Discoid Platelets Research Articles

Three-month effects of MDL 19,660 on the canine platelet and erythrocyte

Nine male and nine female Beagle dogs were divided into three groups and administered orally 0, 15, or 30 mg/kg/day of the antidepressant compound MDL 19,660(5-(4-chlorophenyl)-2,4-dihydro-2,4-dimethyl-3H-1,2,4-triazole-3-thione) for 3 months to determine the long-term effects on hemopoietic cells. Compared to a control platelet range of 353,000–452,000/μl, a thrombocytopenia reached lowest mean levels of 135,000/μl in the 15 mg/kg/day dogs after 2 weeks and 81,000/μl in the 30 mg/kg/day dogs after 1 week. Subsequently, platelet numbers progressively increased and by the end of the study averaged 222,000/μl and 203,000/μl in dogs administered 15 and 30 mg/kg/day. Ultrastructural study of the platelet increase in the 30 mg/kg/day dogs revealed more smaller discoid platelets but no change in percentage platelets with vacuolar degeneration. Histologically, megakaryocyte hyperplasia was present in the sternal marrows and spleens of treated dogs. These observations suggest that increased thrombopoiesis rather than reduced destruction was involved in this partial recovery of platelet numbers during continuous treatment. Concurrently, cyclic formation of reticulocytes and Heinz bodies occurred in dogs given 30 mg/kg/day of MDL 19,660. These dogs had slightly lower erythrocyte counts, hemoglobin levels, and hematocrits in association with hemosiderosis (spleen, liver), extramedullary hematopoiesis (spleen), and bone marrow hypercellularity. These findings indicate that both destructive and regenerative processes followed MDL 19,660-induced Heinz body formation.

Quantitative optical determination of the viability of platelet concentrates

An optical method to assess platelet viability in platelet transfusion concentrates has been developed which is based on the observation that healthy, discoid platelets transmit more light in shear flow than aged, spherical cells. Using a specially designed glass channel, the rise in transmission of light through platelet samples with agitation is referenced against a measurement of transmission without flow. Referencing the measurements may minimize the effects of red blood cell contamination, as well as other variations among platelet packs such as plasma characteristics and plastic container variability. At an optimal frequency of agitation, the ratio of the transmission of light with agitation to that of the sample at rest is found to correlate highly with the concentration of discoid platelets, r = 0.92 to 0.95 ( P < 0.001), for light sources including a HeNe laser and two LEDs. Using this index, the discoid platelet counts of twelve platelet packs are estimated and compared with manual counts. The mean difference between the two methods was 0.009× 10 9 discoid platelets ml −1 with a standard deviation of 0.11× 10 9 cells ml −1. We conclude that the method may be applied to the development of a non-invasive device to monitor the concentration of viable, discoid platelets in storage.

Three-Month Effects of MDL 19,660 on the Canine Platelet and Erythrocyte

Nine male and nine female Beagle dogs were divided into three groups and administered orally 0, 15, or 30 mg/kg/day of the antidepressant compound MDL 19,660 (5-(4-chlorophenyl)-2,4-dihydro-2,4-dimethyl-3H-1,2,4-triazole-3-thione) for 3 months to determine the long-term effects on hemopoietic cells. Compared to a control platelet range of 353,000–452,000/μl, a thrombocytopenia reached lowest mean levels of 135,000/μl in the 15 mg/kg/day dogs after 2 weeks and 81,000/μl, in the 30 mg/kg/day dogs after 1 week. Subsequently, platelet numbers progressively increased and by the end of the study averaged 222,000/μl and 203,000/μl in dogs administered 15 and 30 mg/kg/day. Ultrastructural study of the platelet increase in the 30 mg/kg/day dogs revealed more smaller discoid platelets but no change in percentage platelets with vacuolar degeneration. His tologically, megakaryocyte hyperplasia was present in the sternal marrows and spleens of treated dogs. These observations suggest that increased thrombopoiesis rather than reduced destruction was involved in this partial recovery of platelet numbers during continuous treatment. Concurrently, cyclic formation of retic ulocytes and Heinz bodies occurred in dogs given 30 mg/kg/day of MDL 19,660. These dogs had slightly lower erythrocyte counts, hemoglobin levels, and hematocrits in association with hemosiderosis (spleen, liver), extramedullary hematopoiesis (spleen), and bone marrow hypercellularity. These findings indicate that both destructive and regenerative processes followed MDL 19,660-induced Heinz body formation.

Activation-independent exposure of the GPIIb-IIIa fibrinogen receptor

Following platelet activation, surface receptors for fibrinogen are exposed. On the activated platelet, glycoprotein IIb-IIIa (GPIIb-IIIa) serves as the receptor for fibrinogen. However, the molecular mechanisms which regulate GPIIb-IIIa fibrinogen receptor exposure are unknown. D3GP3 is an IgG 1, κ monoclonal antibody which is specific for glycoprotein IIIa (GPIIIa). The binding of D3GP3 to GPIIIa, in intact GPIIb-IIIa complexes, induces fibrinogen binding and platelet aggregation. To determine if D3GP3 binding to GPIIIa directly caused the exposure of fibrinogen receptors or, secondarily, due to stimulus response coupling, platelet activation parameters were monitored following the addition of D3GP3 to platelets suspensions. D3GP3 binding did not induce detectable Ca ++ mobilization, protein phosphorylation or activation of the pertussis toxin sensitive G-protein subunit α-41. Further, D3GP3-induced aggregation was not blocked by PGE 1, aspirin, apyrase or the combination of all three reagents. Scanning electron microscopy of D3GP3-induced aggregates demonstrated that the aggregates were composed of discoid platelets. These data suggest that the binding of D3GP3 to GPIIIa induced a conformational change in GPIIb-IIIa such that the fibrinogen receptor was exposed in an activation-independent fashion. This provides evidence that conformational changes in the GPIIb-IIIa complex can result in the transformation of the complex to the high affinity binding competent state.

Receptor–ligand complexes are cleared to the open canalicular system of surface‐activated platelets

Human platelets were incubated with gold particles coupled to fibrinogen to label the glycoprotein IIb-IIIa (GPIIb-IIIa) receptor after initial activation of the cells by contact with formvar-coated grid and glass surfaces. Fibrinogen-gold (Fgn-Au) markers were absent on discoid platelets, but diffusely spread over the surface and extended pseudopods of early dendritic cells. Conversion to spread platelets resulted in movement of ligand-receptor complexes away from the cell margin toward cell centres. However, Fgn-Au gold did not concentrate in the central region. Rather, the Fgn-Au, GPIIb-IIIa complexes in the middle of spread platelets appeared to move toward a belt-like, intermediate zone, as did the ligand receptor complexes from the cell margin and pseudopods. The ultimate destination of the mobile receptor-ligand complexes, however, appeared to be channels of the surface-connected open canalicular system (OCS). Fgn-Au was concentrated in OCS channels of most dendritic and a small proportion of spread platelets. The decreased frequency of Fgn-Au filled channels in more transformed platelets may have been due to collapse or evagination of the OCS. Examination of platelets exposed to Fgn-Au after spreading on glass and then prepared for thin sections confirmed that the OCS was the final destination for mobile ligand receptor complexes on surface-activated platelets. Findings of this study are consistent with previous work showing clearance of mobile receptor-ligand complexes to the OCS of platelets activated in suspension.

Aggregated platelets enhance adherence of Candida yeasts to endothelium.

The adherence of Candida albicans yeasts to human umbilical vein endothelium to subendothelial extracellular matrix (ECM) was investigated. Yeasts added to confluent endothelium in citrated platelet-poor plasma adhered on the average of 1 colony forming unit (cfu) per culture well. When platelets were added as platelet-rich plasma, a significant increase of yeast adherence was not seen. However, when endothelium was contracted by treatment with 2 mM EDTA, resulting in exposure of ECM, yeast adherence was increased to 10 cfu/well. When platelets were added with these yeasts, the number of adhering yeasts was further increased to 23 cfu/well (P less than .01). This represented an increase in adherence of yeasts of 230%. When the endothelial cells were completely removed and ECM exposed, platelets were found to likewise augment yeast adherence. Platelets, when added to the ECM, formed aggregates to which the yeasts firmly adhered. Likewise, when platelets were aggregated by adenosine diphosphate and mixed with yeasts, yeasts were shown to bind avidly to aggregated platelets, whereas yeasts did not adhere to unactivated, discoid platelets. Thus, exposed subendothelial ECM induces the aggregation of platelets and yeasts bind avidly to these platelet aggregates.

Measuring platelet function with platelet shape change, an early event in aggregation

Platelet shape change precedes aggregation and is sensitive to significantly less stimulant. In this study, differences in percent light transmission (%T) through stirred suspensions of spheroid or discoid platelets are exploited for studying the response of platelets to Platelet Activating Factor (PAF). Changes in %T between stirred and non-stirred suspensions of unstimulated platelets in Platelet Rich Plasma (PRP) varied with the starting concentration. Aggregation was blocked by the presence of citrate while activation by ADP was minimized by apyrase. However, phosphatidic acid formation, ATP release and minimal serotonin release occurred. A shape change parameter (SCP) was defined which varied with the concentration of PAF. Its dependence on PAF was shifted to lower concentration with washed platelets. The dose-response curves for the SCP were found to be virtually identical for both sheep and human platelets. Human platelets were then used to correlate morphology, as determined by phase contrast microscopy, with spectrophotometric data in the form of the SCP. A linear regression analysis of SCP and percent activation revealed a virtual 1:1 correspondence with a slope of 1.066 and an intercept of 0.009 with the standard error of the estimate equal to 0.053. The correlation coefficient was 0.990, making it significant at p < 0.001. This method provides an alternative to platelet aggregation assays as a convenient, sensitive and reliable measure of platelet activation.

Platelet storage: changes in cytosolic Ca2+ actin polymerization and shape

Platelets gradually lose their disc shape during storage. The authors studied simultaneous changes in platelet cytosolic Ca2+ (Cai) and the polymerization state of actin as related to the shape. Platelet concentrates were stored under blood bank conditions for up to 10 days. Aliquots were removed and analyzed as follows: platelet Cai and increments in Cai induced by adenosine diphosphate (ADP) were determined by fluorescence of fura-2-loaded cells; loss of disc shape was determined by differences in light scattering intensity induced by stirring; and the ratio of globular and total actin (G/T) of platelets in plasma was determined by a modification of the DNase inhibition assay. Globular actin was found to be 86 +/- 3% of total actin in freshly drawn platelets suspended in plasma. The following changes occurred during storage: G/T in platelet concentrates increased from 63 +/- 5 (day 0) to 74 +/- 2% in the first 24 hours then fell to 33 +/- 6% by day 10. The percent discoid platelets also increased from day 0 to day 1 then fell in the ensuing days. There was an initial drop in Cai from day 0 to day 1, after which Cai increased on days 3 and 6. Globular actin polymerization during storage closely correlated with the change in percent discs (r = 0.95). During 6 days of storage Cai was highly correlated with shape change (r = 0.97) and to a lesser extent (r = 0.87) with the ratio of globular actin. The authors conclude that actin polymerization, shape, and Ca2+ change in a related fashion during storage.

Platelet Storage: Changes in Cytosolic Ca2+ Actin Polymerization and Shape

Platelets gradually lose their disc shape during storage. The authors studied simultaneous changes in platelet cytosolic Ca2+ (Cai) and the polymerization state of actin as related to the shape. Platelet concentrates were stored under blood bank conditions for up to 10 days. Aliquots were removed and analyzed as follows: platelet Cai and increments in Cai induced by adenosine diphosphate (ADP) were determined by fluorescence of fura-2-loaded cells; loss of disc shape was determined by differences in light scattering intensity induced by stirring; and the ratio of globular and total actin (G/T) of platelets in plasma was determined by a modification of the DNase inhibition assay. Globular actin was found to be 86 +/- 3% of total actin in freshly drawn platelets suspended in plasma. The following changes occurred during storage: G/T in platelet concentrates increased from 63 +/- 5 (day 0) to 74 +/- 2% in the first 24 hours then fell to 33 +/- 6% by day 10. The percent discoid platelets also increased from day 0 to day 1 then fell in the ensuing days. There was an initial drop in Cai from day 0 to day 1, after which Cai increased on days 3 and 6. Globular actin polymerization during storage closely correlated with the change in percent discs (r = 0.95). During 6 days of storage Cai was highly correlated with shape change (r = 0.97) and to a lesser extent (r = 0.87) with the ratio of globular actin. The authors conclude that actin polymerization, shape, and Ca2+ change in a related fashion during storage.

PRE‐TRANSFUSION NON‐INVASIVE QUALITY ASSESSMENT OF STORED PLATELET CONCENTRATES

The Platelet Monitor System has been designed to assess routinely prepared platelet concentrates (P.C.) during storage. The system is based upon observations of changes in light transmission induced by platelets in intact packs. It is practical, provides constant gentle agitation for P.C., and non-invasively give continuous semi-quantitative evidence of the probable numbers of discoid platelets within each pack. The information yielded correlates both with established in vitro parameters of platelet behaviour, and with in vivo survival of platelets in normal healthy volunteers. The device safely provides the Blood Banker with the means to achieve on-going assessment of methods and improvements--or failings--in P.C. production. To the clinician it offers information throughout and even beyond present arbitrary storage limits, about which P.C. are likely to be of least or greatest value to a thrombocytopenic patient.

Effect of prostacyclin analogue (carbacyclin) on the interaction of platelets with different collagen substrates. Inhibition of camp inccrease by collagens type I, III and IV

We investigated the effects of a stable prostacyclin analogue, carbacylcin, on the interaction of platelets with collagen substrates differing in thier ability to activate platelets: human collagens type I. III, IV and V (CI, CIII, CIV and CV), and commercial calf skin collagen type I (CSC). The total adhension was measured using 51Cr-labelled platelets, and quantitative morphometry of adherent platelets was performed by scanning electron microscopy (SEM). Carbacyclin in the concentrations inducing a 10-fold rise in platelets cAMP did not effect the adhension of platelets to weak substrates, CV and CSV, but reduced the adhesion to strong substrates, CIV and *by 49%) and CI/CIII (by 78%), which stimulated massive spreading and formation of surface-boud aggregates respectively. Carbacyclin inhibited all morphological manifestations of platelet activation associated with adhension: conversion of native discoid platelets to spherical ones on CSC; massive spreading on CIV; and aggregate formation on CI/CIII. Massive spreading and aggregation on a weak substrate (CSC) stimulated by arachidonic acid and thrombin was also inhibited by carbacyclin. Under the same concentration of angonists aggregation of platelets was more sensitive to the action of carbacyclin, than spreading. Strong collagen substrates CI, CIII and CIV, but not CV and gelatin, inhibited the carbacyclin-induced rise in platelet cAMP.

A rate equation for blood platelet aggregation

A kinetic scheme for agonist-induced aggregation of blood platelets was formulated in terms of the kinetics of agonist interaction with the nonaggregable, discoid platelets, formation of aggregable forms by shape-change reactions, and interactions among shape-changed forms. Taking into account the relative magnitudes of the rate constants of the different steps and assuming aggregation to be by hydrophobic forces, an equation similar in form to the Michaelis-Menten equation was derived to characterize aggregation kinetics. The kinetic formulation could account for several empirical observations and may be used to interpret kinetic effects of antiplatelet drugs more informatively than at present.

Correlative video enhanced DIC-LM, HVEM, and low-voltage high-resolution SEM in the study of surface and internal ultrastructure

Previously we have found high voltage electron microscopy (HVEM) and conventional scanning electron microscopy (SEM) to be useful in investigating relationships between surface and subsurface structure. The use of directly conjugated 5nm and 18nm colloidal gold-ligand or colloidal gold-antibody has been useful with HVEM and SEM to determine the location of surface membrane associated glycoproteins relative to surface and internal ultrastructure. Using the platelet as a model system, we have demonstrated cytoskeletal rearrangement coincident with the activation-associated shape change response. Furthermore the activity and location of the receptor (membrane glycoprotein complex IIb IIIa) for fibrinogen and several other RGD sequence-containing “adhesive” proteins is related to the structure and cytoskeletal organization of the platelet. The receptor is present, but unable to bind whole ligand, on discoid and spherical platelets (small ligand fragments and monoclonal antibody or peptide sequences specific for the active site can, however, bind).

Pre-transfusion non-invasive quality assessment of stored platelet concentrates.

The Platelet Monitor System has been designed to assess routinely prepared platelet concentrates (P.C.) during storage. The system is based upon observations of changes in light transmission induced by platelets in intact packs. It is practical, provides constant gentle agitation for P.C., and non-invasively give continuous semi-quantitative evidence of the probable numbers of discoid platelets within each pack. The information yielded correlates both with established in vitro parameters of platelet behaviour, and with in vivo survival of platelets in normal healthy volunteers. The device safely provides the Blood Banker with the means to achieve on-going assessment of methods and improvements--or failings--in P.C. production. To the clinician it offers information throughout and even beyond present arbitrary storage limits, about which P.C. are likely to be of least or greatest value to a thrombocytopenic patient.

Ultrasound-induced orientation of discoid platelets and simultaneous changes in light transmission: preliminary characterisation of the phenomenon.

Discoid platelet suspensions were subjected to ultrasonic fields, in the 1-10 MHz frequency range and at estimated acoustic pressures in the range of 0.05-7.6 X 10(4) Pa. Absolute definition of the acoustic field/pressures was precluded by the small dimensions, and internal reflective surfaces, of the experimental configurations employed. Direct microscopy revealed platelet flow, in patterns consistent with theory for ultrasonically induced streaming. This ultrasonic effect was presumed to be accompanied by platelet orientation since it could be quantitated by simultaneous light transmission measurements. The preliminary characterisation of this phenomenon, defined as ultrasound induced light transmission change (USILTC) is reported, and indicates that it may be readily quantitated in a reproducible manner, but which shows a complex relationship both to pressure and frequency. In addition, qualitative characteristics of the response traces are apparent under certain conditions, particularly 5 MHz above 1 X 10(4) Pa, and may relate to platelet viability. Preliminary evidence is provided and discussed, which suggests that USILTC may be a useful model for studying certain ultrasound bioeffects, and may be a useful tool for indirectly assessing platelet morphology and viability for quality assurance purposes.

Studies of the minimum temperature at which human platelets can be stored with full maintenance of viability.

Platelet concentrates from normal donors were stored for 3 days under identical conditions except for the temperature of storage, which was maintained at 21 +/- 0.5, 19.5 +/- 0.5, or 18 +/- 0.5 degrees C. Immediate posttransfusion recovery of the stored platelets determined by 51Cr labeling averaged 47, 47, and 48 percent after storage at 21, 19.5, and 18 degrees C, respectively (differences not significant). Mean life span of the transfused platelets, however, was 8.12, 5.21, and 1.85 days at 21, 19.5, and 18 degrees C, respectively. The difference between mean life span following storage at 21 degrees C was significantly different from that after storage at 18 degrees C (p less than 0.03). Reduction in viability after storage at the lower temperature correlated with the reduction in the number of discoid platelets. These findings indicate that platelet viability is compromised after storage for 3 days at 18 degrees C and, possibly, at 19.5 degrees C, and illustrate the need for quality control of temperature in short-term platelet storage.

Specialized membrane areas in non-activated and thrombin-activated platelets.

Filipin, a complex of polyene antibiotics, forms morphologically distinctive complexes with cholesterol in cell membranes under proper experimental conditions. When applied to non-activated, discoid platelets, filipin-induced lesions (FIL) occurred in rows at the platelet equator, suggesting a specialized membrane organization at the platelets' largest circumference. In some thrombin-activated platelets we observed surface membrane blebbing and release of lipid vesicles that predominantly originated from the plasma membrane proper, but some originated from (unidentified) platelet granules. FIL were initially present in high numbers over the entire bleb, they accumulated later at the neck of blebs, while the released vesicle was free of FIL. Absence of intramembrane protein particles (IMP) from the membranes of blebs and vesicles suggests that released vesicles are essentially without cholesterol and intrinsic membrane proteins and may consist predominantly of phospholipids. Membrane blebbing and vesicle release may represent unmasking and release of procoagulant platelet factor 3 activity.

Preparation of washed platelets from non-anti coagulated human blood

Sodium citrate is almost always used to anticoagulate blood for the preparation of washed platelet suspensions. Several adverse effects of citrate on platelet functional responses have been reported. We investigated the extent of activation of platelets and plasmatic coagulation during the preparation of washed platelets from human blood to which no anticoagulant was added. Washed platelets from native blood (PNB) were prepared by passing freshly-drawn human blood rapidly through a mixed Sephadex G-25/G-50 column to remove divalent cations. Gel-filtered blood (GFB), diluted by the elution medium containing 0.35% albumin and 2U/ml apyrase, was obtained within 5 minutes of vene-puncture. Using CaCI 2, the GFB was found to contain a mean of 1.65 x 10 mM calcium. Fibrinopeptide A measurements indicated less activation of plasmatic coagulation in GFB than in citrated blood. Measurements of β-thromboglobulin and platelet factor 4 during the various stages of the preparation of PNB showed no platelet activation. No platelet aggregates, as measured by the platelet aggregate ratio method, were observed in GFB. Transmission electron microscopy showed intact discoid platelets similar to those in platelet-rich plasma. The reduced activation of platelets and of plasmatic coagulation was due to the more effective removal of calcium ions from the blood by gel filtration than chelation by citrate. PNB may thus provide a model for studying the requirements for calcium in platelet function in the absence of any citrate-platelet interactions.

Organization of the cytoskeleton in resting, discoid platelets: preservation of actin filaments by a modified fixation that prevents osmium damage.

This study evaluates the structural organization of the cytoskeleton within unactivated, discoid platelets. Previously, such studies have been difficult to interpret because of the ease with which platelets are stimulated, the sensitivity of actin filaments to cell extraction buffers, and the general problem of preserving actin filaments with conventional fixatives, compounded by the density of the cytoplasm in the platelet. In this study we have employed a new fixative containing lysine, which protects actin filaments against damage during fixation and thin-section processing. We used thick (0.25-micron) sections and conventional thin sections of extracted cells (fixed and lysed simultaneously by the addition of 1% Triton X-100 to the initial fixative) as well as thin sections of whole cells to examine three preparations of human platelets: discoid platelets washed by sedimentation; discoid platelets isolated by gel filtration; and circulating platelets collected by dripping blood directly from a vein into fixative. In all of these preparations, long, interwoven actin filaments were observed within the platelet and were particularly concentrated beneath the plasma membrane. These filaments appeared to be linked at irregular intervals to the membrane and to each other via short, approximately 20- to 50-nm-long cross-links of variable width. Although most filaments were outside the circumferential band of microtubules and the cisternae of the open canalicular system, individual filaments dipped down into the cytoplasm and were found between the microtubules and in association with other membranes. The ease with which single actin filaments can be seen in the dense cytoplasm of the human platelet after lysine/aldehyde fixation suggests the great potential of this new fixative for other cells.

Freeze-Fracture Studies of Human Blood Platelets Activated by Thrombin Using Rapid Freezing

In this study the influence of thrombin activation on human blood platelets has been followed by freeze-fracturing electron microscopy using rapid freezing in order to catch the initial changes in shape and the morphological alterations during the process of exocytosis of secretory granules. We found that isolation of the platelets by itself leads to some degree of shape change, which made it impossible to study the resting discoid platelet by rapid freezing. Activation of the platelets by thrombin induced dilation of the "surface connecting system (SCS)" with formation of large vacuoles as a result of fusion of the secretory granules with SCS. No intermediary fusion stages or structures were observed even using rapid freezing. Volcano-like protrusions and the corresponding complementary pits were seen at the SCS. These structures were interpreted by us as fractures through protoplasmic channels crossing the SCS. These channels originate during the swelling of the SCS as a result of the fusion of secretory granules with the SCS.