Einleitung: Besonders schwere Formen der Schwangerschafts-assoziierten Erkrankung Präeklampsie (PE) werden mit der endothelialen Dysfunktion sowie der Bildung synzytiotrophoblastärer Mikropartikel (STBM) in Verbindung gebracht. Diesen wird die Fähigkeit zugeschrieben, inflammatorischen Prozesse am Endothelium zu provozieren. Das thrombogene Potential dieser STBMs ist jedoch noch nicht weitgehend aufgeklärt und dessen Untersuchung demensprechend Ziel dieser Studie.

Hintergrund: Das Bild kritisch kranker Patienten mit schwerer Sepsis oder septischen Schock auf Intensivstationen (ITS) wird von Immobilität und Bettruhe bestimmt. Eine wesentliche Folge ist die zunehmende physische Dekonditionierung der Patienten während ihres ITS-Aufenthaltes, die den klinischen aber auch ambulanten Krankheitsverlauf erheblich beeinflusst. Um dieser Entwicklung entgegen zu wirken, stellt die frühe physiotherapeutische Intervention (PTI) eine Therapiestrategie in der Behandlung dieser Patienten dar. Ziel: In zwei unabhängigen Studien sollte erfasst werden, inwieweit die frühe PTI in das Behandlungskonzept bei septischen Patienten am Universitätsklinikum in Jena implementiert ist und ob die Häufigkeit der PTI den akuten Krankheitsverlauf positiv beeinflussen kann (Studie I, retrospektiv). Darüber hinaus wurde erhoben, welche physiotherapeutischen Maßnahmen angewendet und welche Therapieziele dabei verfolgt werden (Studie II, prospektiv). Ergebnisse: Retrospektiv wurden 16.041 Patienten analysiert, wobei 999 die Kriterien für schwere Sepsis oder septischen Schock erfüllten. Die Mortalitätsrate septischer Patienten betrug 30,8% und die mittlere ITS-Liegedauer 12 Tage. Insgesamt wurden 9.476 PTI angewendet, die auf 77% der Patienten entfielen. Die PTI-Rate (PTI/Liegedauer) bei Patienten mit schwerer Sepsis oder septischen Schock betrug 42%. Regressionsanalysen zeigten auf, dass die PTI-Rate ein starker Prädiktor für die ITS-Mortalität ist. Als wichtigste Therapieziele konnten prospektiv die Prophylaxe/Therapie von Kontrakturen und Pneumonien erhoben werden, wobei passive Mobilisierungsmaßnahmen, die Atemtherapie und Lagerungstherapie am häufigsten angewendet wurden. Schlussfolgerung: Erstmalig wurde die Implementierung von physiotherapeutischen Maßnahmen und deren mögliche Auswirkungen bei Patienten mit Sepsis analysiert. Hieran sollten sich weitere Studien anschließen.

Background In patients with sepsis and renal failure, extracorporeal blood flow during renal replacement therapy may lead to the deposition of bacteria on artificial membranous surfaces, which might be suitable for the detection of pathogens. We studied whether discarded dialysis hemofilters can be used for the detection of bacteremia in patients with sepsis and renal failure.Methods Hemofilters of 16 ICU patients with sepsis were sampled. The hemofilters were incubated with soy broth and dehisced under sterile conditions. Samples were plated on blood agar and analyzed. Patient's characteristics were assessed.Results Despite the use of antibiotics in 87.5 % (14/16), a true positive detection rate of 31.3 % (5/16) for bacteremia was found by using cultures from hemofilters. The overall true positive rate of blood cultures was significantly lower (10.7 %, 8/75, p = 0.048). Bacteria detected in hemofilters were similar to those found in blood cultures or by cultures from other sources of infection in 80 % (4/5).Conclusions Cultures from used hemofilters of patients with sepsis and renal failure provide the opportunity to identify pathogenic microorganisms as an add-on approach. Further studies should investigate whether this method is applicable in clinical practice to enhance the sensitivity of microbiological diagnostics.

IntroductionIn 2002, the Surviving Sepsis Campaign defi ned a strategy that aimed to reduce the high mortality due to sepsis.One point of this strategy was a recommendation to recognize that sepsis is a frequent cause of death and high economic costs in the pediatric intensive care unit.Knowledge of the disease is the fi rst step to impact it.There are few studies on pediatric sepsis epidemiology in the world and none in Colombia.Hypothesis The epidemiological features of Colombian children are diff erent from other countries.Methods We constructed a website where 14 intensive care units across the country reported in a prospective way the epidemiological features of children with sepsis using an electronic process [1].We asked for sociodemographics, microbiological data, sepsis classifi cation, complications, and outcome. ResultsWe collected 253 patients from March to May 2009.Fifty-fi ve percent of the cases were male and 45% were female; 53% were less than 1 year old.A total of 67.2% came from urban areas and 33% came from rural villages.Eighty-fi ve percent were very poor (score 1 and 2 over 6 used in Colombia as socioeconomic classifi cation).Forty-fi ve percent have governmentsupported insurance.In total, 23.72% of the population presented with sepsis; 30.04% with severe sepsis; and 46.5% with septic shock.The infection origin was respiratory in 54.55%, followed by abdominal in 17.39%.In 50.2% no cause was identifi ed.A total of 75.1% required mechanical ventilation.The mortality rate was 20.4%.Conclusions Sepsis, severe sepsis, or septic shock is a common diagnosis in Colombian intensive care units.The majority of pediatric patients are 2 years or younger and from the poorest communities.It aff ected males more.In the majority, the process starts in the respiratory system.We had diffi culty identifying the cause.The disease causes high mortality and cost for a developing society.We need a complete survey to fi nd a correct approach to the problem.Reference 1. Sepsis en Columbia [www.sepsisencolombia.com]P2 Randomized controlled trials are not designed to prove the safety of third-generation hydroxyethyl starch for resuscitation: results from a systematic review CS Hartog, M Kohl, K Reinhart

Physical stress, such as that induced by strenuous exercise, is held responsible for activation of platelets and formation of platelet–leukocyte conjugates as well as increasing the risk of vascular thrombotic events, and primary cardiac arrest [1]. It has previously been shown that intense exercise increases the plasma level of von Willebrand factor (VWF) [2]. Platelet aggregation associated with an enhanced expression of adhesion molecules on platelets such as P-selectin (CD62P) and glycoprotein (GP) IIb/IIIa is also increased after the prolonged exercise [3]. However, the underlying mechanisms of exercise-induced changes on platelet aggregability remain unclear. Also, it is well documented that exhaustive exercise leads to activation of several ‘stress hormones’. Epinephrine and vasopressin are key regulators of the stress response via activation of adrenergic and V1 receptors, respectively. The magnitudes of the responses are modulated by both the relative intensity and the duration of exercise: in general, the higher the intensity and the duration of exercise, the more intensive the hormonal release [4]. Epinephrine and vasopressin trigger the activation of endothelial cells [5], which may result in the release of ultralarge VWF multimers (ULVWF), that in turn may induce platelet activation and thrombus formation under the condition of high shear stress, that is, in particular in the microcirculation [6]. The biological activity of the platelet-activating ULVWFM is regulated by a specific plasma metalloprotease ADAMTS-13 (A Disintegrin And Metalloprotease with Thrombospondin-1 Repeats) [7]. Recently, there is a compelling body of evidence, that support the concept of a reciprocal behavior of the proteolytic activity of ADAMTS-13 and plasma VWF levels [8, 9]. A similar association could be shown for various (patho)physiological conditions, including systemic inflammation following the endotoxin challenge and stimulation of endothelial VWF release by desmopressin [10, 11]. VWF is a well-documented acute phase protein, and the pro-inflammatory cytokine interleukin (IL)-6 has been reported to cause an irreversible inhibition by proteolytic fragmentation of ADAMTS-13 [12]. We recently showed that physical stress results in a gene expression pattern in circulating blood leukocytes that is very similar to that observed in systemic inflammation [13]. Keeping this in mind, the present study was performed to analyze the response of the VWF/ADAMTS-13 system to standardized strenuous and long-term physical stress of healthy individuals. We serially measured the plasma activity of ADAMTS-13 and VWF:Ag, soluble CD62P (sCD62P) and IL-6 concentration in plasma samples of healthy, moderately trained individuals before and after a standardized exercise test on a treadmill ergometer with 90% of the individual anaerobic threshold for 90 min. Six volunteers were consecutively enrolled, all of them gave their written consent to the study, which was approved by the institutional ethical committee. To explore whether any changes of either ADAMTS-13 activity or VWF, the only thus far known substrate, were a result of the stress response observed under these conditions, blood samples were taken by clean venipuncture from an antecubital vein under the controlled venous stasis after 30 min rest (baseline) and immediately, 2, 6 and 24 h after exercise using a 20 gauche canula and 10 mL vacutainers containing 1 mL of 0.13 mol L−1 sodium citrate. Plasma was obtained by centrifugation (1500 × g, 20 min, 4 °C within 30 min after venipuncture), divided into aliquots and stored at −80°C until assayed. ADAMTS-13 activity was measured as the cleavage of a fluorescence-labeled substrate representing the D1596 to R1668 region of VWF [14]. Levels of VWF, IL-6 and sCD62P were determined by ELISA techniques. Continuous variables are presented as medians and inter quartile range (IQR). A general linear model analysis of variance (anova) was performed for statistical analysis. In a variety of studies it was shown that the type of exercise model used in this study, the duration and intensity as well as the chosen time points for plasma sampling is appropriate for the investigation of inflammatory changes after physical stress [15]. As shown in Fig. 1, physical exercise caused a time-dependent increase in the plasma levels of VWF, IL-6 and sCD62P as well as a decrease in the ADAMTS-13 activity. In comparison to basal pre-exercise measurements, immediately after exercise, VWF, sCD62P as well as IL-6 reached values of 177%, 167%, and 196%, respectively. The values declined during the postexercise phase and returned to basal levels within 6 h. ADAMTS-13 activity was significantly decreased 2 h after exercise by about 22% and returned to the basal range at 6 h after exercise. Collagen-binding activity of VWF as a measure for high molecular weight multimers reflected the changes in ADAMTS-13 activity, but the increase of about 15% did not reach significance. Moreover, platelet count was enhanced by 41% after exercise. Time changes of ADAMTS-13, VWF:Ag, sCD62P, IL-6 and platelet count in volunteers undergoing physical stress. B indicates baseline values prior to exercise, 0, 2, 4, 24 denote samples taken immediately, 2, 6 and 24 hours after exercise. Significant differences (P < 0.05) as determined by anova testing is indicated by an asterisk. The increase of the VWF level that was observed in the early poststress period is probably a result of an endothelial cell stimulation brought about by an increased IL-6 release as shown here and/or of stress hormones such as vasopressin or epinephrine [16]. Stimulation of endothelial cells may also be reflected by the increase in the level of sCD62P, which is a marker of both platelet and endothelial cell activation [17]. The inverse time course of VWF level (increase) and the activity of its degrading enzyme ADAMTS-13 (decrease) as observed here is consistent with other reports, where both parameters had been measured under various clinical and experimental conditions such as endotoxinemia or desmopressin application [9-11]. Thus far, the mechanism(s) regulating ADAMTS-13 activity is not well understood. In a recent study, we could not demonstrate any transcriptional regulation of ADAMTS-13 under various experimental conditions [18]. It was discussed by others that ADAMTS-13 activity could be exhausted when VWF is present in extremely high concentrations [9] or is removed from circulation by binding to VWF, which is immobilized on the surface of activated endothelial cells [19]. Furthermore, the increase of plasma IL-6 level that was observed in our study could also contribute to the decrease in ADAMTS-13 activity as a direct inactivation by IL-6 because of fragmentation of the enzyme molecule has been reported [12]. Recently, Padillo et al. [20] could demonstrate that CD62P anchors ULVWF to the surface of endothelial cell thereby facilitating the ULVWF cleavage by ADAMTS-13. It can be speculated that ULVWF binding to endothelial cells and thus its effective proteolytic cleavage might be disturbed in the presence of increased levels of sCD62P. The increase in sCD62P level after physical stress as it was observed in our study may not only reflect an activation of endothelial cells, but also an activation of platelets [21]. It is not clear yet whether the physical stress-induced platelet activation as observed here and elsewhere [22-24] is mediated by a platelet–VWF interaction or is because of a exercise-induced increase of stress hormones, that is, epinephrine and vasopressin which both are known to activate platelets [25]. Overall, the observed imbalance between VWF level and ADAMTS-13 activity may predispose healthy individuals for an increased platelet aggregation as observed following the extensive physical stress. On the other hand, the effect of ‘stress hormones’ such as epinephrine and vasopressin seems to play a critical role in activation of hemostasis via the regulation of ADAMTS-13 and VWF. Vasopressin – not only binding to V1 receptors on vascular smooth muscle cells [26] and on endothelial cells [27], but also to platelet V1 receptors [25] – may function as an alternative pathway affecting the platelet activation status and ADAMTS-13/VWF balance. In this context, it is known that vasopressin stimulates the expression of activation-dependent platelet antigens such as CD62 [25] and secretion of VWF in vitro via V1 receptor [28]. In summary, our data provide further evidence of an altered hemostasis after physical stress that is similar to those observed in case of a systemic inflammatory reaction. Moreover, prolonged exercise may serve as a valuable, non-invasive tool to investigate the balance between the release of VWF because of endothelial cell activation and ADAMTS-13 activity and its role in hemostasis and systemic inflammatory response. The study was supported in part by a grant from the IZKF Jena, TP 4.8, Thrombotic Microangiopathy in Sepsis. CL Bockmeyer has received financial support from the Friedrich-Schiller-University of Jena ‘Förderverein’ in form of the ‘Loder-Grant’ for young investigators. Neither the sponsor nor any pharmaceutical company was involved in study design, data collection, data analysis and interpretation, manuscript preparation, or the decision for submission. No conflicting interests of the authors.

Numerous studies support the notion that an activation of sphingomyelinases and a subsequent increase of the concentration of the bioactive lipid mediator ceramide are critical in the concert of inflammatory stimuli and to the induction of apoptosis during inflammation. Here we show that patients with severe sepsis exhibit an enhanced sphingolytic activity in comparison with controls [ 262 pmol/(mlxh) vs. 123.6 pmol/( mlxh), P < 0.005]. During the clinical course, a further increase was paralleled by the severity of illness and by fatal outcome. Moreover, we show that oxidative stress may partially account for the increased activity through posttranslational modification of the enzyme. In a murine endotoxic shock model, administration of a low molecular weight inhibitor diminished the rise in enzymatic activity and improved the survival rate. In liver specimen, inhibition of activity correlated with a reduced rate of hepato-cellular apoptosis. Our data support the concept that activation of the plasmatic isoform of sphingomyelinase may play a critical role in the development of apoptosis and organ failure in sepsis. An inhibition of the secreted isoform of sphingomyelinase should be explored further as a potential target in the complicated puzzle of sepsis.

A guideline for neurological expert testimony is proposed covering medical as well as juridicial aspects including recent court rulings, intended to support medical experts and to protect both probands and mandators against arbitrary or scientifically invalid statements. The role of the testimoning expert and the main requirements for testifying in different fields of insurances and legal procedures is being described. Likewise problems of experts liability and payment are touched.

SummaryThe metalloproteinase ADAMTS13 cleaves VWF multimers instantaneously when they are released from endothelial cells. Absent or manifestly diminished proteolytic activity of ADAMTS13 results in the appearance and accumulation of ultralarge VWF multimers (ULVWFM) in plasma, characterised by the manifestation of Thrombotic Thrombocytopenic Purpura (TTP). Despite congenital defects, infections and the actions of drugs such as cyclosporine A, doxycycline and corticosteroids apparently are involved in its development. To examine the possibility of transcriptional regulation of ADAMTS13 activity, we analyzed RNA levels in various cell culture systems and the response to known and assumed modulators of gene expression. We demonstrate the expression of ADAMTS13 in liver homogenates and a parenchyma liver cell culture system Hep3B, supporting the hypothesis that liver is an important source of plasma ADAMTS13, whereas there was no alteration in gene expression after stimulation of liver cells with proinflammatory stimuli such as endotoxin, TNF-α, IL-6, IL-1β as well as immuno-suppressive agents, such as cyclosporine A, a variety of steroids as well as doxycycline. Therefore, we analysed the ADAMTS13 gene for binding sites of transcription factors in silico and compared the data with those found in two sets of 24 genes considered either as differentially regulated by prototypic inflammatory regulation or as unvaried under various conditions. On the basis of these data, the promotor of ADAMTS13 features the characteristics of a gene, which remains unvaried under a variety of conditions. To our knowledge, the current data demonstrate for the first time, that an alteration in transcriptional activity is negligible in accounting for diminished proteolytic activity as observed under various experimental and, in particular, clinical conditions.

Objective: Platelet-activating factor (PAF) is a potent proinflammatory mediator in systemic inflammation and sepsis and is inactivated by the enzyme PAF-acetylhydrolase (PAF-AH). Recently, a large phase III clinical trial using recombinant PAF-AH to treat patients with severe sepsis was performed but failed to reduce 28-day mortality rate. To get more information on the activity of PAF-AH in sepsis, we repeatedly measured its activity in plasma in critically ill patients compared with healthy controls. Design: Retrospective cohort study. Setting: Intensive care unit. Patients: Two hundred thirty-one patients who were admitted to an operative intensive care unit within 1 yr were enrolled and evaluated daily for American College of Chest Physicians/Society of Critical Care Medicine criteria. PAF-AH activity was measured as the release of [3H]-acetate from [3H]-acetyl-PAF. Interventions: Analysis of plasma samples. Measurements and Main Results: At the day of admission, PAF-AH activity of patients was below controls but markedly increased over time. Higher activities were seen in patients with severe sepsis or septic shock compared with those without organ failure. With respect to the clinical outcome, lower values were found in nonsurvivors only as long as they had not developed organ failure. In severe sepsis/septic shock, values of nonsurvivors exceeded those of survivors. PAF-AH activity was positively correlated with plasma levels of inflammatory mediators such as neopterine and tumor necrosis factor-α but not with acute phase reactants such as C-reactive protein, interleukin-6, or PCT. In addition, parenteral nutrition with lipid emulsions was seemingly associated with low PAF-AH activity compared with enteral nutrition. Conclusion: The data indicate severity- and time-dependent changes in PAF-AH activity and may help to explain the failure of recombinant PAF-AH treatment strategies that were not based on activity measurements.

s of Papers Presented at the 10th Erfurt Conference on Platelets, 20–23 June 2004, Augustiner-Kloster, Erfurt, Germany Organising committee: Wolfgang Lösche Universitätsklinikum Jena, Klinik für Anästhesiologie und Intensivtherapie, Jena, Germany Peter Spangenberg Fachhochschule Jena, Fachbereich Medizintechnik, Jena, Germany Stan Heptinstall Institute of Clinical Research, University of Nottingham, UK Erika Glusa Institut für Pharmazie, Friedrich-Schiller-Universität Jena, Germany Günter Syrbe Asklepios Fachklinikum Stadtroda, Germany Karsten Schrör Heinrich-Heine-Universität Düsseldorf, Institut für Pharmakologie und Toxikologie, Düsseldorf, Germany The organisers thank the following for sponsoring the conference: American Diagnostica, Aventis Pharma Deutschland, Baxter Deutschland, BD Bioscience, Bayer Health Care, Biotest Pharma, Dade Behring Marburg, Dynabyte medical, Forschungsgruppe Herz-Kreislauf e.V., Gesellschaft für Thromboseund Hämostaseforschung e.V., Mitsubishi Pharma Deutschland, Octapharma, SIRS-Lab, Wyeth Pharma, ZLB Behring Platelets (December 2004) 15(8), 479–517 ISSN 0953-7104 print/ISSN 1369-1635 online/04/080479-39 2004 Taylor & Francis Ltd DOI: 10.1080/09537100412331272587 EVIDENCE FOR DIFFERENTIAL REGULATION OF THE b3 INTEGRINS aVb3 AND aIIBb3 I.G. Ahrens, M. Schwarz, C. Hagemeyer, N. Moran, D.J. Fitzgerald, C. Bode, K. Peter Department of Cardiology and Angiology, University of Freiburg, Germany and Department of Clinical Pharmacology Royal, College of Surgeons, Dublin, Ireland Background: Integrins are heterodimeric transmembrane cell adhesion molecules consisting of an aand b-subunit, forming large extracellular domains and short cytoplasmic tails. The cytoplasmic tails, and in particular the highly conserved GFFKR motif in the b-cytoplasmic tail, are known to be crucial for the regulation of integrin function. Integrin aVb3 is a major surface receptor on endothelial cells. It is involved in many processes such as migration of smooth muscle cells, tumor metastasis, angiogenesis, wound healing and arterial restenosis. Previously, we demonstrated that a deletion of the GFFKR-region locks integrin aIIbb3 in a high affinity state. Here we investigated the role of the GFFKR motif in the functional regulation of the other b3 integrin aVb3. Methods: We generated a double alanine-substitution (GAAKR) and a GFFKR-deletion in the cDNA coding for the aV cytoplasmic tail and stably expressed it together with the b3 subunit in CHOK1 cells. Characterization of the stably expressing cells was performed by flow cytometry and RTPCR followed by sequencing. The activity state of aVb3 was assessed with static adhesion assays and a flow chamber assay with increasing shear rates. Cytoskeletal anchorage was examined by immunofluorescence microscopy and signal transduction was investigated by immunoprecipitation of phosphorylated proteins followed by detection of pp125. Results: Adhesion of transfected cells, carrying either the GFFKR-deletion or the AA-substitution, to immobilized vitronectin or fibrinogen was reduced to the background adhesion of mock transfected CHO-K1 cells. For the flow chamber experiments cells were allowed to interact with the fibrinogen coated matrix for 5min followed by perfusion of the chamber with increasing shear rates. Wildtype aVb3 expressing cells were able to spread even at high shear rates (15 dyne/cm). Cells containing the GFFKRdeletion or AA-substitution did not spread and, if not lifted off at 15 dyne/cm, adhered to the matrix with single cytopods. Immunofluorescence of cells adhering to fibrinogen showed that introducing a GFFKR-deletion or AA-substitution leads to the inability to form actin stress fibers in these cells and immunoprecipitation of phosphorylated proteins revealed that during outside-in signalling phosphorylation pp125 of was impaired in these cells. Conclusion: Unlike in the integrin aIIbb3, in aVb3 the GFFKR-motif is necessary for adhesion to immobilized ligands, spreading under conditions of shear stress, cytoskeletal anchorage, and outside-in signal transduction via phosphorylation of pp125. These findings provide evidence for a differential functional regulation of these integrins via their membrane proximal b-cytoplasmic domains. PLATELET RESPONSE TO CLOPIDOGREL EVALUATED BY THE PFA-100 IN ASPIRIN RESPONDERS AND NON-RESPONDERS K. Andersen, I. Seljeflot, M. Hurlen, H. Arnesen Medical department, Central Hospital of Hedmark, Hamar, Norway and Clinical Cardiovascular Research Unit, Ullevål University Hospital, Oslo, Norway Background: Several studies have shown individual variations in the response to aspirin treatment, thus the concept of aspirin resistance or nonresponsiveness has been introduced. We studied platelet response to aspirin and clopidogrel therapy with the PFA-100 (Dade Behring), a method more suitable for screening than formerly available methods. Clopidogrel may have a special potential in patients not responding to aspirin. The purpose of this study was to investigate the response of clopidogrel utilizing the PFA-100 in aspirin responders and non-responders. Methods: Platelet function was evaluated in fasting condition 24 h after the last dose of medication in 40 patients on chronic aspirin therapy (160mg daily), 4 years after an AMI. CT-measurements were performed using a collagen/epinephrine and a collagen/ADP cartridge. Eighteen patients were classified as aspirin non-responders (CTEPI below the 95 percentile of the CTEPI’s in patients not treated by any platelet inhibitor; 196 s). Twenty patients were randomized to change from aspirin to clopidogrel 75mg daily, the others continued aspirin. Results: At randomization, median (25.75 percentiles) CTEPI was 300 (288, 300) in the responders and 167 (137, 176) in the non-responders ( p<0.001). After 4weeks the CTEPI’s in the patients still on aspirin had not changed significantly. In those on clopidogrel, the CTEPI’s were 127 (109, 264) in the responders and 122 (91, 136) in the nonresponders ( p1⁄4 0.305). CTADP at randomization was 95 (78,119) in the responders and 83 (72, 94) in the non-responders ( p1⁄4 0.032). After 4weeks the CTADP’s in the patients still on aspirin did not differ significantly. In those on clopidogrel, the CTADP’s were 98 (83, 114) in the responders and 90 (69, 102) 480 ABSTRACTS

The effects of the GPIIb-IIIa antagonists abciximab and MK-852 on platelet-leukocyte interactions in vitro were studied and the results compared with those obtained with a combination of aspirin, dipyridamole and AR-C69931 (Asp/Dip/AR-C). Platelet-monocyte (P/M) and platelet-neutrophil (P/N) conjugate formation increased when blood was stirred or a platelet agonist was added. Leukocyte activation also occurred as judged by expression of surface tissue factor antigen and CD11b. Abciximab and MK-852 potentiated P/M, especially when collagen was used. They also increased the amount of tissue factor on the monocytes, but not CD11b. The Asp/Dip/AR-C did not enhance P/M or tissue factor exposure. Augmented tissue factor expression on monocytes in the presence of a GPIIb-IIIa antagonist may be relevant to the increased mortality associated with trials of such antagonists when given orally in patients with vascular disease. The Asp/Dip/AR-C was superior to abciximab and MK-852 in inhibiting platelet and leukocyte function.

For rehabilitation training it is recommended that the intensity of exercise should be distinctly below the individual anaerobic threshold (IAT). We investigated platelet activity, reactivity and platelet-leukocyte conjugate formation following a stardardized treadmill (TR) ergometer test at 90% IAT for 60-120 min. Seventeen healthy male non-smokers underwent TR. Blood samples were taken after a 30-min rest, immediately after exercise, and 2 h after exercise completion. Platelets were detected flow cytometrically by CD41 in whole blood, activated platelets by CD62P. In addition, stimulation of platelets in vitro with 7.5 microM TRAP-6 was performed. For testing platelet-leukocyte conjugates, antibodies against CD45 and CD41 were used. After TR the percent of non-stimulated CD62P-positive platelets (%PC) remained unchanged (1.65 +/- 0.56 to 1.73 +/- 0.79%PC) (mean +/- SD). In contrast, an increase (P<0.05) from 31.9 +/- 13.5 to 37.4 +/- 15.0 %PC in CD62P, TRAP-6 stimulated and enhanced (P<0.01) platelet-leukocyte conjugates (11.7 +/- 3.7 to 16.1 +/- 6.9, CD41-%PC) after TR were observed. Both changes were independent of thrombin generation measured by F1+2 and TAT, and reversible after 2 h. Long-term exercise (90% IAT) on a treadmill ergometer only leads to a moderate increase of platelet reactivity and platelet-leukocyte conjugates. The determination of platelet-leukocyte conjugates may offer the possibility to detect an early activation stage of platelets in vivo.

SummaryTissue factor (TF) is the most important initiator of intravascular coagulation. Platelets contribute to TF exposure on monocytes, but the mechanism is not completely understood. Here we examined the possibility that platelets may release TF that can be transferred to monocytes by platelet-derived microvesicles. When human citrated platelet-rich plasma was incubated with collagen there was an increase in the plasma levels of TF and CD62P. Incubation of plasma obtained from collagen-stimulated PRP with a sediment of red and white blood cells resulted in an increase in the number of monocytes that express TF, CD62P and the platelet-specific antigen CD42a on their surface. This transfer of platelet-derived antigens to monocytes was reduced when CD62P was blocked by a specific antibody or when platelet-derived microvesicles were removed from the plasma either by high speed centrifugation (17,500 X g for 30 min) or by filtration (pore size 0.2 µm). The data indicate that platelet-derived microvesicles that are released from collagen-stimulated platelets may carry TF, CD62P and CD42a and may transfer these antigens to the surface of monocytes. The interaction of platelet-derived microvesicles with monocytes and the transfer of TF to monocytes strongly depend on CD62P.

Measurement of platelet aggregation in platelet-rich plasma (PRP) is a fundamental tool in platelet studies, despite the fact that the technique required for this is time-consuming, may need large volumes of blood, and require particular skill and special equipment. The use of a microplate reader seems useful to perform platelet aggregation more rapidly and with less material. So, the aim of the present study was to validate a simple and rapid method which enables performance of kinetic measurements of platelet aggregation directly in a microtiter plate reader. Platelet aggregation was carried out in 96-well, flat-bottomed microtiter plates. Samples of PRP (140 w l/well) were placed in a microtiter plate. Agonists (10 w l/well) were added using an electronic multichannel dispenser directly before the reading was started. Measurements of the optical density were performed at 650 nm using a THERMOmax™ microplate reader (Molecular Devices, Sunnyvale, USA). During the run time the plate was incubated at 37°C and was mixed with the automix function of the reader. The technique was verified by comparing dose-response curves of platelet agonists and glycoprotein IIb/IIIa antagonists, obtained with the standard aggregometer and with the microtiter plate reader. Platelet aggregation in microtiter plates is simple and rapid. It offers the advantages of lowering the test volumes and the possibility to perform about 90 tests simultaneously. The method was successfully applied to measure platelet inhibition by glycoprotein IIb/IIIa antagonists.

Aims: The aim of this study was to assess the inter- and intra-laboratory variation of the concentration–response to the GPIIb/IIIa-antagonists abciximab and eptifibatide on platelet aggregometry and to compare results with flow cytometric tests as well as the rapid platelet function analyser (RPFA). Methods: In five different laboratory sites, blood from three to five healthy donors was spiked with abciximab or eptifibatide, followed by the assessment of: (1) aggregometry (anticoagulant: sodium citrate 3.18% or hirudin 5 μg/ml); (2) flow cytometry (fibrinogen binding or PAC1-expression), or (3) RPFA. Dose–response curves were established on the basis of a sigmoidal Imax-model [I=(Imax*Cγ)/(IC50γ+Cγ)]. Results: For citrated blood, aggregation induced by 20 μM ADP was blocked up to 100% by both GPIIb/IIIa-antagonists, IC50 values varied between 0.11–0.22 μg/ml for eptifibatide and 1.25–2.3 μg/ml for abciximab. Imax of the response to 5 μg/ml collagen ranged from 46% to 100%, and IC50 values varied between 0.28–0.34 μg/ml for eptifibatide and 2.3–3.8 μg/ml for abciximab. In hirudinized blood, IC50 values for eptifibatide were 1.5- to 3-fold higher than those obtained with citrated plasma. Inhibition of PAC1-expression by abciximab (IC50 0.84 μg/ml) showed results similar those of the RPFA (approx. 1.0 μg/ml); larger differences between PAC1 and RPFA results were observed for eptifibatide. Based on aggregometry, eptifibatide concentrations for 80% inhibition varied from 0.27 to 0.55 μg/ml, and were considerably less when the RPFA was taken as basis (0.15 or 0.22 μg/ml). A similar pattern was observed for abciximab. Conclusions: We found quite a low inter- and intra-laboratory variation in the in vitro pharmacodynamic characterization of GPIIb/IIIa-antagonists by aggregometry, making results of these tests obtained from different laboratories during clinical trials at least comparable. The RPFA exhibits a higher sensitivity to inhibitory GPIIb/IIIa-effects, in keeping with the “real” inhibition of the activated receptor (PAC1) as assessed with more elaborate flow cytometry.

Tethering of PMNL by platelets via CD62P has been shown to cause PMNL activation. Co-incubation of purified PMNL with platelets that were activated with thrombin and then fixed and washed, resulted in the formation of platelet-PMNL conjugates as well as in a generation of reactive oxygen species that were measured as luminol-enhanced chemiluminescence. When platelets were thrombin activated in the presence of RGDS to prevent binding of fibrinogen to membrane receptors, they had a reduced capacity to adhere to PMNL, but ROS generation was enhanced. In samples of citrated whole blood RGDS as well as the more specific platelet fibrinogen receptor antagonist GR144053F or a dissociation of the platelet glycoprotein IIb/IIIa complex markedly enhanced ROS generation that was induced by stirring the samples for 10 min at 1000 rpm, by 175%, 95% and 138%, respectively. Removal of platelets from the whole blood samples also resulted in an enhancement of stirring-induced ROS generation, which was inversely correlated to the platelet count. These data provide some evidence that platelets are capable of inhibiting ROS generation in PMNL by a mechanism that involves platelet-bound fibrinogen and probably depends on fibrinogen-mediated platelet-PMNL contact.

Platelet-activating factor (PAF) and its inactivating enzyme PAF-acetyl-hydrolase (PAF-AH) are implicated in the development of sepsis and its sequela septic shock. It has been shown that the administration of rPAF-AH has a beneficial effect on the outcome of sepsis in animals as well as in humans.