Thrombocyte aggregation in plasma and whole blood during hyperventilation (hypocapnia) in cats

Platelet aggregation in platelet rich plasma (PRP) and whole blood was simultaneously studied in acute experiments on cats in hypocapnic conditions. ADP-induced aggregation increase was determined in PRP and whole blood. Contradictory results were obtained during platelet aggregation induced by collagen and arachidonic acid: increased aggregation in PRP and decreased aggregation in whole blood. The data obtained suggest that ADP is a risk factor for the onset of intravascular thrombosis.     

Platelet aggregation and thromboxane A2 release in primary biliary cirrhosis and effect of D-penicillamine treatment

Platelet function was assessed in 28 patients with primary biliary cirrhosis (PBC), of whom 10 were receiving D-penicillamine. Patients not on D-penicillamine treatment had platelet aggregation similar to that in the healthy control group; the group treated with D-penicillamine showed significantly enhanced platelet aggregation in response to threshold doses of adrenaline and collagen but not ADP. Median thromboxane B2 production was also higher in D-penicillamine treated patients than in controls or untreated patients; this difference did not reach statistical significance. The addition of D-penicillamine in vitro to platelet rich plasma from normal subjects was shown to enhance adrenaline- and collagen-induced platelet aggregation. Abnormalities of platelet function in PBC patients did not correlate with serum cholesterol concentration or with liver function tests but were related to the stage of disease. The present study emphasises the need to consider the aetiology, disease stage and type of treatment when assessing platelet function and prostanoid release in liver disease.     

Real-time monitoring of adhesion and aggregation of platelets using thickness shear mode (TSM) sensor

Hemostasis is required to maintain vascular system integrity, but thrombosis, formation of a clot in a blood vessel, is one of the largest causes of morbidity and mortality in the industrialized world. Novel clinical and research tools for characterizing the hemostatic system are of continued interest, and the object of this research is to test the hypothesis that clinically relevant platelet function can be monitored using an electromechanical sensor. A piezoelectric thickness shear mode (TSM) biosensor coated with collagen-I fibers to promote platelet activation and adhesion was developed and tested for sensitivity to detect these primary events. Magnitude and frequency response of the sensor were monitored under static conditions at 37 degrees C, using platelet-rich plasma (PRP), and PRP with adenosine diphosphate (ADP), a clinical aggregation inhibitor (abciximab), or a collagen binding inhibitor. Sensors loaded with PRP exhibited a 3-stage response; no significant change in response for the first 20 min (Stage-1), followed by a larger drop in response (Stage-2) and subsequently, response gradually increased (Stage-3). Exogenous ADP stimulated an immediate Stage-2 response, while abciximab delayed and reduced the magnitude change of Stage-2. In the presence of collagen inhibitor, Stage-2 response was similar to that of control but was delayed by an additional 20 min. The obtained results, supported by epifluorescence and complementary SEM studies, demonstrated the selective sensitivity of TSM electromechanical biosensors to monitor platelet function and inhibition, particularly aggregation.     

Changes in thromboxane A2 generation and plasma lipid pattern in pseudomenopause induced by gonadotropin releasing hormone (GnRH) analogue buserelin

An increased risk of cardiovascular disease has been found in postmenopausal women in comparison to premenopausal women. The aim of this study was to investigate platelet function, blood clotting and plasma lipid levels in 12 women with a condition of hypoestrogenism, similar to the postmenopausal status induced by treatment with the GnRH analogue buserelin for uterine leiomyoma. Platelet aggregation in whole blood and platelet-rich plasma (PRP), serum thromboxane (TX) B2 production, fibrinopeptide A (FPA) plasma levels and plasma lipid pattern were measured before and after 13 weeks of buserelin treatment. No changes of platelet aggregability were found either in whole blood or PRP. Serum TXB2 generation increased significantly after 13 weeks of therapy (p less than 0.001). No signs of increased thrombin generation were found, as indicated by unchanged FPA plasma levels. Total cholesterol plasma levels were found increased after 13 weeks, LDL cholesterol levels showed a tendency to increase although not significantly. HDL cholesterol and triglyceride concentrations were unaffected. The changes of arachidonic acid metabolism and lipid pattern suggest that buserelin treatment may induce a condition of increased thrombotic risk even if the lack of enhanced thrombin generation and increased platelet aggregability indicates that no blood clotting activation occurs.     

Pharmacology of UK-38485 (dazmegrel), a specific inhibitor of thromboxane A2 synthetase

Thromboxane A2 (TXA2) synthesis in rabbit and human platelet rich plasma (PRP) was inhibited in a dose-dependent manner by UK-38485 (dazmegrel) when the PRP was aggregated with collagen, arachidonic acid and ADP. The level of inhibition was time-dependent. That is, the dose-response curves shifted to lower concentrations with increasing incubation times with UK-38485 prior to addition of aggregation agents. Following bolus intravenous injections of UK-38485 in rabbits, the elimination from serum fitted a 3-exponential curve. The terminal elimination phase had a half-life of 69.8 +/- 3.8 min. Oral treatment of rabbits with UK-38485 for 2 weeks showed that animals with serum concentrations of 0.358 +/- 0.091 microgram/ml of the inhibitor had TXA2 synthesis inhibited in serum by 83.8 +/- 7.1%. This corresponded to animals which were treated with 20 mg/kg/day of the inhibitor.     

Wnt5a Potentiates U46619-Induced platelet aggregation <Emphasis Type="Italic">via</Emphasis> the PI3K/Akt pathway

Platelet aggregation plays crucial roles in the formation of hemostatic plugs and thrombosis. Although it was recently shown that canonical Wnt signaling negatively regulates platelet aggregation, the role of non-canonical Wnt signaling remains unknown. Here, we observed that Wnt5a, one of the non-canonical Wnts, positively regulated platelet aggregation. Platelet aggregation was potentiated by the addition of Wnt5a to collagen-or U46619-induced rat platelet rich plasma (PRP). Treatment with Wnt5a to U46619-stimulated PRP resulted in an increase in the level of phosphorylated Akt, whereas phosphorylation of PKCδ and JNK1 was unaffected. In addition, inhibition of PI3K blocked the potentiating effect of Wnt5a. Taken together, these results suggest that Wnt5a potentiates U46619-induced platelet aggregation via the PI3K/Akt pathway.     

Platelet serotonin content and uptake in spontaneously hypertensive rats

Platelet serotonin (5-HT) content and uptake were studied in male SHR and WKY at various ages. Blood was withdrawn from the carotid artery under anesthesia and 5-HT levels determined from platelet rich plasma (PRP) using a HPLC technique coupled with an electrochemical detection method. Platelet 5-HT uptake was studied by incubating PRP at 37 degrees C for 10 sec with increasing concentrations of 3H-5HT. Lineweaver- Burk plots of 3H-5HT uptake were linear suggesting simple Michaelis- Menten uptake kinetics. The SHR had more platelets than age-matched controls and consequently a higher blood circulating pool of 5-HT. Nevertheless, the 5-HT platelet levels were similar to those of their age-matched rats. The 5 week-old SHR and WKY had greater numbers of platelets and higher 5-HT platelet levels than the older rats of both strains. The affinity constants (Km) and the maximal velocities (Vmax) of platelet 5-HT uptake did not differ significantly between the 12 week- and the 6 month-old SHR and WKY. These data suggest that the SHR do not show the same impairment in platelet 5-HT metabolism as observed in essential hypertension in man.     

Presence of factors that activate platelet aggregation in mitral stenotic patients' plasma

BACKGROUND: Although the association between mitral stenosis (MS) and increased coagulation activity is well recognized, it is unclear whether enhanced coagulation remains localized in the left atrium or whether this represents a systemic problem. To assess systemic coagulation parameters and changes in platelet aggregation, we measured fibrinogen levels and performed in vitro platelet function tests in plasma obtained from mitral stenotic patients' and from healthy control subjects' peripheral venous blood. METHODS: Sixteen newly diagnosed patients with rheumatic MS (Group P) and 16 healthy subjects (Group N) were enrolled in the study. Platelet-equalized plasma samples were evaluated to determine in vitro platelet function, using adenosine diphosphate (ADP), collagen and epinephrine in an automated aggregometer. In vitro platelet function tests in group N were performed twice, with and without plasma obtained from group P. RESULTS: There were no significant differences between the groups with respect to demographic variables. Peripheral venous fibrinogen levels in Group P were not significantly different from those in Group N. Adenosine diphosphate, epinephrine and collagen-induced platelet aggregation ratios were significantly higher in Group P than in Group N. When plasma obtained from Group P was added to Group N subjects' platelets, ADP and collagen-induced, but not epinephrine-induced, aggregation ratios were significantly increased compared to baseline levels in Group N. CONCLUSION: Platelet aggregation is increased in patients with MS, while fibrinogen levels remain similar to controls. We conclude that mitral stenotic patients exhibit increased systemic coagulation activity and that plasma extracted from these patients may contain some transferable factors that activate platelet aggregation.     

Platelet inhibition by nitrite is dependent on erythrocytes and deoxygenation

BACKGROUND: Nitrite is a nitric oxide (NO) metabolite in tissues and blood, which can be converted to NO under hypoxia to facilitate tissue perfusion. Although nitrite is known to cause vasodilation following its reduction to NO, the effect of nitrite on platelet activity remains unclear. In this study, the effect of nitrite and nitrite+erythrocytes, with and without deoxygenation, on platelet activity was investigated. METHODOLOGY/FINDING: Platelet aggregation was studied in platelet-rich plasma (PRP) and PRP+erythrocytes by turbidimetric and impedance aggregometry, respectively. In PRP, DEANONOate inhibited platelet aggregation induced by ADP while nitrite had no effect on platelets. In PRP+erythrocytes, the inhibitory effect of DEANONOate on platelets decreased whereas nitrite at physiologic concentration (0.1 μM) inhibited platelet aggregation and ATP release. The effect of nitrite+erythrocytes on platelets was abrogated by C-PTIO (a membrane-impermeable NO scavenger), suggesting an NO-mediated action. Furthermore, deoxygenation enhanced the effect of nitrite as observed from a decrease of P-selectin expression and increase of the cGMP levels in platelets. The ADP-induced platelet aggregation in whole blood showed inverse correlations with the nitrite levels in whole blood and erythrocytes. CONCLUSION: Nitrite alone at physiological levels has no effect on platelets in plasma. Nitrite in the presence of erythrocytes inhibits platelets through its reduction to NO, which is promoted by deoxygenation. Nitrite may have role in modulating platelet activity in the circulation, especially during hypoxia.     

Platelet-vessel wall interactions: effects of platelets and plasma on the antiaggregatory activity and 6 keto-PGF1 alpha production in isolated perfused aortas

An experimental model for the study of platelet-vessel wall interactions has been developed, based on perfusion of rat platelet rich plasma (PRP) through isolated rat aortas. In the perfused PRP, platelet aggregation was inhibited and levels of 6 Keto PGF1 alpha and cAMP were elevated over the values found in non perfused PRP. When PPP or buffer were perfused through the isolated artery, elevations of 6 Keto PGF1 alpha levels in the perfusate were smaller (in perfused PPP) or of shorter duration (in both perfused PPP and buffer). The presence of platelets in the perfusion fluid thus enhanced the formation of Prostacyclin by the arterial wall. Levels of 6 Keto PGF1 alpha in PRP obtained from aspirin-treated animals and in PRP from normal animals, both perfused through normal aortas, were the same, and also levels of the above metabolite in normal PRP perfused through aortas of aspirin-treated animals did not differ from those found in non perfused PRP. It is concluded, from these data, that PRP does not stimulate PGI2 formation in perfused aortas by providing cyclic endoperoxides. The experimental model developed allows the study of interactions between normal platelets and aortas from experimentally treated animals or viceversa.     

Influence of propranolol on platelet aggregation and thromboxane B2 production from platelet-rich plasma and whole blood

The beta-adrenoceptor antagonist propranolol is used in the therapy of hypertension and ischemic heart disease. The aim of our study was to evaluate the effects of this drug on platelet aggregation and on synthesis of thromboxane B2 (the stable metabolite of Thromboxane A2) from platelet rich plasma (PRP), whole blood samples and during spontaneous clotting. The results indicate that propranolol at concentrations near the therapeutic range, significantly inhibit collagen and thrombin-induced platelet aggregation and TxB2 synthesis from PRP. Furthermore the drug demonstrates inhibitory activity on B-TG release and TxB2 production from whole blood samples and on spontaneous clotting. The results suggest that some benefits of propranolol in the treatment of patients with coronary artery disease or cardiovascular conditions associated with platelet hyperaggregability may also be related to interference with platelet activation "in vivo" and with TxA2 generation.     

Platelet function testing in the pony

Platelet isolation techniques and platelet function were evaluated in 35 adult ponies. Platelet recovery from whole blood was consistent and the preparation of platelet rich plasma was facilitated by an enhanced erythrocyte sedimentation rate. All platelet samples aggregated in response to 10 microM ADP. However, concentrations of ADP as high as 100 microM did not elicit significant 14C-serotonin release. Collagen induced irreversible platelet aggregation and 14C-serotonin release in all samples. The threshold dose for collagen in most ponies was 1.5 micrograms. Arachidonic acid (500 microM) failed to induce irreversible platelet aggregation or 14C-serotonin release in any of the samples evaluated. Pony platelets were nonresponsive to epinephrine (5.5 microM).     

Long-range interactions in mammalian platelet aggregation. I. Evidence from kinetic studies in brownian diffusion.

The Smoluchowski theory describing aggregation in suspensions of spherical colloidal particles due to Brownian diffusion-controlled two-body collisions, was used to obtain collision efficiencies, alpha B, for adenosine diphosphate (ADP)-induced platelet aggregation in citrated platelet-rich plasma (PRP) from humans, dogs, and rabbits. For these diffusion studies, PRP was stirred with 10 microM ADP for 0.5 s, then kept nonstirred at 37 degrees C for varying times before fixation; the percent aggregation was computed from the decrease in particle concentration with time measured with a resistive particle counter. Up to 20% of rabbit platelets formed microaggregates within 60 s of ADP addition to such nonstirred suspensions, corresponding to mean alpha B values of approximately 0.9. However, human and dog platelets aggregated approximately 10 times and 2-3 times faster than rabbit platelets within the first 60 s of ADP addition, corresponding to alpha B approximately 8 and 2, respectively. These high alpha B (much greater than 1) for human platelets were independent of initial platelet count and were equally observed with the calcium ionophore A23187 as activator. In about one-third of human, dog, or rabbit PRP, comparable and lower values of alpha B (less than 0.5) were obtained for a slower second phase of aggregation seen for the nonstirred PRP over 60-300 s post ADP-addition. Platelet aggregability in continually stirred PRP was distinct from that observed in Brownian diffusion (nonstirred) because comparable aggregation was observed for all three species' stirred PRP, whereas greater than 3-8 times more ADP is required to yield 50% of maximal rates of aggregation for nonstirred than for stirred PRP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of platelet aggregation by 6-keto-PGE1; lack of an effect on cyclic GMP levels

The effects of 6-keto-PGE₁ on aggregatory responses to arachidonic acid (AA), adenosine diphosphate (ADP) and collagen were studied in human platelet-rich plasma (PRP). In addition, experiments were carried out to determine if these effects correlate with changes in platelet cyclic AMP and cyclic GMP levels. 6-Keto-PGE₁ incubated in PRP produced dose-related increases in platelet cyclic AMP levels whereas platelet cyclic GMP levels were unchanged. Control aggregations induced by AA and ADP did not alter cyclic AMP and cyclic GMP levels whereas control aggregations induced by collagen elevated cyclic GMP levels while cyclic AMP levels were unchanged. 6-Keto-PGE₁ produced a dose-dependent inhibition of platelet aggregation induced by AA, ADP and collagen and this inhibition correlated with a dose-related increase in cyclic AMP levels. Since 6-keto-PGE₁ does not consistently alter cyclic GMP levels in human PRP, the present data support previous studies suggesting that 6-keto-PGE₁ produces inhibition of platelet aggregation through the stimulation of cyclic AMP accumulation.     

Improvement of platelet aggregation abnormalities in thrombocytosis after thrombocytopheresis

Platelet function tests were performed in three patients with thrombocytosis in myeloproliferative disorders before and after a swift reduction of platelet count by thrombopheresis. The decrease of platelet count obtained after the procedure was reversed in six days. In two patients with platelet aggregation defects, the normalization of aggregation abnormalities was observed after pheresis, followed by a progressive decrease of platelet response until the pre-pheresis values on 6th day. In the third patient with normal platelet aggregation, a progressive increase of platelet aggregation response was noted on the days following thrombopheresis with ischaemic symptoms of a foot toe. In all three patients, the changes of platelet aggregation were accompanied by a related increase of megathrombocytes. In the two patients with platelet aggregation abnormalities, plasma and platelet beta-thromboglobulin levels were related to changes in platelet count and aggregation.     

Role of arachidonic acid in platelet aggregation induced by S. typhimurium endotoxin

The platelet aggregation reaction was used to assess the influence of arachidonic acid (AA), endotoxin (E) S. typhimurium and ADP on platelet aggregation properties. All the three substances induced platelet aggregation. A higher degree of aggregation was attained by the application of E combined with AA and ADP as compared with the effects produced by E and ADP alone. Prolonged incubation of platelet-rich plasma (PRP) samples with E led to an essential decrease of the aggregation degree on ADP addition. Incubation of PRP samples with E and ADP did not evoke any analogous decrease in the platelet aggregation degree. The data obtained indicate that AA stimulates platelet aggregation induced by E and ADP.     

Does the magnetic field of a magnetic stirrer in an optical aggregometer affect concurrent platelet aggregation?

Platelets are subjected to extremely low frequency electromagnetic fields during standard aggregometry measurements owing to the use of a magnetic stir bar in the instrument. This study evaluates the effects of this magnetic field exposure on platelet aggregation by comparing the results obtained in a modified aggregometer. Blood samples from healthy volunteers were anticoagulated using citrate or heparin. Platelet‐rich plasma (PRP) samples were prepared. A mechanical stirring device was attached to the aggregometer instead of the magnetic stir bar system. The PRP samples were stirred using a stirring rod tip that did not produce any magnetic fields in one channel of the aggregometer; in the other channel, a stirring rod carrying a small magnet at its tip was used. As a result, a magnetic field in the extremely low frequency range and in the amplitude range of 1.9–65 mT was applied to the platelets assigned to the channel where the magnetic stirring rod tip was used. Aggregation was induced using adenosine diphosphate (ADP), collagen, or epinephrine. The slopes, maximum aggregation values, and areas under the aggregation curves were compared between the magnetic and neutral stirring rod tip groups. For samples stirred with the magnetic stirring rod tip, a significant decrease was observed in 12 of the 14 parameters evaluated for aggregations induced with ADP or collagen compared to the neutral stirring rod tip, regardless of the method used for anticoagulation. This observation indicates that the magnetic stir bars used in standard aggregometry may significantly alter aggregation parameters and platelets may be possible targets of electromagnetic fields. Bioelectromagnetics 34:349–357, 2013. © 2012 Wiley Periodicals, Inc.     

Platelet aggregability in patients with common carotid artery ligation

Increased platelet aggregability is regarded as being a sensitive indicator of the initiation of thrombotic processes. Platelet aggregation was analysed in blood taken from the common carotid artery before and 30 min after its ligation in 3 patients, as well as in the venous blood of 14 patients in the late postoperative period. No tendency towards increasing platelet aggregation was observed in either of the groups investigated.     

Characterization of liposomes carrying von Willebrand factor-binding domain of platelet glycoprotein Ibalpha: a potential substitute for platelet transfusion.

Platelet glycoprotein (GP) Ib/IX/V complex is a receptor for von Willebrand factor (vWf), which plays a crucial role in primary hemostasis by mediating platelet adhesion to injured blood vessels. We have expressed in CHO cells a fragment of GPIba that retained a vWf-binding function. The recombinant fragment (rGPIba) was incorporated into liposomes and evaluated their functions in vitro. rGPIba on the liposome surface was detectable by flow cytometric analysis. Addition of vWf and ristocetin caused specific agglutination of rGPIbalpha-liposomes, as evaluated by an aggregometer or a fluorescent microscopy. When ristocetin was added to platelet-rich plasma (PRP) pre-mixed with rhodamine-labeled rGPIbalpha-liposomes, platelets aggregated and rhodamine-fluorescence was strongly positive in the platelet thrombi, suggesting that heterologous aggregation (attachment of liposomes to platelets) occurred. Platelet aggregation in PRP at low platelet concentration (20-80 x 10(6)/ml) was enhanced by rGPIbalpha-liposomes in a dose-dependent manner. Thus, rGPIbalpha-liposomes may accumulate on vWf-exposed subendothelial tissues and enhance platelet function in vivo, supporting hemostasis in thrombocytopenic individuals.     

Effect of salvianolic acids from Radix Salviae miltiorrhizae on regional cerebral blood flow and platelet aggregation in rats.

This study was conducted to observe the effect of salvianolic acids (SA) on regional cerebral blood flow (rCBF) in rats and on platelet aggregation in vitro and in vivo. Cerebral ischemia was produced in rats by occluding of the right middle cerebral artery, together with the right common carotid artery. rCBF was monitored by H2 clearance method with a tissue blood-flow meter. Platelet aggregation induced by collagen, ADP, and AA was measured in vitro and in vivo by platelet aggregometer. Doses of SA at 6 and 10 mg/kg body wt. (i.v.) improved rCBF in rats after ischemia, but had no obvious effect on normal rCBF. In vitro, SA inhibited significantly the platelet aggregation induced by collagen, ADP, and AA with IC50 values of 0.197, 2.22 and 3.29 x 10(3) mg/l, respectively. In vivo, doses of SA at 6 and 10 mg/kg body wt. inhibited significantly the platelet aggregation induced by collagen, and SA at 10 mg/kg body wt. inhibited remarkably platelet aggregation induced by ADP. The results suggest that SA could improve rCBF in the ischemic hemisphere and inhibit platelet aggregation in rats.