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Ultrastructural morphometric analysis of human blood platelets exposed to minimal handling procedures.

A detailed morphometric study of normal human blood platelets is described. The purpose has been to evaluate the morphological characteristics of platelets exposed to minimal handling procedures in order to obtain an optimal basis for the appraisal of platelets in disease. Blood from 10 healthy volunteers was collected directly into buffered glutaraldehyde and processed for electron microscopy. This platelet fixation procedure resulted in excellent preservation of resting platelet ultrastructure with one exception: the dense bodies. Compared to platelets fixed following washing procedures, our directly fixed platelets comprised fewer pseudopods and contained more glycogen. An unexpected feature of the open canalicular system was the apparent release of blisters interpreted as microvesicles. Employing a computerized image analyzer, 300 of the platelets were examined morphologically. The morphometric data thus obtained were analyzed statistically, resulting in a set of standard values for morphological characteristics of human platelets which we have found useful in subsequent evaluations of platelet morphology in disease. Significant inter-individual variance was, however, detected in two instances, in the section area of the alpha granules, as well as the area fraction of platelet sections occupied by channels of the open canalicular system (OCS). This should be taken into consideration when appraising platelet ultrastructure in health and disease.

Adult↗

Blood platelets and angiotensin II.

To investigate whether human blood platelets contain angiotensin II, platelets were isolated from platelet-rich plasma and resuspended in buffer solution. No angiotensin was detectable by radio-immunoassay in this suspension. However, after sonification all samples appeared to contain significant amounts of angiotensin II. This amount was not reduced by the addition of angiotensinase inhibitors, thus ruling out an assay artefact due to breakdown of tracer. Subsequent experiments showed that the immunoreactive material behaved similarly to standard angiotensin II in several respects.

Angiotensin II↗

[Fatty acid and lipid levels in the blood platelets of patients with alcoholism].

The fatty acid content of thrombocytic phospholipids was assayed in 10 patients suffering from alcoholism in a state of the alcoholic abstinence syndrome, after egress from that state and following detoxication and tonic therapy. Ten donors served as control. The patients were also examined for lipid content of blood platelets before and after the treatment. It is established that in patients being in a state of the alcoholic abstinence syndrome, the fatty acid content of thrombocytic phospholipids markedly differed from control. 100% of the patients manifested a material decrease in the content of linoleic acid, dihomo- gamma-linolenic and arachidonic acids. The content of some acids belonging to the omega-3 group was also reduced. During that period, the lipid content also considerably differed from the control: the content of phospholipids noticeably decreased whereas that of free cholesterol rose. The improvement of the patients' clinical status after egress from the alcoholic abstinence syndrome was coupled with a noticeable normalization of the fatty acid content of thrombocytic phospholipids. A clear-cut tendency towards normalization of the fatty acid content was recorded only after desintoxication and tonicizing therapy. Meanwhile the lipid content considerably differed from normal even after the treatment. The changes in the lipid and fatty acid content of blood platelets in patients with alcoholism may produce disorders in prostanoid synthesis and functional properties of blood platelets, leading to the impairment of the coagulation and anticoagulation blood systems.

Alcoholism↗

High voltage cryomicroscopy of human blood platelets.

We have analyzed the ultrastructure of human blood platelets using ultrarapid freezing techniques followed by either freeze-substitution fixation or direct imaging of frozen hydrated specimens in a high voltage electron microscope. Freeze substitution preserved a regularity in the arrangement of microtubules when compared with chemically fixed specimens, as identified by a preferred center-to-center distance of 30 nm. Platelets imaged in the frozen hydrated state during different stages of adhesion and spreading were rich in cytoplasmic detail and revealed novel features not previously reported. Different granule populations were distinguished in the unstained samples, and the morphology of the cells was dramatically different from that of dried cells. Membrane channels formed as the cells spread, increasing in size and number as the number of granules in the cell decreased, suggesting a fusion of granule membranes with one another or an additional membrane system to release their contents. The data support the idea that direct cryoimaging is an optimum method for characterizing rapid structural changes of labile organelles in whole cells.

Blood Platelets↗

Human plasma epidermal growth factor/beta-urogastrone is associated with blood platelets.

Human epidermal growth factor (hEGF) has previously been isolated from urine and probably is identical to human beta-urogastrone (hUG). Immunoreactive hEGF/UG has been found in the plasma of normal subjects. In this study, using immunoaffinity chromatography to extract hEGF/UG from plasma, we found that immunoreactive hEGF/UG in blood was associated with blood platelets. It was present in platelet-rich, but not platelet-poor plasma and serum, and was found predominantly in the platelet fraction of whole blood. Sephadex G-50 Fine gel-exclusion chromatography of an extract of outdated blood bank platelets revealed two hEGF/UG components, one of which eluted in the void volume, and the other of which coeluted with purified standard hEGF/UG. The former hEGF/UG component was a high-molecular weight form that was cleaved into hEGF/UG by incubation with either mouse EGF/UG-associated arginine esterase or trypsin. It appeared to be identical to the high-molecular weight hEGF/UG previously reported in human urine, except for its apparently equal activities in radioimmunoassay and radioreceptor assay. The latter hEGF/UG component was immunologically, biologically, and physiochemically indistinguishable from highly purified hEGF/UG from human urine and was immunologically different from purified human platelet-derived growth factor. Platelet-associated hEGF/UG may account for the mitogenic activity of serum in cell lines in which platelet-derived growth factor is not active. Since hEGF/UG appears to be liberated from platelets during coagulation, platelet-associated EGF/UG may be involved in normal vascular and tissue repair and in the pathogenesis of atherosclerotic lesions. The discovery that the EGF/UG in plasma is associated with blood platelets raises important new possibilities for its role in human health and disease.

Blood Platelets↗

Uptake of 5-hydroxytryptamine by blood platelets in Huntington's chorea and Alzheimer type of presenile dementia.

Kinetics of 5-HT uptake by blood platelets was studied om eleven patients with Huntington's chorea and in ten patients with presenile dementia of Alzheimer type. In both groups of patients 5-HT uptake was unchanged in comparison to that of respective controls of the same age. The results do not confirm earlier reports of an increased 5-HT uptake by blood platelets in Huntington's chorea. Platelet 5-HT uptake does not seem to serve as biological test in either disease.

Adult↗

[Changes in platelet aggregation and cAMP levels in isolated blood platelets in rats after 5.0 Gy and 10.0 Gy whole-body irradiation doses].

Rats were subjected to whole-body irradiation with doses of 5.0 Gy and 10.0 Gy. After intervals of 1.5 hours, 3, 6, 12 and 24 hours following irradiation the authors tested the aggregation capacity of thrombocytes after induction with adenosine--5--diphosphate and the cAMP level in isolated thrombocytes. Statistically significant differences between experimental and control groups were found particularly at some time intervals in rats irradiated with 10.0 Gy. The authors found a significant acceleration of the time of maximal aggregation at time intervals of 3.6 and 24 hours following irradiation with 10.0 Gy. Only at the time interval of 3 hours after irradiation with 10 Gy a statistically significant reduction of the maximum aggregation, as compared with the control group, occurred, and similarly the tang alpha value indicates that also the initial rate of aggregation was lower. The cAMP level in isolated thrombocytes was markedly reduced in particular after 5.0 Gy already during the early intervals after irradiation. A temporary increase of the cAMP levels was recorded 3 hours after irradiation with 10.0 Gy which may explain the reduced aggregation capacity of thrombocytes found during the same time interval. The results of both tests are not sufficient evidence of a hypercoagulation state after irradiation with the mentioned doses within 24 hours after irradiation.

Animals↗

The distribution of peroxidase in phagocytizing human blood platelets, electron microscopy findings.

On the basis of a previously elaborated method for testing the phagocytic ability of human blood platelets we have undertaken studies for testing the distribution of peroxidase in phagocytizing platelets. Our findings confirmed the distribution of peroxidase in the dense tubular system and in granules in regular blood platelets. In the course of phagocytosis we noted the presence of peroxidase less evidently in both subcellular structures during the first of 5 minutes. In the next steps we observed the localization of this enzyme mainly near the phagolysosome. The change of this localization and the role of this enzyme in unspecific immunity against bacteria and neoplasia is briefly discussed.

Blood Platelets↗

Glucose-6-phosphatase activity in liver and blood platelets of two patients with glycogen storage disease type I.

Glucose-6-phosphatase (G-6-Pase) activity in liver and blood platelets of two patients with glycogen storage disease (GSD) type I is described. Both patients had a reduced activity of G-6-Pase in liver. The km value for glucose 6-phosphate (G-6-P) of residual activity in liver of both patients was similar to that of control liver. We could not demonstrate any reduced activity of platelet G-6-Pase in the patients. Platelet G-6-Pase with our assay method seems to represent a nonspecific phosphatase activity. Our observation suggests that it is necessary to examine platelet G-6-Pase of many other patients with GSD type I to confirm that G-6-Pase deficiency can be diagnosed by enzyme assay performed on blood platelets.

Alanine↗

Stored blood platelets and microaggregate formation.

The structure and function of platelets in stored blood were tested for 21 days of storage with both light and electron microscopy as well as determination of platelet aggregation response to adenosine diphosphate. Observations were made for a period of 21 days of storage at 4 degrees C. These observations demonstrate the appearance of spontaneous platelet aggregates in stored blood which differ morphologically from aggregates induced by the addition of adenosine diphosphate during the first ten days of storage. The appearance of spontaneous platelet aggregates paralleled a decrease in platelet count and progressive decline in the number of morphologically normal appearing platelets. The platelets that were induced to aggregate with adenosine diphosphate formed more closely packed aggregates that those that formed spontaneously and that the adenosine diphosphate-induced aggregates could still be induced through the tenth day of storage, at which time few morphologically normal platelets could be clearly identified in the plasma. Spontaneous platelet aggregates showed no fibrin in the microaggregates but rather significant numbers of leukocytes contained in the aggregates which degenerated progressively beyond a storage period of five days.

Adenosine Diphosphate↗

A study of protein phosphorylation in shape change and Ca++-dependent serotonin release by blood platelets.

Upon treatment with agents such as thrombin, collagen or concanavalin A, blood platelets change shape, secrete serotonin and phosphorylate two proteins having molecular weights of approximately 20,000 and 40,000. We have analyzed the relationship of this protein phosphorylation to shape change and release aided by the fact that while shape change occurs independently of extracellular calcium, release of serotonin displays a rather strict calcium requirement. Under limited calcium conditions, where virtually no serotonin release occurs, (Con A)-stimulated phosphorylation is uninhibited. Divalent cations (Mg++, Co++ and Zn++) also inhibit release but not phosphorylation. The microtubule effectors colchicine and D2O show concomitant effects on release and phosphorylation, indicating a microtubule involvement prior to phosphorylation. Papaverine inhibits release and phosphorylation while not strongly influencing shape change, suggesting that shape change does not require phosphorylation. We therefore conclude that phosphorylation of these proteins takes place after shape change but prior to release, and although it may be required for secretion to occur, the two processes are easily separated. Thus phosphorylation of these proteins is not likely to be an integral component of the release mechanism.

Animals↗

Seasonal variability in blood platelet 3H-imipramine binding in healthy controls: age and gender effects.

Binding of 3H-imipramine to blood platelet membranes was determined four times (once each season) in 26 healthy volunteers (11 men and 15 women), over the course of 1 year to determine possible seasonal variations. Blood platelets were obtained in April-May, July-August, October-November, and January-February. Significant seasonal variations in the maximum number of binding sites were found in women but not in men, with circannual peak in summer and a nadir in spring. The pattern of seasonal variations was not the same in men and women. The present results highlight the importance of monitoring for gender and season in binding studies. We found no significant correlation between 3H-imipramine binding parameters and age.

Adult↗

Modulation of the low affinity Ca2+-binding sites of skeletal muscle and blood platelets Ca2+-ATPase by nordihydroguaiaretic acid.

The antioxidant nordihydroguaiaretic acid (NDGA) inhibited the different sarco/endoplasmic reticulum Ca2+-ATPase isoforms found in skeletal muscle and blood platelets. For the sarcoplasmic reticulum, but not for the blood platelets Ca2+-ATPase, the concentration of NDGA needed for half-maximal inhibition was found to vary depending on the substrate used and its concentration in the assay medium. The phosphorylation of the sarcoplasmic reticulum Ca2+-ATPase by ATP and by Pi were both inhibited by NDGA. In leaky vesicles, measurements of the ATP<-->Pi exchange showed that NDGA increases the affinity for Ca2+ of the E2 conformation of the enzyme, which has low affinity for Ca2+. The effects of NDGA on the Ca2+-ATPase were not reverted by the reducing agent dithiothreitol nor by the lipid-soluble antioxidant butylated hydroxytoluene.

ATP Synthetase Complexes↗