The effects of elongational stress exposure on the activation and aggregation of blood platelets.

Hemodynamic shear is known to stimulate blood and endothelial cells and induce platelet activation. Many studies of shear-induced platelet stimulation have employed rotational viscometers in which secondary flow effects are assumed to be negligible. Shear induced platelet activation occurs at elevated shear rates where secondary flows may contribute a significant percentage of the total hydrodynamic force experienced by the sample. Elongational stress, one component of this secondary flow, has been shown to alter transmembrane ion flux in intact cell and the permeability of synthetic membrane preparations. Elongational flow also occurs in the vasculature at sites of elevated shear stress. Secondary flow components may contribute to platelet activation induced during shear stress application in rotational viscometry. A unique 'constrained convergence' elongational flow chamber was designed and fabricated to study platelet response to elongational stress exposure. The elongational flow chamber was capable of producing an elongation rate of 2.1 s-1 with a corresponding volume averaged shear rate of 58.33 s-1. Significant changes were observed in the total platelet volume distribution and measured response to added chemical antagonists after elongational stress exposure. The total platelet volume histogram shifted toward larger particle sizes, suggesting the formation of large aggregates as a result of elongational stress exposure. Platelets exposed to elongational stress demonstrated a dose dependent decrease in added ADP-induced aggregation rate and extent of aggregation.     

MicroRNA 320a and Membrane Antigens as Tools to Evaluate the Pathophysiology of Platelets Stored in Blood Banks

Our research group, through the analysis of miRNomes in platelet concentrates (PCs) stored in blood banks, identified and validated the miR-127 and miR-320a miRNAs as biomarkers of platelet storage lesions (PSLs) in PCs. In order to validate the miRNAs 127 and 320a methodologically, as PSL biomarkers in a large number of PC bags, we also evaluated important immunological markers involved in the platelet activation/aggregation process—the CD62P receptor (P-selectin), the surface glycoproteins (GP) IIb/IIIa, and the purinergic P2Y12 receptor—via flow cytometry. The miRNAs miR-127 and miR-320a were quantified by real-time quantitative PCR (RT-qPCR). To carry out this study, 500 collection tubes were used at the upper edge of the PC bags containing platelets. Each tube was divided into seven equal parts (totaling 3500 samples) for platelet analysis from 7 different storage days, where the 1st day represents the high-quality control, and the 7th day corresponds to the low-quality control of the platelets. After analyzing all parameters during storage days, it was concluded that the relative quantification of miR-320a below 0.50 and the CD62P receptor below 27.92% are reliable indicators of the absence of storage lesions in blood banks. We believe that the values found in the expression of the CD62P receptor legitimize the use of the miR-320a and miR-127 miRNAs to build a kit capable of accurately measuring whether the stored platelets are suitable for transfusion.     

Electronic particle size measurements of platelet aggregates formed in vitro.

The aggregation of human platelets induced by adenosine diphosphate (ADP) was used to evaluate electronic particle size analyzer measurements of platelet aggregates in plasma. As platelets began to clump in plasma, the total volume and the diameter of individual aggregates increased; after a time dependent on experimental conditions, the diameter increased but the total volume remained unchanged. Similar but opposite changes in size distribution occurred during platelet deaggregation. The total volume of aggregates formed in plasma varied (linear correlation coefficient = 0.99) with the total volume of platelets which were available to clump and with simultaneous changes in optical density. The diameter of the aggregates varied with the concentration of, and time of exposure to, ADP and with the total volume of platelets and aggregates in plasma was not different from that of control platelets in untreated plasma, the individual platelets aggregated without an accompanying increase in size. This study demonstrates that platelet aggregation can be characterized by electronic measurements of the size distribution of platelet aggregates.     

Storage of apheresis platelets in ethylene-vinyl acetate copolymer bags: relationship between the bag size and the number of platelets maintaining aerobic metabolism

The present study was designed to determine whether a bag made from ethylene-vinyl acetate copolymer (EV) with superior flexibility at subzero temperature is suitable for a storage container of single-donor apheresis platelets. Apheresis platelets were stored with 100 ml plasma in 1-liter bags made of EV or standard polyvinyl chloride (PVC) plastic at 22 degrees C with constant agitation. The oxygen permeability of the 1-liter EV bag averaged 1447 nmol/min/atm, which was about 1.5 times higher than that of PVC bags. The partial oxygen tension (PO2) of platelet concentrates (PC) has linearly decreased to 16 mm Hg with increasing platelet counts. The level of the partial carbon dioxide was always higher in EV bags than in PVC bags. Oxygen consumption rates of platelets stored in EV and PVC bags with a sufficient oxygen supply averaged 1.25 and 1.20 nmol/min/10(9) platelets, respectively. The rates of glucose consumption and lactate production were not changed in two bags. Ninety percent of the total ATP production of about 8 nmol/min/10(9) platelets were generated through the aerobic metabolism. The platelet counts in the 1-liter EV and PVC bags, at which PO2 is 16 mm Hg, were 2.2 and 1.5 x 10(11) platelets, respectively. The study indicates that apheresis platelets stored in EV bags at 22 degrees C have no different metabolic changes when compared with those of PVC bags. In addition, the number of platelets maintaining the aerobic metabolism is 1.5 times higher than that of PVC bags.     

Leaching of plasticizers from and surface characterization of PVC blood platelet bags.

The leaching of phthalate plasticizers from four types of blood platelet bags was investigated. The anticoagulant solutions used in the blood collection bags had pH values of 5.64 +/- .04 and contained no detectable amounts of phthalates. Platelet bag materials from each bag were soaked in normal salines for up to 5 days. The salines were tested for the leached phthalates from the bags but none could be found. However, di-(2-ethylhexyl) phthalate (DEHP) leached out of the PL-146 and Terumo bags into bovine calf serum used for soaking the bag materials. There was an increase in the amount of DEHP leached from about 1.1 mg at the end of one day to about 3.3 mg per gm of bag material at the end of a five day extraction with the serum. In PL-732 sets, a platelet bag made of a specialty polyolefin, the amount of DEHP leached out was less than 0.02 mg per g of bag material. CLX bags, which contained tri-(2-ethylhexyl) trimelliate (TETM) as a plasticizer, showed a negligible amount of it leaching into the calf serum. Infra-red spectra showed that PL-146 bags had been coated with a layer of a fatty acid amide while the Terumo bags contained a layer of a silicone fluid on their inner surfaces. CLX bags showed a coating of stearates, which were probably soaps of calcium or zinc. Scanning electron microscopy (SEM) showed that the inner surfaces of each brand of the bag were distinctly different morphologically. The two PVC bags were very similar whereas the surfaces morphology of PL-732 was rougher. Terumo bag had a different surface morphology than those of the other bags whereas the CLX bags had a very regular surface pattern. The exact significance of the surface morphology is not certain but excessively rough surfaces may not be desirable for the bags.     

The miRNA Profile of Platelets Stored in a Blood Bank and Its Relation to Cellular Damage from Storage

Millions of blood products are transfused each year, and many lives are directly affected by transfusion. Platelet concentrate (PC) is one of the main products derived from blood. Even under good storage conditions, PC is likely to suffer cell damage. The shape of platelets changes after 5 to 7 days of storage at 22°C. Taking into consideration that some platelet proteins undergo changes in their shape and functionality during PC storage. Sixteen PC bags were collected and each PC bag tube was cut into six equal pieces to perform experiments with platelets from six different days of storage. Thus, on the first day of storage, 1/6 of the tube was used for miRNA extraction, and the remaining 5/6 was stored under the same conditions until extraction of miRNAs on each the following five days. Samples were sequenced on an Illumina Platform to demonstrate the most highly expressed miRNAs. Three miRNAs, mir127, mir191 and mir320a were validated by real-time quantitative PCR (RQ-PCR) in 100 PC bags tubes. Our method suggests, the use of the miRNAs mir127 and mir320a as biomarkers to assess the "validity period" of PC bags stored in blood banks for long periods. Thus, bags can be tested on the 5th day of storage for the relative expression levels of mir127 and mir320a. Thus, we highlight candidate miRNAs as biomarkers of storage damage that can be used as tools to evaluate the quality of stored PC. The use of miRNAs as biomarkers of damage is unprecedented and will contribute to improved quality of blood products for transfusions.     

An integrated proteomics and genomics analysis to unravel a heterogeneous platelet secretion defect

Eight patients with clinical bleeding problems have evidence for platelet storage pool disease as they present with impaired platelet aggregation and secretion with low concentrations of ADP and collagen and an absence of second phase aggregation with epinephrine. Electron microscopy analysis further showed a reduced but not absent amount of platelet dense granules, and CD63 staining was decreased compared to healthy controls. The presence of alpha granules and CD62P expression after platelet activation was normal. This work aimed at identifying differentially expressed proteins in the platelet releasate and its remaining pellet after activation with A23187 and TRAP in patients and controls using DIGE-based proteomic technology. We identified 44 differentially expressed proteins in patients and the altered expression for some of them was confirmed by immunoblot analysis. Most of these proteins belong to the class of cytoskeleton-related proteins. In addition, 29 cytoskeleton-related genes showed an altered expression in platelet mRNA from patients using a real-time PCR array. In conclusion, our study shows that the dense granule secretion defect in patients with platelet storage pool disease is highly heterogeneous with evidence of an underlying cytoskeleton defect.     

Relationships between Platelets and Blood Cells in Postprandial Glycemia in Healthy Individuals

The relationships between platelet aggregation and blood cell amount were studied in healthy subjects after fasting and after a standard carbohydrate breakfast. Positive relationships were revealed between certain parameters of platelet aggregation and the amounts of monocytes and eosinophils. Negative relationships were revealed between most parameters of platelet aggregation and the amount of platelets. Along with the well-known food leukocytosis and increased fibrinogen, postprandial conditions were characterized by an increase in certain parameters of platelet aggregation, destruction of the relationship between the fibrinogen concentration and platelet aggregation, impairment of the inverse relationship between the amount and aggregation of platelets, and moderate direct or inverse relationships between certain parameters of the first and the second stages of platelet aggregation and the erythrocyte parameters. As a disturbing factor, postprandial conditions activated platelet hemostasis by modulating the initial stages of aggregation and substantially changed the interrelations between blood cells.     

Proteolytic degradation of vimentin and glial fibrillary acidic protein in rat astrocytes in primary culture

The receptor for ADP on the platelet membrane, which triggers exposure of fibrinogen-binding sites and platelet aggregation, has not yet been identified. Two enzymes with which ADP interacts on the platelet surface, an ecto-ATPase and nucleosidediphosphate kinase, have been proposed as possible receptors for ADP in ADP-induced platelet aggregation. In the present study, experiments were conducted with washed human platelets to examine if a relationship existed between platelet aggregation, fibrinogen binding and the enzymatic degradation of ADP. With 12 different platelet suspensions, a good correlation (P less than 0.01) was found between the extent of platelet aggregation and the amount of 125I-fibrinogen bound to platelets after ADP stimulation. No correlation was found between these parameters and the rate or extent of transformation of [14C]ADP to [14C]ATP or [14C]AMP. The binding of fibrinogen to platelets was inhibited in parallel with aggregation when ADP stimulation was impaired by the enzymatic degradation of ADP by the system creatine phosphate/creatine phosphokinase, or by the use of specific antagonists, such as ATP and AMP. These antagonists also influenced the enzymatic degradation of ADP. This effect occurred at lower concentrations of ATP or AMP than those required to inhibit ADP-induced platelet aggregation and fibrinogen binding. Our results demonstrate that ATP and AMP may be used as specific antagonists of the ADP-induced fibrinogen binding to platelets. They do not provide evidence to suggest that enzymes which metabolize ADP on the platelet surface are involved in the mechanism of ADP-induced platelet aggregation.     

Effects of diamide and iodoacetamide on the expression of the glycoprotein IIb/IIIa complex on blood platelets

We have examined the effects of two agents that alter platelet thiol-disulphide status on platelet aggregation and on the ability of platelets to bind a monoclonal antibody (M148) that is directed toward an epitope on the glycoprotein IIb/IIIa complex. The immediate effect of both diamide and iodoacetamide is to enhance aggregation but after further incubation diamide, but not iodoacetamide, inhibits platelet aggregation. Incubation of platelets with diamide, but not iodoacetamide, is accompanied by a marked increase in the amount of M148 that binds to platelets. This is presumably a reflection of an altered distribution of glycoproteins on the platelet surface. It is known that diamide, but not iodoacetamide, leads to polymerisation of cytoskeletal proteins in platelets. Thus evidence is provided that agents that interact with the cytoskeleton inhibit platelet behaviour via an effect on surface glycoproteins.     

The use of acridine orange for testing blood platelet integrity

Two processes are involved in the accumulation of acridine orange in human blood platelets. One follows a diffusion like kinetics and is independent of the ATP level whereas the second one can be completely abolished by ATP depletion. The acridine orange incorporation rate seems to be a suitable parameter for testing platelet integrity. It reflects very sensitively the influence of the preparation method as well as of anticoagulating substances used on the stability of platelet suspensions. The rates of acridine orange incorporation and of aggregation were measured in platelet-rich plasma and in saline suspended platelets after gel filtration, respectively, over a period of 120 min storage. Both rates are influenced to a different degree by anticoagulating agents such as citrate, heparin and EDTA. When contact with anticoagulating agents during platelet preparation is avoided, platelets show a constant acridine orange incorporation and aggregation during storage and the smallest morphological alteration.     

Platelet lysis and aggregation in shear fields.

A rotational viscometer was used to study the effects of shear stress on platelets in human platelet-rich plasma (PRP). For 5-min exposure times, shear stresses above 160 dynes/cm2 induced platelet lysis (as determined by release of platelet lactic dehydrogenase). For 30-s exposure times, shear stresses greater than 600 dynes/cm2 were required to induce platelet lysis. The platelet counts of sheared PRP were decreased to as low as one-fifth the original count due largely to shear-induced aggregation. The count is a minimum at intermediate stress levels (200-400 dynes/cm2). Higher stresses induce disaggregation as well as lysis. The diminution in the counts was partially reversed in 2 h incubation after cessation of shearing. Experiments were carried out with three different viscometer configurations so that the shear stress and the solid surface area access could be varied independently. Surface access was not a significant variable in the conditions of the experiments. Thus aggregation and lysis may be induced by stress effects alone as well as by solid surface effects. The results also show that the response of platelets to shear stress is strongly dependent on exposure time. Platelets are much less resistant to shear stress than red cells for relatively long exposure times. However, the converse is true for very short exposure times.     

Aggregation and disaggregation kinetics of human blood platelets: Part II. Shear-induced platelet aggregation.

A population balance equation (PBE) mathematical model for analyzing platelet aggregation kinetics was developed in Part I (Huang, P. Y., and J. D. Hellums. 1993. Biophys. J. 65: 334-343) of a set of three papers. In this paper, Part II, platelet aggregation and related reactions are studied in the uniform, known shear stress field of a rotational viscometer, and interpreted by means of the model. Experimental determinations are made of the platelet-aggregate particle size distributions as they evolve in time under the aggregating influence of shear stress. The PBE model is shown to give good agreement with experimental determinations when either a reversible (aggregation and disaggregation) or an irreversible (no disaggregation) form of the model is used. This finding suggests that for the experimental conditions studied disaggregation processes are of only secondary importance. During shear-induced platelet aggregation, only a small fraction of platelet collisions result in the binding together of the involved platelets. The modified collision efficiency is approximately zero for shear rates below 3000 s-1. It increases with shear rates above 3000 s-1 to about 0.01 for a shear rate of 8000 s-1. Addition of platelet chemical agonists yields order of magnitude increases in collision efficiency. The collision efficiency for shear-induced platelet aggregation is about an order of magnitude less at 37 degrees C than at 24 degrees C. The PBE model gives a much more accurate representation of aggregation kinetics than an earlier model based on a monodispersed particle size distribution.     

Flow cytometric studies of platelet responses to shear stress in whole blood

The objective of this work was to evaluate quantitatively the effects of flow on platelet reactions using a flow cytometric technique. Whole blood was exposed to well defined, laminar shear stress in a cone-and-plate viscometer in the absence of added agonists. Blood specimens were fixed with formaldehyde and incubated with two monoclonal antibodies. Antibody 6D1, specific for platelet membrane glycoprotein Ib (GPIb), was used to identify and enumerate platelets and platelet aggregates on the basis of their characteristic forward scatter and 6D1-FITC fluorescence profiles. Anti-CD62 antibody, specific for the granule membrane protein-140 (GMP-140), was used to measure platelet activation. Results showed platelet aggregation increasing with increasing shear stress with marked increase in this response for a pathophysiological stress level of 140 dyn/cm² and higher. This stress level also was the apparent threshold for formation of large platelet aggregates (“large” refers to particles larger than 10 μm in equivalent sphere diameter). These platelet responses to shear stress were insensitive to aspirin, but strongly inhibited by agents that elevate platelet cyclic adenosine monophosphate (cAMP) levels. Moreover, pre-incubation of whole blood with monoclonal antibodies that inhibit von Willebrand factor binding to GPIb or von Willebrand factor and fibrinogen binding to GPIIb/IIIa inhibited platelet aggregation. Aggregation induced by shear at 37° C was less in extent than at 23° C. At physiological shear stresses, whole blood was more susceptible to shear-induced platelet aggregation than platelet-rich plasma. This study reaffirms that flow cytometric methods have several important advantages in studies of shear effects on platelets, and extends the methodology to whole blood unaltered by cell separation methods.     

Molecular mechanisms of platelet activation and aggregation induced by breast cancer cells

Tumor cell-induced platelet aggregation represents a critical process both for successful metastatic spread of the tumor and for the development of thrombotic complications in cancer patients. To get further insights into this process, we investigated and compared the molecular mechanisms of platelet aggregation induced by two different breast cancer cell lines (MDA-MB-231 and MCF7) and a colorectal cancer cell line (Caco-2). All the three types of cancer cells were able to induce comparable platelet aggregation, which, however, was observed exclusively in the presence of CaCl₂ and autologous plasma. Aggregation was supported both by fibrinogen binding to integrin αIIbβ3 as well as by fibrin formation, and was completely prevented by the serine protease inhibitor PPACK. Platelet aggregation was preceded by generation of low amounts of thrombin, possibly through tumor cells-expressed tissue factor, and was supported by platelet activation, as revealed by stimulation of phospholipase C, intracellular Ca²⁺ increase and activation of Rap1b GTPase. Pharmacological inhibition of phospholipase C, but not of phosphatidylinositol 3-kinase or Src family kinases prevented tumor cell-induced platelet aggregation. Tumor cells also induced dense granule secretion, and the stimulation of the P2Y12 receptor by released ADP was found to be necessary for complete platelet aggregation. By contrast, prevention of thromboxane A₂ synthesis by aspirin did not alter the ability of all the cancer cell lines analyzed to induce platelet aggregation. These results indicate that tumor cell-induced platelet aggregation is not related to the type of the cancer cells or to their metastatic potential, and is triggered by platelet activation and secretion driven by the generation of small amount of thrombin from plasma and supported by the positive feedback signaling through secreted ADP.     

An Inhibition of p38 Mitogen Activated Protein Kinase Delays the Platelet Storage Lesion

Background and Objectives

Platelets during storage undergo diverse alterations collectively known as the platelet storage lesion, including metabolic, morphological, functional and structural changes. Some changes correlate with activation of p38 mitogen activated protein kinase (p38 MAPK). Another MAPK, extracellular signal-related kinase (ERK), is involved in PLT activation. The aim of this study was to compare the properties of platelets stored in plasma in the presence or absence of p38 and ERK MAPK inhibitors.

Materials and Methods

A single Trima apheresis platelet unit (n = 12) was aliquoted into five CLX storage bags. Two aliquots were continuously agitated with or without MAPK inhibitors. Two aliquots were subjected to 48 hours of interruption of agitation with or without MAPK inhibitors. One aliquot contained the same amount of solvent vehicle used to deliver the inhibitor. Platelets were stored at 20–24°C for 7 days and sampled on Days 1, 4, and 7 for 18 in vitro parameters.

Results

Inhibition of p38 MAPK by VX-702 leads to better maintenance of all platelet in vitro storage parameters including platelet mitochondrial function. Accelerated by interruption of agitation, the platelet storage lesion of units stored with VX-702 was diminished to that of platelets stored with continuous agitation. Inhibition of ERK MAPK did not ameliorate decrements in any in vitro platelet properties.

Conclusion

Signaling through p38 MAPK, but not ERK, is associated with platelet deterioration during storage.     

Aggregation kinetics of human mesenchymal stem cells under wave motion

Human mesenchymal stem cells (hMSCs) are primary candidates in cell therapy and regenerative medicine but preserving their therapeutic potency following culture expansion is a significant challenge. hMSCs can spontaneously assemble into three‐dimensional (3D) aggregates that enhance their regenerative properties. The present study investigated the impact of hydrodynamics conditions on hMSC aggregation kinetics under controlled rocking motion. While various laboratory methods have been developed for hMSC aggregate production, the rocking platform provides gentle mixing and can be scaled up using large bags as in wave motion bioreactors. The results show that the hMSC aggregation is mediated by cell adhesion molecules and that aggregate size distribution is influenced by seeding density, culture time, and hydrodynamic conditions. The analysis of fluid shear stress by COMSOL indicated that aggregate size distribution is inversely correlated with shear stress and that the rocking angle had a more pronounced effect on aggregate size distribution than the rocking speed due to its impact on shear stress. hMSC aggregates obtained from the bioreactor exhibit increased stemness, migratory properties, and expression of angiogenic factors. The results demonstrate the potential of the rocking platform to produce hMSC aggregates with controlled size distribution for therapeutic application.     

Aggregation and disaggregation kinetics of human blood platelets: Part III. The disaggregation under shear stress of platelet aggregates.

In the preceding two papers (1, 2), a population balance equation (PBE) mathematical model was developed, validated, and applied to the analysis of platelet aggregation kinetics under the influence of hydrodynamic shear stress. The present work involves the application of the model to the analysis of platelet reactions under shear stress in circumstances where disaggregation processes are of dominant importance: the disaggregation of aggregates formed in response to added agonists. Aggregation-disaggregation experiments were performed in the constant shear field of a rotational viscometer, and the evolution of the particle size distribution was determined by use of an electronic particle counter. The PBE model was used to simulate the experimental results. Exploratory calculations made it possible to reduce a rather complete, complex model to a more tractable form which retains the capability of simulating the experimental observations. For the experimental conditions studied, disaggregation by a splitting mechanism was found to be of dominant importance. The surface erosion mechanism can be neglected without significant impact on results. Physical reasoning confirmed by exploratory calculations showed that a discontinuous form of the breakage rate expression which incorporates a minimum friable particle size, gives significantly better results than a continuous expression. A simple step function void fraction parameter was found to be at least as successful as a more complicated, continuous function. The resulting simplified model has the potential of increasing our understanding of kinetics and mechanisms of platelet reactions, and of characterizing the state of platelet activity. Hence, it may be useful in efforts to understand thrombotic and hemostatic processes.     

Adiponectin inhibits hyperlipidemia-induced platelet aggregation via attenuating oxidative/nitrative stress

Adiponectin acts as an endogenous antithrombotic factor. However, the mechanisms underlying the inhibition of platelet aggregation by adiponectin still remain elusive. The present study was designed to test whether adiponectin inhibits platelet aggregation by attenuation of oxidative/nitrative stress. Adult rats were fed a regular or high-fat diet for 14 weeks. The platelet was immediately separated and stimulated with recombinant full-length adiponectin (rAPN) or not. The platelet aggregation, nitric oxide (NO) and superoxide production, endothelial nitric oxide synthase (eNOS)/inducible NOS (iNOS) expression, and antioxidant capacity were determined. Treatment with rAPN inhibited hyperlipidemia-induced platelet aggregation (P<0.05). Interestingly, total NO, a crucial molecule depressing platelet aggregation and thrombus formation?was significantly reduced, rather than increased in rAPN-treated platelets. Treatment with rAPN markedly decreased superoxide production (-62 %, P<0.05) and enhanced antioxidant capacity (+38 %, P<0.05) in hyperlipidemic platelets. Hyperlipidemia-induced reduced eNOS phosphorylation and increased iNOS expression were significantly reversed following rAPN treatment (P<0.05, P<0.01, respectively). Taken together, these data suggest that adiponectin is an adipokine that suppresses platelet aggregation by enhancing eNOS activation and attenuating oxidative/nitrative stress including blocking iNOS expression and superoxide production.     

Improvement of platelet aggregation abnormalities in thrombocytosis after thrombocytopheresis

Platelet function tests were performed in three patients with thrombocytosis in myeloproliferative disorders before and after a swift reduction of platelet count by thrombopheresis. The decrease of platelet count obtained after the procedure was reversed in six days. In two patients with platelet aggregation defects, the normalization of aggregation abnormalities was observed after pheresis, followed by a progressive decrease of platelet response until the pre-pheresis values on 6th day. In the third patient with normal platelet aggregation, a progressive increase of platelet aggregation response was noted on the days following thrombopheresis with ischaemic symptoms of a foot toe. In all three patients, the changes of platelet aggregation were accompanied by a related increase of megathrombocytes. In the two patients with platelet aggregation abnormalities, plasma and platelet beta-thromboglobulin levels were related to changes in platelet count and aggregation.