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PubMed2005

Agonists of toll-like receptor (TLR)2 and TLR4 are unable to modulate platelet activation by adenosine diphosphate and platelet activating factor.

Inappropriate platelet activation is a feature of acute and chronic diseases such as disseminated intravascular coagulation (DIC) and atherosclerosis. Since proinflammatory microbial-derived agonists can be involved in the pathogenesis of these diseases, we examined the potential role of TLR4 (mediating responses to LPS) and TLR2 (which responds to bacterial lipopeptides) in platelet activation. Our data suggested low-level expression of TLR2 and TLR4 on platelets, determined by flow cytometry, and we also observed expression of TLR4 on a megakaryocytic cell line by both flow cytometry and immunohistochemistry. Stimulation of the platelets with the TLR4 agonist LPS, and the synthetic TLR2 agonist Pam3CSK4, resulted in no platelet aggregation, no increase in CD62P surface expression and no increase in the cytosolic concentration of Ca2+. The TLR agonists were also unable to directly activate platelets primed with epinephrine, or pretreated with a low concentration of ADP or PAF. Pretreatment of platelets with LPS or Pam3CSK4 also failed to modulate the platelet response to submaximal concentrations of the classical platelet agonists ADP and PAF. We conclude that the TLR agonists LPS and Pam3CSK4 have no direct effect on platelet activation and that platelet TLRs may be a remnant from megakaryocytes. TLR2 and TLR4 agonists are thought to have a significant role in diseases such as atherosclerosis and DIC, but our research suggests that this is through a mechanism other than direct platelet activation or by modification of platelet responses to other agonists.

Adenosine Diphosphate↗

PubMed1997

Evaluation of platelet activation and platelet-neutrophil aggregates in ponies with alimentary laminitis.

OBJECTIVES: To determine whether platelets are hyperaggregable or form platelet-neutrophil aggregates during the prodromal stages of acute laminitis of ponies. ANIMALS: Healthy adult ponies: 8 experimental and 6 control. PROCEDURES: Acute laminitis was induced by oral administration of corn starch and wood flour to 8 ponies, and indices of platelet activation were evaluated. Blood samples were collected before and at 4, 8, 12, 24, 28, and 32 hours after carbohydrate administration, and PCV, total plasma protein concentration, platelet count, activated clotting time, whole blood recalcification time, spontaneous platelet aggregation, ex vivo platelet aggregation responses, and platelet-neutrophil aggregates were determined. When lameness was first detected, ponies were euthanatized and arteriography and histologic examination of hooves were performed. RESULTS: Carbohydrate overload was associated with hyperaggregability of platelets throughout the prodromal stages of laminitis and increased numbers of platelet-neutrophil aggregates. Reduction of blood supply to affected hooves was variable, and blood clots were found in 6 of 11 laminitis-affected hooves. CONCLUSIONS: Platelets were hyperaggregable throughout the prodromal stages of carbohydrate-induced laminitis and formed platelet-neutrophil aggregates. Platelet-neutrophil aggregates may initiate or contribute to development of acute laminitis. CLINICAL RELEVANCE: Anti-platelet therapy may be useful for treatment of acute alimentary laminitis in horses.

Acute Disease↗

PubMed2006

Releasing growth factors from activated human platelets after chitosan stimulation: a possible bio-material for platelet-rich plasma preparation.

INTRODUCTION: Thrombin is commonly used for activating the platelets and releasing the growth factors on the application of platelet-rich plasma (PRP). We have previously reported that chitosan can enhance rabbit platelet aggregation. In this study, the effects of chitosan on the subsequent growth factors release after human platelets activation were examined to evaluate the possibility of chitosan being used as a substitute for thrombin during PRP preparation. MATERIAL AND METHODS: Human platelet activation was determined by aggregation, adhesion and alpha-granule membrane glycoprotein expression. Platelet aggregation was measured by the turbidimetric method, the adhesion was directly examined on chitosan-coated glass plates under light microscope and scanning electron microscope (SEM), and the alpha-granule membrane glycoprotein was detected by fluorescent isothiocyanate (FITC)-conjugated anti-CD61 antibody through flow cytometry. The subsequent epidermal growth factor (EGF), platelet-derived growth factor (PDGF)-AB and transforming growth factor (TGF)-beta1 release from platelets were assayed by ELISA after mixing with chitosan. RESULTS: The enhancing effects on the platelet adhesion and the aggregation from chitosan were observed. Under both microscopes, the adhesive platelets on the chitosan-coated plates were not only greater in number but also earlier in activation than those on the control plates. With flow cytometry, increased glycoprotein IIIa expression in platelets was detected after chitosan treatment. Greater concentrations of growth factors were measured from PRP after chitosan treatment than after the solvent treatment. CONCLUSION: Because of the observations of growth factors releasing from activated human platelets after chitosan stimulation, we suggest that chitosan may be an appropriate substitute for thrombin in PRP preparation.

Biocompatible Materials↗

PubMed1990

The inhibition of platelet-activating factor-induced platelet activation by oleic acid is associated with a decrease in polyphosphoinositide metabolism.

In an earlier study (Miwa, M., Hill, C., Kumar, R., Sugatani, J., Olson, M. S., and Hanahan, D. J. (1987) J. Biol. Chem. 262, 527-530) it was shown that an inhibitor of platelet-activating factor (PAF), a powerful endogenous mediator of platelet aggregation, was present in freeze-clamped perfused livers. Subsequently, we determined that this substance was a mixture of unsaturated free fatty acids (FFA). Among these FFA, oleic acid between 10 and 100 microM was found to be a potent inhibitor of PAF-induced platelet aggregation and serotonin secretion. Consequently, in order to understand the molecular mechanism of oleic acid action, we investigated the effects of this FFA on several biochemical events associated with platelet aggregation induced by PAF. The effect of oleic acid and/or PAF on the level of [32P]phosphatidylinositol 4-phosphate (PIP) and [32P]phosphatidylinositol 4,5-bisphosphate (PIP2) was examined by using platelets labeled with [32P]phosphate. Oleic acid induced a dose-dependent decrease in the levels of [32P]PIP and [32P]PIP2; a maximal decrease in [32P]PIP and [32P]PIP2 of approximately 50 and 25%, respectively, was observed within seconds after the addition of 20 microM oleic acid and persisted for at least 15 min. Oleic acid did not induce the formation of [3H]inositol phosphates in platelets prelabeled with [3H]inositol, suggesting that the decrease in [32P]PIP and [32P]PIP2 was not due to a stimulation of phospholipase C. In contrast to oleic acid, PAF induced a dose-dependent increase in the [32P]PIP level, reaching a maximum of approximately 200% 3 min after the addition of 1 nM PAF to the platelets. This increase in [32P]PIP was accompanied by platelet aggregation and secretion, and a close correlation was established between the [32P]PIP level and the degree of aggregation. Oleic acid and PAF, when added together to the platelets, interacted by affecting the level of [32P]PIP and [32P]PIP2 in an opposite way since the decrease in the level of [32P]PIP and [32P] PIP2 induced by oleic acid was partially reversed by an excess of PAF. The decrease in the levels of [32P] PIP and [32P]PIP2 caused by oleic acid was associated with an inhibition of platelet aggregation induced by PAF. Interestingly, oleic acid did not block [3H]PAF binding to platelets but inhibited the PAF-induced phosphorylation of platelet proteins of 20 kDa and 40 kDa. These results suggest that inhibition of the PAF response by oleic acid may be at one of the steps in the signal transduction.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1982

Platelet lipoxygenase-dependent oxygen burst. Evidence for differential activation of lipoxygenase in intact and disrupted human platelets.

The metabolism of arachidonic acid in platelets by both cyclooxygenase and lipoxygenase involves the rapid consumption of molecular oxygen. However, selective inhibition of cyclooxygenase completely abolishes the arachidonate-induced oxygen burst in intact platelets. This is in contrast to platelet lysates, in which approximately 50% of the arachidonate-induced oxygen burst remains detectable following inhibition of cyclooxygenase with acetylsalicylic acid. This lipoxygenase oxygen burst is blocked by preincubation of the platelets with ETYA, which inhibits both cyclooxygenase and lipoxygenase. In cell-free 100000 x g supernatants of platelet lysates, which contain only lipoxygenase activity, arachidonate induces an oxygen burst which is not blunted by preincubation with aspirin but is completely abolished by preincubation with ETYA. The finding of a lipoxygenase-dependent oxygen burst in platelet lysates but not in intact platelet suspensions suggests differential activation or differential availability of platelet lipoxygenase in intact and disrupted platelets. This was confirmed by a 5 min lag in the generation of [14C]HETE (the major lipoxygenase product) from [14C]arachidonic acid in intact platelets, but an almost immediate initiation of [14C]HETE production in platelet lysates. In contrast, the synthesis of [14C]thromboxane B2 (the major cyclooxygenase product) from [14C]arachidonic acid began immediately in both intact and disrupted platelet preparations and peaked within 5 min. These observations provide new insight into factors controlling platelet hydroxy acid production and help to explain the nature of the platelet oxygen burst.

Arachidonic Acid↗

PubMed1996

Alterations in individual molecular species of human platelet phospholipids during thrombin stimulation: electrospray ionization mass spectrometry-facilitated identification of the boundary conditions for the magnitude and selectivity of thrombin-induced platelet phospholipid hydrolysis.

Although the rapid thrombin-induced release of arachidonic acid in human platelets has been known for over 20 years, the amount of arachidonic acid mass mobilized and the source of the released arachidonic acid has remained a subject of intense controversy. Herein, we exploit the analytic power and sensitivity of electrospray ionization mass spectrometry to identify plasmenylethanolamines as the largest source of arachidonic acid mass released during thrombin stimulation and to demonstrate the presence of multiple novel molecular species of plasmenylethanolamines in human platelets. Specifically, 90 s after thrombin stimulation a total of 60.1 nmol of arachidonic acid-containing phospholipids/10(9) platelets was hydrolyzed which included the loss of 31.8 nmol/10(9) platelets from ethanolamine glycerophospholipids (hydrolysis of plasmenylethanolamines represented 63% of the mass lost from the ethanolamine glycerophospholipid pool) but only 10.9 nmol/10(9) platelets from choline glycerophospholipids. Human platelet phosphatidylserine and phosphatidylinositol pools contained similar amounts of arachidonic acid mass in resting platelets (approximately equal to 20 nmol/10(9) platelets), and each pool contributed 8.7 nmol/10(9) platelets after thrombin stimulation. From these results, a lower boundary for the rate of thrombin-induced arachidonic acid mobilization in human platelets can be set at > 60 nmol/10(9) platelets, thereby identifying specific kinetic characteristics and substrate selectivities of the phospholipase(s) activated during platelet stimulation. Collectively, these results underscore the importance of plasmenylethanolamines as the major storage depot of arachidonic acid in resting platelets and as the major source of arachidonic acid mobilized after thrombin stimulation of human platelets.

Blood Platelets↗

PubMedDate not supplied

Enhancement of the indirect anti-platelet antibody test and its application to platelet immunology.

Comparisons in the same patients of platelet-bound anti-platelet antibody (APA) levels (direct test) with serum APA levels (indirect test) frequently do not give the same results. Indirect test results frequently are negative or marginally elevated even though platelet-bound antibody is greatly increased on direct testing. The most likely cause for these differences between the two tests is insufficient binding of serum antibody to test platelets in vitro. In an effort to enhance in vitro antibody binding, we examined four test modifications affecting platelet attachment. Rabbit anti-human platelet antisera (absorbed with their specific human donor leukocytes to remove HLA- and granulocyte-specific antibodies) were combined under various conditions with human platelets and tested in a modified antiglobulin consumption test utilizing rabbit IgG. The test conditions varied: incubation time of the serum APA-platelet mixture at 37 degrees C; age of the test platelet pool; ionic strength of the mixture (varied through incorporation of a low-ionic-strength solution (LISS); and vortex agitation for 30 seconds prior to the addition of sera. Optimal attachment in standard phosphate-buffered saline took place in 60 minutes using test platelets stored for either 3 or 30 days, and yielded mean values for rabbit immune sera of 9.9 fg of IgG per platelet. Preincubation controls yielded a mean of 0.7 to 1.5 fg per platelet. Incorporation of LISS or vortexing of test platelets did not affect the results of control, but both modifications substantially increased test values of immune sera to 15.8 and 17.3 fg of IgG per platelet, respectively. Combining LISS with vortexing did not further increase values.

Animals↗

PubMed1991

The in vivo kinetics of 111In- and 51Cr-labelled platelets: a comparative study using both stored and fresh platelets.

The in vivo kinetics of simultaneously injected 51Cr- and 111In-labelled platelets was investigated in 20 healthy male volunteers. The studies were carried out using both fresh platelets and platelets stored for 5 d at 22 degrees C; the disappearance of platelet-bound radioactivity was measured on whole blood samples as well as platelet pellets. Compared to 111In, the labelling efficiency was notably lower for 51Cr, and a higher amount of 51Cr was bound to contaminating red cells. As regards the fresh platelets, only small dissimilarities were observed in the in vivo kinetics obtained with the two labels. 51Cr yielded a slightly higher platelet recovery and longer T1/2 than 111In, when whole blood samples were used for calculations; no differences were seen when using platelet pellets. When stored platelets were studied, 51Cr gave significantly longer T1/2 and mean life-span (MLS) than did 111In. This difference was present for all mathematical models used for the calculation of MLS, and when whole blood samples as well as platelet pellets were employed. It was demonstrated that the estimation of MLS was also highly dependent upon techniques of blood sampling and curve fitting. Calculation, in which the initial data points were excluded, gave consistently longer MLS (P less than 0.0001), as compared to when all data points were included. Furthermore, when all survival studies were grouped together, calculations using platelet pellets gave a significantly (P less than 0.001) shorter platelet MLS than calculations using whole blood samples. It is concluded that both 51Cr and 111In are acceptable as radiolabels for both fresh and stored platelets. However, it appears that the viability of stored platelets may be influenced by the choice of label, and caution must be taken when these isotopes are used together in dual tracer experiments. Also, our results show that a meticulously standardized processing of blood samples and experimental data is required to enable interlaboratory comparisons of the results.

Adult↗

PubMed1993

Elevated platelet-associated IgG in SLE patients due to anti-platelet autoantibody: differentiation between autoantibodies and immune complexes by ether elution.

The level of platelet-associated IgG (PAIgG) is reported to be elevated in patients with systemic lupus erythematosus (SLE). However, the nature of PAIgG is unclear. We have investigated whether the PAIgG of SLE consists of anti-platelet autoantibodies or immune complexes (IC). The PAIgG values measured by flow cytometry were elevated in 11/25 patients with SLE. 3/6 SLE patients with thrombocytopenia had a high level of PAIgG (the mean fluorescence intensity > 10). We used an ether elution technique to determine whether elevated PAIgG consists of anti-platelet antibodies or IC. Preliminary experiments showed that the eluates prepared from platelets sensitized with anti-HPA-4a antibody reacted with normal platelets, while the eluates prepared from platelets sensitized with heat-aggregated IgG or model IC failed to react with normal platelets. These results indicate that the reactivity of eluates can distinguish between platelet-bound antibody and IC. We applied this technique to analysis of the PAIgG of SLE platelets. The eluates from SLE platelets (the mean fluorescence intensity > 10) reacted with normal platelets, indicating that the PAIgG of SLE platelets has the nature of antiplatelet autoantibodies. Furthermore, we investigated the target antigens which bind PAIgGs of SLE, using the direct immunoprecipitation procedure and modified antigen capture ELISA (MACE). Both methods identified GPIIb/IIIa as the target antigens. We conclude that the ether elution technique can distinguish between anti-platelet antibodies and IC, and that the PAIgGs of SLE with a high PAIgG value and thrombocytopenia have the nature of anti-platelet autoantibodies.

Antigen-Antibody Complex↗

PubMed1995

Platelet-induced neutrophil activation: platelet-expressed fibrinogen induces the oxidative burst in neutrophils by an interaction with CD11C/CD18.

Mutual contacts and platelet-expressed fibrinogen seem to be required for the stimulation of neutrophils by activated platelets. The beta 2-integrins CD11b/CD18 and CD11c/CD18 are potential receptors for fibrinogen on neutrophils. In order to investigate whether binding of fibrinogen to these integrins is involved, monoclonal antibodies (MoAbs) and Gly-Pro-Arg-Pro (GPRP) peptide that inhibits fibrinogen binding to CD11c/CD18 were checked for their effects on the interaction of activated platelets and neutrophils. The luminol-amplified chemiluminescence (CL) as a measure for the oxidative burst of neutrophils was recorded simultaneously to the platelet aggregation in mixed cell suspensions. The adhesion of platelets and neutrophils was determined microscopically. The thromboxane A2 mimetic U46619 was used as a potent platelet agonist but that does not stimulate neutrophils. aggregation and a strong CL of neutrophils. The platelet-induced activation of neutrophils required added fibrinogen which fibronectin or thrombospondin could not substitute for. Cytochalasin D (Cyto D) that blocks actin polymerization totally abrogated the platelet-induced Cl of neutrophils. The MoAb OKM1 against CD11b, which blocks fibrinogen binding to CD11b/CD18 as well as the MoAbs IOT16 and IOT18 directed against CD11a and CD18, respectively, had no effect. In contrast, the MoAb LeuM5 which inhibits the binding of fibrinogen to CD11c/CD18 revealed a strong inhibition. Furthermore, GPRP peptide which CD11c/CD18 recognizes on the A alpha-chain of fibrinogen also strongly inhibited the platelet-induced CL of neutrophils, whereas control peptides such as Gly-His-Arg-Pro (GHRP) or Gly-Pro-Gly-Gly (GPGG) had no effect. In contrast to the platelet-induced CL of neutrophils, Cyto D, MoAb against CD11c and GPRP peptide did not inhibit the CL induced by FMLP and PAF in pure neutrophil suspensions. They also did not affect U46619-induced platelet aggregation. The adhesion of platelets and neutrophils was neither dependent on added fibrinogen nor inhibited by Cyto D, MoAb against CD11c and the GPRP-peptide. Therefore fibrinogen and actin polymerization seem not to be required for the adhesion of neutrophils to platelets. However, the activation of neutrophils depends on the interaction of CD11c/CD18 with the A alpha-chain of platelet-expressed fibrinogen and the contractile system of neutrophils.

Blood Platelets↗

PubMed1993

IgG inhibits the increase of platelet-associated C3 stimulated by anti-platelet antibodies.

We investigated the increase of platelet-associated IgG and complement component 3 (C3) caused by the in vitro action of anti-platelet MoAbs, and the effect of mouse and human IgG on these events. Anti-glycoprotein IIb/IIIa and anti-glycoprotein Ib MoAbs caused a slight increase of C3, but not of platelet-associated IgG. In contrast, anti-CD9 and anti-Fc gamma II receptor MoAbs caused an increase of both platelet-associated C3 and IgG. In particular, three MoAbs which activated the complement system caused a marked increase of C3. When platelet-rich plasma was treated with aspirin and prostaglandin E1 before incubation with antibodies, the increase of platelet-associated IgG was inhibited in all cases. In contrast, the increase of platelet-associated C3 was scarcely influenced. These results suggest that the binding to platelets of platelet-activating antibodies caused the increased expression of IgG molecules on the platelet surface and a possible increase of platelet-associated IgG. However, the increase of platelet-associated C3 appeared to depend on specific characteristics of the antibodies tested, such as a complement-activating effect. In addition, intact mouse or human IgG inhibited the increase of platelet-associated C3 caused by complement-activating antibodies, while F(ab')2 mouse or human IgG had no such effect. This suggested that the Fc portion of IgG may block the increase of C3 mediated by anti-platelet antibodies.

Animals↗

PubMed1977

On the analysis of platelet survival curves and the calculation of platelet production and destruction.

Platelet survival, platelet production and megakaryocyte quantifications were studied in control subjects and in patients with idiopathic thrombocytopenic purpura (ITP) and primary thrombocythaemia (PT). A platelet death probability function (DPF), which gives the expected probability of a randomly chosen platelet to be destroyed within the nextcoming short time interval, was constructed on the basis of the present knowledge of platelet function and the factors that influence its survival. Starting from this function platelet age and life span distributions and the function for the survival curve were derived. Platelet mean life span (MLS) was in controls 6.9d, in PT 5.2Dd and in ITP 0.44d. Determinations of platelet life span and platelet age distributions revealed a considerable variation in platelet life spans about these means and a great difference in platelet age compositions between the 3 groups of subjects studied. The function for the platelet survival curve, derived from the DPF, was employed in the calculation of platelet production and there was a highly significant relationship between platelet production and megakaryocyte volume per microliter bone marrow giving mutual confirmation to the 2 determinations and to the correctness of the death probability function.

Adolescent↗

PubMed1972

Heterogeneity of human platelets. V. Differences in glycolytic and related enzymes with possible relation to platelet age.

Human platelets were separated by desity-centrifugation into heavy and light populations. Heavy platelets have an average volume approximately twofold greater than light platelets, and have previously been shown to be young platelets. All 11 enzymes of the Embden-Meyerhof pathway plus the five related enzymes: phosphoglucomutase, glucose-6-P dehydrogenase, 6-P-gluconic dehydrogenase, alpha-glycerol-P dehydrogenase, and glutathione reductase (TPNH) were examined in cell lysates from total, heavy, and light platelet populations. Apparent Km for individual enzymes were measured in a total platelet population. Empirical V(max) of the individual enzymes were measured in total, heavy, and light platelet populations. The three apparent rate-limiting enzymes for glycolysis were hexokinase, phosphofructokinase, and glyceraldehyde-3-P dehydrogenase. Heavy platelets contained approximately twofold greater enzyme activity (per gram wet weight) than light platelets for 7 of the 16 enzymes measured: hexokinase, phosphohexoisomerase, phosphofructokinase, glyceraldehyde-3-P dehydrogenase, phosphoglycerokinase, lactic dehydrogenase, and phosphoglucomutase. Heavy platelets also contained 1.9-fold greater reduced glutathione (GSH), 1.7-fold greater DPNH, and 1.2-fold greater TPNH than light platelets. Heavy platelets contained 1.8-fold less lipid peroxidation products (malonyl aldehyde equivalents) than light platelets and were 2.4-fold more resistant to lipid peroxidation catalyzed by 0.1 mM FeCl(3). Sterile incubation of heavy platelets, in vitro for 17 hr, resulted in a significant loss of enzyme activity for the "elevated" seven enzymes when compared with the remainder. Reducing agents such as GSH (0.1 mM), ascorbic acid (0.1 mM), and dithiothreitol (0.01 mM), when added to the incubation mixture, significantly reduced the in vitro loss of activity. In vitro incubation was also associated with a significant loss of GSH and DPNH and a 1.8-fold increase in lipid peroxidation products.

Ascorbic Acid↗

PubMed2006

Lipopolysaccharide from enterohemorrhagic Escherichia coli binds to platelets through TLR4 and CD62 and is detected on circulating platelets in patients with hemolytic uremic syndrome.

This study presents evidence that human platelets bind lipopolysaccharide (LPS) from enterohemorrhagic Escherichia coli (EHEC) through a complex of toll-like receptor 4 (TLR4) and CD62, leading to their activation. TLR4 colocalized with CD62 on the platelet membrane, and the TLR4 specificity of LPS binding to platelets was confirmed using C57BL/10ScN mice lacking Tlr4. Only platelets from TLR4 wild-type mice bound O157LPS in vitro. After in vivo injection, O157LPS bound to platelets from wild-type mice, which had lower platelet counts than did mice lacking TLR4. Mouse experiments confirmed that O157LPS binding to TLR4 is the primary event leading to platelet activation, as shown by CD40L expression, and that CD62 further contributes to this process. Activation of human platelets by EHEC-LPS was demonstrated by expression of the activated GPIIb/IIIa receptor, CD40L, and fibrinogen binding. In perfusion experiments, platelet activation on endothelial cells was TLR4 and CD62 dependent. O157LPS was detected on platelets from 12 of 14 children with EHEC-associated hemolytic uremic syndrome (HUS) and on platelets from 2 children before the development of HUS but not on platelets of EHEC-infected children in whom HUS did not develop (n = 3). These data suggest that O157LPS on platelets might contribute to platelet consumption in HUS.

Adolescent↗

PubMed1991

Hemostasis in Glanzmann's thrombasthenia (GT): GT platelets interfere with the aggregation of normal platelets.

The transfusion requirements for a 6-year-old Glanzmann's thrombasthenia (GT) patient undergoing tonsillectomy and adenoidectomy were studied. Transfusion of pheresed platelets from a single normal donor increased the platelet count by 63 x 10(9)/L but did not correct the bleeding time. Since the ratio of normal:GT platelets in vivo was approximately 1:5, it was possible that GT platelets interfered with the function of normal platelets. To test this hypothesis, mixtures of platelet-rich plasma (PRP) from a normal donor and the patient were studied to determine a ratio of normal:GT platelets that would yield acceptable in vitro aggregation. Normal:GT ratios of 1:4 and 3:2 resulted in 25% and 59% aggregation, respectively. Mixtures of normal and nonfunctional ethylene glycol tetra-acetic acid-treated platelets gave similar results. Aggregates from mixtures of normal and patient platelets were also examined morphologically by light microscopy and were proportional in size to the normal:GT platelet ratio. Transfusion of platelets from the pheresis of four donors increased the patient's platelet count by greater than 300 x 10(9)/L (normal:GT ratio 1:1), produced 53% aggregation, and resulted in satisfactory postoperative hemostasis. The platelet transfusion requirement for this GT patient was much greater than would have been expected in the absence of aggregation-defective platelets.

Antibodies, Monoclonal↗

PubMed1991

Identification of alloimmunized patients: use of radiolabeled allogeneic platelet kinetic measurements and platelet antibody tests.

In a group of stable, nonthrombocytopenic leukemia patients awaiting bone marrow transplantation, results of paired allogeneic radiolabeled platelet kinetic measurements were correlated with the results of several different platelet and lymphocytotoxic antibody tests to determine which parameters could be used to identify patients who were alloimmunized to platelets. Seven patients with acute leukemia who had been transfused during induction therapy were used as the test group, and, as a control group, five untransfused patients with chronic myelogenous leukemia were also studied. Concurrent fibrinogen survival measurements were performed in all patients to assess whether hemostatic factor consumption (ie, disseminated intravascular coagulation) was present. Allogeneic platelet survival measurements were reduced from normal in all 12 study patients. In 8 of 12 patients, fibrinogen and platelet survival measurements were comparably reduced, suggesting disease-related platelet consumption. In four heavily transfused patients with acute leukemia, allogeneic platelet survivals were markedly reduced to less than or equal to 2.1 days, compared with the 3.5- to 7.4-day platelet survival measurements found in the other eight patients. The disproportionately short platelet survivals compared with fibrinogen survival measurements in these four patients, combined with documented positive antibody tests to their donors' platelets in the three patients with evaluable tests, suggested that these patients had become alloimmunized to platelets because of their prior transfusions. There was substantial concordance between the two radiolabeled allogeneic donor platelet survival measurements performed in each of these patients, suggesting that host rather than donor factors have a major influence on transfusion outcome (r = .93, P less than .001). The platelet cross-match tests, using the radiolabeled protein Staph A assay combined with the IgG enzyme-linked immunosorbent assay test, had the best correlation with the posttransfusion recovery and survival of the donors' platelets.

ABO Blood-Group System↗

PubMed1988

5HT-kinetics and sensitivity of human blood platelets: variations with age, gender and platelet number.

Platelet shape change and aggregation as well as serotonin (5-hydroxytryptamine; 5HT) content in platelets, spontaneous release of 5HT, 5HT plasma levels, urinary excretion of 5-hydroxyindoleacetic acid (5HIAA) and plasma beta-thromboglobulin (beta-TG) were investigated in 45 healthy subjects (27 men, 18 women). Platelets from women were more susceptible to aggregation (both by 5HT and ADP) than platelets from men. However, in men, 5HT-induced shape change and aggregation as well as plasma beta-TG concentration increased with age. In men, 5HT-induced platelet aggregation correlated positively with 5HT plasma levels. An inverse relationship was found in men between platelet number on the one hand and platelet 5HT content, 5HT release, 5HT plasma levels and urinary 5HIAA excretion rates on the other hand. In all subjects 5HT-induced platelet aggregation correlated negatively with platelet number. These results indicate that age and gender must be considered in designing clinical studies on 5HT and in evaluating human platelet 5HT kinetics. The platelet number seems to be related to 5HT kinetics of platelets and their reaction to 5HT in men. An age-dependent change in the reactivity of platelets to 5HT (rather than their absolute 5HT sensitivity) may contribute to the enhanced platelet turnover and higher incidence of cardiac events in older men.

Adult↗

PubMed1987

Platelet antithrombin defect in malignancy: platelet protein alterations.

Sixty-eight patients with malignant disease were divided into two groups based on the results of the platelet antithrombin test (PAT). The normal group had a PAT clotting time ranging from 21.4 to 29.8 seconds, which was equivalent to 25% to 65% inactivation of the 2 U of thrombin added to the test system. The other group showed abnormal PAT clotting time, less than 21.4 seconds or less than 25% thrombin inactivation. The polypeptide composition of platelets from the two patient groups was analyzed by sodium dodecyl sulfate (SDS)-electrophoresis on 7.5% polyacrylamide gels. A polypeptide of 180,000 apparent mol wt was decreased or absent in both Coomassie blue- and Alcian blue-stained gels of the platelets from patients whose PAT was abnormal; this polypeptide comigrated with purified platelet thrombospondin. Tritium labeling of platelet surface glycoproteins by the periodate-borohydride method followed by two-dimensional electrophoresis was performed on platelets of seven patients with abnormal PAT. When they were compared with ten patients with normal PAT, a glycoprotein of 140,000 apparent mol wt with a pl of 4.5 to 5.2 was decreased in platelets of all seven patients with abnormal PAT. Nitrocellulose replicas of one-dimensional gels of platelets from 13 of 14 patients with abnormal PAT showed decreased reaction with an anti-human platelet glycocalicin antiserum. Platelets of these same patients also showed a decreased or absent platelet agglutination induced by ristocetin. Patients with normal PAT had a mean agglutination slope of 1.25 +/- 0.6 (n = 26) as compared with 0.37 +/- 0.34 (n = 26) for the abnormal PAT group (P less than .001). Results indicate that platelets from a subpopulation of tumor patients characterized by decreased platelet antithrombin activity have alterations in two platelet glycoproteins, identified as GPIb and thrombospondin.

Antithrombins↗