[Therapy of inherited diseases of platelet function. Interdisciplinary S2K guideline of the Permanent Paediatric Committee of the Society of Thrombosis and Haemostasis Research (GTH e. V.)]. / Therapie hereditärer Thrombozytopathien. Interdisziplinäre S2K-Leitlinie der Ständigen Kommission Pädiatrie der Gesellschaft für Thrombose- und Hämostaseforschung e. V.

Inherited disorders of platelet function are a heterogeneous group. For optimal prevention and management of bleeding, classification and diagnosis of the underlying defect are highly recommended. An interdisciplinary guideline for a diagnostic approach has been published (AWMF # 086-003 S2K; Hämostaseologie 2014; 34: 201-212). Underlying platelet disorder, platelet count, age and clinical situation modify treatment. Exclusive transfusion of platelet concentrates may be inappropriate as potentially adverse effects can outweigh its benefit. A stepwise and individually adjusted approach for restitution and maintenance of haemostasis is recommended. Administration of antifibrinolytics is generally endorsed, but is of particular use in Quebec disease. Restricted to older children, desmopressin is favourable in storage pool disease and unclassified platelet disorders. Although licensed only for patients with Glanzmann thrombasthenia and alloantibodies, in clinical practice rFVIIa is widely used in inherited platelet disorders with severe bleeding tendency. This guideline aims at presenting the best available advice for the management of patients with inherited platelet function disorders.

[Diagnosis of inherited diseases of platelet function. Interdisciplinary S2K guideline of the Permanent Paediatric Committee of the Society of Thrombosis and Haemostasis Research (GTH e. V.)]. / Diagnose angeborener Störungen der Thrombozytenfunktion. Interdisziplinäre S2K-Leitlinie der Ständigen Kommission Pädiatrie der Gesellschaft für Thrombose- und Hämostaseforschung e. V.

Congenital disorders of platelet function are a heterogeneous group of disorders that are often not detected until bleeding occurs. In clinical settings only a few methods have proven to be useful for identification and classification of inherited platelet disorders. For a rational diagnostic approach, a stepwise algorithm is recommended. Patient history and clinical investigation are mandatory. Von Willebrand disease and other coagulation disorders should always be ruled out prior to specific platelet testing. Platelet count, size, volume (MPV) and morphology may guide further investigations. The PFA-100® CT is suited for screening for severe platelet defects. Platelet aggregometry allows assessment of multiple aspects of platelet function. Flow cytometry enables diagnosis of thrombasthenia Glanzmann, Bernard-Soulier syndrome and storage pool defects. Molecular genetics may confirm a putative diagnosis or pave the way for identifying new defects. We present an unabridged version of the interdisciplinary guideline.

Screening for bacteremia in sepsis and renal failure using hemofilters for renal replacement therapy.

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.

PP055. Preliminary results of the more prepared study (microparticle orientated risk evaluation in the prediction of preeclampsia among risk gravidas): A multicenter prospective prognostic marker study.

INTRODUCTION: Preeclampsia (PE) is a potentially dangerous pregnancy pathology contributing to a higher worldwide mortality and morbidity. The negative influence of syncytiotrophoblastic microparticles (STBMs) on the placenta and maternal endothelia is thought to play a key role in generating the inflammatory effects that lead to PE symptoms. Doppler sonography of the uterine arteries assists in identifying a risk population, however, the positive predictive value for this method is low. OBJECTIVES: Aim of this study is to evaluate whether STBMs can serve as an accessory marker to conventional Doppler sonography to better identify pregnant women who will actually develop PE. METHODS: Pregnant women between 19-21 gestational weeks (GW) with abnormal uterine perfusion were enrolled into this prospective study. Plasma samples were taken at inclusion (baseline) and at two further visits at 8 week intervals to follow STBM concentration alterations during pregnancy. The primary endpoint assessed is PE and/or hemolysis, elevated liver, low platelets (HELLP) syndrome. Other PE-associated pathologies (intrauterine growth retardation [IUGR], intrauterine fetal demise [IUFD], placental abruption, premature delivery) constitute the secondary endpoints. Maternal STBM concentrations were measured using a home made Enzyme Linked Sorbent Assay (ELSA) which specifically measures STBMs. The receiver operating characteristics (ROC) for baseline measures are graphically displayed and area under curve (AUC) is estimated including 95% confidence levels. RESULTS: Of the 73 women included in the study, 16 developed PE (cases) and 56 did not (control). After analyses of mid-gestational probes, the ROC curve was in close proximity to the line of no-discrimination. CONCLUSION: Our preliminary results indicate that the maternal STBM concentration at mid-gestation does not predict the development of PE or associated pregnancy pathologies. Further analysis is underway to assess whether STBM measurements at later gestational time points can predict PE shortly before onset of disease.

Validation of a procedure to assess ASA-response in patients with decreased, initial TRAP induced aggregation.

Whole blood aggregometry on the is a simple, fast and standardized method and it is widely used to assess platelet function under antiplatelet therapy. Reference ranges and a cut-off value as a measure of ASA response were established by measuring arachidonic acid induced aggregation (ASPI-test) in healthy volunteers and cardiac patients after and used to classify patients as ASA responders or non-responders. However, assessing the platelet function is highly affected by pre-analytical and analytical conditions and often reduced aggregation by TRAP induced aggregation (TRAP-test) is seen, rendering the samples difficult for interpretation of the ASPI-test and the responder status to ASA. We hypothesised that in this simplified model any preanalytical factor has the same effect on TRAP-testing as on ASPI-testing and that by calculating the ratio between a defined, normal TRAP-test result and the TRAP-test result measured for the individual patient this ratio could be applied to the measured ASPI-test thereby reaching a more valid discrimination between ASA responders and -non-responders. TRAP- and ASPI-test were performed in blood from ASA-treated volunteers and controls on Multiplate before an after pneumatic tube delivery as a model to stimulate shear stress induced platelet activation and aggregation. The calculated, normalised ASPI test result after tube delivery did not differ significantly from the initial ASPI test result although tube delivery had a significant impact on the measured ASPI test result. If applied to patients samples a definite judgement on the ASA response status of patients with reduced "general platelet activatability" could be given. Normalisation of the ASPI-test result using the TRAP-test result may provide a method to judge on the ASA response status in patients with decreased initial "general platelet activatability".

The balance between von-Willebrand factor and its cleaving protease ADAMTS13: biomarker in systemic inflammation and development of organ failure?

PURPOSE: This review investigates and highlights the activity of Willebrand factor (VWF) and its cleaving protease as biomarkers of the development of multiple organ dysfunction in infectious and noninfectious systemic inflammatory response syndrome. STATE OF THE ART: Ultra-large VWF (ULVWF) multimers activate platelets resulting in a prothrombotic situation. Systemic inflammation is associated with increased ULVWF plasma level and a decreased ADAMTS13 activity. The potential role of ADAMTS13 as a diagnostic and prognostic marker of disseminated intravascular coagulopathy is largely underestimated. SUMMARY: VWF is an acute phase protein and its plasma level increases in systemic inflammation. When released from endothelial cells and platelets, the native multimeric glycoprotein is mostly present in the ultralarge form (ULVWF), which may have a major clinical significance under proinflammatory conditions. ULVWF-multimers may activate endothelial cells and platelets simultaneously. The multimers undergo limited proteolysis by a specific plasma metalloprotease known as ADAMTS13 (a disintegrin and metalloprotease with thrombospondin motif), thus, in healthy individuals only marginal amounts of circulating ULVWF are detectable. Severe hereditary or acquired ADAMTS13 deficiency causes thrombotic thrombocytopenic purpura (TTP), which contributes to prothrombotic coagulation abnormalities preceding organ dysfunction systemic inflammatory response syndrome (SIRS). In proinflammatory conditions, ADAMTS13 activity decreases due to various mechanisms, (i) down regulation on a transcriptional level, (ii) proteolytic degradation, and (iii) consumption due to the high substrate level. Marked dysbalance as found in patients with severe sepsis or septic shock results in substantial amounts of plasma ULVWF. This level of dysbalance is negatively correlated with platelet count and positively correlated with the severity of inflammation and the degree of organ failure.

Platelets and tissue factor.

Tissue factor (TF) is the most important initiator of intravascular coagulation. This article will review published evidence on the contribution of platelets to TF exposure to the circulating blood. The following mechanisms will be discussed: decryption of monocyte TF by platelets, contribution of platelets to TF expression in leukocytes, platelet-derived TF and its procoagulant activity, and activation of circulating TF by platelets.

Lipoprotein-associated phospholipase A2 and plasma lipids in patients with destructive periodontal disease.

OBJECTIVES: Periodontitis is believed to be an independent risk factor of cardiovascular disease (CVD) and to be associated with a moderate systemic inflammatory reaction and hyperlipidaemia. Lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) is an enzyme that has been shown to be a risk factor of CVD and that is involved in the degradation of the phospholipid mediator platelet-activating factor (PAF), a potent mediator of inflammation. MATERIAL AND METHODS: In the present study, we measured concentrations of plasma lipids and plasma activity of Lp-PLA(2) in 32 patients (mean age 43+/-11 years) with moderate-to-severe periodontitis before and 3 months after local treatment. RESULTS: Periodontal therapy resulted in a significant reduction of local inflammation and tissue destruction as reflected in reduced pocket depths and reduced bleeding indices. Pre- and post-treatment plasma lipid levels were (median and range, mmol/l): total cholesterol (C) 5.01 (3.94-7.15) and 4.91 (3.32-8.01); low-density lipoprotein-cholesterol (LDL-C) 3.14 (2.40-4.84) and 2.96 (1.39-5.04); HDL-C 1.27 (0.73-2.17) and 1.25 (0.74-2.55); triglycerides 1.37 (0.48-5.11) and 1.14 (0.38-792). Using the Wilcoxon's rank test, neither parameter showed a significant change. In contrast to the lacking response of plasma lipids, we observed a significant reduction in the activity of Lp-PLA(2). Local treatment lowered the enzyme activity by about 10% from 3.61+/-0.99 to 3.29+/-0.94 micromol/ml/h (mean+/-SD; p<0.001). The pre-treatment values of Lp-PLA(2) and LDL-C significantly correlated with clinical parameters of inflammation and periodontal destruction. CONCLUSION: This study indicates that treatment of periodontitis significantly reduces the serum activity of Lp-PLA(2), which is believed to be an independent cardiovascular risk factor.

Monitoring the effects of platelet glycoprotein IIb/IIIa antagonists with a microtiter plate method for detection of platelet aggregation.

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 microl/well) were placed in a microtiter plate. Agonists (10 microl/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 degrees 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.

Inhibition of leukocyte chemiluminescence by platelets: role of platelet-bound fibrinogen.

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.

In vitro dose response to different GPIIb/IIIa-antagonists: inter-laboratory comparison of various platelet function tests.

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 microg/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)*Cg)/(IC50g + Cg)]. RESULTS: For citrated blood, aggregation induced by 20 microM ADP was blocked up to 100% by both GPIIb/IIIa-antagonists, IC50 values varied between 0.11-0.22 microg/ml for eptifibatide and 1.25-2.3 microg/ml for abciximab. I(max) of the response to 5 microg/ml collagen ranged from 46% to 100%, and IC50 values varied between 0.28-0.34 microg/ml for eptifibatide and 2.3-3.8 microg/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 microg/ml) showed results similar those of the RPFA (approx. 1.0 microg/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 microg/ml, and were considerably less when the RPFA was taken as basis (0.15 or 0.22 microg/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.

Plasma lipid and blood glucose levels in patients with destructive periodontal disease.

Hyperlipidaemia and hyperglycaemia are major risk factors for cardiovascular disease. In recent years, some evidence has been presented that periodontal disease is associated with an increased risk of cardiovascular disease. To further elucidate this association, we have studied standard blood chemistry variables known as risk markers for cardiovascular disease in periodontally diseased and healthy subjects. We have measured levels of plasma lipids and fasting blood glucose in 39 subjects with moderate periodontal disease (age 50-60 years) and compared the results with those obtained in 40 age- and sex-matched controls. Both groups were systemically healthy according to their medical history. Total cholesterol, low density lipoprotein cholesterol and triglycerides were significantly higher in periodontally diseased subjects by about 8% (p<0.03), 13% (p<0.003) and 39% (p<0.001), respectively, when compared to controls. Although subjects with diabetes were excluded from the study, we found significantly higher blood glucose levels in the patient than in the control group (85 +/- 25 versus 73 +/- 17 mg/dl; p<0.02). There was also a significantly higher frequency of pathological plasma lipid profiles in the patient than in the control group. The results indicate that hyperlipaemia and pre-diabetes may be associated with periodontal disease in systemically healthy subjects. These data do not allow us to decide, whether periodontal disease causes an increase in hyperlipaemia and in a prediabetic state or whether periodontal disease and cardiovascular disease share hyperlipidaemia and the prediabetic state as common risk factors.

[Billing fraud from the viewpoint of the federal social court]. / Abrechnungsbetrug aus Sicht des Sozialgerichts.

The intended settlement fraud as it is found in business criminality does not play a role in the social jurisdiction (of Berlin). A physician is endangered to be prosecuted if his treatments become uneconomically as defined by the social law regarding health insurances. These medical procedures are characterized as being excessively extended. The border of settlement fraud may then be reached easily.

Activation of leukocytes in whole blood samples by N-formyl-methionyl-leucyl-phenylalanine (FMLP) enhances platelet aggregability but not platelet P-selectin exposure and adhesion to leukocytes.

Adhesion of platelets to neutrophils and monocytes is believed to play an important role in intercellular communication. Evidence has been provided that such heterotypic cell-cell contacts via adhesion molecules may be directly involved in intercellular signal transduction as well as facilitate the action of soluble signal transmitters, e.g. cathepsin G, PAF or nitric oxide. With respect to platelet activation, stimulatory and inhibitory effects of leukocytes have been reported, and the results obtained seem to be influenced by the experimental conditions. We investigated the effect of leukocyte stimulation on platelet behaviour in samples of human citrated whole blood. Adding the chemotactic peptide FM LP, which stimulates neutrophils and monocytes but not lymphocytes and platelets, to stirred whole blood samples resulted in a significant enhancement ( P < 0.01) of spontaneous as well as ADP-induced platelet aggregation (25 vs 33% and 66 vs 69% , respectively). In contrast stirring-induced as well as ADP-induced increase of P-selectin exposure (33 and 107% , respectively) was not affected by FMLP. In unstirred whole blood samples, about 10 to 20% of neutrophils and monocytes had bound platelets to their surfaces, and the number of these heterotypic conjugates was enhanced about twofold during spontaneous platelet aggregation. Addition of FMLP significantly reduced the stirring-induced formation of platelet-neutrophil conjugates but not of platelet-monocyte conjugates. These results indicate that neutrophil and/or monocyte activation in whole blood may enhance platelet aggregation, but not secretion (CD62P exposure) and formation of heterotypic platelet-leukocyte conjugates.

GPIIb-IIIa antagonists cause rapid disaggregation of platelets pre-treated with cytochalasin D. Evidence that the stability of platelet aggregates depends on normal cytoskeletal assembly.

Platelet activation is accompanied by changes in the composition of the platelet cytoskeleton with rapid incorporation and displacement of certain proteins. Here we have inhibited cytoskeletal assembly by pretreating platelets with cytochalasin D (CyD) and investigated the effect on the stability of the aggregates that form. The experiments were performed in both citrated and hirudinized platelet-rich plasma (PRP) and aggregation was induced by adenosine diphosphate (ADP), collagen, the TXA2-mimetic U46619 and adrenaline. Platelets in the aggregates that formed, underwent rapid disaggregation on addition of EDTA or a GpIIb-IIIa antagonist such as MK-852 and GR144053F, all of which are agents that interfere with the ability of fibrinogen to interact with GpIIb-IIIa. This was the case irrespective of the aggregating agent used and occurred in both citrated and hirudinized PRP. In contrast, the rate of disaggregation brought about by some other agents, iloprost and ARL 66096, appeared to be unaffected by CyD. Information was also obtained on the effects of CyD on the cytoskeletal changes brought about by ADP and the effects on the cytoskeleton of subsequent addition of M K-852. The results show that CyD retards the incorporation of certain proteins (actin, myosin, alpha -actinin, actin binding protein and a 66 K protein) into the cytoskeleton and that subsequent addition of MK-852 results in rapid displacement of some of these with re-incorporation of a 31 K protein. The results suggest that the early changes in the cytoskeleton following platelet activation contribute to the stability of the aggregates that form, and that interference with these early changes results in aggregates that are easily disassembled by agents that interfere with GpIIb-IIIa-fibrinogen complex formation.

Role of GPIIb-IIIa in platelet-monocyte and platelet-neutrophil conjugate formation in whole blood.

Platelets in stirred whole blood can be induced to form aggregates and also to form heterotypic platelet-monocyte (P/M) and platelet-neutrophil (P/N) conjugates. Here we have investigated the effects of three GPIIb-IIIa antagonists (GR144053F, MK-852 and Reopro, a CD62P-blocking antibody, GA6, and EDTA on the conjugate formation that occurs on stirring whole blood and in response to adding ADP and PAF. We have confirmed the identities of the conjugates by light microscopy after cell sorting. Platelet aggregation was measured by platelet counting. Monocytes, neutrophils, P/M and P/N were detected and quantitated using immunofluorescence and flow cytometry. Stirring whole blood resulted in both platelet aggregation and formation of P/M but not P/N. Adding ADP or PAF to whole blood caused rapid platelet aggregation and generation of both P/M and P/N. All of the GPIIb-IIIa antagonists studied had similar effects: inhibition of stirring-induced platelet aggregation and P/M formation, and inhibition of ADP-induced platelet aggregation and P/N formation. In contrast, they accelerated ADP induced-P/M conjugate formation and PAF-induced formation of both P/M and P/N. Both EDTA and GA6 completely inhibited P/M and P/N, which is commensurate with CD62P being involved in platelet-leucocyte conjugate formation. The results of these investigations suggest that GPIIb-IIIa has a dual role in determining the interaction between platelets and leukocytes.