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

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.

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

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.

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

Blood ◽  
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.

scholarly journals Effect of Diazepam on Platelet Function

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.

MECHANISMS FOR THE IN VIVO ANTIPLATELET EFFECTS OF ORGANIC NITRATES

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.

Enhancement of ADP-induced Platelet Aggregation by Adrenaline in Vivo and Its Prevention

1973 ◽  
Vol 29 (02) ◽  
pp. 490-498 ◽  
Author(s):  
Hiroh Yamazaki ◽  
Itsuro Kobayashi ◽  
Tadahiro Sano ◽  
Takio Shimamoto
Keyword(s):  
Platelet Count ◽  
Platelet Aggregation ◽  
Platelet Rich Plasma ◽  
The Other ◽  
Endothelial Surface ◽  
Important Interaction ◽  
Blood Coagulability ◽  
The Difference

SummaryThe authors previously reported a transient decrease in adhesive platelet count and an enhancement of blood coagulability after administration of a small amount of adrenaline (0.1-1 µg per Kg, i. v.) in man and rabbit. In such circumstances, the sensitivity of platelets to aggregation induced by ADP was studied by an optical density method. Five minutes after i. v. injection of 1 µg per Kg of adrenaline in 10 rabbits, intensity of platelet aggregation increased to 115.1 ± 4.9% (mean ± S. E.) by 10∼5 molar, 121.8 ± 7.8% by 3 × 10-6 molar and 129.4 ± 12.8% of the value before the injection by 10”6 molar ADP. The difference was statistically significant (P<0.01-0.05). The above change was not observed in each group of rabbits injected with saline, 1 µg per Kg of 1-noradrenaline or 0.1 and 10 µg per Kg of adrenaline. Also, it was prevented by oral administration of 10 mg per Kg of phenoxybenzamine or propranolol or aspirin or pyridinolcarbamate 3 hours before the challenge. On the other hand, the enhancement of ADP-induced platelet aggregation was not observed in vitro, when 10-5 or 3 × 10-6 molar and 129.4 ± 12.8% of the value before 10∼6 molar ADP was added to citrated platelet rich plasma (CPRP) of rabbit after incubation at 37°C for 30 second with 0.01, 0.1, 1, 10 or 100 µg per ml of adrenaline or noradrenaline. These results suggest an important interaction between endothelial surface and platelets in connection with the enhancement of ADP-induced platelet aggregation by adrenaline in vivo.

A Comparison of the Effect of Decorsin and Two Disintegrins, Albolabrin and Eristostatin, on Platelet Function

1995 ◽  
Vol 74 (05) ◽  
pp. 1316-1322 ◽  
Author(s):  
Mary Ann McLane ◽  
Jagadeesh Gabbeta ◽  
A Koneti Rao ◽  
Lucia Beviglia ◽  
Robert A Lazarus ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Function ◽  
Sequence Similarity ◽  
Platelet Aggregation Inhibitors ◽  
Antiplatelet Drug ◽  
Rgd Sequence ◽  
Fibrinogen Receptor ◽  
Activated Platelets

SummaryNaturally-occurring fibrinogen receptor antagonists and platelet aggregation inhibitors that are found in snake venom (disintegrins) and leeches share many common features, including an RGD sequence, high cysteine content, and low molecular weight. There are, however, significant selectivity and potency differences. We compared the effect of three proteins on platelet function: albolabrin, a 7.5 kDa disintegrin, eristostatin, a 5.4 kDa disintegrin in which part of the disintegrin domain is deleted, and decorsin, a 4.5 kDa non-disintegrin derived from the leech Macrobdella decora, which has very little sequence similarity with either disintegrin. Decorsin was about two times less potent than albolabrin and six times less potent than eristostatin in inhibiting ADP- induced human platelet aggregation. It had a different pattern of interaction with glycoprotein IIb/IIIa as compared to the two disintegrins. Decorsin bound with a low affinity to resting platelets (409 nM) and to ADP-activated platelets (270 nM), and with high affinity to thrombin- activated platelets (74 nM). At concentrations up to 685 nM, it did not cause expression of a ligand-induced binding site epitope on the (β3 subunit of the GPIIb/IIIa complex. It did not significantly inhibit isolated GPIIb/IIIa binding to immobilized von Willebrand Factor. At low doses (1.5-3.0 μg/mouse), decorsin protected mice against death from pulmonary thromboembolism, showing an effect similar to eristostatin. This suggested that decorsin is a much more potent inhibitor of platelet aggregation in vivo than in vitro, and it may have potential as an antiplatelet drug.

Interactions of Staphylokinase with Human Platelets

1995 ◽  
Vol 73 (03) ◽  
pp. 472-477 ◽  
Author(s):  
H R Lijnen ◽  
B Van Hoef ◽  
D Collen
Keyword(s):  
Platelet Aggregation ◽  
Platelet Function ◽  
Specific Binding ◽  
Platelet Rich Plasma ◽  
Human Platelets ◽  
Normal Plasma ◽  
Atp Secretion ◽  
Platelet Disaggregation ◽  
Activation Rate

SummaryThe interactions of recombinant staphylokinase (SakSTAR) with human platelets were investigated in a buffer milieu, in a human plasma milieu in vitro, and in plasma from patients with acute myocardial infarction (AMI) treated with SakSTAR.In a buffer milieu, the activation rate of plasminogen by SakSTAR or streptokinase (SK) was not significantly altered by addition of platelets. Specific binding of SakSTAR or SK to either resting or thrombin- activated platelets was very low. ADP-induced or collagen-induced platelet aggregation in platelet-rich plasma (PRP) was 94 ± 2.7% or 101 ± 1.7% of control in the presence of 0.1 to 20 μM SakSTAR, with corresponding values of 95 ± 2.8% or 90 ± 4.6% of control in the presence of 0.1 to 4 μM SK. No effects were observed on platelet disaggregation. ATP secretion following collagen-induced platelet aggregation was 4.3 ± 0.26 μM for SakSTAR (at concentrations of 0.1 to 20 μM) and 4.4 ± 0.35 μM for SK (at concentrations of 0.1 to 4 μM), as compared to 3.4 ± 0.70 μM in the absence of plasminogen activator.Fifty % lysis in 2 h (C50) of 60 μl 125I-fibrin labeled platelet-poor plasma (PPP) clots prepared from normal plasma or from plasma of patients with Glanzmann thrombasthenia and immersed in 0.5 ml normal plasma, was obtained with 12 or 16 nM SakSTAR and with 49 or 40 nM SK, respectively. C50 values for lysis of 60 μl PRP clots prepared from normal or patient plasma were also comparable for SakSTAR (19 or 21 nM), whereas SK was 2-fold more potent toward PRP clots prepared from Glanzmann plasma as compared to normal plasma (C50 of 130 versus 270 nM).No significant effect of SakSTAR on platelet function was observed in plasma from patients with AMI treated with SakSTAR, as revealed by unaltered platelet count, platelet aggregation and ATP secretion.Thus, no effects of high SakSTAR concentrations were observed on human platelets in vitro, nor of therapeutic SakSTAR concentrations on platelet function in plasma.

Comparison of the effect of coagulation and platelet function impairments on various mouse bleeding models

2014 ◽  
Vol 112 (08) ◽  
pp. 412-418 ◽  
Author(s):  
Nima Vaezzadeh ◽  
Ran Ni ◽  
Paul Y. Kim ◽  
Jeffrey I. Weitz ◽  
Peter L. Gross
Keyword(s):  
Platelet Aggregation ◽  
Platelet Function ◽  
Saphenous Vein ◽  
Ex Vivo ◽  
Arterial Injury ◽  
Inhibitory Antibody ◽  
Study Objective ◽  
Tail Tip

SummaryHaemostatic impairments are studied in vivo using one of several murine bleeding models. However it is not known whether these models are equally appropriate for assessing coagulation or platelet function defects. It was our study objective to assess the performance of arterial, venous and combined arterial and venous murine bleeding models towards impaired coagulation or platelet function. Unfractionated heparin (UFH) or αIIbβ3 inhibitory antibody (Leo.H4) were administered to mice, and their effects on bleeding in saphenous vein, artery, and tail tip transection models were quantified and correlated with their effects on plasma clotting and ADP-induced platelet aggregation, respectively. All models exhibited similar sensitivity with UFH (EC50 dose = 0.19, 0.13 and 0.07 U/g, respectively) (95% CI = 0.14 – 0.27, 0.08 – 0.20, and 0.03 – 0.16 U/g, respectively). Maximal inhibition of ex vivo plasma clotting could be achieved with UFH doses as low as 0.03 U/g. In contrast, the saphenous vein bleeding model was less sensitive to αIIbβ3 inhibition (EC50 = 6.9 µg/ml) than tail transection or saphenous artery bleeding models (EC50 = 0.12 and 0.37 µg/ml, respectively) (95% CI = 2.4 – 20, 0.05 – 0.33, and 0.06 – 2.2 µg/ml, respectively). The EC50 of Leo.H4 for ADP-induced platelet aggregation in vitro (8.0 µg/ml) was at least 20-fold higher than that of the tail and arterial, but not the venous bleeding model. In conclusion, venous, arterial and tail bleeding models are similarly affected by impaired coagulation, while platelet function defects have a greater influence in models incorporating arterial injury.

Arginine vasopressin release from human platelets after irreversible aggregation

1990 ◽  
Vol 78 (1) ◽  
pp. 113-116 ◽  
Author(s):  
Giovanni Anfossi ◽  
Elena Mularoni ◽  
Mariella Trovati ◽  
Paola Massucco ◽  
Luigi Mattiello ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Arginine Vasopressin ◽  
Platelet Rich Plasma ◽  
Human Platelets ◽  
Vasopressin Release ◽  
Irreversible Aggregation ◽  
Local Release

1. The release of arginine vasopressin from human platelets was investigated in platelet-rich plasma after irreversible aggregation induced by adenosine 5′-pyrophosphate, collagen, sodium arachidonate, thrombin and adrenaline in vitro. 2. Arginine vasopressin levels were significantly higher in the supernatant from stimulated platelet-rich plasma than from unstimulated samples, reaching 3.5 × 10−12 (range 1.6–12.5 × 10−12) mol/l in the absence of an aggregating agent, 8.8 × 10−12 (range 4.2–17.5 × 10−12) mol/l after adenosine 5′-pyrophosphate, 13.7 × 10−12 (2.2–63.2 × 10−12) mol/l after collagen, 7.8 × 10−12 (2.2–14.6 × 10−12) mol/l after sodium arachidonate, 7.8 × 10−12 (2.2–16.3 × 10−12) mol/l after thrombin and 12.2 × 10−12 (4.8–32.1 × 10−12) mol/l after adrenaline. 3. An arginine vasopressin level of 18 × 10−12 mol/l, which can be achieved physiologically, increased the sensitivity of platelets to adenosine 5′-pyrophosphate and collagen in vitro; the same concentration of arginine vasopressin caused a potentiation of the effect of catecholamines on the response of platelets to sodium arachidonate. 4. These results indicate that intraplatelet arginine vasopressin is released during aggregation and suggest that a local release of arginine vasopressin could occur after complete platelet aggregation in vivo.

Inhibition of Human Platelet Aggregation by the Proteolytic Effect of Streptokinase (SK). Role of the Factor VIII Related Antigen

1975 ◽  
Author(s):  
R. Castillo ◽  
S. Maragall ◽  
J. A. Guisasola ◽  
F. Casals ◽  
C. Ruiz ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Factor Viii ◽  
Platelet Rich Plasma ◽  
Gel Filtration ◽  
Inhibitory Effect ◽  
Factor Viii Related Antigen ◽  
Human Factor Viii ◽  
Induced Aggregation

Defective ADP-induced platelet aggregation has been observed in patients treated with streptokinase. This same effect appears “in vitro” when adding SK to platelet rich plasma (PRP). Classic hemophilia and normal platelet poor plasmas (PPP) treated with SK inhibit the aggregation of washed platelets; plasmin-treated normal human serum also shows an inhibitory effect on platelet aggregation. However, von Willebrand SK-treated plasmas do not inhibit the aggregation of washed platelets. The same results appear when plasmas are previously treated with a rabbit antibody to human factor VIII.This confirms that the antiaggregating effect is mainly linked to the digested factor VIII related antigen.The inhibition of ADP-induced platelet aggregation has been proved in gel filtration-isolated and washed platelets from SK-treated PRP.Defective ristocetin-induced platelet aggregation has also been observed- This action does not appear in washed platelets from SK-treated PRP in presence of normal PPP, but it does in presence of SK-treated PPP, which suggests that the inhibition of the ristocetin-induced aggregation is due to the lack of factor VIII and not to the factor VIII-related products.Heparin, either “in vivo” or “in vitro”, has corrected the antiaggregating effect of SK.

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