Endotoxin-induced platelet activation in human whole blood in vitro

The effect of purified bacterial endotoxin was studied on human platelets in vitro. In adding up to 1 microgram/mL of a highly purified endotoxin, we found neither aggregation nor ATP release in heparinized or citrated human platelet-rich plasma. On the other hand; endotoxin at concentrations as low as a few ng/mL (as may be found in septic patients) caused platelet aggregation in both heparinized and citrated human whole blood, as monitored by change in impedance, free platelet count, and size. Unlike collagen, the platelet aggregation with endotoxin occurred after a long lag phase, developed slowly, and was rarely coupled with measurable release of ATP. The platelet aggregating effect of endotoxin was dose-dependent and modified by exposure of the endotoxin to ionizing radiation. Thus, the activation of human platelets by "solubilized" endotoxin in plasma requires the presence of other blood cells. We propose that the platelet effect is mediated by monocytes and/or neutrophils stimulated by endotoxin.     

Molecular aspects of cloricromene (AD6) distribution in human platelets and its pharmacological effects

Cloricromene (AD6) is an investigational drug which inhibits platelet aggregation and release reaction. We studied the relationship between its action and its distribution and metabolism in platelets. Incubation of anticoagulated whole blood or platelet-rich plasma (PRP) without exogenous aggregating agents resulted in a progressive decrease of platelet count with a concomitant increase of beta-thromboglobulin (BTG) release. AD6 (20-50 mumols/l), but not acetylsalicylic acid (ASA), incubated with whole blood or PRP, prevented the fall in platelet count and the release of BTG for at least 150 min. Moreover, incubation of PRP with AD6 (50 mumols/l) and subsequent stimulation by ADP at threshold concentrations resulted in a significant reduction (about 30%) in aggregation for at least 90 min. AD6 (20 mumols/l) added to PRP was rapidly metabolized by hydrolysis of an ester bond to AD6 acid, a stable catabolite pharmacologically inactive in platelets. Significant amounts of AD6 acid (up to 13.26 +/- 2.80 pmol/10(6) platelets) were associated with the platelets after incubation either at 37 degrees C or 4 degrees C. The amount of AD6 acid in the platelet pellet was proportional to AD6 concentration (2 to 100 mumols/l). PRP incubation with AD6 acid (20 mumols/l) resulted in very low levels (less than 1 pmol/10(6) platelets) of the same compound in the platelet pellet after 1, 5 or 30 min. These data suggest that AD6 is taken up as an ester and converted to its acid catabolite with a consequent long-lasting inhibition of platelet function.     

Effects of any epoxymethano stable analogue of prostaglandin endoperoxides (U-46619) on human platelets

U-46619 is a stable epoxymethano analogue of cyclic endoperoxide PGH2. We studied platelet aggregation, 14C-5HT release, LDH extrusion and prostaglandin and thromboxane production induced by this compound in platelet-rich plasma samples from 15 healthy volunteers. Each subject was tested both before and 90 min after aspirin (500 mg) ingestion. The threshold aggregating concentration (TAC) of U-44619 ranged between 0.18 and 0.90 micro M. Aggregation was maximal between 40 and 60 min after venipuncture and was concentration-dependent. At concentrations below the TAC, U-44619 induced primary reversible aggregation with minimal 14C-5HT release. At TAC or higher concentrations aggregation and release proceeded as parallel events. Neither prostaglandin or thromboxane production nor LDH loss could be detected in any of the situations tested. Aspirin ingestion did not modify the pattern of platelet responses. In unstirred, not aggregated platelet samples 14C-5HT release by U-46619 occurred to a similar extent as in stirred, aggregated platelet samples. Addition to citrated PRP of 0.3 mM Na2 EDTA blocked both aggregation and release induced by U-46619. This compound, however, aggregated washed platelets resuspended in Ca++-free-tyrode-albumin containing fibrinogen. The mechanism by which U-46619 activates platelets differs from that of all other common aggregating agents.     

Collagen-induced rat platelet reactivity is enhanced in whole blood in both the presence and absence of dense granule secretion.

Collagen induced aggregation, ATP secretion and thromboxane (TxB2) generation of storage pool deficient platelets were compared to normal platelets of closely related rat strains. Platelet function was monitored in citrated-platelet-rich-plasma (PRP) and citrated whole blood. Wistar (W) and fawn-hooded (FH) rat strains and their F2 hybrids were utilized. The W strain, which is ancestral to the FH strain, is not storage pool deficient while the FH strain is. This was manifested by the total lack of collagen induced ATP secretion from platelets of the FH strain while the platelets of the W strain secreted normally. Utilizing platelets from the F2 generation of WxFH matings, the absence of dense granule secretion (ATP) from the FH platelets, as well as other platelet defects of FH rats, were shown to be associated with homozygosity for the red-eyed dilution gene [r]. The non-secreting FH platelets were utilized to determine the effects of secreted dense granule constituents upon collagen induced aggregation and TxB2 generation. The non-secreting storage pool deficient platelets did aggregate and did generate TxB2 upon stimulation with collagen; however, the storage pool deficient FH platelets demonstrated less TxB2 generation and did not aggregate as effectively as the normally secreting platelets of the W strain. When evaluating collagen induced platelet function in whole blood as compared to PRP, the storage pool deficient platelets remained less reactive than normally secreting platelets, but both platelet types demonstrated enhanced aggregation and increased TxB2 generation in whole blood.     

Newborn platelet dysfunction: a storage pool and release defect.

Per cent aggregation, release and content of adenine nucleotides, and specific radioactivity were evaluated in citrated platelet-rich plasma (PRP) prepared from paired samples of maternal and cord blood. Platelets of newborn infants aggregated normally in response to highdose ADP (20 muM), strong collagen suspensions, and thrombin; however, when compared with PRP from the mothers or from normal adults, per cent aggregation in response to lower concentrations of ADP (2 muM), weak collagen, and part particularly epinephrine was markedly reduced. Nucleotide release after stimulation of the newborns' PRP with the latter two inducers was also impaired. ATP and ADP content of the newborns' platelets was also significantly less than that of their mothers or of normal adults, but specific activity was normal. The data suggest that the impairment of ADP release in the platelets of newborn infants is due to decreased sensitivity to external stimuli. Since metabolic ATP is necessary for the platelet release reaction, it is postulated that the platelet dysfunction results from a lack of metabolic ATP.     

An assessment of whole blood impedance aggregometry using blood from normal subjects and haemodialysis patients

Impedance aggregometry allows the measurement of platelet responses in whole blood as well as in PRP. The variability of haematocrit values encountered when applying this technique to haemodialysis patients prompted an investigation of the effects of red cells on platelet aggregation in whole blood. Collagen induced aggregation was measured in both PRP and whole blood from haemodialysis patients and healthy controls, Platelets from haemodialysis patients were less aggregable than those from the controls when tested in PRP, but more aggregable when tested in whole blood. Blood samples with a range of haematocrit values were prepared by mixing PRP and autologous red cells, and used to study the effect of haematocrit on platelet aggregation. In blood from control subjects aggregation rate was reduced by rising haematocrit but no reduction of maximum aggregation occurred until haematocrit exceeded 40%. In contrast uraemic platelets showed increased responses in the presence of red cells. In a limited cross-over study no significant difference was found in the effect on platelet aggregation of washed erythrocytes from uraemic and non-uraemic donors. It is concluded that red cell presence influences platelet aggregation by complex mechanisms during impedance aggregometry and that this effect must be considered when interpreting results.     

Selective PF4 release in vitro induced by heparin and related glycosaminoglycans (GAGs)--correlation with beta-TG release and platelet aggregation

We studied human platelet aggregation and beta-TG/PF4 release induced by heparin and related GAGs in vitro both in normal PRP and in PRP after aspirin. In our experimental conditions, heparin and related GAGs always caused PF4 release in vitro from normal platelets, whether or not there was measurable platelet aggregation in the aggregometer. Significant beta-TG release was induced only by the mucosal heparin preparation (which also induced platelet aggregation in some citrated PRP). Therefore, while beta-TG release in vitro seems to correlate with platelet aggregating activity of heparin, the selective PF4 release, caused by heparin and related GAGs also in conditions in which neither platelet aggregation nor beta-TG are measurable, is probably associated with the high affinity of PF4 for heparin. The degree of affinity of GAGs for PF4 (heparin greater than DeS greater than HS) seems to correlate with PF4 release. Moreover, the significant reduction in PF4 release in vitro after aspirin suggests that GAGs-induced PF4 release is related to a cyclooxygenase-dependent activation process.     

The effect of human plasma on platelet function in familial hypercholesterolemia

Using a platelet-rich plasma (PRP) preparation, platelets from subjects with familial hypercholesterolemia (FH) were found to be more reactive to the aggregating agents epinephrine, ADP and thrombin than platelets obtained from normal individuals. Gel-filtered platelets (GFP)i.e. platelets free of any plasma constituents also showed increased activation as determined by platelet aggregation and ¹⁴C-serotonin release. On incubating washed platelets from normal subjects with plasma obtained from FH subjects the platelet response to aggregating agents was significantly increased. Incubation of washed platelets from the FH patients with normal plasma, however, resulted in a significant decrease in platelet activity. Our data suggest that the increased platelet activation in FH patients is the result of a change in the platelets induced by abnormal plasma constituents.     

Endotoxin-mediated inhibition of human platelet aggregation

A variety of endotoxins, when added to human platelet-rich plasma (PRP) or to suspensions of washed platelets (WP), demonstrated an inhibitory effect on platelet aggregation induced by various aggregating agents. Endotoxin blocked the release of 14C serotonin from platelets but had no influence on cyclic AMP production. Endotoxin did not interfere with thromboxane generation by platelets. However, endotoxin-treated platelets failed to respond to added thromboxane. The inhibitory effect of endotoxin on platelet aggregation was more pronounced in the presence of ionophore A23187 as compared to other aggregating agents and was effectively reversed by calcium but not by magnesium, another divalent cation. Furthermore, endotoxin failed to inhibit the ristocetin-induced agglutination of formaldehyde-fixed platelets; a non-calcium dependent phenomenon. These findings appear to suggest that endotoxin-mediated inhibitory activity of platelet aggregation is related to the interference in the role of calcium. The antiaggregatory activity of endotoxin appears to be due to a direct and rapid action on platelets and not due to a non-specific binding, as the effect was not abolished by washing the endotoxin-incubated platelets. Endotoxin-mediated alteration of platelet function may contribute to bleeding diathesis in septecemic and endotoxemic patients.     

Changes of spontaneous and induced platelet aggregation in the presence of a human endothelial cell monolayer

A new, simple method has been developed for the investigation of platelet aggregation in the presence of a cultured confluent human endothelial cell monolayer, in disk shaped rotating cuvettes, without magnetic stirring. The endothelial cells inhibited the aggregating effect of several inducers in a concentration dependent manner. At a platelet count of 5 x 10(5)/microliter in PRP e.g. the aggregating effect of 1 microgram/microliter thrombin was completely abolished. Spontaneous aggregation was also prevented by the EC-monolayer. A correlation could be established between the inhibitory effect of ECs and the number of platelets. In PRP with a platelet count of 7 x 10(-5)/microliters the inhibitory effect of the endothelial cell monolayer significantly decreased.     

The effect of thromboxane A2 synthesis inhibitors on platelet aggregation in whole blood

Aggregatory responses to arachidonic acid and collagen in vitro were compared in blood (single platelet counting) and PRP (light aggregometry) from four species. Sensitivity of mouse, rat and rabbit PRP to these agonists was not predictive of respective potency in whole blood, whereas for human platelets, responses in blood did not differ significantly from PRP. Indomethacin (3-300 microM) inhibited arachidonic acid-induced aggregation in each species, and collagen responses in all except mouse. In contrast, the thromboxane synthase inhibitors dazoxiben (3.7-372 microM) and SC 38249 (2.6-260 microM) demonstrated activity only in rabbit and human blood. However, since in many experiments drug efficacy decreased significantly in blood compared to corresponding PRP, the concentrations of each agent necessary to inhibit responses were above those at which selectivity has previously been demonstrated against isolated enzyme preparations or in PRP. A fundamental reappraisal of both the potency and selectivity of these inhibitors in whole blood appears essential before their mechanism of action can be firmly established.     

Potentiation of ibudilast inhibition of platelet aggregation in the presence of endothelial cells

Although communications between platelets and endothelial cells or other blood cells are important in in vivo thrombus formation, laboratory platelet function tests are usually performed in isolation from these surrounding cells. In this study, we evaluated the effect of an antiplatelet drug, ibudilast (3-isobutyryl-2-isopropylpyrazolo[1,5-a]pyridine), on platelet aggregation in the presence and absence of human umbilical vein endothelial cells (HUVECs) and with the use of platelet-rich plasma (PRP) or whole blood as platelet samples. Stimulation-dependent platelet aggregation was weakened in the presence of HUVECs, which was especially prominent when the thrombin receptor-activating peptide SFLL (compared with ADP and epinephrine) was used as an aggregating agent. Ibudilast hardly affected SFLL-induced platelet aggregation (in PRP), while this antiplatelet agent was found to clearly inhibit this SFLL-induced response in a concentration-dependent manner, in the presence of HUVECs. Ibudilast tended to inhibit ADP- or epinephrine-induced platelet aggregation in the presence of HUVECs, but the effects were not statistically significant. Enhanced inhibition by ibudilast of SFLL-induced platelet aggregation (in the presence of HUVECs) was reproduced with the use of whole blood samples when a screen filtration pressure method was employed. It is suggested that the platelet aggregation studies in the presence of endothelial cells and/or other blood cells provide us with valuable information on platelet reactivity in vivo and improvement of antiplatelet therapy.     

A method of testing platelet aggregation in native whole blood

A method of testing collagen induced platelet aggregation and ATP release in native (= non anticoagulated) whole blood by monitoring the electrical impedance in the Chrono Log Whole Blood Aggregometer is reported. It is the first simple method by which aggregation of human platelets can be measured in their natural environment. In normal individuals lower threshold collagen concentrations could induce platelet aggregation as determined with this method than in the other tested methods (impedance method with citrated blood, optical method in platelet rich plasma). The aggregation response was not inhibited by hirudin or heparin in therapeutic dose. The luminescence channel of the Whole Blood Aggregometer permits measurements of ATP release in native whole blood.     

Iloprost and tissue-type plasminogen activator differentially affect platelet aggregation in platelet-rich plasma and in whole blood.

Platelet aggregation in platelet-rich plasma (PRP) is more sensitive to agonists and more resistant to antagonists than that in whole blood. In this study, we show that the presence of neutrophils in whole blood accounts at least in part for diminished platelet aggregation in whole blood. The platelet-inhibitory effect of neutrophils relates to the release of nitric oxide and not to elastase or adenosine. Furthermore, two unrelated platelet inhibitors, iloprost and tissue-type plasminogen inhibitor, decrease platelet aggregation to a greater extent in whole blood than in PRP. However, these agents exert cumulative or synergistic inhibitory effects with neutrophils on platelet aggregation in PRP. Thus, the inhibition of platelet aggregation in vivo may relate to the interaction between neutrophils and platelet-inhibitory agents.     

Aspirin and dazoxiben as inhibitors of platelet behaviour: modification of their effects by agents that alter cAMP production

The effects of aspirin and dazoxiben were determined on platelet behaviour in platelet-rich plasma (PRP) from 20 volunteers. Dazoxiben prevented aggregation and the release reaction induced by arachidonic acid (AA) in nine of the samples; in the other eleven aggregation and the release reaction still occurred. Aspirin always prevented aggregation and release but higher concentrations were needed in some of the samples of PRP than with others. When the platelets were sensitive to dazoxiben they were relatively sensitive to aspirin; when they were insensitive to dazoxiben they were relatively insensitive to aspirin. The effects of agents that alter production of cAMP on the sensitivity of platelets to aspirin and dazoxiben were determined. Increasing the intracellular level of cAMP rendered platelets more sensitive to the inhibitory effects of both aspirin and dazoxiben; lowering the level of cAMP made the platelets less sensitive to both agents.     

Verapamil is a potent inhibitor of 5-HT-induced platelet aggregation

We have studied the effects of verapamil, diltiazem and amlodipine on 5-HT-induced platelet aggregation and compared the results with those obtained for other platelet aggregating agents. Experiments were carried out using both human whole blood and platelet-rich plasma (PRP). Verapamil (but not diltiazem or amlodipine) inhibited 5-HT-induced platelet aggregation at much lower concentrations (IC50 = about 1 microM) than were required for inhibition of aggregation induced by other aggregating agents. Like some other selective inhibitors of 5-HT-induced platelet aggregation, it was not possible to completely overcome the inhibition by increasing the concentration of 5-HT. The antiaggregatory effects of verapamil were similar, but not identical, in whole blood and PRP. These results show that the Ca2+ channel blocker verapamil has some selectivity as an inhibitor of 5-HT-induced platelet aggregation and that this behaviour as a 5-HT antagonist should be taken into account when interpreting any therapeutic benefit ascribed to this drug.     

Pulsed Radiofrequency Neuromodulation Contributes to Activation of Platelet-Rich Plasma in In Vitro Conditions

ObjectivesRecent years have brought new developments in interventional chronic pain management, namely regenerative orthopedics utilizing platelet-rich plasma (PRP) as well as further evolution of pulsed radiofrequency neuromodulation (PRF). Both methods have been used separately. Here, we investigated whether PRF may potentiate the activation of platelets in PRP samples when both these techniques are combined together in in vitro conditions.Materials and MethodsStudies were performed on concentrated PRP samples (PRPs) obtained from acid citrate dextrose-treated blood taken from 11 healthy volunteers. PRPs were divided into four groups: 1) nonactivated PRP; 2) thrombin-activated PRP as a positive control for maximal platelets activation; 3) PRF-treated PRP exposed for 20 min to PRF energy generated by neurotherm radio frequency generator at 500 kHz, with a voltage of 40 V and maximal temperature of 42°C; and 4) a combination of groups 2 and 3.ResultsPRF-induced platelet activation measured by platelet factor 4 (PF4) and ATP release from PRPs was significantly higher compared to nonactivated PRPs, and similar to PF4 and ATP release from thrombin-activated PRPs. Thrombin activation did not potentiate PF4 release in PRF samples and even reduced ATP level. Additionally, PRF neither induced any platelet membrane damage measured by lactic dehydrogenase release from PRP nor modified any platelets viability or metabolism measured by MTT.ConclusionsWe confirmed that PRF may activate PRP without additional platelet activators. So, a combination of both methods PRF and PRP application may provide a more effective opportunity for tissue regeneration in dentistry, surgery, dermatology, or in orthopedics.     

Collagen-induced platelet aggregation:--evidence against the essential role of platelet adenosine diphosphate

The hypothesis that platelet ADP is responsible for collagen-induced aggregation has been re-examined. It was found that the concentration of ADP obtaining in human PRP at the onset of aggregation was not sufficient to account for that aggregation. Furthermore, the time-course of collagen-induced release in human PRP was the same as that in sheep PRP where ADP does not cause release. These findings are not consistent with claims that ADP alone perpetuates a collagen-initiated release-aggregation-release sequence. The effects of high doses of collagen, which released 4-5 microM ADP, were not inhibited by 500 microM adenosine, a concentration that greatly reduced the effect of 300 microM ADP. Collagen caused aggregation in ADP-refractory PRP and in platelet suspensions unresponsive to 1 mM ADP. Thus human platelets can aggregate in response to collagen under circumstances in which they cannot respond to ADP. Apyrase inhibited aggregation and ATP release in platelet suspensions but not in human PRP. Evidence is presented that the means currently used to examine the role of ADP in aggregation require investigation.     

Exercise-induced changes in platelet aggregation; a comparison of whole blood and platelet rich plasma techniques

Studies have been performed to assess the effect of exercise on spontaneous platelet aggregation in shaken whole blood, and on agonist-induced platelet aggregation in whole blood and platelet rich plasma (PRP). Spontaneous platelet aggregation in shaken whole blood was increased following exercise compared to pre-exercise values. The increase in spontaneous aggregation after exercise correlated inversely with the increase in white cell count in whole blood. Platelet sensitivity in whole blood to adrenaline, collagen and adenosine diphosphate (ADP) was increased following exercise. Changes in platelet sensitivity to adrenaline following exercise correlated with increases in plasma noradrenaline levels but not with changes in blood cell counts. In PRP, platelet sensitivity to ADP and to collagen was increased following exercise when the pre and post-exercise PRP platelet counts were not corrected to allow for the increase in platelet count which occurred with exercise. When the PRP platelet counts were corrected, no changes in platelet sensitivity to any agonist after exercise were observed.     

The nature of interactions between tissue-type plasminogen activator and platelets

To elucidate interactions responsible for inhibition of aggregation of platelets in platelet-rich plasma (PRP) harvested from whole blood preincubated with t-PA, experiments were performed with PRP and washed platelets under diverse conditions of preincubation. Both ADP and collagen induced aggregation were inhibited in PRP unless aprotinin had been added to the preincubated whole blood concomitantly with t-PA. However, in washed platelets prepared after the same exposure aggregation was intact. When washed platelets were supplemented with fibrinogen degradation products (FDPs) in concentrations simulating those in whole blood preincubated with t-PA, aggregation induced with either ADP or collagen was inhibited. Thus, the inhibition in PRP depended on generation of FDPs by activated plasminogen. The functional integrity of surface glycoprotein (GP) IIb/IIIa receptors in washed platelets was documented by autoradiography after SDS-PAGE of surface labeled GPs and by fibrinogen binding despite preincubation of the whole blood or washed platelets themselves with t-PA and plasminogen as long as exogenous calcium (greater than or equal to 0.1 microM) was present. In contrast, when calcium was absent, the platelet GP IIb/IIIa receptor was rendered susceptible to degradation by plasmin, and aggregation was inhibited by preincubation at 37 degrees C even if aprotinin was present when aggregation was being assayed. These observations reconcile disparate results in the literature from studies in vivo and in vitro by demonstrating that inhibition of aggregation of platelets in PRP and in whole blood reflects indirect effects of plasminogen activation rather than direct effects of t-PA or plasmin on the platelets themselves.