Physical stress as a model to study variations in ADAMTS-13 activity, von Willebrand factor level and platelet activation.
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ADAM Proteins↗
Biomedical subjects
W Lösche
Publications and source records attributed to W Lösche.
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.
Animals↗
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.
1-Alkyl-2-acetylglycerophosphocholine Esterase↗
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.
Abciximab↗
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.
Blood Platelets↗
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.
Adenosine Diphosphate↗
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.
Aged↗
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.
Journal Article↗
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.
Journal Article↗
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.
Journal Article↗
Measurement of platelet activation and adhesion to leukocytes during haemodialysis.
In this study we used fluorescent-labelled antibodies to measure the extent of platelet adhesion to polymorphonuclear leukocytes (PMNLs), monocytes and lymphocytes. The activation of platelets, PMNLs and monocytes were also measured during the course of haemodialysis treatment using flow cytometric techniques established in our laboratory. Twenty patients were treated with either a cellulose membrane (TFU) or a polycarbonate filter (Pro 500). Blood samples were taken from the output line of the dialyser 2, 15, 30 and 180 min after commencing dialysis and just before starting treatment. Compared with the pre-dialysis sample, there was a marked increase in platelet-PMNL conjugate formation at 2 min, followed by a decrease in conjugates at 15 and 30 min, and a slight increase at 180 min. During extracorporeal circulation PMNLs become activated as measured by a CD11b upregulation at 15, 30 and 180 min, but not at 2 min. Platelet binding to monocytes was increased above 15 min after starting dialysis, and monocyte activation was slightly increased above basal levels during the same period. The activation state of circulating free platelets, as measured by surface P-selectin exposition, initially decreased slightly, but then returned to basal levels over the 3-h period. Changes in cell counts were also detected: there was a massive decrease in circulating PMNLs and monocytes, and a small decrease in circulating platelets, at 15 and 30 min. These reverted to basal values by the end of the 3-h period. There was no change in the number of circulating lymphocytes or erythrocytes. These results show that flow cytometric studies on whole blood samples may provide important information on the behaviour of circulating blood cells, which could supplement conventional clinical measurements, to give a better insight into changes that occur in the circulation during haemodialysis.
Journal Article↗
Monocyte tissue factor (CD142) and Mac-1 (CD11b) are increased in septic patients.
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Adult↗
[Billing fraud from the viewpoint of the federal social court].
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.
Fees, Medical↗
Platelets inhibit the activity of platelet-activating factor acetylhydrolase in monocyte-derived macrophages.
Blood platelets are capable of interacting with monocytes and macrophages and of enhancing various functions of these cells, which are believed to play a role in thrombosis and inflammation. An increase in the uptake of oxidised low density lipoprotein (LDL), in the synthesis of procoagulant tissue factor, thrombospondin and leukotrienes, as well as stimulation of oxygen radical production by platelets has been described (1-5). In circulating blood, a substantial proportion of monocytes was found to be associated with platelets, but the pathophysiological significance of such platelet-monocyte conjugates is not yet clear (6,7). Immigration of monocytes into the arterial intima and their differentiation into macrophages are initial steps in the development of an atherosclerotic lesion (8). During differentiation, there is a tremendous increase in the activity and secretion of the enzyme PAF acetylhydrolase (PAF = platelet-activating factor = 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) (9,10), and there is some evidence that this enzyme may contribute to the development of atherosclerosis. It cleaves PAF, and the remaining lyso-PAF is chemotactic for monocytes (11). Furthermore it also acts on oxidised low density lipoproteins and enhances their uptake into macrophages (12,13). We were therefore interested in investigating whether platelets may modulate the differentiation of monocytes into macrophages and the activity of PAF acetylhydrolase.
1-Alkyl-2-acetylglycerophosphocholine Esterase↗
Formation of platelet-leukocyte conjugates in whole blood.
The purpose of this investigation was to obtain information on platelet-leukocyte conjugate formation in whole blood and on factors that affect it. We also measured platelet and leukocyte activation by quantitating the expression of CD62P and CD11b. In both cases a flow cytometric approach was used. The results show that platelet-monocyte and platelet-polymorphonuclear leukocyte (PMNL) conjugate formation is enhanced by simply stirring blood, with optimum conjugate formation occurring after 10 min. In the case of monocytes,conjugate formation was enhanced by adenosine diphosphate (ADP). Both monocyte and PMNL conjugate formation was enhanced by phorbol myristate acetate (PMA), but L-formyl methionyl lysyl proline (FMLP) was either without effect (monocytes) or inhibitory (PMNL). EDTA also inhibited conjugate formation (implying involvement of divalent cations), as did dextran sulphate (implying involvement of P-selectin = CD62P). Interestingly GR144053F, which acts at GpIIb-IIIa on platelets to interfere with fibrinogen binding, and also glycyl prolyl arginyl proline (GPRP), a peptide that interferes with the interaction between CD11c on leukocytes and fibrinogen, did not inhibit platelet-monocyte conjugate formation, but did inhibit the platelet-PMNL interaction; this indicates that GpIIb-IIIa on platelets and CD11c on leukocytes and fibrinogen are involved in mediating the interaction between platelets and PMNL but not platelets and monocytes. Surprisingly arginyl-glycyl aspartyl serine (RGDS) inhibited the formation of both types of conjugate but this may be because it also inhibited both platelet and leukocyte activation as measured by CD62P and CD11b exposure and/or interferes with the binding of adhesion molecules other than fibrinogen. The results show that a flow cytometric procedure can be effective in obtaining rapid information on platelet-leukocyte conjugate formation in whole blood and on factors that are involved in its regulation. It is suggested that the technique may be applicable to the study of platelet-leukocyte conjugate formation in whole blood in disease, and also to study the effects of drugs interfering with conjugate formation.
Journal Article↗
Contact-induced modulation of neutrophil elastase secretion and phagocytic activity by platelets.
It has been reported that platelets stimulate generation of reactive oxygen species in neutrophils and monocytes by a mechanism that requires mutual cell-cell contact and the presence of P-selectin on the platelet surface. In the present study we investigated the effect of platelet-neutrophil contacts on neutrophil elastase secretion and phagocytic activity. Non-activated or thrombin-activated platelets were fixed with formaldehyde, washed and incubated with neutrophils in the absence or presence of various neutrophil agonists. Elastase secretion was determined by measuring the enzyme activity in cell-free supernatants using a chromogenic substrate. Platelet-neutrophil adhesion and ingestion of zymosan particles by neutrophils were quantitated by light microscopy. Platelets significantly reduced elastase secretion from neutrophils but had no effect on the elastase activity in the supernatant of neutrophil lysates. When neutrophils were stimulated with the ionophore A23187 or the chemotactic peptide FMLP, thrombin-activated platelets were more potent to inhibit elastase secretion when compared with non-activated platelets. Neutrophils that were not able to bind platelets to their surface had a significantly lower phagocytic activity when compared with neutrophil with adherent platelets or neutrophils that were incubated in the absence of platelets. The results indicate that platelet-neutrophil contacts may also lead to an inhibition of neutrophil functions and that such inhibition could be due to a transient contact rather than due to a firm platelet-neutrophil adhesion.
Blood Platelets↗
Studies on the effects of agonists and antagonists on platelet shape change and platelet aggregation in whole blood.
The shape change that occurs when platelets are stimulated with an agonist can be quantitated by monitoring changes in their forward-scatter/side-scatter profile using a flow cytometer. Here we have stimulated platelets in citrated whole blood with several agonists and determined the time-course and extent of the shape change that occurs. In some experiments parallel investigations of shape change and aggregation were performed. Aggregation was measured by monitoring the fall in number of single platelets using a Whole Blood Platelet Counter. Some agents (ADP, PAF, U46619 and 5HT) produced a strong and rapid change in platelet forward-scatter/side-scatter that was maximal within 10 s. Others (A23187 and collagen) produced a strong but slower response. Adrenaline produced only a weak response that was also slow to develop, and PMA did not produce any response. The concentrations of each of ADP, PAF, U46619 and 5HT needed to induce a shape change were lower than those required for aggregation. Selective PAF, TXA2 and 5HT antagonists (WEB 2086, sulotroban and MCI-9042) clearly inhibited both the shape change and the aggregation induced by the appropriate agonist; in each case the effect of the antagonist was to move the dose-response curve to the right. These results are consistent with the shape change and aggregation brought about by each of these agonists being mediated via a single receptor. In contrast, a selective P2T purinoceptor antagonist (ARL 66096) markedly inhibited the aggregation induced by ADP but was found to have little or no effect on shape change. This is consistent with these platelet responses to ADP being mediated by different receptors, with P2T receptors mediating only the aggregation response.
15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
ADP-induced platelet aggregation and actin polymerization. Involvement of GpIIb/IIIa and the effect of Mg2+.
We have investigated the effects of Mg2+ (added to platelet rich plasma [PRP] as 10mM MgCl2 or MgSO4) on the platelet aggregation and actin polymerization that occurs in response to adenosine diphosphate (ADP). The PRP was prepared from blood containing hirudin as anticoagulant. Mg2+ added before 1 microM ADP completely inhibited aggregation and markedly inhibited actin polymerization. Mg2+ (10mM) added before 10 microM ADP converted irreversible aggregation into a reversible response; similarly, apparently irreversible actin polymerization was converted to a reversible response in which polymerization was followed by some actin depolymerization. Mg2+ added after inducing platelet aggregation with 10 microM ADP produced parallel disaggregation of platelets and actin depolymerization. Actin polymerization occurs immediately on adding ADP to PRP (in association with shape change) and further polymerization occurs in association with platelet aggregation. When aggregation (and the actin polymerization associated with this) was prevented by adding M148, a monoclonal antibody directed at the GpIIb/IIIa complex, or simply by avoiding stirring the sample, Mg2+ had no effect on actin polymerization/depolymerization. Thus Mg2+ only affected the changes in actin that were associated with the aggregation response. This was in contrast to iloprost (which acts at the PGI2 receptor to stimulate adenylate cyclase) which induced rapid actin depolymerization when added after ADP stimulation of platelets under circumstances where platelet aggregation was avoided. These results show that Mg2+ affects actin polymerization as well as platelet aggregation, and that it affects the actin polymerization associated with aggregation but not that associated with shape change (in contrast to iloprost which inhibits both).(ABSTRACT TRUNCATED AT 250 WORDS)
Actins↗