Factors influencing platelet aggregation in whole blood

In order to evaluate the interference of blood cells on platelet aggregation, spontaneous platelet aggregation (SPA), ADP, and collagen-induced platelet aggregation were investigated in whole blood by the impedance method and in platelet-rich plasma (PRP) by densitometric and impedance aggregometers. Stirring of the sample induced a significant decrease of neutrophils (P less than 0.001) but no changes of red blood cell (RBC) and platelet count. After collagen addition, a further decrease of neutrophils was observed, while RBC count was unmodified. The occurrence of SPA was not different in whole blood and in PRP. Platelet number and hematocrit did not affect either spontaneous or collagen-induced aggregation. A significant linear relation (r = -0.60, P less than 0.01) between neutrophil count and collagen whole blood platelet aggregation was found. Collagen- and ADP-induced aggregation were significantly higher and lower, respectively, in whole blood than in PRP using the densitometric method. No differences were observed in SPA and collagen platelet aggregation according to age and sex.     

Ability of whole blood aggregometer to detect platelet hyperaggregability

In this study the reliability of platelet aggregation in whole blood (WB) was investigated in clinical conditions associated with thromboembolic complications. Spontaneous (SPA) and collagen-induced platelet aggregation were evaluated both in whole and diluted blood by the impedance method and in platelet-rich plasma (PRP) by the Born aggregometer in 18 healthy subjects, 15 patients with ischemic heart disease (IHD), and 15 patients with insulin-independent diabetes. SPA occurred more often in WB than in PRP, and in WB the occurrence of SPA was significantly more frequent in the patient groups (4 of 15 patients with IHD and 6 of 15 diabetic patients) than in the controls (1 of 18). WB aggregation induced by collagen was significantly higher in patients with IHD and in diabetic patients than in controls (P less than 0.01), whereas diluted WB and PRP aggregation were not statistically different from controls either in patients with IHD or in diabetic patients. WB aggregation values were found to be related, although not very closely, to megathrombocyte count (r = 0.31, P less than 0.05) whereas not at all to platelet count or hematocrit. No relationship was observed between WB aggregation and disease severity (angiographic lesions and number of ischemic attacks) in patients with IHD and between WB aggregation and HbAlc values in diabetic patients.     

Platelet aggregation in platelet-rich plasma and whole blood in 120 patients with myeloproliferative disorders

In vitro platelet aggregation in platelet-rich plasma (PRP) and in whole blood (WB) was assessed in 31 patients with idiopathic myelofibrosis, 32 with essential thrombocytosis, 23 with polycythemia vera, and 34 with chronic myelogenous leukemia. In PRP most subjects showed normal or reduced platelet aggregation, whereas in WB the majority of patients showed increased platelet function. Spontaneous platelet aggregation (SPA) was observed frequently in WB, whereas it was seldom observed in PRP. SPA in WB was inhibited by in vitro addition of aspirin and apyrase, and SPA was only partially dependent on high platelet count because it also occurred in samples with normal platelet content (at variance with 13 subjects with reactive thrombocytosis, in which SPA was observed only in samples with high platelet concentration). Platelets from patients with idiopathic myelofibrosis had the highest tendency to undergo SPA.     

Platelet volume parameters as a diagnostic tool: The influence of anticoagulation and storage conditions on platelet impedance volume

Summary Rapid progress has been made in the design of aperture impedance cell counters, and parameters such as mean platelet volume and platelet distribution width have become routinely available to most physicians. Platelet volume is influenced by both platelet production in the bone marrow and platelet activation or sequestration in the circulation. In thrombocytopenic patients, it is often possible to differentiate between megakaryocytic and amegakaryocytic disease states on the basis of platelet volume analysis. In patients with thrombocytosis, a myeloproliferative disorder may be suspected if the platelet distribution width is high. However, the conditions of sample preparation and storage still give rise to considerable inaccuracy in the determination of platelet volume parameters. In this study, platelet impedance volume was strongly influenced by anticoagulation, storage time, and incubation temperature. Changes in platelet volume were more pronounced in whole blood than in platelet rich plasma. However, mainly large platelets were lost during the preparation of platelet rich plasma. Collecting blood directly into a mixture of citrate and low dose glutaraldehyde stabilized platelet volume for up to 2 h after venipuncture at room temperature. This method reduces platelet volume changes in vitro and is in this respect superior to the usual EDTA blood count or the use of platelet-inhibitory agents.Abbreviations CO₂ Carbon dioxide - EDTA Ethylene diamine tetraacetic acid - GTA Glutaraldehyde - LDH Lactate dehydrogenase - MPV Mean platelet volume - PRP Platelet rich plasma - WB Whole blood     

Monomeric and polymeric collagen-induced platelet aggregation in citrated and heparinized platelet-rich plasma

The physical and chemical properties of Type I bovine collagens were studied in relation to their platelet aggregating activity in citrated and heparinized human platelet-rich plasmas (PRP). Despite close similarities in physical and chemical properties, significant differences were found in platelet aggregating potency between two monomeric atelocollagens. Skin atelocollagen was a potent and corneal atelocollagen was a very weak inducer of platelet aggregation in citrated and heparinized PRP. In a polymeric form, however, corneal atelocollagen was a stronger platelet aggregating agent than monomeric skin acid-soluble or atelocollagen. Removal of the telopeptides altered some of the characteristics of the platelet aggregation induced by monomeric skin collagen. The rate and maximum extent of aggregation were the same with skin acid-soluble (intact) and atelocollagens in either type of PRP, but the lag periods and aggregation times were longer in citrated and somewhat shorter in heparinized PRP with skin atelocollagen than with acid-soluble collagens. The possible mechanisms leading to the differences observed in platelet aggregating activity of collagens in different physical states and from different tissues, and their distinct platelet aggregation patterns in differently anticoagulated PRP, are discussed.     

Optimized preparation method of platelet-concentrated plasma and noncoagulating platelet-derived factor concentrates: maximization of platelet concentration and removal of fibrinogen

Platelet-rich plasma (PRP) has been clinically used as an easily prepared growth factor cocktail that can promote wound healing, angiogenesis, and tissue remodeling. However, the therapeutic effects of PRP are still controversial, due partly to the lack of optimized and standardized preparation protocols. We used whole blood (WB) samples to optimize the preparation protocols for PRP, white blood cell-containing (W-PRP), platelet-concentrated plasma (PCP), and noncoagulating platelet-derived factor concentrate (PFC). PRP and W-PRP were most efficiently collected by 10 min centrifugation in a 15-mL conical tube at 230-270 g and 70 g, respectively. To prepare PCP, platelets were precipitated by centrifugation of PRP at >2300 g, 90% of supernatant plasma was removed, and the platelets were resuspended. For preparation of noncoagulating PFC, the supernatant was replaced with one-tenth volume of saline, followed by platelet activation with thrombin. Platelet (before activation) and platelet-derived growth factor (PDGF)-BB (after activation) concentrations in PCP were approximately 20 times greater than those in WB, whereas PFC contained a 20-times greater concentration of platelets before platelet activation and a 50-times greater concentration of PDGF-BB without formation of a fibrin gel after platelet activation than WB. Surprisingly, total PDGF-BB content in the PFC was twice that of activated WB, which suggested that a substantial portion of the PDGF-BB became trapped in the fibrin glue, and replacement of plasma with saline is crucial for maximization of platelet-derived factors. As an anticoagulant, ethylene di-amine tetra-acetic acid disodium inhibited platelet aggregation more efficiently than acid citrate dextrose solution, resulting in higher nonaggregated platelet yield and final PDGF-BB content. These results increase our understanding of how to optimize and standardize preparation of platelet-derived factors at maximum concentrations.     

Comparison of platelet aggregation and adenosine triphosphate secretion in whole blood and platelet-rich plasma from normal dogs

Platelet aggregation and adenosine triphosphate (ATP) secretion were measured in whole blood and platelet-rich plasma (PRP) from normal dogs using electrical impedance and turbidimetric techniques. General appearance of the aggregation curves, ATP secretion, and aggregation rate were similar in PRP in response to adenosine diphosphate (ADP) or collagen using both techniques. In response to ADP, aggregation was detected sooner while the maximum aggregation response decreased at a relatively greater rate with decreasing agonist concentrations using the turbidimetric technique. Shape change was consistently detected using the turbidimetric, but not the impedance, technique. Using electrical impedance, there were differences between whole blood and PRP in aggregation and ATP secretion responses which depended on the agonise used to activate the platelets. In response to ADP, aggregation responses were lower in whole blood relative to PRP. In response to platelet-activating factor (PAF), maximum aggregation was greater in whole blood while aggregation rate and ATP secretion were greater in PRP. In response to collagen, aggregation response and ATP secretion were lower in PRP. Dilution of PRP with buffer instead of platelet-poor plasma (PPP) lessened many of the differences between whole blood and PRP samples. These findings suggest that plasma constituents and blood cells other than platelets affect aggregation and secretion in an agonist-dependent manner.     

Attempts for aspirin monitoring with a new assay system, Ultegra Rapid Platelet Function Assay (RPFA), based on turbidimetric platelet agglutination of whole blood samples

Platelet aggregation measured by the optical density method has been applied for the assessment of platelet functions. However, as the method has to use platelet-rich plasma, it requires centrifugation of blood samples, which takes a considerable period of time. Using whole blood as samples has advantage because there is no pre-treatment of samples before measurement of platelet functions. Additionally, it would be desirable to have a bedside assay that reflects hyper-function of platelets and can monitor inhibitory effects of anti-platelet drugs. Rapid Platelet Function Assay (RPFA) is a qualitative test to aid the detection of platelet dysfunction due to anti-platelet drug ingestion, which uses citrated whole blood for sample in point of care or laboratory settings. The RPFA is a turbidimetric analysis, based on an optical detection system which measures platelet agglutination as an increase in light transmission. The aim of this study is to assess the accuracy and reproducibility of RPFA and to determine whether RPFA can monitor the effects of anti-platelet drugs. During the first 30 minutes after blood collection, Aspirin Reaction Units (ARU) determined with RPFA gradually increased, and reached its plateau after 30 minutes. The ARU values remained almost constant thereafter until 6 hours after blood collection (Fig. 3). These findings suggest that platelet function is unstable immediately after blood collection. Therefore, in this study ARU measurement was performed 60 minutes after blood collection. The reproducibility of ARU is very good both before and after aspirin intake. Seven days after daily uptake aspirin, ARU was decreased as compared with the control (440.8 +/- 39.4 vs. 663.4 +/- 2.4 ARU). RPFA measurement provides rapid information on platelet function that mirrors turbidimetric platelet agglutination and reflects COX1-depedent platelet activity.     

The effect of thrombocytopenia on the determination of platelet aggregation.

Platelet aggregation has been widely assumed to be unmeasurable in thrombocytopenic samples. Using a sensitive differential self differential self-calibrating aggregometer, and standard aggregating agents, aggregation was measured in serially diluted platelet-rich plasma. Aggregation induced by ADP or collagen was reproducible at platelet counts as low as 50,000 per cu. mm., and with epinephrine aggregation was reproducible at platelet counts as low as 75,000 per cu. mm.     

Platelet thrombopathy in asthmatic patients with elevated immunoglobulin E

Abnormalities of second-wave platelet aggregation were demonstrated in 17 of 33 asthmatic patients in whom drug and diet intake were controlled in the hospital. Mean abnormal responses were significantly greater after epinephrine- (p < 0.001), adenosine diphosphate-(<0.001), collagen- (p = 0.01), and thrombin- (p < 0.001) induced platelet aggregation in patients with immunologically mediated asthma and serum IgE levels >250 U/ml as compared to patients without immunologic factors and/or normal controls. Mean pollen-specific radioallergosorbent (RAST) binding was also significantly higher in patients with abnormal aggregation as compared to normal platelet responders (p = 0.02). Release of serotonin generally reflected abnormal aggregation patterns in asthmatic patients. Platelet factor 4 release was significantly decreased in the same groups of patients. These results suggest that the allergic state may affect platelet membrane responsiveness to multiple aggregating agents.     

Current status and problems of platelet function tests]

Platelet function tests have been utilized for a long time as a useful tool for the diagnosis of qualitative platelet disorders. However, recently reports suggest that platelet function tests currently available in routine laboratory do not necessarily reflect in vivo hemostatic states. First of all, bleeding time could sometimes be non-informative for the following reasons; 1) Duke method, the most popular method in Japan, has poor reproducibility, 2) there is no appropriate method for monitoring of aspirin ingestion, 3) prolongation of bleeding time does not correlate with the amount of blood loss during surgery. Platelet adhesion is still measured by the ability of platelets to be retained on glass beads. However, this test is unable to detect selectively platelet adhesion. Thus, the test in which platelets can exclusively adhere to subendothelial components such as collagen, may be required. Platelet aggregation has been mostly analyzed by the changes in light transmittance in platelet rich plasma (PRP) with a platelet aggregometer. However, this test totally depends on the platelet count in PRP or duration of time after taking blood from patients. Moreover, platelet aggregation in this system is optimally observed and is hardly detectable when the in vivo Ca++ concentration has been chelated. Furthermore, the test could not detect small platelet aggregates, thus being unable to measure the early phase of platelet aggregation. These observations suggest that more simple and specific tests should be developed and become available in routine laboratory.     

Interaction between platelet-aggregating response and vasoconstrictive response to platelet activating factor.

It is very important to know under medical treatment which kinds of platelet agonists participate in abnormal platelet-blood vessel interactions. The present study, focusing on platelet activating factor (PAF) was undertaken in an attempt to investigate its action on platelet aggregating response and vasocontractile response to noradrenaline (NA-R). We used autologous platelets and isolated perfused arterial segments from Japanese white rabbits. Firstly, typical tracings of platelet aggregating response to PAF were increased in a dose-dependent fashion, which remained constant and long-lasting. Secondly, noradrenaline (NA) at 5 to 25 ng elicited an initially augmented response in the presence of platelet rich plasma (PRP) with PAF, followed by gradually attenuated responses. Based on the light transmission intensity, platelet aggregation did not seem to be directly or strictly linked to vasocontractile response. Pretreatment with either dibutyryl cyclic AMP (DBcAMP) or indomethacin (IM, a cyclo-oxygenase inhibitor) clearly caused reductions in NA-R as well as platelet aggregation in the presence of PRP with collagen, whereas platelet aggregation and NA-R in the presence of PRP with PAF were scarcely influenced by pretreatment with either DBcAMP or IM. Thus, it seems reasonable to conclude that, in contrast to the response to collagen, platelet aggregation response to PAF was almost indifferent to the adenylate cyclase-cyclic AMP system and the cyclo-oxygenase metabolic pathway.     

Spontaneous platelet aggregation in a hereditary giant platelet syndrome (MPS)

The characteristics of spontaneous platelet aggregation (SPA) in a hereditary giant platelet syndrome (Montreal platelet syndrome, MPS) are examined. SPA was quantitated by microscopy from the decrease in single platelets in platelet-rich plasma (PRP). In contrast to normal donors, a significant proportion (20-50%) of platelets in MPS whole blood and PRP occurred in microaggregates typically containing 2-6 disk-shaped platelets. Stirring MPS-PRP at 1000 rpm for 10 minutes further increased the fraction of platelets in aggregates by 10-170%, the percentage increase not being correlated to the donor's platelet count (5000-220,000 microliters-1). Normal platelets resuspended in MPS platelet-poor plasma (PPP) did not undergo SPA, whereas MPS platelets resuspended in normal PPP or Ca2+-free, fibrinogen-free Tyrode's continued to show SPA. The increase in SPA could be inhibited by 10 microM prostaglandin (PG) E1, 150 mM ASA or glutaraldehyde or formaldehyde fixation; however, it was not inhibited by 10 nM PGI2 and was only partially inhibited by 1 microM 2-chloroadenosine and 1-10 units/ml apyrase. SPA in Acid-citrate-dextrose-PRP was much less than in PRP; however, SPA reoccurred on returning the platelets to platelet-free plasma or Tyrode's. Platelet aggregation (PA) could be increased over that due to SPA alone by the addition of adenosine diphosphate, adrenaline, collagen, ionophore A-23187, arachidonic acid and ristocetin, with results suggesting that the response to these agents is normal. The ristocetin-induced increase in PA was completely blocked by an IgG specific for Bernard-Soulier syndrome. In contrast, MPS platelets had a reduced sensitivity to thrombin, which appeared to be more pronounced at low platelet counts. There was no correlation between the thrombin insensitivity and the extent of SPA. Total adenosine triphosphate (ATP) and thrombin-induced release of ATP and platelet factor 4 appeared normal for MPS platelets. The ultrastructural features of MPS platelets were within normal limits except for an increased frequency of giant granules. SPA was observed for 5/5 MPS donors, but only one of three MPS donors' platelets evaluated for glycoprotein I and sialic acid content showed any measurable reduction as compared with normal controls. The above observations point to the existence of an as yet undetermined anomaly of MPS plasma membrane related to a fibrinogen and Ca2+ independent form of platelet aggregation.     

Use of native or platelet count adjusted platelet rich plasma for platelet aggregation measurements

BACKGROUND: It is still not clear whether native or platelet count adjusted platelet rich plasma (PRP) should be used for platelet aggregation measurements.AIM: To evaluate the necessity of using adjusted PRP in platelet function testing. METHODS: Platelet aggregation with native PRP and adjusted PRP (platelet count: 250/nl, obtained by diluting native PRP with platelet poor plasma) was performed on the Behring Coagulation Timer (BCT(R)) using ADP, collagen, and arachidonic acid as agonists. Healthy subjects, patients on antiplatelet treatment, and patients with thrombocytosis (platelet counts in PRP > 1250/nl) were investigated. RESULTS: No significant differences in the maximum aggregation response were seen when using either native or adjusted PRP from healthy subjects and patients on antiplatelet treatment. Nevertheless, some patients taking aspirin or clopidogrel showed reduced inhibition of ADP and arachidonic acid induced aggregation in adjusted PRP but not in native PRP. The maximum velocity of healthy subjects and patients on antiplatelet treatment varied significantly as a result of the degree of dilution of the adjusted PRP. Surprisingly, the BCT provided good results when measuring platelet aggregation of native PRP from patients with thrombocytosis, whereas commonly used aggregometers could not analyse platelet aggregation of native PRP in these patients. CONCLUSION: The time consuming process of PRP adjustment may not be necessary for platelet aggregation measurements. Moreover, using adjusted PRP for monitoring aspirin or clopidogrel treatment may falsify results. Therefore, it may be better to use native PRP for platelet aggregation measurements, even in patients with thrombocytosis.     

Platelet aggregation in atopic and normal patients

Platelet aggregation was used as a model to evaluate the proposed beta blockade in subjects with allergic rhinitis and asthma. Studies using epinephrine, adenosine diphosphate (ADP), and streptococcal M protein as aggregating agents showed that there was no significant difference in platelet aggregation between atopics and controls. Furthermore, aggregation induced by all three agents was inhibited by propranolol and phentolamine, as well as by caffeine and dibutyryl cyclic adenosine monophosphate (AMP). This inhibition was evidenced with platelets from both atopics and controls. The results suggest that with platelet aggregation the atopic population taken as a whole does not differ from the normal population. Although certain atopic subjects exhibit unique aggregation patterns, pharmacologic manipulation of this system fails to document that these unique patterns are due to beta blockade.     

Disaggregation and reaggregation of ‘irreversibly’ aggregated platelets: A method for more complete evaluation of anti-platelet drugs

Anti-platelet drugs are generally screened by evaluating their ability to influence initiation and development of irreversible aggregation of platelets. However, to fully characterize the inhibitory effects of an agent, it is essential to determine if it can also disperse irreversibly aggregated cells, whether drug-treated, dispersed platelets are refractory, sensitive to some but not all aggregating agents, as sensitive to all agents as before initial exposure, hypersensitive or whether they can be restored to a sensitive state by modulation of the platelet membrane. We have developed an_vitro system for evaluating the influence of a variety of agents on disaggregation and reaggregation of aggregated blood platelets. Results demonstrate that some agents which inhibit aggregation can also cause disaggregation, while others cannot. The ability to disaggregate is often selective, revealing a dependence on the nature of the agent causing aggregation, and the time after irreversible aggregation that the inhibitor is added. Agents that elevate intracellular cyclic adenosine monophosphate levels were potent inducers of platelet dissociation in aggregates caused by adenosine diphosphate. On the other hand, antimalarials, calmodulin complexing agents and phospholipase inhibitors caused dissociation of platelet aggregates, irrespective of the agonist used. By and large, the dissociated cells were refractory to the action of agonists. Restoration of the sensitivity of disaggregated platelets by treatment with epinephrine demonstrate an ability of inhibitor treated, refractory platelets to recover full functional capacity almost immediately. Thus, careful study of the effects of inhibitors on disaggregation, recovery and reaggregation may reveal features critical to the selection of anti-platelet drugs for clinical utilization.     

Platelet aggregation by laminar shear and Brownian motion

Rates of platelet aggregation in citrated PRP were measured under conditions of a well-defined Couette shear flow at 25°C. Smoluchowski's coagulation theories for Brownian motion and for simultaneous Brownian and shear motion were combined with the Rayleigh-Gans theory of light scattering to measure Brownian and shear collision efficiency factors for systems of aggregating platelets. This approach permits a considerably more systematic and readily interpretable data base for analyzing normal and abnormal platelet aggregation phenomena than was heretofore possible. This technique was also used to investigate the effects of varying ADP and PGE₁ concentrations on platelet aggregation rates. Slight enhancement of platelet aggregation was observed in PRP suspensions in which red cell ghosts were added.     

Function of human platelets during extracorporeal circulation.

The interaction between human platelets and nonbiologic surfaces was studied during in vitro recirculation of 500 ml of fresh, heparinized human blood in four different perfusion circuits. Circuits differed in surface area (0.1 m2 or 0.9 m2) and in surface composition. No important differences were observed between standard silicone-rubber and filler-free, silicone-rubber surfaces. Platelet counts decreased to 85% of control in 0.1- m2 circuits, but retained normal sensitivity to aggregating agents and released only small amounts of platelet factor 4 (PF4). In contrast, platelet counts in 0.9-m2 circuits decreased to 20% of control within 2 min and platelet sensitivity was depressed out of proportion to the fall in platelet count. Plasma PF4 progressively increased and platelet PF4 content progressively decreased during 6 h of recirculation. The results indicate that human platelets may exist in three conditions during extracorporeal circulation. Some platelets are unaltered, some are less sensitive to aggregating agents, and others have undergone extensive release. The ratio of blood volume to surface area appears to be an important determinant of platelet-surface interaction.     

富血小板血浆中血小板浓度对血小板聚集的影响

目的探讨富血小板血浆（PRP）血小板浓度对血小板聚集的影响。方法随机收集107名门诊体检健康志愿者静脉血浆,分离PRP和PPP（贫血小板血浆）后,用血液分析仪测定PRP血小板浓度,按PRP浓度用自身PPP配成50×109/L～400×109/L浓度梯度的血浆,用血浆比浊法测血小板聚集率。结果 PRP血小板浓度在50×109/L～400×109/L,二磷酸腺苷（ADP）和花生四烯酸（AA）诱导的血小板聚集率分别为（10.4±8.7）%至（55.3±9.9）%和0%至（75.2±10.1）%,血小板聚集率随血小板浓度的增高而增高,血小板浓度为250×109/L时,聚集率增幅减缓,血小板浓度小于150×109/L时,血小板聚集率明显降低。结论 PRP血小板浓度对血小板聚集的影响明显,血小板聚集试验PRP血小板浓度大于150×109/L;血浆比浊法血小板聚集试验适宜的血小板浓度为250×109/L。     

Endothelin does not affect aggregation of human platelets

Endothelin (ET-1) is a recently discovered endothelial-derived peptide with pronounced vasoconstrictor activity. The present study addressed whether ET-1, in analogy with several other vasoactive agents, can induce or modulate aggregation of human platelets in vitro. Venous blood from healthy donors was collected in citrate or heparin and platelet-rich plasma (PRP) was prepared. Portions of the PRP were added to drugs, and platelet aggregation was recorded according to Born & Cross (1963). ET-1 added to the PRP (final concentrations 1-100 nM) did not induce aggregation of platelets, either in citrate- or heparin-containing plasma. Adenosine-diphosphate (0.5-2 microM) or thrombin (0.1-0.4 NIH units ml-1) induced dose-dependent aggregation of platelets in citrate- or heparin-containing PRP; such aggregation was, however, not affected by ET-1 (1-100 microM) either. We conclude that ET-1, in contrast to other endothelial-derived vasoactive agents, lacks direct effect on platelet aggregation in vitro.