a stenosed artery the platelets are activated by increased shear stress and blood turbulence with release of platelet serotonin causing positive feedback activation of more platelets. At present, there does not seem to be a bench in vitro test that accurately predicts in vivo efficacy in stenosed artery occlusive thrombosis.

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C57BL/6JOlaHsd Mice with Tandem Deletion of the Multimerin 1 and Alpha-Synuclein Genes Have Impaired Platelet Function in Vivo and in Vitro That Can Be Corrected by Multimerin 1

Blood ◽

10.1182/blood.v112.11.3926.3926 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3926-3926 ◽

Cited By ~ 1

Author(s):

Subia Tasneem ◽

Adili Reheman ◽

Heyu Ni ◽

Catherine P.M. Hayward

Keyword(s):

Platelet Aggregation ◽

Platelet Function ◽

Knockout Mice ◽

Platelet Adhesion ◽

Thrombus Formation ◽

Alpha Synuclein ◽

Type I ◽

Significant Difference

Abstract Studies of mice with genetic deficiencies have provided important insights on the functions of many proteins in thrombosis and hemostasis. Recently, a strain of mice (C57BL/6JOlaHsd, an inbred strain of C57BL/6J) has been identified to have a spontaneous, tandem deletion of the multimerin 1 and α-synuclein genes, which are also adjacent genes on human chromosome 4q22. Multimerin 1 is an adhesive protein found in platelets and endothelial cells while α-synuclein is a protein found in the brain and in blood that is implicated in neurodegenerative diseases and exocytosis. In vitro, multimerin 1 supports platelet adhesion while α-synuclein inhibits α-granule release. We postulated that the loss of multimerin 1 and α-synuclein would alter platelet function and that recombinant human multimerin 1 might correct some of these abnormalities. We compared platelet adhesion, aggregation and thrombus formation in vitro and in vivo in C57BL/6JOlaHsd and C57BL/6 mice. Thrombus formation was studied by using the ferric-chloride injured mesenteric arteriole thrombosis model under intravital microscopy. We found that platelet adhesion, aggregation and thrombus formation in C57BL/6JOlaHsd were significantly impaired in comparison to control, C57BL/6 mice. The number of single platelets, deposited 3–5 minutes after injury, was significantly decreased in C57BL/6JOlaHsd mice (P <0.05, platelets/min: C57BL/6 = 157 ± 15, n=16; C57BL/6JOlaHsd = 77 ± 13, n=17). Moreover, thrombus formation in these mice was significantly delayed. Thrombi in C57BL/6JOlaHsd were unstable and easily dissolved, which resulted in significant delays (P<0.001) in vessel occlusion (mean occlusion times: C57BL/6 = 15.6 ± 1.2 min, n=16; C57BL/6JOlaHsd = 31.9 ± 2.1 min, n=17). We further tested platelet function in these mice by ADP and thrombin induced platelet aggregation using platelet rich plasma and gel-filtered platelets, respectively. Although no significant differences were seen with ADP aggregation, thrombin-induced platelet aggregation was significantly impaired in C57BL/6JOlaHsd mice. Platelet adhesion to type I collagen (evaluated using microcapillary chambers, perfused at 1500 s−1 with whole blood) was also impaired in C57BL/6JOlaHsd mice. However, platelets from C57BL/6JOlaHsd mice showed a normal pattern of agonist-induced release of α-granule P-selectin. Multimerin 1 corrected the in vitro aggregation and adhesion defects of C57BL/6JOlaHsd platelets. Furthermore, the transfusion of multimerin 1 into C57BL/6JOlaHsd mice corrected the impaired platelet deposition and thrombus formation in vivo. No significant difference was found in tail bleeding time between the two groups of mice. As α-synuclein knockout mice have a shortened time to thrombus formation (Circulation2007;116:II_76), the effects of multimerin 1 on impaired platelet function in C57BL/6JOlaHsd mice provide supportive evidence that multimerin 1 contributes to platelet adhesion and thrombus formation at the site of vessel injury. The findings suggest multimerin 1 knockout mice will be useful to explore platelet function. The first two authors and participating laboratories contributed equally to this study.

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Recovery Of Platelet Function After Aspirin Ingestion: In Vivo And In Vitro Studies

10.1055/s-0038-1652098 ◽

1981 ◽

Author(s):

B A Bradlow ◽

N Chetty ◽

M Birnbaum

Keyword(s):

Platelet Function ◽

Recovery Of Function ◽

Platelet Rich Plasma ◽

Recovery Period ◽

In Vitro Studies ◽

In Vivo Studies ◽

Partial Recovery ◽

Mean Values

Blood was taken from normal volunteers before and two hours after a 600mg oral dose of aspirin (ASA). Platelet rich plasma (PRP) was prepared from both samples. Mixtures of normal and ASA treated PRP were tested for aggregation responses and for production of thromboxane B2 (TXB2) by RIA (New England Nuclear). Normal aggregation responses were found when the mixture contained 26% of normal platelets (NP) with ADP, 40% NP with adrenalin, 54% NP with collagen and 52% NP with arachidonic acid (AA)(Mean Values). TXB2 production in the mixtures increased when the proportion of normal platelets was raised above 10 to 30%. These findings indicate partial restoration of platelet function by an admixture of 10-20% NP to ASA treated platelets although 50% NP was necessary to restore all functions tested. In vivo studies on seven volunteers given 300mg ASA showed partial recovery of malondialdehyde production after 72 hours, partial return of aggregation responses to collagen, ADP, adrenalin and AA at 96 hours and partial or full return of aggregation responses in all subjects by 120 hours. Since new platelets enter the circulation at the rate of 10-15% per day the results of our in vitro studies suggest that some recovery of platelet function might occur as soon as 24 hours after a dose of ASA and full recovery within 3-5 days. The longer recovery period indicated by the in vivo studies may be attributable to the effect of ASA on megakaryocyte cyclooxygenase.Conclusion: ASA administered once every 24 hours may be sufficient to suppress platelet function adequately and continuously but partial recovery of function might occur 48-72 hours after ASA ingestion.

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GSK3β Is a Negative Regulator of Platelet Function In Vitro and In Vivo.

Blood ◽

10.1182/blood.v108.11.395.395 ◽

2006 ◽

Vol 108 (11) ◽

pp. 395-395

Author(s):

Donna S. Woulfe ◽

Shelley August ◽

Dongjun Li

Keyword(s):

Platelet Aggregation ◽

Platelet Function ◽

Ferric Chloride ◽

Arterial Thrombosis ◽

Platelet Rich Plasma ◽

Human Platelets ◽

Negative Regulator ◽

Response Curves

Abstract GSK3β is a ser-thr kinase that is itself phosphorylated on ser9 by the kinase Akt. Because Akt has recently been shown to regulate platelet aggregation and arterial thrombosis in mice, we sought to identify Akt substrates in platelets that may play important roles in platelet function. We show here that the Akt effector, GSK3β, is present in platelets and becomes phosphorylated after treatment of mouse or human platelets with ADP or thrombin receptor-activating peptides (TRAP). Agonist-dependent phosphorylation of GSK3β is reduced by pre-treatment of mouse or human platelets with the PI3K inhibitor LY294002 and is also reduced in platelets from Akt2−/−Akt1+/− mice relative to non-littermate controls, suggesting that agonist-induced GSK3β phosphorylation is partially PI3K- and Akt-dependent. To determine whether GSK3β plays a role in platelet function, aggregation and secretion of dense granule contents were evaluated in human platelets treated with the GSK3 inhibitors, LiCl or SB216763. The dose-response curves for agonist-induced platelet aggregation and secretion were left-shifted in the presence of either inhibitor compared to untreated control platelets, suggesting that GSK3 activity suppresses platelet aggregation. Comparative immunoblots suggest that GSK3β is more highly expressed in platelets than GSK3α. Therefore, to confirm that GSKβ plays a suppressive role in platelet function, the aggregation of platelet-rich plasma (PRP) from GSK3β+/− mice was compared to that of non-littermate controls (GSK3β −/− mice die in utero). PRP from GSK3β+/− mice showed enhanced aggregation and secretion in response to U46619 or TRAP compared to control PRP. TRAP-induced binding of AlexaFluor-fibrinogen to platelet surfaces was also enhanced in washed platelets from GSK3β+/− mice compared to control platelets. Finally, the effect of GSK3β on platelet function in vivo was evaluated using two thrombosis models: a ferric chloride injury model of arterial thrombosis and a collagen-induced model of disseminated thrombosis. In the arterial thrombosis model, all GSK3β+/− mice (n=5) formed stable occlusive thrombi after ferric chloride injury to the carotid artery, whereas the majority of wildtype mice (67%) formed no thrombi, 27% formed stable occlusive thrombi, and 7% formed unstable thrombi under the same conditions (n=15). In a model of disseminated thrombosis, injection of a combination of collagen (170 μg/kg) and epinephrine (350 μM/kg) resulted in reduced survival of GSK3β+/− mice 10 minutes post-injection relative to wildtype mice (20%, n=5 versus 83%, n=6, respectively). Histological examination of lung sections suggested that all mice that died did so due to pulmonary embolism. These data suggest that removal of a single allele of GSK3β in mice confers enhanced sensitivity to thrombotic insult. Taken together, these results suggest that GSK3β is a substrate of Akt-dependent phosphorylation in platelets and, in contrast to the function of Akt, acts as a negative regulator of platelet function in vitro and in vivo.

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A nucleus-like compartment shields bacteriophage DNA from CRISPR-Cas and restriction nucleases

10.1101/370791 ◽

2018 ◽

Cited By ~ 8

Author(s):

Senén D. Mendoza ◽

Joel D. Berry ◽

Eliza S. Nieweglowska ◽

Lina M. Leon ◽

David A. Agard ◽

...

Keyword(s):

Type I ◽

Type Ii ◽

Immune Systems ◽

Dna Targeting ◽

Rna Targeting ◽

Immune Pathways ◽

Type V ◽

Restriction Modification

All viruses require strategies to inhibit or evade the immunity pathways of cells they infect. The viruses that infect bacteria, bacteriophages (phages), must avoid nucleic-acid targeting immune pathways such as CRISPR-Cas and restriction endonucleases to replicate efficiently1. Here, we show that a jumbo phage infecting Pseudomonas aeruginosa, phage ΦKZ, is resistant to many immune systems in vivo, including CRISPR-Cas3 (Type I-C), Cas9 (Type II-A), Cas12 (Cpf1, Type V-A), and Type I restriction-modification (R-M) systems. We propose that ΦKZ utilizes a nucleus-like shell to protect its DNA from attack. Supporting this, we demonstrate that Cas9 is able to cleave ΦKZ DNA in vitro, but not in vivo and that Cas9 is physically occluded from the shell assembled by the phage during infection. Moreover, we demonstrate that the Achilles heel for this phage is the mRNA, as translation occurs outside of the shell, rendering the phage sensitive to the RNA targeting CRISPR-Cas enzyme, Cas13a (C2c2, Type VI-A). Collectively, we propose that the nucleus-like shell assembled by jumbo phages enables potent, broad spectrum evasion of DNA-targeting nucleases.

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Effect of Diazepam on Platelet Function

10.1055/s-0039-1680490 ◽

1977 ◽

Author(s):

A. C. Carvalho ◽

R. W. Colman ◽

R. Vaillancourt ◽

R. Cabrai ◽

R. Anaya

Keyword(s):

Platelet Aggregation ◽

Platelet Function ◽

Platelet Rich Plasma ◽

Normal Subjects ◽

Serotonin Release ◽

Significant Inhibition ◽

Function Evaluation ◽

Induced Aggregation

Diazepam (Valium) is one of the most prescribed medications in the world. Patients on Diazepam may need platelet function evaluation. Therefore, a study of its effect on both in vivo and in vitro platelet function was undertaken in 8 normal volunteers. Diazepam (10–40μg/ml) was incubated in vitro with platelet rich plasma (250,000/μl) at intervals of 15, 30, 60, 120, and 240 minutes followed by determination of platelet aggregation and 14C-serotonin release. Fifty percent inhibition of platelet aggregation and release by Diazepam was obtained at 1 hr with epinephrine (p<0.01) and at 2 hrs with ADP (p<0.01), but no significant effect was noted with collagen. The Diazepam inhibitory effect on platelet aggregation and release was overcome by high concentrations of aggregating agents, suggesting that its primary effect is not mediated by inhibition of prostaglandin synthesis.Following oral ingestion of 5mg of Diazepam, platelet aggregation and 14C-serotonin release were determined serially (2, 4, 8, 12, 24, and 48 hours) in the 8 normal subjects. After 8 hours, Diazepam inhibited ADP-induced aggregation and release by 39% (p<0.01) and epinephrine by 50% (p<0.01). No significant inhibition of collagen was observed. Forty-eight hours after Diazepam intake, platelet function returned to normal in all subjects.Our data show that Diazepam impairs both platelet aggregation and release in vitro and in vivo. Although the effect of Diazepam on in vivo hemostasis is still uncertain, our results suggest caution in the interpretation of platelet function testing in patients on this drug.

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In vivo N-Terminomics Highlights Novel Functions of ADAMTS2 and ADAMTS14 in Skin Collagen Matrix Building

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2021.643178 ◽

2021 ◽

Vol 8 ◽

Author(s):

Cédric Leduc ◽

Laura Dupont ◽

Loïc Joannes ◽

Christine Monseur ◽

Dominique Baiwir ◽

...

Keyword(s):

Mouse Skin ◽

Collagen Matrix ◽

Extracellular Proteins ◽

Surface Proteins ◽

Type I ◽

Skin Collagen ◽

A Disintegrin And Metalloproteinase ◽

Type V

A disintegrin and metalloproteinase with thrombospondin type I motif (ADAMTS)2 and ADAMTS14 were originally known for their ability to cleave the aminopropeptides of fibrillar collagens. Previous work using N-terminomic approach (N-TAILS) in vitro led to the identification of new substrates, including some molecules involved in TGF-β signaling. Here, N-TAILS was used to investigate the substrates of these two enzymes in vivo, by comparing the N-terminomes of the skin of wild type mice, mice deficient in ADAMTS2, in ADAMTS14 and in both ADAMTS2 and ADAMTS14. This study identified 68 potential extracellular and cell surface proteins, with the majority of them being cleaved by both enzymes. These analyses comfort their role in collagen matrix organization and suggest their implication in inflammatory processes. Regarding fibrillar collagen, this study demonstrates that both ADAMTS2 and ADAMTS14 are involved in the processing of the aminopropeptide of alpha1 and alpha2 type V collagen. It also revealed the existence of several cleavage sites in the Col1 domain and in the C-propeptide of type I collagens. In addition to collagens and other extracellular proteins, two major components of the cell cytoskeleton, actin and vimentin, were also identified as potential substrates. The latter data were confirmed in vitro using purified enzymes and could potentially indicate other functions for ADAMTS2 and 14. This original investigation of mouse skin degradomes by N-terminomic highlights the essential role of ADAMTS2 and ADAMTS14 in collagen matrix synthesis and turnover, and gives clues to better understand their functions in skin pathophysiology. Data are available via ProteomeXchange with identifier PXD022179.

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MECHANISMS FOR THE IN VIVO ANTIPLATELET EFFECTS OF ORGANIC NITRATES

10.1055/s-0038-1643435 ◽

1987 ◽

Author(s):

R De Caterina ◽

D Giannessi ◽

W Bernini ◽

A Mazzone

Keyword(s):

Platelet Aggregation ◽

Platelet Function ◽

Isosorbide Dinitrate ◽

Platelet Rich Plasma ◽

Secondary Wave ◽

Organic Nitrates ◽

Inhibit Platelet Aggregation ◽

Threshold Doses

Organic nitrates (nitroglycerin, isosorbide dinitrate) are inhibitors of platelet function more effective in vivo than in vitro (Am J Cardiol 1984; 53:1683), the in vivo effect requiring concentrations 10-100 times lower than in vitro. We have previously excluded that such difference is due to elicitation by nitrates of prostacyclin synthesis in human endothelial cells or vascular fragments (Circulation 1985; 71:176). In the present study we evaluated alternative explanations: that the difference is due (1) to generation of more active drug metabolites; (2) to synergism between nitrates and prostacyclin in the inhibition of platelet function. Isosorbide dinitrate (ISDN) and its two main in vivo metabolites, isosorbide-2-mononitrate (IS-2-MN) and isosorbide-5-mononitrate (IS-5-MN), were compared in their ability to inhibit platelet aggregation and thromboxane (TX) B2 formation (RIA) in respone to threshold doses of ADP, adrenaline, collagen, arachidonic acid and thrombin in citrated platelet-rich plasma. The same tests were performed in 10 healthy volunteers before, during (at 5, 15 and 30 min) and after infusion of the three drugs at 8 mg/h for 30 min in 3 different days. Finally, the concentration of prostacyclin (and its stable analogue Iloprost) added in vitro to platelets, and required to inhibit platelet aggregation by 50% (IC50) after 5 min pre-incubation of platelets with nitrates was determined. In vitro incubation of platelets with IS-2-MN resulted in greater inhibition of both aggregation and TX formation (by ADP and adrenaline) than with ISDN and IS-5-MN. At 10−7M, only IS-2-MN significantly inhibited aggregation (−12%, P< 0.05) and TX formation (from 9.2±1.8 to 5.9± 0.6 ng/ml) by ADP, while minimum effective concentrations were 10−7M for ISDN and 10−7m for IS-5-MN. These in vitro differences are unlikely to be the explanation of in vivo findings, since IS-2-MN, ISDN and IS-5-MN were equipotent when administered in vivo (complete abolition of secondary wave after ADP and adrenaline at 30 min of infusion). At supra-threshold doses of all the aggregating agents, all three drugs, at 10−7M, decreased IC50 for prostacyclin from 2.9± 1.3 to 0.32± 0.18 nM (P< 0.01). Synergim with prostacyclin is most likely to account, at least partially, for in vivo antiplatelet effects by organic nitrates.

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