Effects of short-term hypoxia on platelet counts of mice

Recent studies have shown that long-term hypoxia causes decreased platelet counts in mice and short-term hypoxia increased platelet counts. In an attempt to explain the mechanism that increases platelet counts of mice after exposure to short-term hypoxia, we measured platelet counts, total circulating platelet counts (TCPC), total circulating platelet masses (TCPM), percentages of 35S incorporation, and platelet sizes. Platelet counts, as well as TCPC and TCPM of mice, increased after 1-3 days of hypoxia, but these values were decreased after 6-7 days of hypoxia. Although platelet counts were increased in hypoxic mice, the percentage 35S incorporation into platelets and platelet sizes did not show a concurrent increase. After 6 days of hypoxia, average platelet diameters began to increase as platelet counts decreased. Splenic release did not account for the increase in platelet counts of mice after short-term hypoxia. It seems possible, therefore, that megakaryocytes "shed" platelets into the circulation in response to hypoxia. The platelets that enter the circulation in response to short-term hypoxia are smaller and incorporate less 35S than platelets that are produced in response to acute thrombocytopenia.     

Effect of exercise on pulmonary function tests in normal first degree relations of asthmatic subjects.

The prevalence of exercise-induced asthma (EIA) after six minutes of standard exercise test on bicycle ergometer was studied in thirty healthy first degree relations of asthmatic subjects (group II) and was compared with that in thirty healthy controls (group I). Pulmonary function tests (PFT) measuring FVC, FEV1, FEF25-75% and PEFR were performed on each subject prior of exercise, immediately following exercise period and serially at 5 minute interval for 25 minutes thereafter. Bronchial lability was noted in 7% and 27% of the subjects in groups I and II respectively. Of all the parameters measured, FEF25-75% exhibited most striking changes when expressed as % of the baseline value.     

The effect on skin blood flow of short-term venous hypertension in normal subjects.

Plugging of skin capillaries by activated white blood cells is one of the proposed mechanisms by which skin damage may be initiated in chronic venous insufficiency. The aim of this study was to determine whether a microcirculatory deficit was induced in the skin by raising the venous pressure proximally for thirty minutes. Seventeen subjects with no evidence of venous or arterial disease had laser Doppler velocimetry performed in the goiter region of the leg; 8 different subjects had the measurement done on the dorsum of the hand. Peak hyperemic response following three minutes of ischemia was measured before and after a thirty-minute period of sustained venous hypertension applied by a proximal tourniquet inflated to 80 mm Hg. A decrease in the peak flow: baseline flow ratio (median ratio 2.25 before, 1.70 after, p less than 0.02) and an increase in the time taken to reach maximal hyperemia (median time ten seconds before, twenty seconds after, p less than 0.01) were observed after the period of venous hypertension in the lower limb. The second parameter, but not the first, was significantly affected in the upper limb. The authors conclude that a microvascular deficit in the skin is demonstrable after a short period of venous hypertension. This is consistent with the white-cell-trapping theory, but other possible explanations are discussed.     

Effect of mental stress on platelet function in normal subjects and in patients with coronary artery disease.

We studied the effect of emotional stress (mental arithmetic for 10 min) in 10 postinfarction patients and in 10 age-matched apparently healthy subjects as controls. Blood samples for platelet function studies and for the determination of epinephrine levels in serum were taken in basal conditions, at the end of mental stress and after 30 min of recovery. Patients were studied twice, in washout of medications and after oral administration of dipyridamole, 200 mg twice a day for 6 consecutive days. Mental stress induced in patients significant increments in different hemodynamic parameters (heart rate, systolic blood pressure and diastolic blood pressure) and in serum epinephrine levels. Concomitantly, the test produced a significant increase in platelet aggregation (induced by 3 microM ADP or 1 microgram/ml collagen), the formation of circulating platelet aggregates and an increase in plasma thromboxane B2 levels. Hemodynamic parameters and platelet function tests returned to baseline values after 30 min. Similar activation of hemodynamic parameters, similar increase in epinephrine levels and lower increase in platelet function by emotional stress were observed in control subjects. Treatment of patients with dipyridamole had no effect on stress-induced increase in hemodynamic parameters and epinephrine levels, but decreased stress-related platelet activation. These data can contribute to a better understanding of the complex relationships between psychosocial factors, the hemostatic system and vascular disease.     

Standardization of platelet function tests

Methods for the standardization of several currently used platelet function tests (bleeding time, capillary fragility, platelet retention, platelet aggregation, platelet factor 3, and platelet volume profiles) are presented. Different variables that may interfere with the reproducibility of the results of each assay were identified and standardized. Using the standardized techniques, the range of normal values for each test was determined in a large population of normal volunteers and used to identify disease states by comparing patient results with those of the normal population. A format for presenting the entire profile of platelet function parameters is proposed.     

Effect of domperidone on gastro-oesophageal function in normal human subjects

The aim of the study was to investigate gastro-oesophageal function in normal volunteers after oral administration of domperidone as a single dose (20 mg). The study was designed as a double-blind cross-over investigation. Ten healthy men (aged 27-50 years; median, 30) joined the study. Oesophageal function tests were performed, starting 1.5 h after intake of domperidone or placebo. Gastro-oesophageal sphincter pressure was measured with a perfused catheter system and a continuous pull-through technique. No changes in sphincter pressure were found. Peristaltic pressure amplitude in the body of the oesophagus and the duration and velocity of peristalsis were measured after wet swallows (bolus 5 ml of water). No changes were found. Intragastric pH did not change significantly after domperidone. No effect of domperidone on the results of a standard acid-clearing test could be found. Plasma concentrations of domperidone did not correlate with any of the other variables. In conclusion, domperidone given orally in a dose that has been shown to accelerate gastric emptying does not influence gastro-oesophageal sphincter pressure or peristaltic activity in the normal oesophagus.     

Low- vs high-dose aspirin. Effects on platelet function in hyperlipoproteinemic and normal subjects

Low-dose aspirin may be inadequate for inhibition of platelet function in hyperlipoproteinemics due to increased platelet reactivity. Platelet function was studied in 18 type II hyperlipoproteinemic and 12 normal subjects after at least ten days of treatment with placebo and with low-dose (0.45 mg/kg/day) and high-dose (900 mg/day) aspirin. In the normal and hyperlipoproteinemic subjects, low-dose aspirin produced near maximal (90%) inhibition of platelet thromboxane generation, significant prolongation of the bleeding time, and significant inhibition of platelet aggregation, similar in degree to the inhibition produced by high-dose aspirin. There was no significant difference between hyperlipoproteinemic and normal subjects in any of the platelet function measures before and after aspirin treatment. Thus, a daily 0.45-mg/kg aspirin dose (20 to 45 mg) effectively inhibited platelet function in type II hyperlipoproteinemics, who do not appear to have an increased dose requirement for aspirin.     

Gender,race and diet affect platelet function tests in normal subjects,contributing to a high rate of abnormal results

To assess sources of variability in platelet function tests in normal subjects, 64 healthy young adults were tested on 2–6 occasions at 2 week intervals using four methods: platelet aggregation (AGG) in platelet‐rich plasma (PRP) in the Bio/Data PAP‐4 Aggregometer (BD) and Chrono‐Log Lumi‐Aggregometer (CL); and AGG in whole blood (WB) in the CL and Multiplate Platelet Function Analyser (MP), with ATP release (REL) in CL‐PRP and CL‐WB. Food and medication exposures were recorded prospectively for 2 weeks prior to each blood draw. At least one AGG abnormality was seen in 21% of 81 drug‐free specimens with CL‐PRP, 15% with CL‐WB, 13% with BD‐PRP and 6% with MP‐WB, increasing with inclusion of REL to 28% for CL‐PRP and 30% for CL‐WB. Epinephrine AGG and REL were significantly reduced in males (P < 0·0001). Ristocetin AGG and collagen and thrombin REL were significantly reduced in Blacks (P < 0·0001). One‐third of specimens drawn following flavonoid‐rich food exposures had aberrant results, compared to 8·5% of specimens without such exposures (P = 0·0035). PRP tests had less intra‐individual variation than WB tests. Gender, race, diet and test system affected results of platelet function testing in healthy subjects, suggesting caution when interpreting the results of platelet function testing in patients.     

Effect of cisapride on the gastro-oesophageal function in normal human subjects

The aim of this study was to investigate oesophageal peristalsis and gastro-oesophageal function in normal subjects after 4 days of cisapride 10 mg p.o. 3 times/day and 10 mg 1.5 h before the investigation. The study was carried out in a double-blind cross-over design with coded cisapride or placebo tablets. Basal sphincter pressure increased after cisapride (p less than 0.002). The peristaltic pressure amplitude in the oesophageal body as well as the duration and velocity of the peristaltic pressure wave were measured after wet swallows. No changes were found. Intragastric pH was unchanged after cisapride. No effect was found on the result of a standard acid clearing test. Plasma concentration of cisapride did not correlate with any of the other variables. Oral cisapride increases fasting gastro-oesophageal sphincter pressure, but does not influence oesophageal peristalsis, acid clearing or intragastric pH in normal subjects.     

Effect of laryngeal anesthesia on pulmonary function testing in normal subjects

Pulmonary function tests (PFT) were performed on 11 normal subjects before and after topical anesthesia of the larynx. The PFT consisted of flow volume loops and body box determinations of functional residual capacity and airway resistance, each performed in triplicate. After the first set of tests, cotton pledgets soaked in 4% lidocaine were held in the pyriform sinuses for 2 min to block the superior laryngeal nerves. In addition, 1.5 ml of 10% cocaine was dropped on the vocal cords via indirect laryngoscopy. PFT were repeated 5 min after anesthesia. Besides routine analysis of the flow volume loops, areas under the inspiratory (Area I) and expiratory (Area E) portions of the loops were calculated by planimetry. Area I, peak inspiratory flow (PIF), as well as forced inspiratory flow at 25, 50, and 75% forced vital capacity (FVC), decreased after anesthesia. Peak expiratory flow decreased after anesthesia, but Area E and forced expiratory flow at 25, 50, and 75% FVC were unchanged. This protocol also was performed in 12 normal subjects with isotonic saline being substituted for the lidocaine and cocaine. In this group, no significant differences were observed when flow volume loop parameters were compared before and after topical application of saline. In 5 spontaneously breathing anesthetized dogs, posterior cricoarytenoid muscle and afferent superior laryngeal nerve activity were recorded before and after laryngeal anesthesia performed with the same procedure used in the human subjects. Laryngeal anesthesia resulted in a substantial decrease or a complete disappearance of afferent SLN activity recorded during unobstructed and obstructed respiration. The data suggest that laryngeal receptors help modulate upper airway patency in man.     

Arachidonic acid causes lysis of blood cells and ADP-dependent platelet activation responses in platelet function tests

The use of arachidonic acid (AA) to stimulate platelets is considered as a specific approach to study aspirin treatment efficacy. However, very high concentrations of AA are used, and it has been previously reported that AA can induce cell lysis in other settings. Several clinical studies have reported decreased responses to AA in whole blood tests in the presence of clopidogrel.

Our aim was to investigate whether unspecific effects contribute to AA-induced aggregation and platelet activation in light transmission aggregometry (LTA) in platelet-rich plasma (PRP), and in assays using whole blood, multiple electrode aggregometry (MEA, Multiplate®), and flow cytometry.

We report that cell lysis, especially of red blood cells, does occur at concentrations of AA used in the clinical tests and that ADP is very important for the AA-induced platelet activation responses. In flow cytometry, very limited platelet activation was detected before reaching AA concentrations in the millimolar range, where cell lysis also occurred, making it problematic to develop a reliable flow cytometry assay using AA as reagent.

We conclude that cell lysis and ADP release contribute to AA-induced platelet responses, most markedly in whole blood assays. This finding could potentially explain some differences between studies comparing methods using whole blood and PRP and also how clopidogrel treatment could influence AA-induced aggregation results in previously published studies. Our findings highlight some issues with AA as reagent for platelet activation, which also have an impact on how platelet activation assays using AA should be interpreted.     

The value of platelet function tests

Platelet function tests are used to detect patients with abnormal platelet function, which may be inborn or acquired or to detect increased platelet activation which may be accompanied by an increased risk of thrombosis. Platelet function tests are also used to monitor platelet function inhibitors as aspirin, clopidogrel or platelet membrane glycoprotein IIb/IIIa-inhibitors. Incorrect blood sampling is a major source of error in measuring platelet function. Global tests besides platelet count and bleeding time are thrombelastography, the platelet function analyzer (PFA) and possibly in the future the new Impact-system. Specific tests measure platelet spreading and adhesion to defined surfaces. In a series of methods platelets are counted before and after passage of a filter. Some of these tests are partially standardized. The most frequently measured platelet function is aggregation induced by ADP, collagen or other substances as first described by Gustav Born. Some newer methods to perform aggregometry are described. Platelet activation can be detected by measuring spontaneous aggregation as in the PAT-test. Prospective trials with this test have shown that enhanced spontaneous aggregation is a risk factor for new vascular occlusions in diabetics and for myocardial infarctions in healthy individuals. The Wu and Hoak-test and the measurement of released platelet factor 4 or betaglobulin are of limited value. Flow cytometric methods are frequently used to measure platelet activation markers as CD 62 and others. Platelet induced thrombin generation is an interesting function to measure drug effects. None of the presently available platelet function tests is well standardized, so there is much room for improvement.     

Effects of dietary fish oil on platelet function and plasma lipids in hyperlipoproteinemic and normal subjects

We studied the effects of dietary supplementation with an encapsulated fish oil concentrate (Maxepa) on platelet function, fibrinolysis, and plasma lipids and lipoproteins in 9 normal subjects, 10 patients with type IV hyperlipoproteinemia, and 6 with type IIB hyperlipoproteinemia. After a baseline period, the subjects crossed over randomly between treatment periods with Maxepa (providing 3.24 g eicosapentaenoic acid and 2.16 g docosahexaenoic acid per day) and safflower oil (used as a control), given for 6 weeks each. Administration of Maxepa led to a slight prolongation of the bleeding time in all groups and to modest inhibition of platelet aggregation in the type IV hyperlipoproteinemics and normal subjects, with partial (41%) inhibition of thromboxane synthesis from baseline levels noted in the normal group. Plasma total fibrinolytic actively did not change significantly in any group. Maxepa treatment resulted in a marked decrease in triglyceride and VLDL-cholesterol and a slight increase in HDL-cholesterol was noted after Maxepa in the type IV hyperlipoproteinemics (4.11 +/- 0.13 mmol/l vs. 3.10 +/- 0.16 mmol/l, Maxepa vs. safflower oil). We conclude that dietary supplementation with fish oil results in a relatively minor degree of inhibition of platelet function in normal and hyperlipoproteinemic subjects, and a potentially adverse increase in LDL-cholesterol in type IV hyperlipoproteinemics.     

Anorectal function in normal human subjects: Effect of gender

Multiport anorectal manometry and external anal sphincter (EAS) and internal anal sphincter (IAS) electromyography were conducted in 15 males (41±3 years) and 20 females (43±2 years; 5 nulliparous) during rest, maximum conscious sphincter contraction, rectal distension and increases in intra-abdominal pressure. The basal pressure declined within 15 minutes of insertion of the manometric probe to a stable plateau, 55±4% of the initial value. The maximum basal (91±5 vs 61±6 cm water; mean±SEM), minimum basal (43±7 vs 27±3 cm water) and the maximum squeeze pressures (257±20 vs 107±13 cm water) were higher (p<0.05) in males than females. Distension of a rectal balloon caused a reduction in pressure in all anal channels, that increased in depth and duration as the distending volume was increased. These anal relaxations were associated with rectal contractions and transient increases in the electrical activity of the EAS. Upon deflating the balloon, the anal pressure increased to values that exceeded the pre-inflation values. The pre-inflation (89±4 vs 49±4 cm water), post-inflation (104±9 vs 62±7 cm water) and residual (47±4 vs 30±2 cm water) pressures during rectal distension were significantly higher in males than in females (p<0.05). The higher residual pressure in males was associated with a higher EAS index during rectal distension (0.94±0.10 vs 0.65±0.10 mv s;p<0.05). The lowest volume required to cause a desire to defaecate was significantly higher in males than in females (76±7 vs 48±6 ml;p<0.05) and only 13% of males compared with 55% females (p<0.01) felt pain during rectal distension with 100 ml. During increases in intra-abdominal pressure, all subjects showed increases in pressures in the outermost anal channels, that were associated with increases in the electrical activity of the EAS and were significantly higher in males compared with females (188±17 vs 98± 9 cm water;p<0.05). In conclusion, the data suggest that males have stronger internal and external anal sphincters than females, while females have greater rectal sensitivity.     

Effect of hypoxia on the sensation of dyspnea during hypercapnic ventilatory response in normal subjects]

We examined, in 32 normal adults, the effect of hypoxia on the sensation of dyspnea during hypercapnic ventilatory response (HCVR). The tests were conducted under two different levels of inspiratory O2 content, either hyperoxia (PETO2 greater than 150 Torr) or hypoxia (PETO2 50-55 Torr), with simultaneous assessment of dyspnea sensation by visual analogue scaling (VAS). The sensation was evaluated either in relation to VE standardized by predicted MVV (the slope of VAS-VE regression line or VAS at VE 40%) or in relation to PETCO2 (the slope of VAS-PETCO2 line or VAS at PETCO2 55 Torr). Concomitant hypoxia significantly enhanced both the mean value of delta VE/delta PETCO2 and that of delta P0.1/delta PETCO2. The sensation of dyspnea did not differ between the two conditions when it was evaluated in relation to ventilation, whereas it was markedly greater during hypoxic HCVR when it was evaluated in relation to PETCO2. The hypoxic augmentation of the sensation, compared at PETCO2 55 Torr, could be explained by increase of the motor output from the respiratory center, since it was positively correlated with the relative change of VE, VTTI, and delta P0.1/delta PETCO2 (r = 0.70, p less than 0.0001; r = 0.63, p less than 0.0001; r = 0.40, p less than 0.05, respectively). From these findings, we conclude that hypoxia does not have a direct dyspnogenic effect, at least in normal subjects.