CONGENITAL MACROTHROMBOCYTOPENIA, LEUCOCYTE INCLUSIONS, DEAFNESS AND PROTEINURIA: FUNCTIONAL AND ELECTR0NMICROSCOPIC OBSERVATIONS ON PLATELETS AND MEGAKARYOCYTES

1987 ◽  
Author(s):  
D Blockmans ◽  
M J Heynen ◽  
J Vermylen ◽  
R Verwilghen
Keyword(s):  
Whole Blood ◽  
Plasma Membranes ◽  
Platelet Rich Plasma ◽  
Shape Change ◽  
Central Zone ◽  
Peripheral Zone ◽  
Impedance Method ◽  
Functional Studies ◽  
Ultrastructural Studies ◽  
Granulocyte Function

We report here a female patient of 33 years with a variant of Alport's syndrome (macrothrombocytopenia, leucocyte inclusions, deafness and proteinuria). The bleeding problems consisted of ecchymoses and menorrhagia, the deafness was of the ^ensorineural type. The platelet count in whole blood was 14.109/I, the mean platelet volume 22.8 μm3 . The template bleeding time exceeded 30 minutes. Ultrastructural studies of the peripheral blood revealed giant spheroid platelets with a high density of organelles, an abundance of vacuoles and an apparently disorganized microtubular system. In addition, unusual granule free areas were observed in the neutrophils of the patient and her mother. Granulocyte function was normal, except for a low myeloperoxidase content.Functional studies of the platelets in platelet rich plasma showed normal aggregation curves related to the low platelet number, although no shape change could be elicited. Platelet aggregation studies in whole blood (impedance method) gave supernormal aggregation curves; this suggests the limited usefulness of this technique in patients with such large platelets.The bone marrow contained numerous dysplastic megakaryocytes. In the mature granular megakaryocytes vacuoles and cysternae were organized in a radiating pattern demarkating elongated platelet territories. The platelet producing megakaryocytes showed fragmentation of the central zone and discharge of platelets through openings of the peripheral zone. These megakaryocytes had an immunological phenotype resembling that of very young cells (TR 14%, GP IIa 17% and GP IIIa 7%). The conversion of the elongated platelet territories into giant spheroid platelets probably results from remodelling within the circulation. The internalisation of plasma membranes would give rise to the extended invaginated canalicular system. Further studies are needed to explain the exact pathogenesis of this syndrome.

scholarly journals Ultrastructural Studies of Platelet Aggregates From Human Subjects Receiving Clopidogrel and From a Patient With an Inherited Defect of an ADP-Dependent Pathway of Platelet Activation

1996 ◽  
Vol 16 (12) ◽  
pp. 1532-1543 ◽  
Author(s):  
Michel Humbert ◽  
Paquita Nurden ◽  
Claude Bihour ◽  
Jean-Max Pasquet ◽  
Joëlle Winckler ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Platelet Activation ◽  
Platelet Rich Plasma ◽  
Shape Change ◽  
High Dose ◽  
Platelet Response ◽  
Platelet Surface ◽  
Ultrastructural Studies ◽  
Contact Points ◽  
Platelet Aggregates

Our study investigated the effect of the antithrombotic drug clopidogrel (75 mg/d for 7 days) on the ultrastructure of platelet aggregates induced by ADP or 2-methylthio-ADP (2-MeS-ADP) in citrated platelet-rich plasma and examined the activation state of the GP IIb/IIIa complexes. Results were compared with those obtained for patient M.L., who has a congenital disorder characterized by a reduced and reversible platelet response to ADP. When untreated normal platelets were stimulated with high-dose ADP, electron microscopy revealed large and stable aggregates often surrounded by a layer of what appeared to be degranulated platelets. The reversible aggregates of platelets from subjects receiving clopidogrel or from patient M.L. did not show this layer. Electron microscopy showed that in both situations, the aggregates were composed of loosely bound platelets with few contact points. Immunogold labeling of ultrathin sections of Lowicryl-embedded aggregates formed by ADP or 2-MeS-ADP showed a much decreased platelet surface staining by (1) a polyclonal anti-fibrinogen antibody and (2) AP-6, a murine anti–ligand-induced binding site monoclonal antibody specific for GP IIb/IIIa complexes occupied with fibrinogen. Similar findings were seen after disaggregation, when many single platelets were present that showed no signs of secretion. Flow cytometry confirmed that the number of ligand-occupied GP IIb/IIIa complexes was much lower on platelets stimulated with ADP or 2-MeS-ADP after clopidogrel treatment. As expected from previous studies, ADP-induced platelet shape change and Ca 2+ influx were unaffected by clopidogrel. These results agree with the hypothesis that platelet activation by ADP is biphasic and highlight a receptor-induced activation pathway affected by clopidogrel (or congenitally impaired in patient M.L.) that is necessary for the full activation of GP IIb/IIIa and the formation of stable macroaggregates.

The active metabolite of prasugrel inhibits ADP-stimulated thrombo-inflammatory markers of platelet activation: Influence of other blood cells, calcium, and aspirin

2007 ◽  
Vol 98 (07) ◽  
pp. 192-200 ◽  
Author(s):  
Joseph Jakubowski ◽  
You FuLi ◽  
Marc Barnard ◽  
Marsha Fox ◽  
Matthew Linden ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Whole Blood ◽  
Blood Cells ◽  
Inflammatory Markers ◽  
Active Metabolite ◽  
Platelet Rich Plasma ◽  
Shape Change ◽  
Flow Cytometric Analysis ◽  
Platelet Surface

SummaryThe novel thienopyridine prodrug prasugrel, a platelet P2Y12 ADP receptor antagonist, requires in vivo metabolism for activity. Although pharmacological data have been collected on the effects of prasugrel on platelet aggregation,there are few data on the direct effects of the prasugrel’s active metabolite, R-138727, on other aspects of platelet function. Here we examined the effects of R-138727 on thrombo-inflammatory markers of platelet activation, and the possible modulatory effects of other blood cells, calcium, and aspirin. Blood (PPACK or citrate anticoagulated) from healthy donors pre- and post-aspirin was incubated with R-138727 and the response to ADP assessed in whole blood or platelet-rich plasma (PRP) by aggregometry and flow cytometric analysis of leukocyte-platelet aggregates,platelet surface P-selectin, and GPIIb-IIIa activation. Low-micromolar concentrations of R-138727 resulted in a rapid and consistent in-hibition of these ADP-stimulated thrombo-inflammatory markers.These rapid kinetics required physiological calcium levels, but were largely unaffected by aspirin. Lower IC50 values in whole blood relative to PRP suggested that other blood cells affect ADP-induced platelet activation and hence the net inhibition by R-138727. R-138727 did not inhibit P2Y12-mediated ADP-induced shape change, even at concentrations that completely inhibited platelet aggregation, confirming the specificity of R-138727 for P2Y12. In conclusion, R-138727, the active metabolite of prasugrel, results in rapid, potent, consistent, and selective inhibition of P2Y12-mediated up-regulation of thromboinflammatory markers of platelet activation.This inhibition is enhanced in the presence other blood cells and calcium,but not aspirin.

GUARANA (Paullinia cupana) INHIBITS AGGREGATION IN WHOLE BLOOD

1987 ◽  
Author(s):  
D A F Chamone ◽  
M Ivany-Silva ◽  
C Cassaro ◽  
G Bellotti ◽  
C Massumoto ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Whole Blood ◽  
Ex Vivo ◽  
Platelet Rich Plasma ◽  
Oral Intake ◽  
Inhibitory Effect ◽  
Impedance Method ◽  
Paullinia Cupana ◽  
Induced Aggregation

Guarana, a methylxanthine obtained from the seeds of Paullinia cupana has been largely used in the Amazon region by native indians during centuries as stimulant. We evaluated the effect of guarana on ex-vivo and in vitro platelet aggregation induced by adenosine-5-diphosphate (ADP) in human and rat whole blood with an impedance (Chrono-Log, model 500) and in their platelet rich plasma (PRP) with an optical aggregometer (Chrono-Log, model 440). Ex-vivo studies were carried out after single oral intake of guarana. Seven healthy volunteers (5 male and 2 female) aged 19-26 years who had taken no drugs for 10 days before, ingested 8gm of crude powder of guarana. Blood samples were drawn before and 1 hour after guarana intake. We observed a significative inhibition of platelet aggregation in whole blood meanwhile PRP was un changed as compared to basal values. In vitro studies were performed in whole blood and PRP from human volunteers and male Wis-tar rats. The combined effect of guarana and adenosine was also studied. A control aggregation was always run with saline. The results demonstrated an inhibition statistically significative (p < 0.001) of platelet aggregation in whole blood. Differently from whole blood the PRP with the same concentration of guarana did not result in inhibition of ADP induced aggregation when eva luated with the impedance method. The blood incubation with adenosine and guarana resulted in synergistic inhibitory effect that was much more strinking in whole blood than in PRP. Guarana fails to inhibit aggregation of rat platelets.Our results demonstrate that guarana prevents platelet aggregation in whole blood which depends on red blood cells, probably involving adenosine.

scholarly journals The Effect of Tirofiban on Fibrinogen/Agonist-Induced Platelet Shape Change and Aggregation

2008 ◽  
Vol 14 (3) ◽  
pp. 295-302 ◽  
Author(s):  
I. Anita Jagroop ◽  
Dimitri P. Mikhailidis
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Whole Blood ◽  
Platelet Rich Plasma ◽  
Shape Change ◽  
Adenosine Diphosphate ◽  
Vascular Events ◽  
Spontaneous Platelet Aggregation ◽  
Induced Aggregation ◽  
Platelet Shape

There is evidence linking raised plasma fibrinogen (fib) and platelet hyperactivity with vascular events. One way to inhibit platelets is to block the platelet membrane glycoprotein (GP) IIb/IIIa receptor, which binds circulating fib or von Willebrand factor and cross-links platelets at the final common pathway to platelet aggregation. Tirofiban is a potent and specific fib receptor antagonist, used in the treatment of unstable angina. The authors assessed the effect of tirofiban on spontaneous platelet aggregation (SPA), fib-induced, serotonin (5HT)-induced, and adenosine diphosphate (ADP)-induced aggregation in whole blood by calculating the percentage free platelet count. These various agonists were used alone and in combination. The authors also measured the effect of tirofiban on agonists-induced (ADP, 5HT) platelet shape change (PSC). The effect of fib on PSC was also evaluated in platelet-rich plasma using a high-resolution (0.07 fL) channelyzer. Tirofiban significantly inhibited SPA, fib (2, 4, 8 g/L), ADP, ADP + fib combination, and 5HT-induced aggregation. Tirofiban had no effect on agonist-induced PSC. There was no apparent change in platelet volume with fib. In conclusion, tirofiban does not appear to have an effect on PSC, an early phase of platelet activation. Tirofiban seems to be a nonspecific and an effective inhibitor of platelet aggregation (a later phase of platelet activation) in whole blood. The clinical significance of these findings remains to be established.

FACTORS AFFECTING PLATELET VOLUME ANALYSIS

1987 ◽  
Author(s):  
A Wehmeier ◽  
W Schneider
Keyword(s):  
Incubation Time ◽  
Whole Blood ◽  
Storage Temperature ◽  
Platelet Rich Plasma ◽  
Impedance Method ◽  
Blood Collection ◽  
Volume Distribution ◽  
Dependent Manner ◽  
Platelet Volume ◽  
Volume Analysis

Parameters of platelet volume have become widely available with the introduction of automated platelet counters. However, variant sample processing and in vitro platelet activation have prevented standardization of platelet volume analysis. We investigated the influence of anticoagulation, storage, temperature, and the presence or absence of RBC on platelet volume. Mean platelet volume (MPV) and the mode of the distribution were calculated from the platelet volume distribution curve recorded with the impedance method and plotted in 27 classes between 1.2 and 22 fl. The effects of EDTA (.335%), citrate (.38%), citrate (.38%)/glutaraldehyde (.125%) and a cocktail containing citrate, forskolin, indomethacin and theophyllin were determined 10, 30, 60, 120 and 240 min after blood collection. Tests were made at 4, 20° and 37°C in whole blood and platelet-rich plasma (PRP)from 6 healthy subjects. Platelet volume was strongly dependent on the anticoagulant in a time-dependent manner. MPV was lowest with citrate/glutaraldehyde and highest with EDTA. The maximum difference was 30% at 60 min both in whole blood and PRP. However, this was true only at 4 and 20°C. At 37°C, there was a constant rise in MPV using citrate/glutaraldehyde exceeding volume changes seen with the other anticoagulants. Platelet volume was higher in whole blood as compared to PRP. The difference was dependent on the anticoagulant used and the incubation time (1.8 fl for EDTA and 1.35 fl for citrate/glutaraldehyde, at 60 min). To determine the influence of platelet loss due to PRP preparation on this effect, we determined platelet volume in parallel from whole blood, PRP and the platelet population separated from whole blood by a linear Percoll gradient (n=5, recovery 94%). MPV was 7.6 fl in whole blood, 5.4 fl in PRP and 6.8 fl from the gradient (anticoagulant: citrate/glutaraldehyde). Platelet volume parameters highly depend on anticoagulation, incubation time and temperature. For clinical studies we recommend anticoagulation with citrate/glutaraldehyde and measurement within 2 hr at room temperature in whole blood.

Platelet Volume Measurements: The Importance Of Anticoagulation

1981 ◽  
Author(s):  
C B Thompson ◽  
D D Diaz ◽  
P G Quinn ◽  
M Lapins ◽  
S B Kurtz ◽  
...  
Keyword(s):  
Platelet Count ◽  
Whole Blood ◽  
Mean Platelet Volume ◽  
Room Temperature ◽  
Platelet Rich Plasma ◽  
Shape Change ◽  
Inverse Correlation ◽  
Platelet Volume ◽  
Important Indicator ◽  
Platelet Counts

Recent advances in electrical cell sizing have made mean platelet volume (MPV) routinely available in most clinical laboratories. To study the importance of anticoagulation on platelet size stability, blood was collected in 7 different anticoagulants and stored at room temperature for up to 8 hours. Platelet counts and platelet sizing were performed using whole blood on a Coulter S+ and using platelet-rich plasma on a Coulter H4 Channelyzer. The results suggest that both calcium chelation and acidification were required to inhibit platelet shape change and aggregation. Electrolyte composition, pH, and tonicity of the anticoagulant all influenced the stability of the MPV. As a result of these studies, an anticoagulant combining ACD and Na2EDTA at a pH of 5.0 and an osmotic strength of 308 m0sm/1 was used to study platelet volume and counts in whole blood on a Coulter S+ used in the hematology laboratory of our hospital. Platelet counts with ACD-Na2EDTA anticoagulant were no different from routine Na2EDTA platelet counts and exhibited 2.9% error in reproducibility and a 4.4% variability over the 8 hours of storage. Mean platelet volumes were reproducible to within 3% and had less than 1% variability over the 8 hours of storage. Platelets anticoagulated with ACD-Na2EDTA remained discoid in shape and could be shown to undergo a shape change on stimulation with ADP up to at least 8 hours after collection, when the pH was adjusted to 7.4 and the Ca++ concentration restored.These data demonstrate that platelet count and platelet volumes remained stable in blood collected in ACD-Na2EDTA anticoagulant for up to 8 hours at room temperature. In 52 volunteers studied, an inverse correlation (r=.72, p<0.001) was observed between platelet count and MPV, suggesting that the circulating platelet mass may be a more important indicator of platelet homeostasis than either the platelet count or the mean platelet volume alone.

The Mechanism of Cold-Induced Platelet Hyperfunction.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1651-1651
Author(s):  
Qiang Han ◽  
Junmei Chen ◽  
Yuandong Peng ◽  
Angela L. Bergeron ◽  
Li Liu ◽  
...  
Keyword(s):  
Whole Blood ◽  
Platelet Rich Plasma ◽  
Thrombus Formation ◽  
Cold Treatment ◽  
Flow Chamber ◽  
Type Iii Collagen ◽  
Clotting Time ◽  
Functional Studies ◽  
Lower Shear ◽  
Induced Aggregation

Abstract Refrigerated platelets function better when transfused than do platelets stored at room temperature, although chilled and rewarmed platelets do not survive long in the circulation. Here, we describe the one possible mechanism of this increase in the hemostatic function of platelets upon cold treatment. Cold exposure has been demonstrated to cluster GP Ib-IX complexes on platelets, enabling platelet recognition and phagocytosis by macrophages. We find that, in addition to increasing the binding to the leukocyte integrin Mac-1 (aMb2), clustering of GP Ib-IX complexes also augments von Willebrand factor (VWF)-related functions. Before the functional studies, platelets were chilled to 4°C for 2 hr and rewarmed to 37°C for 30 min. Chilled, rewarmed platelets, whether in platelet-rich plasma (PRP), whole blood, or reconstituted whole blood (all anticoagulated with Cirtate Sodium) perfused through a parallel-plate flow chamber (36 dynes/cm2) attached in greater numbers to VWF-coated surfaces than did platelets from the same donor kept at 37°C or 22°C. The increased function was also reflected in increased thrombus number and size on a surface of type III collagen. The surface coverage, an indicator of thrombus formation, increased from 10% to 25% upon cold. Chilled, rewarmed platelets also responded to lower doses of a VWF-specific agonist, ristocetin Chilled, rewarmed platelets showed 80% aggregation while control platelet only had 10% aggregation at low concentration of ristocetin (0.5mg/ml) treatment, and displayed a decreased threshold for shear-induced aggregation, aggregating at lower shear rates than control platelets at both 22°C and 37°C. Finally, when tested in a platelet function analyzer (PFA-100), chilled, rewarmed platelets demonstrated significantly shorter closure times than control platelets using both epinephrine/collagen and ADP/collagen cartridges. The epinephrine/collagen cartridges clotting time of chilled platelets decreased from normal 180 s to 162 s and ADP/collagen cartridges clotting time decreased from 118s to 99s. To exclude that the observed functional increases were not due to changes in GP Iba expression or prior platelet activation, we evaluated by flow cytometry the surface levels of GP Iba, P-selectin, and activated αIIbβ3.(antibody PAC-1 binding). We found no differences in any of these parameters between chilled, rewarmed platelets and control platelets. These results suggest that, if their rapid clearance can be prevented, cold-stored platelets may have enhanced hemostatic function of potential benefit in the bleeding patient. The results also caution, however, that trials will be necessary to exclude potential thrombotic consequences of infusing hyperfunctional, refrigerated platelets.

Ultrastructural Studies on the Surface Coat of Human Platelet Aggregated by Polylysine and Dextran

1978 ◽  
Vol 40 (01) ◽  
pp. 011-023 ◽  
Author(s):  
Y Taketomi ◽  
A Kuramoto
Keyword(s):  
Platelet Aggregation ◽  
Cell Surface ◽  
Plasma Membranes ◽  
Platelet Rich Plasma ◽  
Ion Concentration ◽  
Surface Coat ◽  
Release Reaction ◽  
Ultrastructural Studies ◽  
Induced Aggregation ◽  
Positively Charged

SummaryPositively charged macromolecule, polylysine (mol. wt. 15,000; 23,000; 180,000) could induce the platelet aggregation in low concentration but high concentration was required in the case of neutral macromolecule, dextran (mol. wt. 40,000; 250,000; 2,000,000). The larger molecules of polylysine and dextran were more effective in inducing platelet aggregation. In the dextran-induced aggregation, positively charged Thorotrast particles on the cell surface did not decrease significantly. On the other hand, the surface membranes of platelets aggregated by polylysine were essentially devoid of bound particles. Heparin in hibited the poly lysine-induced platelet aggregation but not the dextran-induced aggregation. These findings suggested that polylysine induced aggregation more effectively than dextran by reducing the negative surface charge and giving stronger adsorption force on cell surface.In platelet-rich plasma, polylysine elicited the release reaction of 14C-serotonin but dextran did not. Possible mechanism by which polylysine could elicit the release reaction is the formation of more tightly packed platelet aggregate than that by dextran in the presence of the low calcium ion concentration in citrated platelet-rich plasma. Average distance between plasma membranes of aggregated platelets, however, did not vary with the degrees of polymerization of these macromolecules.

Platelet Shape Change in Whole Blood: Differential Effects of Endotoxin

1994 ◽  
Vol 71 (05) ◽  
pp. 646-650 ◽  
Author(s):  
M L Nystrom ◽  
M A Barradas ◽  
J Y Jeremy ◽  
D P Mikhailidis
Keyword(s):  
Whole Blood ◽  
Platelet Rich Plasma ◽  
Shape Change ◽  
Platelet Activating Factor ◽  
Final Concentration ◽  
Platelet Volume ◽  
Differential Effects ◽  
Platelet Shape

SummaryThe effect of endotoxic lipopolysaccharide (LPS) on platelet shape change (PSC; a preaggregation event) was investigated. PSC is accompanied by an increase in median platelet volume (MPV), which was measured using a channelyzer. In whole blood, but not in platelet rich plasma (PRP), LPS (final concentration 80 mg/1) caused an increase in MPV that could be detected for 2 h. When PRP (prepared from LPS- and saline-pretreated whole blood) was incubated for 40 min, the LPS-mediated increase in MPV could no longer be detected. Taken together, these data imply that PSC is both initiated and maintained by a labile factor(s) present in whole blood, but not in PRP.PRP was prepared from LPS-pretreated whole blood and incubated for 40 min to allow reversal of the LPS-induced PSC; further stimulation with LPS caused PSC. Platelets from LPS-pretreated whole blood also showed enhanced PSC with seioloum (J-HT), diminished PSC with platelet activating factor (PAF), and no change of response to ADP and collagen. Hence, LPS pretreatment of whole blood differentially alters responses of platelets to further stimulation with LPS and other agonists. A specific PAF antagonist completely abolished the effect of LPS on MPV. These data may contribute to an understanding of the cascading thrombotic events and thrombocylopeuia assoeialed wiib septicaemia.

The plaque lipid lysophosphatidic acid stimulates platelet activation and platelet-monocyte aggregate formation in whole blood: involvement of P2Y1 and P2Y12 receptors

Blood ◽  
2004 ◽  
Vol 103 (7) ◽  
pp. 2585-2592 ◽  
Author(s):  
Nadine Haserück ◽  
Wolfgang Erl ◽  
Dharmendra Pandey ◽  
Gabor Tigyi ◽  
Philippe Ohlmann ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Lysophosphatidic Acid ◽  
Whole Blood ◽  
Platelet Rich Plasma ◽  
Shape Change ◽  
Receptor Activation ◽  
Aggregate Formation ◽  
Serotonin Secretion ◽  
Induced Aggregation ◽  
Platelet Shape

Abstract Despite the fact that lysophosphatidic acid (LPA) has been identified as a main platelet-activating lipid of mildly oxidized low-density lipoprotein (LDL) and human atherosclerotic lesions, it remains unknown whether it is capable of activating platelets in blood. We found that LPA at concentrations slightly above plasma levels induces platelet shape change, aggregation, and platelet-monocyte aggregate formation in blood. 1-alkyl-LPA (16:0 fatty acid) was almost 20-fold more potent than 1-acyl-LPA (16:0). LPA directly induced platelet shape change in blood and platelet-rich plasma obtained from all blood donors. However, LPA-stimulated platelet aggregation in blood was donor dependent. It could be completely blocked by apyrase and antagonists of the platelet adenosine diphosphate (ADP) receptors P2Y1 and P2Y12. These substances also inhibited LPA-induced aggregation of platelet-rich plasma and aggregation and serotonin secretion of washed platelets. These results indicate a central role for ADP-mediated P2Y1 and P2Y12 receptor activation in supporting LPA-induced platelet aggregation. Platelet aggregation and platelet-monocyte aggregate formation stimulated by LPA was insensitive to inhibition by aspirin. We conclude that LPA at concentrations approaching those found in vivo can induce platelet shape change, aggregation, and platelet-monocyte aggregate formation in whole blood and suggest that antagonists of platelet P2Y1 and P2Y12 receptors might be useful preventing LPA-elicited thrombus formation in patients with cardiovascular diseases.

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