scholarly journals A New Model for Antiplatelet Drug Discovery

1977 ◽  
Author(s):  
R.N. Saunders ◽  
T.S. Burns ◽  
E.R. Waskawic ◽  
M.R. Stelzer
Keyword(s):  
Platelet Count ◽  
Platelet Rich Plasma ◽  
Antiplatelet Agents ◽  
Adenosine Diphosphate ◽  
Male Rats ◽  
Antiplatelet Drug ◽  
Preliminary Evaluation ◽  
Transient Ischemic Attacks

Circulating platelet aggregates (CPA) have been observed by Wu and Hoak (Lancet 2: 294, 1974) in the blood of patients with transient ischemic attacks, acute myocardial infarction and acute peripheral arterial insufficiency. We have discovered that retired breeder (RB) male rats have spontaneous CPA which respond to therapy with clinically proven antiplatelet drugs. CPA are described as the ratio of the platelet count from blood drawn into a citrate/formalin solution compared to the platelet count from blood drawn into a citrate solution. A platelet aggregate ratio (PAR) of 1.0 is indicative of the absence of CPA. The PAR of 24 virgin male rats (0.94 ± .02, S.E.M.) was significantly greater (P<0.01) than that of 34 male RB rats (0.77 ± .02)-indicating the presence of increased CPA in the RB rats. Acetylsalicylic acid (ASA) and sulfinpyrazone (S) have been successfully used clinically in patients with CPA and transient ischemic attacks (Stroke 6: 521, 1975).RB rats treated with ASA or S (100 mg/kg/ day, i.g. for 8 days) demonstrated significantly (P < 0.01) increased PAR of 0.88 ± 0.02 and 0.99 ± 0.02 respectively. In vitro responses of RB platelets in citrate-treated platelet-rich plasma to adenosine diphosphate (0.4 yM to 3 uM/109 platelets) and collagen (420 μg to 1470 μg/109 platelets) were similar in sensitivity to platelets from virgin rats. This suggests that the hyperactivity of RB rat platelets expressed as CPA is related to an increased aggregate stimulation in vivo. The results indicate that the male RB rat may be a useful model for the preliminary evaluation of therapeutically useful antiplatelet agents.

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.

The Effects Of Prostacyclin (PGI2) On Intravascular Platelet Aggregation

1981 ◽  
Author(s):  
G G Duncan ◽  
G Mallarkey ◽  
G M Smith
Keyword(s):  
Platelet Count ◽  
Guinea Pigs ◽  
Adenosine Diphosphate ◽  
Before And After ◽  
Dependent Inhibition ◽  
Induced Aggregation ◽  
Dose Dependent ◽  
Anaesthetised Rats

Intravascular aggregation can be measured by counting the number of circulating platelets before and after the injection of aggregation agents. The Technicon Autocounter was modified to count platelets continuously and connected via a double cannula in a carotid artery to an anaesthetised animal.Adenosine diphosphate (ADP) and collagen gave dose- dependent falls in the circulating platelet count when injected into rats, guinea pigs and rabbits. This enabled aggregation to be accurately quantitated in vivo.The infusion of PGI2 (0.25-1 ug/kg/min) in anaesthetised rats and rabbits produced a dose-dependent inhibition of the fall in platelet count produced by ADP and collagen. The formation of PGI2 can be inhibited in vitro by 15- hydroperoxyarachidonic acid (15HPAA). When 20 ug/kg/min of 15HPAA was infused into rats, aggregation produced by collagen was significantly increased suggesting that PGI2 is continuously formed by the rat vascular endothelium. This observation was confirmed by infusing 6-keto PGF1α antiserum. This antibody also prevented the inhibitory activity of PGI2 on collagen-induced aggregation. The study of continuous platelet counting in guinea pigs has been hampered by the occurrence of thrombocytopenia in certain animals. When 2 ug/kg/min of PGI2 was infused for 10 mins, a rise in the circulating platelet count to a steady plateau 4-5 × 105 platelets occurredThese experiments have shown that PGI2 will prevent aggregation by ADP and collagen and will reverse spontaneous thrombocytopenia and that PGI2 is continuously released from the vessels of anaesthetised rats.

scholarly journals Do patients with thromboembolic disease have circulating platelet aggregates?

Blood ◽  
1984 ◽  
Vol 64 (1) ◽  
pp. 205-209 ◽  
Author(s):  
FH Kohanna ◽  
MH Smith ◽  
EW Salzman
Keyword(s):  
Platelet Rich Plasma ◽  
Venous Blood ◽  
Adenosine Diphosphate ◽  
Normal Subjects ◽  
Thromboembolic Disease ◽  
Circulating Platelet Aggregates ◽  
Platelet Aggregates ◽  
Lower Ratio

Reports of circulating platelet aggregates (ie, microemboli) in thromboembolism and other vascular disorders are based on a method (Wu and Hoak , 1974) in which venous blood is collected via scalp vein needle and tubing into either formaldehyde, which fixes aggregates, or EDTA, which disperses them. The ratio of platelet counts in platelet- rich plasma (PRP) from the two blood samples after centrifugation is interpreted as a measure of platelet aggregates in the circulation in vivo. We compared this standard Wu and Hoak technique with a modified one, in which blood was drawn directly into a syringe, and with a third method that avoided centrifugation by counting single platelets in whole blood. Both modified techniques could detect aggregates generated in vitro with adenosine diphosphate (ADP). In 12 normal subjects, the three methods were equivalent, but in 37 patients with thromboembolic disorders, the standard Wu and Hoak method gave a lower ratio than the other methods. Similar results were found in a subset of eight patients with myocardial infarction. Heparin treatment of patients did not influence the results. The data suggest that formation of platelet aggregates occurred during venipuncture. Platelets may be hyperactive in patients with thromboembolic disease and may form aggregates in vitro during collection, but the concept of chronic microembolism in such patients should be reassessed.

Hydrogen Peroxide (H2O2) an Inhibitor of the Platelet Release Reaction

1975 ◽  
Author(s):  
M. J. Stuart ◽  
H. Holmsen ◽  
F. A. Oski
Keyword(s):  
Steady State ◽  
Platelet Rich Plasma ◽  
Adenosine Diphosphate ◽  
Minute Period ◽  
Bacterial Interaction ◽  
Steady State Levels ◽  
In Vivo Effects ◽  
Induced Aggregation

The effect of H2O2 on platelet metabolism, aggregation and release was studied by the in vitro exposure of 3H-adenine labelled platelet rich plasma (PRP) to H2O2. On incubation of PRP with H2O2 (100 to 500 μM final cone.) over a 30 minute period, there was a mean drop of 38% in the baseline steady state levels of radioactive metabolic ATP, the fall occurring in the first 3 minutes of incubation, with a corresponding increase in the levels of radioactive inosinemonophosphate and hypoxanthine. This was not a nonspecific lytic effect on the platelet since no extracellular leakage of platelet nucleotides occurred during the incubation. Further, mean decreases of 8 to 38% in steady state levels of platelet metabolic ATP were observed during incubations of PRP with 5 to 500 μM H2O2 respectively. Finally, the action of H2O2 on adenosine diphosphate (ADP) induced biphasic aggregation and release was studied in PRP preincubated for 3 minutes with H2O2 (100 to 500 μM). Partial inhibition of the primary wave, and complete inhibition of the second wave of ADP induced aggregation was observed in the H2O2 pretreated platelets, con-commitant with inhibition of release of platelet non-metabolic ATP and ADP, when compared to the control saline preincubated platelets. Since H2O2 is generated in vivo by bacteria and leucocytes during phagocytosis, the in vivo effects of the described inhibition of H2O2 and its possible role in platelet-leucocyte-bacterial interaction requires further elucidation.

scholarly journals Efficacy of a Semi Automated Commercial Closed System for Autologous Leukocyte- and Platelet-Rich Plasma (l-prp) Production in Dogs: A Preliminary Study

Animals ◽  
2020 ◽  
Vol 10 (8) ◽  
pp. 1342 ◽  
Author(s):  
Roberta Perego ◽  
Eva Spada ◽  
Luciana Baggiani ◽  
Piera Anna Martino ◽  
Daniela Proverbio
Keyword(s):  
Platelet Count ◽  
Closed System ◽  
Whole Blood ◽  
Platelet Rich Plasma ◽  
In Vivo Studies ◽  
High Concentration ◽  
Commercial System ◽  
Thrombin Activation

Background: To characterize the cellular composition (platelets, erythrocytes, and leukocytes) and determine platelet-derived growth factor isoform BB (PDGF-BB) concentration in canine leukocyte- and platelet rich plasma (L-PRP) produced using a commercial semi-automated closed system. Methods: Twenty milliliters of citrated whole blood were obtained from 30 healthy un-sedated canine blood donors and processed using a semi-automated completely closed commercial system (CPUNT 20, Eltek group, Casale Monferrato, Alessandria, Italy) according to the manufacturer’s instructions. Erythrocyte, leukocyte, and platelet counts were determined in both whole blood (WB) and resultant L-PRP. The PDGF-BB concentration was evaluated after bovine thrombin activation of 10 L-PRP samples. Results: This commercial system produced on average 2.3 ± 0.7 mL of L-PRP containing a high concentration of platelets (767,633 ± 291,001 μL, p < 0.001), with a 4.4 fold increase in platelet count, lower concentration of erythrocytes (528,600 ± 222,773 μL, p < 0.001) and similar concentration of leukocytes (8422 ± 6346 μL, p = 0.9918) compared with WB. L-PRP had an average of 3442 ± 2061 pg/mL of PDGF-BB after thrombin activation. Neutrophils, lymphocytes and monocytes average percent content in L-PRP was 14.8 ± 13.2, 71.7 ± 18.5 and 10.7 ± 6.4, respectively. Conclusion: Sterile canine L-PRP prepared using this semi-automated closed system is easy to obtain, produces a significant increase in platelet count compared to WB and contains a detectable concentration of PDGF-BB after activation. Additional in vitro and in vivo studies are needed to assess inflammatory markers concentration and the therapeutic efficacy of this L-PRP in dogs.

Platelet Aggregation Evoked In Vitro and In Vivo by Phosphatidic Acids and Lysoderivatives: Identity with Substances in Aged Serum (DAS)

1979 ◽  
Vol 42 (02) ◽  
pp. 631-640 ◽  
Author(s):  
K A Schumacher ◽  
H G Classen ◽  
M Späth
Keyword(s):  
Platelet Count ◽  
Platelet Aggregation ◽  
Guinea Pig ◽  
Pulmonary Vascular Resistance ◽  
Platelet Rich Plasma ◽  
Human Platelets ◽  
Active Components ◽  
Phosphatidic Acids

SummaryIn serum incubated at 36° C for 18-24 hours a factor (DAS) develops which on intravenous injection into cats evokes platelet aggregation followed by an increase in pulmonary vascular resistance (PVR). This change in PVR is mediated via the platelets since it significantly correlates with the preinjection platelet count. There is evidence that phosphatidic acids (PA) and lysophosphatidic acids (LPA) are the active components of DAS. Investigations performed on platelet-rich plasma from man, cat, pig, dog, rabbit, guinea pig, and rat demonstrate that only human and feline platelets exposed to PA or to LPA are aggregated. Feline platelets are more sensitive to either compound than are the platelets from men; however, human platelets exhibit two exceptional properties, a) the sensitivity rapidly declines with time, b) pretreatment with subthreshold concentrations of LPA or PA induces a specific tachyphylaxis.

scholarly journals Do patients with thromboembolic disease have circulating platelet aggregates?

Blood ◽  
1984 ◽  
Vol 64 (1) ◽  
pp. 205-209 ◽  
Author(s):  
FH Kohanna ◽  
MH Smith ◽  
EW Salzman
Keyword(s):  
Platelet Rich Plasma ◽  
Venous Blood ◽  
Adenosine Diphosphate ◽  
Normal Subjects ◽  
Thromboembolic Disease ◽  
Circulating Platelet Aggregates ◽  
Platelet Aggregates ◽  
Lower Ratio

Abstract Reports of circulating platelet aggregates (ie, microemboli) in thromboembolism and other vascular disorders are based on a method (Wu and Hoak , 1974) in which venous blood is collected via scalp vein needle and tubing into either formaldehyde, which fixes aggregates, or EDTA, which disperses them. The ratio of platelet counts in platelet- rich plasma (PRP) from the two blood samples after centrifugation is interpreted as a measure of platelet aggregates in the circulation in vivo. We compared this standard Wu and Hoak technique with a modified one, in which blood was drawn directly into a syringe, and with a third method that avoided centrifugation by counting single platelets in whole blood. Both modified techniques could detect aggregates generated in vitro with adenosine diphosphate (ADP). In 12 normal subjects, the three methods were equivalent, but in 37 patients with thromboembolic disorders, the standard Wu and Hoak method gave a lower ratio than the other methods. Similar results were found in a subset of eight patients with myocardial infarction. Heparin treatment of patients did not influence the results. The data suggest that formation of platelet aggregates occurred during venipuncture. Platelets may be hyperactive in patients with thromboembolic disease and may form aggregates in vitro during collection, but the concept of chronic microembolism in such patients should be reassessed.

Human Platelet Aggregation In Response To Multiple Agonists In Plasma Anticoagulated With Heparin

1981 ◽  
Author(s):  
H A Culliver ◽  
N G Ardlie
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Human Platelet ◽  
Platelet Rich Plasma ◽  
Adenosine Diphosphate ◽  
Human Platelet Aggregation

The lowest concentrations at which epinephrine and vasopressin have been reported to interact positively in causing platelet aggregation in vitro are at least two orders of magnitude greater than the physiological concentrations of these hormones in blood. The aim of this study was to examine the interaction between several agonists of human platelet aggregation. The aggregating agents used were adenosine diphosphate (ADP), epinephrine, norepinephrine, 5-hydroxytryptamine and vasopressin. Platelet-rich plasma (PRP) was prepared from blood anticoagulated with minimal concentrations of heparin in an attempt to more closely reflect the in vivo situation.Aggregation caused by ADP was potentiated by epinephrine at a concentration exceeding the level obtained in circulating blood. When a third agonist (vasopressin) was used in combination with ADP and epinephrine, aggregation was enhanced at concentrations of vasopressin and epinephrine obtained in blood. When used as a fourth agonist norepinephrine and 5-hydroxytryptamine potentiated aggregation at physiological concentrations. The response to multiple agonists was greater in heparinized PRP than citrated PRP. Hirudin decreased the extent of aggregation in heparinized PRP caused by multiple agonists suggesting that thrombin may be involved.Since the concentrations of combined agonists required to induce in vitro platelet aggregation can be obtained in circulating blood these findings may explain why platelet activation occurs in certain pathological states.

scholarly journals Administration of pegylated recombinant human megakaryocyte growth and development factor to humans stimulates the production of functional platelets that show no evidence of in vivo activation

Blood ◽  
1996 ◽  
Vol 88 (9) ◽  
pp. 3288-3298 ◽  
Author(s):  
CJ O'Malley ◽  
JE Rasko ◽  
RL Basser ◽  
KM McGrath ◽  
J Cebon ◽  
...  
Keyword(s):  
Platelet Count ◽  
Growth And Development ◽  
Atp Release ◽  
Adenosine Diphosphate ◽  
In Vitro Assays ◽  
Initial Increase ◽  
Platelet Surface ◽  
Development Factor

This report describes the effect of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) on platelet production and platelet function in humans. Subjects with advanced solid tumors received PEG-rHuMGDF daily for up to 10 days. There was no increase in circulating platelet count at doses of 0.03 or 0.1 microgram/kg/d by day 12 of study. At doses of 0.3 and 1.0 microgram/kg/d there was a threefold median increase (maximum 10-fold) in platelet count by day 16. The platelets produced in vivo in response to PEG-rHuMGDF showed unchanged aggregation and adenosine triphosphate (ATP)-release responses in in vitro assays. Tests included aggregation and release of ATP in response to adenosine diphosphate (ADP) (10, 5, 2.5, and 1.25 mumol/L), collagen (2 micrograms/mL), thrombin-receptor agonist peptide (TRAP, 10 mumol/L) and ristocetin (1.5 mg/mL). Administration of aspirin to an individual with platelet count of 1,771 x 10(3)/L resulted in the typical aspirin-induced ablation of the normal aggregation and ATP-release response to stimulation with arachidonic acid (0.5 mg/mL), collagen, and ADP (2.5 and 1.25 mumol/L). There was no change in the expression of the platelet-surface activation marker CD62P (P-selectin) nor induction of the fibrinogen binding site on glycoprotein IIb/IIIa as reported by the monoclonal antibody, D3GP3. An elevation of reticulated platelets was evident after 3 days of treatment with PEG-rHuMGDF and preceded the increase in circulating platelet count by 5 to 8 days; this reflected the production of new platelets in response to PEG-rHuMGDF. At later time points, the mean platelet volume (MPV) decreased in a manner inversely proportional to the platelet count. Levels of plasma glycocalicin, a measure of platelet turnover, rose 3 days after the initial increase in the peripheral platelet count. The level of plasma glycocalicin was proportional to the total platelet mass, suggesting that platelets generated in response to PEG-rHuMGDF were not more actively destroyed. Thus, the administration of PEG-rHuMGDF, to humans, increased the circulating platelet count and resulted in fully functional platelets, which showed no detectable increase in reactivity nor alteration in activation status.

Storage of Human Blood Platelets

1974 ◽  
Vol 32 (02/03) ◽  
pp. 405-416 ◽  
Author(s):  
M. R Hardeman ◽  
Carina J L. Heynens
Keyword(s):  
Storage Temperature ◽  
Platelet Rich Plasma ◽  
Blood Platelets ◽  
Storage Period ◽  
Serotonin Uptake ◽  
Hypotonic Shock ◽  
Glycolytic Intermediates

SummaryStorage experiments were performed at 4°, 25° and 37° C with platelet-rich plasma under sterile conditions. In some experiments also the effect of storing platelets at 4° C in whole blood was investigated.Before, during and after three days of storage, the platelets were tested at 37° C for their serotonin uptake and response to hypotonic shock. In addition some glycolytic intermediates were determined.A fair correlation was noticed between the serotonin uptake and hypotonic shock experiments. Both parameters were best maintained at 25° C. Also platelet counting, performed after the storage period, indicated 25° C as the best storage temperature. Determination of glycolytic intermediates did not justify any conclusion regarding the optimal storage temperature. Of the various anticoagulants studied, ACD and heparin gave the best results as to the serotonin uptake and hypotonic shock response, either with fresh or stored platelets. The use of EDTA resulted in the lowest activity, especially after storage.The results of these storage experiments in vitro, correspond well with those in vivo reported in the literature.

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