Platelet activation: ultrastructure and morphometry in platelet-rich plasma of horses

This study was conducted to investigate the activation ability of the platelet-rich plasma (PRP) by pharmacological agents, as well as to verify the need or not of this activation for therapeutic use. The PRP was obtained from four healthy crossbred geldings aged 13 to 16 years (15±1years), and was processed for observation and quantification of the platelet morphology by using the transmission electron microscopy. All PRP samples were activated with 10% calcium chloride (CaCl2) solution, pure bovine thrombin or associated with CaCl2. The control (pure PRP) was not pharmacologically activated. In the pure PRP samples, 49% of the platelets were classified as state of activation uncertain, 41% as resting, 9% as fully activated and 1% as irreversibly damaged. Treatment with 10% CaCl2 provided a distribution of 54% platelets in state of activation uncertain, 24% as fully activated, 20% as resting, and 2% as irreversibly damaged. The platelet morphology of the bovine thrombin treated samples did not fit into classification adopted, as showing irregular shape with emission of large filamentous pseudopods, appearance of ruptured and whole granules in the remaining cytoplasm and extracellular environment. There was effect of the treatment on the platelet morphology (P=0.03). The 10% CaCl2 is an adequate platelet-activating agent. However, in cases the use of PRP under its liquid form is necessary, the use of pure PRP is recommended, since besides presenting an adequate percentage of fully activated platelets it also has significant amount of the resting type, which can be activated by substances found in the injured tissue.

Method to obtain platelet-rich plasma from rabbits (Oryctolagus cuniculus )

Abstract: Platelet-rich plasma (PRP) is a product easy and inxpesnsive, and stands out to for its growth factors in tissue repair. To obtain PRP, centrifugation of whole blood is made with specific time and gravitational forces. Thus, the present work aimed to study a method of double centrifugation to obtain PRP in order to evaluate the effective increase of platelet concentration in the final product, the preparation of PRP gel, and to optimize preparation time of the final sample. Fifteen female White New Zealand rabbits underwent blood sampling for the preparation of PRP. Samples were separated in two sterile tubes containing sodium citrate. Tubes were submitted to the double centrifugation protocol, with lid closed and 1600 revolutions per minute (rpm) for 10 minutes, resulting in the separation of red blood cells, plasma with platelets and leucocytes. After were opened and plasma was pipetted and transferred into another sterile tube. Plasma was centrifuged again at 2000rpm for 10 minutes; as a result it was split into two parts: on the top, consisting of platelet-poor plasma (PPP) and at the bottom of the platelet button. Part of the PPP was discarded so that only 1ml remained in the tube along with the platelet button. This material was gently agitated to promote platelets resuspension and activated when added 0.3ml of calcium gluconate, resulting in PRP gel. Double centrifugation protocol was able to make platelet concentration 3 times higher in relation to the initial blood sample. The volume of calcium gluconate used for platelet activation was 0.3ml, and was sufficient to coagulate the sample. Coagulation time ranged from 8 to 20 minutes, with an average of 17.6 minutes. Therefore, time of blood centrifugation until to obtain PRP gel took only 40 minutes. It was concluded that PRP was successfully obtained by double centrifugation protocol, which is able to increase the platelet concentration in the sample compared with whole blood, allowing its use in surgical procedures. Furthermore, the preparation time is appropriate to obtain PRP in just 40 minutes, and calcium gluconate is able to promote the activation of platelets.

Brain ischemia alters platelet ATP diphosphohydrolase and 5'-nucleotidase activities in naive and preconditioned rats

The effects of transient forebrain ischemia, reperfusion and ischemic preconditioning on rat blood platelet ATP diphosphohydrolase and 5'-nucleotidase activities were evaluated. Adult Wistar rats were submitted to 2 or 10 min of single ischemic episodes, or to 10 min of ischemia 1 day after a 2-min ischemic episode (ischemic preconditioning) by the four-vessel occlusion method. Rats submitted to single ischemic insults were reperfused for 60 min and for 1, 2, 5, 10 and 30 days after ischemia; preconditioned rats were reperfused for 60 min 1 and 2 days after the long ischemic episode. Brain ischemia (2 or 10 min) inhibited ATP and ADP hydrolysis by platelet ATP diphosphohydrolase. On the other hand, AMP hydrolysis by 5'-nucleotidase was increased after 2, but not 10, min of ischemia. Ischemic preconditioning followed by 10 min of ischemia caused activation of both enzymes. Variable periods of reperfusion distinctly affected each experimental group. Enzyme activities returned to control levels in the 2-min group. However, the decrease in ATP diphosphohydrolase activity was maintained up to 30 days of reperfusion after 10-min ischemia. 5'-Nucleotidase activity was decreased 60 min and 1 day following 10-min ischemia; interestingly, enzymatic activity was increased after 2 and 5 days of reperfusion, and returned to control levels after 10 days. Ischemic preconditioning cancelled the effects of 10-min ischemia on the enzymatic activities. These results indicate that brain ischemia and ischemic preconditioning induce peripheral effects on ecto-enzymes from rat platelets involved in nucleotide metabolism. Thus, ATP, ADP and AMP degradation and probably the generation of adenosine in the circulation may be altered, leading to regulation of microthrombus formation since ADP aggregates platelets and adenosine is an inhibitor of platelet aggregation.

Determination of serum aluminum, platelet aggregation and lipid peroxidation in hemodialyzed patients

Aluminum (Al3+) overload is frequently associated with lipid peroxidation and neurological disorders. Aluminum accumulation is also reported to be related to renal impairment, anemia and other clinical complications in hemodialysis patients. The aim of the present study was to determine the degree of lipid peroxidation, platelet aggregation and serum aluminum in patients receiving regular hemodialytic treatment. The level of plasma lipid peroxidation was evaluated on the basis of thiobarbituric acid reactive substances (TBARS). Mean platelet peroxidation in patients undergoing hemodialysis was significantly higher than in normal controls (2.7 ± 0.03 vs 1.8 ± 0.06 nmol/l, P<0.05). Platelet aggregation and serum aluminum levels were determined by a turbidimetric method and atomic absorption spectrophotometry, respectively. Serum aluminum was significantly higher in patients than in normal controls (44.5 ± 29 vs 10.8 ± 2.5 µg/l, P<0.05). Human blood platelets were stimulated with collagen (2.2 µg/ml), adenosine diphosphate (6 µM) and epinephrine (6 µM) and showed reduced function with the three agonists utilized. No correlation between aluminum levels and platelet aggregation or between aluminum and peroxidation was observed in hemodialyzed patients.

Effects of simvastatin/ezetimibe on microparticles, endothelial progenitor cells and platelet aggregation in subjects with coronary heart disease under antiplatelet therapy

It is not known whether the addition of ezetimibe to statins adds cardiovascular protection beyond the expected changes in lipid levels. Subjects with coronary heart disease were treated with four consecutive 1-week courses of therapy (T) and evaluations. The courses were: T1, 100 mg aspirin alone; T2, 100 mg aspirin and 40 mg simvastatin/10 mg ezetimibe; T3, 40 mg simvastatin/10 mg ezetimibe, and 75 mg clopidogrel (300 mg initial loading dose); T4, 75 mg clopidogrel alone. Platelet aggregation was examined in whole blood. Endothelial microparticles (CD51), platelet microparticles (CD42/CD31), and endothelial progenitor cells (CD34/CD133; CDKDR/CD133, or CD34/KDR) were quantified by flow cytometry. Endothelial function was examined by flow-mediated dilation. Comparisons between therapies revealed differences in lipids (T2 and T3T1 and T4, P=0.001). Decreased platelet aggregation was observed after aspirin (arachidonic acid, T1platelet aggregation, the amount of circulating endothelial and platelet microparticles, or endothelial progenitor cells. Cardiovascular protection following therapy with simvastatin/ezetimibe seems restricted to lipid changes and improvement of endothelial function not affecting the release of microparticles, mobilization of endothelial progenitor cells or decreased platelet aggregation.

High levels of LDL-C combined with low levels of HDL-C further increase platelet activation in hypercholesterolemic patients

High levels of low-density lipoprotein cholesterol (LDL-C) enhance platelet activation, whereas high levels of high-density lipoprotein cholesterol (HDL-C) exert a cardioprotective effect. However, the effects on platelet activation of high levels of LDL-C combined with low levels of HDL-C (HLC) have not yet been reported. We aimed to evaluate the platelet activation marker of HLC patients and investigate the antiplatelet effect of atorvastatin on this population. Forty-eight patients with high levels of LDL-C were enrolled. Among these, 23 had HLC and the other 25 had high levels of LDL-C combined with normal levels of HDL-C (HNC). A total of 35 normocholesterolemic (NOMC) volunteers were included as controls. Whole blood flow cytometry and platelet aggregation measurements were performed on all participants to detect the following platelet activation markers: CD62p (P-selectin), PAC-1 (GPIIb/IIIa), and maximal platelet aggregation (MPAG). A daily dose of 20 mg atorvastatin was administered to patients with high levels of LDL-C, and the above assessments were obtained at baseline and after 1 and 2 months of treatment. The expression of platelets CD62p and PAC-1 was increased in HNC patients compared to NOMC volunteers (P<0.01 and P<0.05). Furthermore, the surface expression of platelets CD62p and PAC-1 was greater among HLC patients than among HNC patients (P<0.01 and P<0.05). Although the expression of CD62p and PAC-1 decreased significantly after atorvastatin treatment, it remained higher in the HLC group than in the HNC group (P<0.05 and P=0.116). The reduction of HDL-C further increased platelet activation in patients with high levels of LDL-C. Platelet activation remained higher among HLC patients regardless of atorvastatin treatment.

Altered mean platelet volume in patients with polymyositis and its association with disease severity

Polymyositis (PM) is an autoimmune disease characterized by chronic inflammation in skeletal muscle. Mean platelet volume (MPV), a marker in the assessment of systemic inflammation, is easily measured by automatic blood count equipment. However, to our knowledge, there are no data in the literature with respect to MPV levels in PM patients. Therefore, in this study we aimed to investigate MPV levels in patients with PM. This study included 92 newly diagnosed PM patients and 100 healthy individuals. MPV levels were found to be significantly lower compared with healthy controls (10.3±1.23 vs 11.5±0.74 fL, P<0.001). Interestingly, MPV was found to be positively correlated with manual muscle test (MMT) score and negatively correlated with erythrocyte sedimentation rate (ESR) in patients with PM (r=0.239, P=0.022; r=−0.268, P=0.010, respectively). In addition, MPV was significantly lower in active PM patients compared with inactive PM patients (9.9±1.39 vs 10.6±0.92 fL, P=0.010). MPV was independently associated with PM in multivariate regression analyses, when controlling for hemoglobin and ESR (OR=0.312, P=0.031, 95%CI=0.108 to 0.899). The ROC curve analysis for MPV in estimating PM patients resulted in an area under the curve of 0.800, with sensitivity of 75.0% and specificity of 67.4%. Our results suggest that MPV is inversely correlated with disease activity in patients with PM. MPV might be a useful tool for rapid assessment of disease severity in PM patients.

Use of medicines recommended for secondary prevention of acute coronary syndrome

ABSTRACT OBJECTIVE : To analyze if the demographic and socioeconomic variables, as well as percutaneous coronary intervention are associated with the use of medicines for secondary prevention of acute coronary syndrome. METHODS : In this cohort study, we included 138 patients with acute coronary syndrome, aged 30 years or more and of both sexes. The data were collected at the time of hospital discharge, and after six and twelve months. The outcome of the study was the simultaneous use of medicines recommended for secondary prevention of acute coronary syndrome: platelet antiaggregant, beta-blockers, statins and angiotensin-converting-enzyme inhibitor or angiotensin receptor blocker. The independent variables were: sex, age, education in years of attending, monthly income in tertiles and percutaneous coronary intervention. We described the prevalence of use of each group of medicines with their 95% confidence intervals, as well as the simultaneous use of the four medicines, in all analyzed periods. In the crude analysis, we verified the outcome with the independent variables for each period through the Chi-square test. The adjusted analysis was carried out using Poisson Regression. RESULTS : More than a third of patients (36.2%; 95%CI 28.2;44.3) had the four medicines prescribed at the same time, at the moment of discharge. We did not observe any differences in the prevalence of use in comparison with the two follow-up periods. The most prescribed class of medicines during discharge was platelet antiaggregant (91.3%). In the crude analysis, the demographic and socioeconomic variables were not associated to the outcome in any of the three periods. CONCLUSIONS : The prevalence of simultaneous use of medicines at discharge and in the follow-ups pointed to the under-utilization of this therapy in clinical practice. Intervention strategies are needed to improve the quality of care given to patients that extend beyond the hospital discharge, a critical point of transition in care.

Afibrinogenemia following snake bite (Crotalus durissus terrificus)

This paper reports two cases of afibrinogenemia with normal platelet count following Crotalus durissus terrificus, snake bite Both patients presented high output acute renal failure and case two also had increased blood levels of CPK and LDH compatible with the diagnosis of rhabdomyolysis. Case one was given an unknown amount of antivenom and was treated with epsilonaminocaproic acid and a fresh whole blood transfusion and showed recovery of the coagulation disturbance 40 hours following these measures. Case two was given an adequate amount of crotalide antivenom and the coagulation tests performed 12 hours later showed a normal partial thromboplastin time and fibrinogen 86 mg/100ml. Case one presented no haemorrhagic disturbances. Case two presented persistent bleeding following venopuncture and after removal of impetigo crust in the legs. Acute renal failure was treated conservatively and both patients were discharged from the hospital with recovery of the renal function.

Thrombocytopenia and leptospirosis

The present study has intended to contribute to the elucidation of the pathogenic mechanisms, involved in the thrombocytopenia and in the bleeding diathesis seen in the course of Leptospirosis. The group of cases included in the present prospective study consisted of 30 patients with Leptospirosis, admitted to the Infectious and Parasitic Diseases Ward, Hospital das Clínicas, Faculty of Medicine, University of São Paulo. The following possible mechanisms of thrombocytopenia have been considered and therefore investigated: platelet consumption, due to disseminated intravascular coagulation; immune-mediated platelet destruction, due to platelet-associated antibodies and an inhibited platelet production in the bone marrow. Thrombocytopenia occurred in 86.6% of 30 patients and did not seem to be immune-mediated by platelet-associated antibodies. Furthermore it did not seem to be due to a disseminated intravascular coagulation consumption. Although there was a statistically-significant correlation between bone marrow platelet production and platelet counts we think that the static microscopic examination of a bone marrow aspirate cannot accurately depict the dynamic mechanisms of platelet production when these cells are being consumed in peripheral blood. Vasculitis should be considered as the most important factor for the pathogenesis of the bleeding disturbances in Leptospirosis. However, we believe that thrombocytopenia, uremia and coagulation disorders, individually or as a group, should be included among the contributing factors that lead to and worsen bleeding episodes, which represent the leading cause of death in this disease.

Trypanossoma cruzi: course of infection in platelets-depleted mice

The effect of platelet depletion on the course of Trypanosoma cruzi infection in BALB/c mice was investigated. Thrombocytopenia was achieved by inoculation of rabbit anti-platelet IgG during the parasitemic phase of the infection. The number of parasites in the blood of anti-platelet IgG treated was significantly higher than that of non-treated control mice, during the phase of high parasitemia. Cumulative mortality of platelet-depleted mice was consistently but not significantly higher than that of control mice up to the 32nd day of infection; from the 33rd day on they were equivalent, no mortalities occurring from then on, until observations were discontinued on the 60th day. These results suggest that platelets participate of the mechanisms of parasites removal from the bloodstream, but do not have an effective role in the mechanisms of defence against T. cruzi, during the acute phase of infection.

Aggregatory behaviour of platelets incubated with subcellular fractions of normal and chagasic human syncytiotrophoblast

The surface of human syncytiotrophoblast does not induce maternal blood platelet aggregation even though it is not an endothelium. It can be surmised that as occurs in endothelial injury the subcellular components of the syncytiotrophoblast may have pro-or antiaggregatory activity. During congenital Chagas' disease which is associated to trophoblast lesions, platelets may play a role in the development of T. cruzi-induced placentitis. In the present work the aggregatory behaviour of normal human blood platelets was recorded after their challenging with subcellular fractions of syncytiotrophoblast isolated from normal and chagasic women. Nuclear, Mitochondrial, Microsomal and Supernatant fractions isolated from normal and chagasic syncytiotrophoblast failed to induce per se any aggregatory reaction on platelets. When samples of platelet-rich plasma (PRP) were preincubated with normal and chagasic nuclear fractions and then stimulated with collagen at threshold level (CT-PRP) an inhibition of the aggregatory response was observed. Treatment of CT-PRP with normal and chagasic mitochondrial fractions induced inhibition of platelet aggregation whereas only chagasic fraction reduced latency time. Microsornal fraction from normal placentas showed no significant effects on platelet aggregation. It is concluded that subcellular fractions of normal human syncytiotrophoblast do not exhibit any effect on platelet aggregation, whereas those subcellular fractions enriched in intracellular membrane components isolated from chagasic placentas inhibit platelet aggregation.

Evaluation of penicillin therapy in patients with leptospirosis and acute renal failure

The effectiveness of specific antibiotic treatment in severe leptospirosis is still under debate. As part of a prospective study designed to evaluate renal function recovery after leptospirosis acute renal failure (ARF) (ARF was defined as Pcr > or = 1.5 mg/dL), the clinical evolutions of 16 treated patients (T) were compared to those of 18 untreated patients (nT). Treatment or non-treatment was the option of each patient's attending infectologist. The penicillin treatment was always with 6 million IU/day for 8 days. No difference was found between the two groups in terms of age, gender, number of days from onset of symptoms to hospital admission, or results of laboratory tests performed upon admission and during hospitalization, but proteinuria was higher in the treated group. There were no significant difference in the other parameters employed to evaluate patients' clinical evolution as: length of hospital stay, days of fever, days to normalization of renal function, days to total bilirubins normalized or reached 1/3 of maximum value and days to normalization of platelet counts. Dialytic treatment indication and mortality were similar between group T and nT. In conclusion, penicillin therapy did not provide better clinical outcome in patients with leptospirosis and ARF.

Evaluation of hemostasis disorders and anticardiolipin antibody in patients with severe leptospirosis

A prospective study was designed to evaluate disorders of hemostasis and levels of anticardiolipin antibodies (ACL) in 30 patients with severe leptospirosis and acute renal failure (ARF) (ARF was defined as serum creatinine > or = 1.5 mg/dL). The patients had been admitted to the Walter Cantídio University Hospital, São José Infectious Diseases Hospital and General Hospital of Fortaleza, Ceará, from August 1999 to July 2001. They all were male, with a mean age of 32 ± 14 years and with clinical and laboratory diagnoses of ARF leptospirosis. The time elapsed between onset of symptoms and the first hemorrhagic manifestation was 9 ± 4 days. Bleeding was observed in 86% of the patients. Laboratory tests showed significantly high levels of urea (181 ±95 mg/dl), fibrinogen, (515 ± 220 mg/dl), prothrombin time (13.3 ± 0.9 seconds) and low platelet counts (69 ± 65x10³/mm³) on admission. There was no elevation in activated partial thromboplastin time or thrombin time. Levels of IgM and IgG ACL concentrations were significantly increased (p < 0.05) in leptospirosis patients when compared to control patients (28.5 ± 32.4 vs. 11.5 ± 7.9MPL U/ml and 36.7 ± 36.1 vs. 6.5 ± 2.5 GPL U/ml), respectively. Vasculitis, thrombocytopenia and uremia should be considered important factors for the pathogenesis of hemorrhagic disturbances and the main cause of death in severe leptospirosis.

Sanitary conditions of a colony of urban feral cats (Felis catus Linnaeus, 1758) in a zoological garden of Rio de Janeiro, Brazil

The colony of urban stray cats living in the Rio de Janeiro zoological garden was studied in order to develop a population and health control program. As many cats as possible were captured during two months (47 animals) and were classified according to gender, age, weight and coat markings. They were submitted to a general health evaluation, examined for the presence of ectoparasites and sent to a surgical neutering program. All animals had a blood sample drawn for CBC, platelet count, heartworm and retroviruses detection. Capillary blood smears were made for hemoparasites detection. Coat marking and colors were tabby (59.7%), followed by solid black (17%); torbie (10.6%); bicolor (10.6%) and harlequin (2.1%). The only ectoparasites found were fleas, which infested 28% of the animals. The hemoparasites found were Haemobartonella felis (38%) and piroplasmas that could not be differentiated between Cytauxzoon spp. and Babesia spp. (47%). No cat was found infected by Dirofilaria immitis or FeLV (Feline Leukemia Virus), although FIV (Feline Immunodeficiency Virus) antibodies could be detected (21%). There was no correlation between hemoparasites and FIV infections. The estimated total cat population (mark-recapture method) was 59; 68% female and 32% male, suggesting that a neutering program is in fact needed.