Microaggregate Formation Research Articles

Aggregation Phenomena on the Ternary Ionic−Nonionic Surfactant System: Didodecyldimethylammonium Bromide/Octyl-β-d-glucopyranoside/Water. Mixed Microaggregates, Vesicles, and Micelles

The formation of a variety of mixed colloidal aggregates has been investigated on a ternary ionic-nonionic system constituted by (i) a double-chain cationic surfactant with a 12-carbon atom hydrophobic tail, didodecyldimethylammonium bromide (di-C(12)DMAB), (ii) a nonionic single-chain surfactant, octyl-beta-D-glucopyranoside (OBG), and (iii) water. The study has been carried out by means of conductivity, zeta-potential, transmission electron microscopy (TEM), and cryogenic transmission electron microscopy (cryo-TEM) experiments on the highly diluted, very diluted, and moderately diluted regions. The formation of mixed microaggregates, prior to the appearance of mixed vesicles, has been undoubtly confirmed by conductivity, TEM, and zeta-potential results. The concentrations at which these mixed colloidal aggregates form, i.e., the mixed critical microaggregate concentration (CAC), the mixed critical vesicle concentration (CVC), and the mixed critical micelle concentration (CMC), have been determined from conductivity data, while the zeta-potential experiments allow for the characterization of the aggregate/solution interface. The shape and size of the microaggregates and vesicles have been evaluated from TEM and cryo-TEM micrographs, respectively. All of the experimental evidence has been also analyzed in terms of the theoretical packing parameter, P.

Human thiopurine S-methyltransferase (TPMT) pharmacogenetics: Variant allozyme aggresome formation

Background/Aims TPMT*3A, the most common TPMT variant allozyme in Caucasians, has 2 alterations in amino acid sequence, resulting in striking decreases in TPMT protein levels as a result of rapid degradation. “Protein quality control” involves a dynamic balance among protein folding, degradation and aggregation with aggresome formation. We set out to test the hypothesis that, the presence of the proteasome inhibitor MG132, TPMT*3A can form aggresomes in cultured cells. Methods/Results TPMT*3A was highly ubiquinated and associated with hsp90 after transient expression in COS-1 cells. In the presence of MG132, aggresomes formed in cells transfected with TPMT*3A, and ubiquinated TPMT*3A redistributed from the cytosol into the pellet - compatible with aggresome formation. The aggresomes contained vimentin, dynein, γ-tubulin, ubiquitin, hsp70, hsp90 and the deacetylating enzyme HDAC6 that co-localized with TPMT*3A. Aggresome formation could be blocked by the microtubule destabilizing agent, vinblastine. Treatment with the HDAC inhibitor scriptaid inhibited aggresome formation and resulted in TPMT*3A microaggregate formation. Conclusions These results suggest that aggresome formation is one mechanism by which nonsynonymous cSNPs can alter function for proteins of pharmacogenetic importance such as TPMT. Clinical Pharmacology & Therapeutics (2005) 77, P95–P95; doi: 10.1016/j.clpt.2004.12.257

Colloidal Gold Particles Adsorption onto Silanized Glass Surfaces

The adsorption of colloidal gold onto glass surfaces previously treated with bifunctional silanes was studied aiming to enhance the recovery of colloidal gold particles from process water in the mining industry. Colloidal gold samples were prepared via the reduction of gold in HAuCl4 solution by sodium citrate. Glass plates used as model substrates were silanized with an ethanolic solution of (3‐mercaptopropyl)trimethoxysilane (MPS) and subsequently immersed in the colloidal gold dispersion. The characterization methods used were contact angle measurements to evaluate changes in hydrophobicity; UV/visible spectrometry, to estimate the extent of aggregation of the gold particles; atomic absorption, to measure the amount of gold adsorbed onto the glass plates and scanning electron microscopy (SEM), to estimate the number of colloidal gold particles deposited onto the glass plates. The deposition rate of gold particles on silanized glass surfaces was also studied by using a rotating disk system. It was found that, after the initial deposition of nanometric gold particles, the formation of microaggregates started to take place. After 15 min of immersion time, the microaggregates would act as nuclei for the deposition of new individual particles, leading to the formation of agglomerates bigger than 300 nm. The application of the Levich equation to calculate the rate of deposition of the gold particles showed that the process is not controlled only by diffusion but also by the slow adsorption process itself.

Earthworms and management affect organic matter incorporation and microaggregate formation in agricultural soils

Through their feeding activities and cast production, earthworms influence both aggregate turnover and soil organic matter (SOM) dynamics. We studied the impact of earthworm activity on soil macro- and microaggregation and SOM incorporation in different farming systems. Dry-sieved aggregates (4–12.5 mm) of a permanent pasture (PP), a conventional arable field (CA) and an organic arable field (OA) (0–10 and 10–20 cm depth) were separated into different aggregate fractions, which were analyzed for organic C and N. The separation was based on macromorphological characteristics that reflect the dominant process of aggregate formation and the degree of degradation. We distinguished: two classes of biogenic macroaggregates (fresh casts and welded casts); one class of physicogenic macroaggregates (angular to subangular blocky macroaggregates); and an intermediate fraction (rounded to subrounded macroaggregates). The structural arrangement of mineral particles and organic matter and the quantitative contribution of particulate organic matter (POM) and microaggregates were studied in thin sections. Total organic C contents tended to be higher in biogenic than in physicogenic macroaggregates of the PP and OA soils, whereas the reverse was found for the CA soil. Comparison of the different macroaggregate types in thin sections revealed that the worm-made macroaggregates of the PP soil were considerably enriched in fine POM and microaggregates, in which large amounts of organic matter were intimately mixed with fine mineral material. By contrast, worm casts of the CA and OA soils were hardly enriched in POM and microaggregates. Our study demonstrated that earthworms can directly initiate the formation of microaggregates, which in turn affects the physical protection of SOM against microbial decay. Farming practices that stimulate earthworm activity may thus constitute an important aspect of sustainable agricultural management. However, the much smaller amounts of POM and microaggregates present in worm casts of CA and OA than PP indicate a different impact of earthworms on C stabilization depending on land use. Further research is needed to elucidate the exact management conditions that favour the beneficial effects of earthworms on soil microstructure and associated SOM dynamics. In this respect, the use of micromorphological techniques in addition to chemical and physical analyses was shown to be very valuable.

The Cytoplasmic Tail of GPIbß Modulates Signalling Induced by GPIb/VWF Interactions under High Shear Conditions.

The platelet glycoprotein (GP) Ib/IX/V receptor complex controls crucial steps in haemostasis and thrombosis by mediating adhesion to von Willebrand factor (VWF). Nevertheless, the downstream consequences of VWF binding to GPIb remain unclear. The GPIb/IX/V complex consists of four polypeptides GPIbα, GPIbβ, GPIX and GPV. We have shown that a palmitylated (Pal-) peptide based on the sequence of the GPIbβ cytoplasmic domain between R151 and A161 (Pal-RRLRARARARA abbreviated to RARA) increases the velocity of platelets rolling on VWF, and inhibits VWF-dependent adhesion and aggregation.The peptide RARA is delivered into platelets and does not cause any reduction in the surface expression of GPIb-IX-V. We examined the effect of RARA and control peptides (Pal-RAAAARARARA and Pal-RRLRADADADA) on GPIb/IX/V-mediated tyrosine kinase signalling induced by VWF/GPIb interactions under shear conditions using a cone and plate viscometer. Whole platelet lysate phosphotyrosine immunoblots were characterised in the presence and absence of RARA and the control peptides before and after shearing at 120 dynes/cm2 for 5 mins. We then further investigated the effect of RARA on PI-3 kinase signalling, the tyrosine kinase Src and calcium influx under the conditions described above.The platelet permeable peptide RARA alone specifically caused protein tyrosine phosphorylation under resting conditions and caused platelet microaggregate formation with a 35% reduction in single platelets as assessed by flow cytometry. In addition RARA significantly inhibited shear-induced aggregation. When tyrosine phosphorylation was examined in platelets sheared for 5 minutes at 120 dynes/cm2, we observed RARA failed to inhibit tyrosine phosphorylation despite inhibiting shear-induced aggregation.RARA caused Src kinase activation as determined by site-specific phosphorylation of Y416 on Src. Furthermore the Src inhibitor PP1 inhibited RARA induced tyrosine phosphorylation. RARA caused a Src-dependent increase in intracellular [Ca2+] and inhibited any further increase in intracellular calcium induced by thrombin. Pretreatment of platelets with the PI-3 kinase inhibitor wortmannin demonstrated that tyrosine phosphorylation induced by RARA was not PI-3 kinase dependent.These results indicate that a platelet permeable peptide corresponding to specific residues of the cytoplasmic tail of GPIbβ directly stimulates Src, and suggest that the mechanism of its inhibitory effect on VWF-mediated platelet adhesion and aggregation involves the tyrosine kinase-mediated desensitisation of a GPIb/IX/V-triggered signalling pathway. This study demonstrates a novel role for the GPIbβ cytoplasmic tail in mediating Src-dependent shear induced signalling through GPIb/VWF.

Carbon Sequestration in Microaggregates of No‐Tillage Soils with Different Clay Mineralogy

Identification of diagnostic soil organic matter (SOM) fractions and the mechanisms controlling their formation and turnover is critical for better understanding of C dynamics in soils. Enhanced microaggregate formation and stabilization of C due to reduced macroaggregate turnover has been proposed as a mechanism promoting C sequestration in no‐tillage (NT) compared with conventional tillage (CT) systems in temperate soils dominated by 2:1 clay mineralogy. We evaluated the contribution of macroaggregate‐protected microaggregates to total soil organic carbon (SOC) sequestration in NT relative to CT in three soils differing in clay mineralogy: a 2:1 clay‐dominated soil (2:1), a soil with mixed clay mineralogy [2:1 and 1:1] and oxides (mixed), and a soil dominated by (1:1) clay minerals and oxides (1:1). Microaggregates (mM) were isolated from macroaggregates from 0‐ to 5‐ and 5‐ to 20‐cm soil layers. Particulate organic matter (POM) located within the microaggregates (intra‐mM‐POM) was separated from POM outside of the microaggregates (inter‐mM‐POM) and the mineral fraction of the microaggregates (mineral‐mM). In all three soils, total SOC as well as microaggregate‐associated C (mM‐C) was greater with NT compared with CT. Although less than half of the total SOC under NT was associated with the microaggregate fraction, more than 90% of the total difference in SOC between NT and CT was explained by the difference in mM‐C in all three soils. Thus, we identified and isolated a fraction that explains almost the entire difference in total SOC between NT and CT across soils characterized by drastically different clay mineralogy.

Prestorage leukocyte reduction prevents the formation of microaggregates that occlude artificial capillary vessels

Purpose: The passage behavior of stored blood through an artificial microchannel system, as a model of capillary vessels, was examined. Materials and Methods: Using blood obtained from a total of 17 healthy volunteers, untreated and prestorage leukocyte reduced blood, or untreated and prestorage microfiltered (35 μm-pore size) blood were stored, and then the passage behavior through the microchannels was evaluated. Also, using blood from 16 patients stored for about 2 weeks, the effect of a leukocyte reduction filter, microfilter or mesh filter (175–210 μm-pore size) on the passage through the microchannels was examined. Results: Untreated blood passed through the microchannels immediately after blood collection, but after 1 week occlusion of the microchannels occurred. Prestorage leukocyte reduced blood, however, passed through the microchannels for up to 6 weeks. Occlusion of the microchannels occurred with both the untreated and prestorage microfiltered blood. Although filtration through a leukocyte reduction filter provided blood that can pass through the microchannels, occlusion occurred with blood filtered in other way. Conclusions: The present results show that stored blood produces abundant microaggregates, potentially occluding capillary vessels. These microaggregates are not formed after prestorage leukocyte reduction or can be removed by use of a leukocyte reduction filter.

Rapid incorporation of carbon from fresh residues into newly formed stable microaggregates within earthworm casts

SummaryEarthworms play an important role in protecting carbon in the soil, but the exact influence of their activity on the distribution and protection of C is still poorly understood. We investigated the effect of earthworms on the formation of stable microaggregates inside newly formed macroaggregates and the distribution of C in them. We crushed (< 250 µm) soil, and subjected it to three treatments: (i) soil + 13C‐labelled residue + earthworms (these added after 8 days' incubation), (ii) soil + 13C‐labelled residue, and (iii) control (no additions), and then incubated it for 20 days. At the end, we measured the aggregate size distribution, total C and 13C, and we isolated microaggregates (53–250 µm) from macroaggregates (> 250 µm) formed. The 13C in fine particulate organic matter between and within the microaggregates was determined. Earthworms helped to form large macroaggregates (> 2000 µm). These large macroaggregates contained four times more stable microaggregates than those from samples without earthworms. There was more particulate organic matter within and between microaggregates in macroaggregates in the presence of earthworms. The larger amounts of organic matter inside stable microaggregates in casts than in bulk soil after 12 days of incubation (140 mg 13C kg−1 soil compared with 20 mg 13C kg−1 soil) indicates that these microaggregates are formed rapidly around freshly incorporated residues within casts. In conclusion, earthworms have a direct impact on the formation of stable microaggregates and the incorporation of organic matter inside these microaggregates, and it seems likely that their activity is of great significance for the long‐term stabilization of organic matter in soils.

Quantitation of platelet aggregation and microaggregate formation in whole blood by flow cytometry

Platelet aggregation and microaggregate formation were measured in samples of stirred whole blood by flow cytometry. Blood samples were stirred in a multi-sample agitator with ADP, fixed and labelled with a platelet-specific CD42a-FITC fluorescent antibody. The blood was then diluted and applied directly to a flow cytometer. Platelets were identified using a gating procedure based on their expression of CD42a and then quantified. Aggregation was monitored as a fall in the number of single platelets. Both reversible and irreversible aggregation responses to ADP were determined and these were found to correlate directly with aggregation responses determined using a well-established single platelet counting technique using the Ultra-Flo 100 Whole Blood Platelet Counter. We found from flow cytometry that ADP-induced aggregation was coupled with a transient formation of platelet microaggregates over the initial 60 s following ADP addition. Assessment of single platelet loss by flow cytometry was found to be a reliable way of monitoring aggregation responses and provided new information on rapid microaggregate formation in ADP-stimulated blood.

Effect of humic acid, sodium, and calcium additions on the formation of water-stable aggregates in Western Australian wheatbelt soils

The effect of humic acids extracted from coal waste and peat on the formation of water-stable aggregates in kaolinitic soils from the wheatbelt of Western Australia and a prepared kaolinite–quartz mix were assessed. This effect was compared with that produced by adding calcium to the soils. In the case of kaolinite–quartz mix, the treated samples were measured for micro-aggregation since macro-aggregates were not formed under the experimental conditions. The wheatbelt soils were already aggregated and the measurement was of the further macro-aggregation of these aggregates. In kaolinite–quartz, calcium additions alone enhanced the formation of stable micro-aggregates. This effect was amplified with additions of humic acids when up to 30% of the clay-sized particles were aggregated into larger aggregates. This humic acid effect was not observed in the sodium dominated mineral system.In contrast to the kaolinite-quartz samples, the selection of wheatbelt soils used in this study responded little to calcium additions alone. Humic acid increased the size of aggregates in the sodium-treated soils presumably due to the presence of aluminium in the system studied at pH 4.5. The aluminium ions can be expected to act as the bridging cations between the negatively charged clay-sized particles and the anionic groups present in the organic matter. The ineffectiveness of calcium in these soils allowed the positive effect of the humic acids to be expressed clearly. These findings provide another management option for improving the soil structure of salt-affected land because some of these soils are unlikely to respond to gypsum application.

The impact of peripheral arterial disease on circulating platelets

Introduction: To test the hypothesis that circulating platelets display evidence of reversible interactions with atherosclerotic lesions, platelet α-granule content and propensity for microaggregate formation were measured in samples from normal donors ( n=65) and from patients with either peripheral arterial disease ( n=47) or renovascular hypertension ( n=22). To measure the effect of a defined arterial injury on platelet function, platelet samples were compared before and 30 min after elective angioplasty. Materials and methods: P-selectin was measured after strong stimulation of ultra-dilute platelets with thrombin (10 nM). Microaggregation was measured as a platelet count deficit in citrate-anticoagulated platelet-rich plasma (PRP) relative to that predicted from the count in EDTA-anticoagulated blood. Results: Platelet α-granule P-selectin was significantly lower from platelets of patients compared to normal donors. In addition, platelets from patients have a significantly greater propensity to form microaggregates in citrate anticoagulant. In contrast to atherosclerotic renovascular hypertension, platelets from patients with fibromuscular dysplasia, a distinct non-inflammatory cause of arterial stenosis, do not differ significantly from normal donors. Other than the PRP platelet count, which rose transiently following angioplasty, other platelet measures were unchanged by the injury. Conclusions: Atherosclerotic arterial disease is associated with an increased share of platelets unable to express P-selectin and an increased fraction of platelets that microaggregate in citrate anticoagulant. These platelet alterations are not completely explained by either focal arterial injury or abnormal rheology associated with arterial stenosis but appear to be an effect of the atherosclerotic process.

Lipopolysaccharide evokes microaggregation reactions in hemocytes isolated from tobacco hornworms, Manduca sexta

Insect cellular immune reactions to bacterial infection include nodule formation. Eicosanoids mediate several cellular actions in the nodulation process, including formation of hemocyte microaggregates, an early step. In previous work, we reported that isolated hemocytes produce and secrete eicosanoids that influence hemocyte behavior in response to bacterial challenge. We also reported that microaggregate formation in response to challenge was mediated by prostaglandins (PGs), but not by products of the lipoxygenase (LOX) pathways. In this paper we describe experiments designed to test the idea that exposing isolated hemocytes to lipopolysaccharide (LPS) evokes formation of hemocyte microaggregates and this cellular action is mediated by PGs. Results show that isolated hemocyte preparations challenged with LPS formed more hemocyte microaggregates than unchallenged preparations (6.9×10 3 microaggregates/ml hemolymph vs. 2.5×10 3 microaggregates/ml hemolymph). LPS challenge stimulated formation of hemocyte microaggregates in a dose dependent manner. Experimental groups pretreated with cyclooxygenase inhibitors produced fewer hemocyte microaggregates in response to LPS challenge than untreated control groups. The formation of hemocyte microaggregates was not influenced by LOX inhibitors. Furthermore, the influence of dexamethasone was reversed by supplementing the experimental groups with the eicosanoid precursor fatty acid molecule, arachidonic acid and PGH 2. Palmitic acid, which is not substrate for eicosanoid biosynthesis, did not reverse the effects of dexamethasone on the formation of microaggregates. The LOX product 5( S)hydroperoxyeicosa-6 E,8 Z,11 Z,14 Z-tetraenoic acid also did not reverse the effects of dexamethasone. These results are consistent with similar investigations performed with bacterial suspensions. We infer that isolated hemocyte preparations recognize and react to LPS by forming microaggregates and this reaction is mediated by PGs, but not products of the LOX pathway.

Potentiation of platelet activation through the stimulation of P2X1 receptors

The platelet P2X1 purinergic receptor is a ligand-gated ion channel that responds to ATP. The precise role of P2X1 in platelet function is unknown, though stimulation with the P2X1 agonist alpha,beta-Me-ATP is known to result in platelet shape change through elevation of calcium levels. The aim of the present study was to examine further the effects of P2X1 stimulation on platelet activation. Stimulation of P2X1 with alpha,beta-Me-ATP resulted in shape change and small aggregate formation in heparin-anticoagulated platelet preparations. Given the ability of heparin to potentiate platelet activation, subsequent experiments were performed in hirudin. In these platelet preparations, aggregate formation in response to alpha,beta-Me-ATP alone was less than that observed in heparin; however, alpha,beta-Me-ATP significantly potentiated platelet aggregate formation when added in conjunction with other weak platelet agonists [epinephrine or thrombopoietin (TPO)]. Platelet aggregate formation was confirmed by single platelet loss (microaggregate formation), microscopy, and light transmittance studies. Further, the P2X1 antagonist MRS-2159 inhibited platelet shape change and aggregation responses induced by alpha,beta-Me-ATP. Overall, this study demonstrates that P2X1 stimulation can induce/potentiate platelet activation in combination with other platelet agonists. These results are the first demonstration of platelet aggregation mediated through direct P2X1 stimulation, supporting a role for this receptor in regulating platelet activation.

Study 1: unexpected high incidence of coronary artery disease and abnormal platelet reactivity in long-term pediatric kidney transplant recipients

L ong-term outcomes of children who undergo renal transplantation are unknown. However, as they enter adulthood, many will have risk factors for developing coronary artery disease (CAD). Certain noninvasive tests may detect early CAD. Electron-beam computed tomography (EBCT) can measure the amount of coronary artery calcification (CAC), which correlates with the presence of atherosclerotic CAD. In addition, abnormal platelet function has been linked to increased risk for atherogenesis and CAD. The purpose of this study was to determine the incidence of CAD by using EBCT in adults who underwent renal transplantation as children and determine whether presence of accelerated atherogenesis is associated with enhanced platelet reactivity. Eight pediatric kidney transplant recipients with good long-term allograft function ( 10 years) underwent evaluation by EBCT. The CAC score was determined by standardized technique. Platelet reactivity was assessed by measures of dense granule secretory rate, metabolic content, and spontaneous microaggregate formation. Results were compared with those in age-matched controls. The mean age at transplantation was 14 years (range, 10-16). Mean age at evaluation was 45 years (range, 23-48). Mean follow-up in this group was 17 years (range, 10-23). All received cyclosporine-based immunosuppression. Seven had 2 or more clinical risk factors for CAD. All were asymptomatic at the time of evaluation. Seven out of 8 patients had significant CAC detected on EBCT. When compared with ageand gender-matched controls, study patients had CAC scores 99% (n 4), 95% to 99% (n 2), and 75% to 95% (n 1). Platelets from those with CAC were significantly more likely to form microaggregates after mild stimulation compared with those without CAC (P .02). Also, they were more likely to form microaggregates (P .03), had slower dense granule secretion (P .04), and had lower metabolic stores (P .03) compared with controls. Conclusion: The incidence of CAC by EBCT in long-term pediatric kidney transplant recipients is much higher than expected compared with the general population. Besides clinical risk factors, it appears that platelets from these patients are more reactive, which may place them at further increased risk for thrombotic complications of atherosclerosis. Our data suggest that measures to decrease risk factors for CAD should be undertaken as children approach young adulthood. From the William J. von Liebig Transplant Center, Mayo Clinic; and the Department of Radiology; and Division of Cardiovascular Medicine, Mayo Clinic, Rochester, MN. © 2004 Elsevier Inc. All rights reserved. 0955-470X/04/1704-0000$30.00/0 doi:10.1016/j.trre.2003.10.006

Prostaglandins, not lipoxygenase products, mediate insect microaggregation reactions to bacterial challenge in isolated hemocyte preparations

Nodulation is the predominant cellular defense reaction to bacterial challenge in insects. Eicosanoids mediate several steps in the nodulation process, including formation of hemocyte microaggregations. Isolated hemocyte preparations synthesize and secrete eicosanoids, which mediate hemocytic immune reactions. Two major groups of eicosanoids are prostaglandins (products of cyclooxygenase pathways) and various products of lipoxygenase pathways. In this study, we test the hypothesis that prostaglandins, but not lipoxygenase products, mediate hemocyte microaggregation reactions in response to bacterial challenge. Our results indicate that isolated hemocyte preparations pretreated with the cyclooxygenase inhibitors indomethacin and naproxen yielded fewer microaggregates than untreated control groups (3.7×10 5 microaggregates/ml hemolymph vs. 11.0×10 5 microaggregates/ml hemolymph). These inhibitors influence hemocyte microaggregate formation in a dose–dependent manner in treatments ranging from 0 to 200 μM. The lipoxygenase inhibitors esculetin and caffeic acid did not impact the formation of microaggregates in this system. The influence of the phospholipase A 2 inhibitor dexamethasone was reversed by amending experimental (dexamethasone-treated) preparations with prostaglandin H 2, but not prostaglandin D 2, prostaglandin E 2, nor 5( S)-hydroperoxy-6E,8Z,11Z,14Z-eicosatetraenoic acid, a product of the lipoxygenase pathway. We infer that prostaglandins are the primary mediators of microaggregation reactions to bacterial challenge in insect hemocyte preparations.

Differential effect of the GPIIb/IIIa antagonist orbofiban on human platelet aggregate formation in vitro

Platelet microaggregates have been demonstrated in the systemic circulation of patients with atherosclerotic cardiovascular diseases. Glycoprotein IIb/IIIa antagonists have been reported to play roles in platelet activation, which may be associated with pro-thrombotic events. We report the effect of the orally active GPIIb/IIIa antagonist, orbofiban, on human platelet microaggregate formation in vitro. Laser light scatter (LSS) technology was used to monitor small, medium and large platelet aggregates formed in platelet-rich plasma in response to ADP or thrombin receptor activating peptide (TRAP)-6. ADP at 0.5 μM induced only small platelet aggregates that were prevented by orbofiban in a concentration-dependent manner. Likewise, orbofiban dissolved small aggregates after their formation. In marked contrast, in the presence of strong agonist stimulation (20 μM ADP or 3 μM TRAP-6) which generated primarily large platelet aggregates, orbofiban blocked the large aggregates while significantly augmenting small aggregates by three- to sixfold. The data suggest that GPIIb/IIIa antagonists do not induce platelet microaggregation directly but may augment small platelet microaggregate formation indirectly at conditions of strong stimuli. The percentage of the microaggregate population expressing P-selectin remained the same in the presence of orbofiban, indicating that these small microaggregates remain activated, although the mean intensity of expression was diminished, possibly reflecting the reduced size of the particles or density of P-selectin molecules. In summary, an increase in small platelet microaggregates might have contributed to pro-thrombotic events in orbofiban-treated patients.

Effect of recombinant human deoxyribonuclease on the expression of cell adhesion molecules of thawed and processed cord blood hematopoietic progenitors.

The integrity of granulocytic cells and platelets is compromised within cryopreserved stem cell transplants, and consequent DNA release during the thawing procedure can therefore lead to clotting phenomena or microaggregate formation and that in turn may cause loss of progenitor cells. To circumvent this problem a new processing protocol was introduced using recombinant human deoxyribonuclease (rhDNase) to prevent cell aggregate formation. In addition, the impact of this new processing protocol on CD34+ umbilical cord blood (UCB) cells was assessed. Fifty samples derived from 7 buffy coat (BC) volume reduced UCB units were cryopreserved, thawed, and processed with washing solutions that were supplemented with rhDNase in various concentrations. Thereafter, clotting and microaggregate formation was scored microscopically. In addition, expression of the adhesion molecules leukocyte function-associated antigen 1 (LFA-1, n = 6), intercellular adhesion molecule-1 (ICAM-1, n = 11), and L-selectin (n = 11) on CD34+ UCB cells was analyzed by flow cytometry after incubating the samples with either dimethyl sulfoxide (DMSO) 5.5%, rhDNase 10 or 50 U/mL, or a combination of DMSO 5.5% and rhDNase 50 U/mL. At a minimal concentration of 10 U rhDNase/mL, clotting or microaggregate formation could be prevented for all tested samples, whereas cell clots could be observed for concentrations up to 8 U/mL. The expression of adhesion molecules on untreated CD34+ UCB cells (L-selectin: 64.6 +/- 18.8%; LFA-1: 62.6 +/- 7.5%; ICAM-1: 14.8 +/- 4.1%) did not show any significant difference compared with cells that were incubated with up to 50 U/mL rhDNase (L-selectin: 62.2 +/- 19.3%; LFA-1: 63.1 +/- 5.9%; ICAM-1: 17.5 +/- 6.7%). However, after a combined treatment with DMSO 5.5% and rhDNase 50 U/mL, a slight but significant decrease in L-selectin expression could be observed (P < 0.03). The supplementation of rhDNase to a final concentration of 10 U/mL cell suspension proved to be effective in preventing clot formation under the conditions examined and did not lead to decreased expression levels of adhesion molecules. We therefore recommend the use of rhDNase for the prevention of clot formation and cell loss during the processing of thawed UCB transplants.

Microaggregates in agricultural soils and their size distribution determined by X‐ray attenuation

SummaryUnderstanding mechanisms of microaggregate formation in soils requires knowledge of their exact size distribution. With this in mind, we have used X‐ray attenuation to determine the size distributions of microaggregates and primary particles in the range 0.2–63 μm, with a resolution of 100 size increments. Ten arable and grassland soils with organic C contents ranging from 14.7 to 37.7 g kg−1 were analysed. They were subjected to ultrasound at 52 J ml−1 which destroyed most aggregates &gt; 63 μm to give microaggregates in the size range 1–63 μm. The size distribution of microaggregates differed significantly from that of primary particles and was largely independent of their organic C content. Microaggregates were most abundant in 19 of the 100 size increments, contributing to 92% of the major peaks of the size distribution. These preferred increments differed from those of primary particles, but the order for the two was similar. Further analysis of the size distribution revealed a larger mean weight diameter of microaggregates, depending on the size distribution of primary particles. The results suggest a major effect of the size distribution of primary particles on microaggregation, whereas land use seems to have a negligible effect. The proportion of mechanically dispersible clay decreased with increasing C content and indicates structural stability at the microscale.

Effects of the glycoprotein IIb/IIIa antagonist Roxifiban on P-selectin expression, fibrinogen binding, and microaggregate formation in a phase I dose-finding study: no evidence for platelet activation during treatment with a glycoprotein IIb/IIIa antagonist

The hypothesis that glycoprotein (GP) IIb/IIIa antagonists stimulate platelets is controversial. Here, we report the results of flow cytometric measurements of platelet activation markers in a phase I dose optimization study of Roxifiban, an orally active GP IIb/IIIa antagonist. Whole blood was collected at pre-dose and during the dosing interval directly into citrate fixative so that circulating levels of platelet activation could be assessed. P-selectin expression and fibrinogen binding of single platelets were unchanged at any of the dosing intervals compared to the pre-dose values, whereas microaggregate formation was reduced. Blood was also collected in hirudin to maintain physiological calcium concentrations and stimulated with platelet agonists to test whether GP IIb/IIIa antagonists lower the threshold for platelet activation. After stimulation with a concentration range of ADP and TRAP, P-selectin expression was not altered by Roxifiban administration compared to pre-dose levels. Fibrinogen binding and microaggregate formation were reduced by Roxifiban dosing in a dose-dependent manner. Inhibition of both parameters was retained at trough and no increase above pre-dose values was observed at any time. This study provides evidence for a dose-dependent inhibition of platelet functions by an orally active GP IIb/IIIa antagonist and does not detect paradoxical activation of platelets by a GP IIb/IIIa antagonist in humans.

Formation of platelet microaggregates correlates with adverse clinical outcome in patients with coronary artery disease

Platelet activation plays a pivotal role in the pathogenesis of acute coronary syndromes. Laser-light scattering in a platelet aggregometer was used to evaluate aggregate size and number quantitatively. Small platelet aggregates ultimately develop into medium and then large platelet aggregates. Thus the measurement of small platelet aggregates is important in the evaluation of thrombus formation. We examined the relationship between small platelet aggregates and the occurrence of subsequent cardiovascular events. We followed-up 204 patients (149 men and 55 women, mean age 68 +/- 9 years) with coronary artery disease (CAD) for 48 months. Blood sampling to determine platelet aggregation was performed on the day of hospital discharge. The degree of small platelet aggregates [relative risk 4.34, 95% confidence interval (1.62-11.7), p = 0.004] and low left-ventricular ejection fraction [relative risk 2.88, 95% confidence interval (1.23-6.73), p = 0.015] were independent predictors of the occurrence of cardiovascular events in multivariate Cox hazard analysis. In Kaplan-Meier analysis, the degree of small platelet aggregates correlated with the probability of cardiovascular event occurrence. In patients with CAD, an increase in small platelet aggregates is closely correlated with the future occurrence of cardiovascular events