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Biomedical subjects

B Jilma

Publications and source records attributed to B Jilma.

At least 19 recordsLinked to original sources
PubMed2001

Effects of heparin and aspirin on circulating P-selectin, E-selectin and von Willebrand Factor levels in healthy men.

As thrombin stimulates P-selectin expression on platelets and its release into plasma, we hypothesized that enhancing antithrombin activity by unfractionated heparin (UFH) could decrease plasma levels of circulating (c)P-selectin, (c)E-selectin, and von Willebrand Factor (vWF). Hence the effect of UFH and aspirin were examined on these activation markers in healthy volunteers. UFH decreased cP-selectin levels by -10% (CI: -16 - (-4%); P = 0.005) at 24 h, but did not change levels of vWF-Ag. In contrast, aspirin did not affect cP-selectin levels but decreased vWF-Ag levels by -12% (CI: -18 - (-7%); P = 0.005) at 24 h. Neither drug affected cE-selectin levels. Thus, UFH decreases cP-selectin levels, which may reflect decreased platelet activation in vivo. An increase in cP-selectin under UFH therapy should alert the clinician to look for platelet destruction.

Adult↗
PubMed2001

High levels of reticulated platelets and thrombopoietin characterize fetal thrombopoiesis.

To characterize fetal thrombopoiesis, we determined plasma thrombopoietin (TPO) and glycocalicin levels, platelet counts and reticulated platelets (RP) of fetuses and compared them with the respective values of their mothers. Percutaneous umbilical vein sampling in abnormal pregnancies revealed twofold higher thrombopoietin levels and 20-fold higher reticulated platelet counts, but lower levels of glycocalicin in fetuses compared with their mothers (P < 0.05). Neither the expression of platelet glycoprotein Ib and IIb on platelets nor the platelet counts were different between mothers and their fetuses. These data indicate enhanced thrombopoiesis and/or increased platelet turnover in fetuses.

Adult↗
PubMed2001

A new flow cytometric method for simultaneous measurement of residual platelets and RBCs in plasma: validation and application for QC.

BACKGROUND: Guidelines for the preparation of FFP in Austria and Germany require the quantification of residual RBCs in plasma. However, currently there is no validated method for enumeration of RBC counts as low as 1 x 10(9) per L. STUDY DESIGN AND METHODS: A new flow cytometric method was developed for QC, to simultaneously determine if fresh unfrozen plasma contains residual platelets and RBCs. This method uses test tubes (TruCount, Becton Dickinson Immunocytometry Systems) that contain a known number of brightly fluorescent polystyrene beads. Plasma is pipetted into these tubes and mixed with FITC-labeled anti-CD41 and PE-conjugated anti-glycophorin A MoAbs. Acquisition can be performed on a flow cytometer after an incubation period of 15 minutes. RESULTS: Individual standard curves for platelet counts revealed excellent correlation coefficients (r>0.998). Platelet counts were validated against simultaneous measurements by using a cell counter and a hemocytometer. While the flow cytometric method slightly overestimated platelet counts of >40 x 10(9) per L by 10 percent, its precision in the critical range was very good-that is, a deviation of platelets < or = 1 x 10(9) per L from target values, which was even better than microscopic enumeration. The limit of sensitivity was <0.5 x 10(9) per L of platelets. RBC counts were also validated against simultaneous measurements made with a second cell counter. Standard curves for RBC counts also revealed excellent correlation coefficients (r>0.997). The limit of sensitivity was <0.25 x 10(9) RBCs per L. Platelet counts in plasma obtained by plateletpheresis or plasmapheresis ranged from 3 to 60 x 10(9) per L. About 25 percent of all plasma samples had platelet counts greater than the allowed upper limit of 25 x 10(9) per L, while all plasma samples contained fewer RBCs than the upper limit of 6 x 10(9) per L. CONCLUSION: This newly developed method provides a simple, quick, precise, and easily reproducible tool for simultaneous measurement of residual platelets and RBCs in fresh plasma.

Erythrocyte Count↗
PubMed2001

Effects of anticoagulation on thrombopoietin release during endotoxemia.

Several lines of evidence suggest that coagulation may induce the release of thrombopoietin (TPO) into plasma and that TPO levels are higher in disseminated intravascular coagulation. Therefore we set out to illuminate the mechanism of TPO release in the setting of experimental endotoxemia, which induces activation of coagulation and platelets. Endotoxin (lipopolysachharide [LPS], 2 ng/kg) was infused into a total of 54 healthy men in two subsequent studies. Volunteers received infusions of unfractionated heparin, low-molecular-weight heparin, lepirudin, or placebo in a randomized, placebo-controlled fashion after bolus injection of LPS. TPO levels increased on average by 27% to 38% in all groups at 6 hours (P <.05 vs baseline), although all active drugs effectively blocked coagulation. Platelet counts dropped by about 15% at 1 hour after LPS infusion, recovered after 2 days, and exceeded baseline values by 8% to 18% after 7 days (P <.001 vs baseline for all groups). Yet lepirudin blunted the LPS-induced increase in circulating P-selectin by one half (P <.005 vs placebo), whereas both heparins did not diminish the increase in this platelet or endothelial activation marker as compared with placebo. Endotoxemia enhances TPO plasma levels independent of the degree of coagulation induction, which eventually results in increased platelet numbers. Of potential clinical interest is the observation that the direct thrombin inhibitor lepirudin, in contrast to heparins, mitigated LPS-induced platelet activation.

Adult↗
PubMed2001

Effect of intradermal tumor necrosis factor-alpha-induced inflammation on coagulation factors in dermal vessel endothelium. An in vivo study of human skin biopsies.

Inflammatory mediators were shown to exert procoagulant effects on cultured human endothelial cells (EC). In the present study the effect of intradermal application of tumor necrosis factor-alpha (TNF-alpha) on the expression of factors involved in regulation of coagulation at the EC surface, i.e. tissue factor (TF), thrombomodulin (TM) and tissue factor pathway inhibitor (TFPI) was studied in humans in vivo. The endothelial expression of these factors was evaluated immunohistochemically in biopsies taken after intradermal application of 5000 U TNF-alpha in 8 healthy volunteers. After 6 and 22 h biopsies were taken from the injection sites. At TNF-alpha injected sites typical inflammatory changes. e.g. EC upregulation of adhesion molecules and accumulation of leukocytes were detected. In parallel we could document EC expression of TF, downregulation of TM and depletion of tissue factor pathway inhibitor (TFPI) in inflamed areas. Early depletion of endothelial IkappaB alpha at the site of inflammation after application of TNF-alpha points to an activation of the NF-kappaB pathway. Our data suggest that, as shown in in vitro experiments, TNF-alpha activates the NF-kappaB pathway and induces specific procoagulant changes of EC due to expression of TF, down-regulation of TM and depletion of TFPI in vivo in humans. This procoagulant shift in the haemostatic balance on the cell surface, caused by TNF-alpha-induced inflammation, is likely to contribute to thrombosis associated with tissue inflammation in humans.

Adolescent↗
PubMed2000

Inhibitory activity of aspirin on von Willebrand factor-induced platelet aggregation.

The effect of aspirin (ASA) on vWF induced platelet - platelet interaction is unknown. We therefore tested the response of platelets to von Willebrand factor (vWF) coated beads induced platelet aggregation before and after i.v. and oral ASA. 1000 mg ASA was infused to 10 healthy individuals and after a wash-out period 7 volunteers received 100 mg ASA orally over a period of 11 days. Prior to ASA and in regular intervals thereafter we tested the reactivity to vWF-coated beads to assess platelet adhesion/aggregation and the fade-out time of ASA effects on platelets. Considerable interindividual variability in response to vWF-coated beads was observed, both before ASA and after treatment with ASA. The maximal response to vWF-coated beads (Tmax), the time lag, and the slope of the curve were significantly affected by i.v. ASA, whereas 100 mg of ASA had only inconstant effect on Tmax and slope. The absolute reduction of Tmax after ASA depended on the pre-ASA level, while the percentage of the reduction was similar in all individuals. Thus, platelet aggregation induced by vWF-coated beads is impaired by ASA. Furthermore, our data indicate a large interindividual variability of the response to ASA shortly after treatment induction, which becomes more constant after prolonged treatment.

Adult↗
PubMed2000

Endotoxin down-modulates granulocyte colony-stimulating factor receptor (CD114) on human neutrophils.

During infection, the development of nonresponsiveness to granulocyte colony-stimulating factor (G-CSF) may be influenced by the down-modulation of G-CSF receptor (G-CSFR) by cytokines. This down-modulation was studied during experimental human endotoxemia. Healthy volunteers received either 2 ng/kg endotoxin (lipopolysaccharide [LPS], n=20) or placebo (n=10) in a randomized, controlled trial. Endotoxin infusion increased the mean fluorescence intensity of the neutrophil activation marker CD11b >300% after 1 h (P<.001 vs. placebo). LPS infusion down-modulated G-CSFR expression in as early as 60 min (-17%; P=.001 vs. placebo). Down-modulation was almost maximal at 90 min and persisted for 6 h (-50% from baseline; P<.0001 vs. placebo). Plasma levels of G-CSF started to increase only after G-CSFR down-modulation had occurred and peaked 37-fold above baseline at 4 h (P<.0001 vs. placebo). In conclusion, LPS down-modulates G-CSFR expression in humans, which may render neutrophils less responsive to the effects of G-CSF and, thereby, compromise host defense mechanisms.

Adult↗
PubMed2000

Effects of erythropoietin on platelet reactivity and thrombopoiesis in humans.

A recent study in dogs suggested that erythropoietin (EPO) not only promotes the synthesis of increased numbers of reticulated platelets but that these newly produced platelets are hyperreactive compared with controls. Because of the increasing use of EPO in the perioperative setting, we characterized the effects of EPO on platelet reactivity in healthy human volunteers. In a randomized, controlled trial, we studied the effects of EPO on platelet reactivity, thrombopoiesis, and endothelial activation in circumstances similar to those of autologous blood donation. Thirty healthy male volunteers received placebo or EPO (100 or 500 U/kg of body weight given intravenously) three times a week for 2 weeks and underwent phlebotomy on days 8 and 15. Thrombin receptor-activating peptide induced expression of P-selectin, and CD63 increased 2- to 3-fold during EPO treatment. The enhanced platelet reactivity was also reflected by a 50% increase in soluble P-selectin in plasma. Plasma E-selectin levels increased in a dose-dependent fashion by more than 100% during EPO treatment, indicating substantial activation of endothelial cells. A 10% to 20% increase in platelet counts was observed in both EPO groups on day 5. In the placebo group, platelets increased only several days after the first phlebotomy. The increase in platelet counts was not reflected by changes in the amounts of reticulated platelets or circulating progenitor cells. In summary, we found that EPO markedly enhances endothelial activation and platelet reactivity, which may adversely affect patients at cardiovascular risk. However, the increased platelet reactivity could be exploited in patients with platelet dysfunction. (Blood. 2000;95:2983-2989)

Adult↗
PubMed2000

Lepirudin blunts endotoxin-induced coagulation activation.

During sepsis, lipopolysaccharide (LPS) triggers the development of disseminated intravascular coagulation (DIC) via the tissue factor-dependent pathway of coagulation resulting in massive thrombin generation and fibrin polymerization. Recently, animal studies demonstrated that hirudin reduced fibrin deposition in liver and kidney and decreased mortality in LPS-induced DIC. Accordingly, the effects of recombinant hirudin (lepirudin) was compared with those caused by placebo on LPS-induced coagulation in humans. Twenty-four healthy male subjects participated in this randomized, double-blind, placebo-controlled, parallel group study. Volunteers received 2 ng/kg LPS intravenously, followed by a bolus-primed continuous infusion of placebo or lepirudin (Refludan, bolus: 0.1 mg/kg, infusion: 0.1 mg/kg/h for 5 hours) to achieve a 2-fold prolongation of the activated partial thromboplastin time (aPTT). LPS infusion enhanced thrombin activity as evidenced by a 20-fold increase of thrombin-antithrombin complexes (TAT), a 6-fold increase of polymerized soluble fibrin, termed thrombus precursor protein (TpP), and a 4-fold increase in D-dimer. In the lepirudin group, TAT increased only 5-fold, TpP increased by only 50%, and D-dimer only slightly exceeded baseline values (P <.01 versus placebo). Concomitantly, lepirudin also blunted thrombin generation evidenced by an attenuated rise in prothrombin fragment levels (F(1 + 2), P <. 01 versus placebo) and blunted the expression of tissue factor on circulating monocytes. This experimental model proved the anticoagulatory potency of lepirudin in LPS-induced coagulation activation. Results from this trial provide a rationale for a randomized clinical trial on the efficacy of lepirudin in DIC. (Blood. 2000;95:1729-1734)

Adult↗
PubMed2000

The platelet alpha2-integrin (GPIa) nucleotide-807 polymorphism is not associated with a risk for maternal-fetal human platelet antigen-5 incompatibility.

Alloimmunization against human platelet antigen (HPA)-5 may lead to neonatal alloimmune thrombocytopenia. The HPA-5 dimorphism is expressed on the platelet alpha2beta1 integrin. The density of this receptor is associated with another dimorphism of the 2beta1 integrin (nucleotide-807C/T). We hypothesized that anti-HPA-5b-induced neonatal thrombocytopenia is more likely to occur if the receptor is expressed at high than at low levels. Among 933 mother-child pairs, we identified 79 HPA-5aa mothers giving birth to a HPA-Sab offspring. Seventeen mothers had HPA-5b antibodies, but the offspring had a normal platelet count. We genotyped the offspring and mothers for the alpha2-807C/T dimorphism to evaluate its relationship to antibody formation. There was no difference between the frequency of the 807C/T dimorphism among children delivered from alloimmunized mothers and those from mothers without antibodies (P>0.3). The frequency of the 807C/T dimorphism was not different in the two maternal groups. In three maternal-fetal incompatibilities, we observed at delivery normal platelet counts of platelets typed HPA-5b-alpha2807T, despite increasing maternal antibody titers during the pregnancy. Our data do not support the hypothesis that the 807C/T dimorphism in the HPA-5ab children is a predisposing factor to either elicit alloimmunization against HPA-5b or for neonatal alloimmune thrombocytopenia.

Adult↗
PubMed2000

Partial inhibition of nitric oxide synthase primes the stimulated pathway of vWF-secretion in man.

Increased release of von Willebrand factor (vWF) has been linked to the pathogenesis of atherosclerosis. For this complex disease, impairment of endothelium-derived, nitric oxide production and impaired vascular relaxation has also been reported. Since endothelially produced nitric oxide (NO) is known to inhibit secretion of the Weibel-Palade bodies in animals, we hypothesized that NO could mitigate vWF secretion. In a randomized, placebo controlled cross-over trial, eight male volunteers received N-monomethyl-L-arginine (LNMMA) to block endothelial NO production or placebo, and vWF release was stimulated by infusing desmopressin in three cumulative doses (0.05, 0.15, 0.4 microg/kg) in both periods. At a threshold dose of 0.l5 microg/kg desmopressin, concomitant partial blockade of NO production resulted in 20% higher levels of vWF (P<0.04). However, maximal vWF release after 0.4 microg/kg desmopressin was unaffected by L-NMMA (Delta7% between periods, P=0.88). These data show the dampening effect of NO production on vWF release in response to threshold concentrations of secretagogues. This may in part explain the higher vWF levels in cardiovascular diseases associated with impaired endothelial NO generation.

Adult↗
PubMed2000

Granulocyte colony-stimulating factor (G-CSF) downregulates its receptor (CD114) on neutrophils and induces gelatinase B release in humans.

Despite the increasing use of granulocyte colony-stimulating factor (G-CSF) for the mobilization of stem cells and neutrophils, its pharmacodynamic actions are not fully understood. Because of the roles of G-CSF and gelatinase B in leucokinetics, we set out to characterize the interaction of G-CSF with its receptor in humans and its effects on gelatinase B release. G-CSF was infused at bolus doses of 1 microg/kg and 5 microg/kg, and compared to placebo and dexamethasone (1 mg/kg b.i.d), which enhances the plasma levels of endogenous G-CSF. The study was randomized, double-blind, four-way crossover, in eight healthy male volunteers. G-CSF dose-independently induced profound neutropenia (> 95%) within minutes and downregulated its own receptor (CD114) on neutrophils by 75%. The G-CSF/CD114 interaction dose-independently induced degranulation of neutrophils as evidenced by a 300-400% increase in CD11b expression. Degranulation induced up to a 10-fold increase in plasma levels of gelatinase B, an enzyme known to precipitate neutropenia and subsequent neutrophilia in animals. In this study, it was shown that G-CSF downmodulates CD114 expression on the surface of neutrophils in humans and the consequent degranulation enhances gelatinase B release into plasma, which may contribute to mobilization of neutrophils or stem cells.

Antigens↗
PubMed2000

Systemic inflammation increases shear stress-induced platelet plug formation measured by the PFA-100.

The PFA-100 measures platelet plug formation under shear stress and is strongly dependent on von Willebrand Factor (VWF) levels in plasma. We therefore hypothesized that elevated VWF levels, possibly as a result of acute inflammation, adversely influence PFA-100 results. Healthy volunteers received either 2 ng/kg endotoxin or placebo in a randomized controlled trial. Four hours after endotoxin (but not placebo) infusion VWF levels increased by 85%, collagen epinephrine-induced closure time (CT) decreased by 47% and collagen ADP-CT decreased by 38% (P < 0.0001) respectively. In conclusion, systemic inflammation has a major impact on the results obtained by PFA-100 and may confound interpretation of platelet function.

Acute Disease↗
PubMed2000

Paroxetine decreases platelet serotonin storage and platelet function in human beings.

BACKGROUND: Serotonin is a platelet agonist and potent vasoconstrictor that has recently received attention concerning its potential role in acute coronary artery thrombosis. Selective serotonin-reuptake inhibitors, such as paroxetine, are widely used antidepressant agents. We sought to characterize the potential inhibitory effect of paroxetine on platelet function. METHODS: Healthy male volunteers received 20 mg/d paroxetine for 2 weeks in a randomized, double-blind, placebo-controlled, two-way cross-over trial. RESULTS: Paroxetine decreased intraplatelet serotonin concentrations by -83% (P < .01). This inhibited platelet plug formation as reflected by a 31% prolongation of closure time measured with the platelet function analyzer-100 (P < .05). Furthermore, paroxetine lowered expression of the platelet activation marker CD63 in response to two different concentrations of thrombin receptor-activating peptide (P < .01). Plasma concentrations of prothrombin fragment, von Willebrand factor antigen, and circulating P-selectin remained unchanged in either period, indicating that paroxetine does not increase activation of coagulation, endothelium, or platelets in vivo, underlining a favorable safety profile. CONCLUSIONS: Paroxetine substantially decreases intraplatelet serotonin content and thereby reduces platelet plug formation under shear stress, and responsiveness to thrombin receptor activating peptide-induced platelet activation. Further studies will reveal whether these pharmacodynamic effects can be exploited for treatment of thrombotic artery disease.

Adult↗
PubMed2000

Dexamethasone lowers circulating E-selectin and ICAM-1 in healthy men.

Plasma levels of circulating adhesion molecules (AMs) are increased in a number of inflammatory and cardiovascular disorders. Yet the mechanisms regulating the physiologic levels of soluble AMs are largely unknown. It has recently been postulated that glucocorticoids may exert their anti-inflammatory actions partially through the inhibition of cytokine-stimulated expression of E-selectin and intercellular adhesion molecule (ICAM-1). However, it remains controversial whether glucocorticoids affect the basal expression of AMs on resting cells. We have thus evaluated the effects of glucocorticoids by infusing therapeutic doses of dexamethasone (0.04 mg/kg and 1.0 mg/kg twice a day for 2 days) or placebo on plasma levels of circulating E-selectin (cE-selectin), soluble thrombomodulin (sTM), circulating ICAM-1 (cICAM-1), and circulating vascular cell adhesion molecule (cVCAM-1) in 9 healthy men. Plasma was obtained before infusion at 24 and 48 hours. Compared with baseline, levels of cE-selectin decreased by 16% and 22% with the lower and the higher doses, respectively, at 48 hours (P = .007), whereas sTM was unchanged. Both doses of dexamethasone reduced cICAM-1 by about 15% at 48 hours (P = .007), but there were no changes in cVCAM. Dexamethasone time-dependently decreases plasma levels of cE-selectin and cICAM-1 in healthy men. This demonstrates that a glucocorticoid-sensitive mechanism specifically down-regulates normal plasma levels of cE-selectin and cICAM-1 in healthy subjects, which could thus reflect minor baseline inflammation.

Adult↗
PubMed2000

Effects of hyperinsulinemia on plasma levels of circulating adhesion molecules.

Plasma levels of circulating intercellular adhesion molecule-1 (cICAM-1), a potential cardiovascular risk factor, are increased in diabetics. Among other factors, hyperinsulinemia has been proposed to enhance its release into the circulation. Thus, we directly examined the effects of insulin infusion on plasma levels of circulating adhesion molecules, and two other endothelial markers, i.e. von Willebrand factor (vWF) and soluble thrombomodulin (sTM). The study design was balanced, randomized, placebo-controlled, double blind, and cross-over. Twelve healthy male subjects received, on separate study days, a euglycemic hyperinsulinemic clamp (3 mU/kg x min) or placebo over 6 h. Plasma levels of cICAM-1, vascular cell adhesion molecule-1, circulating E-selectin, and sTM were measured by enzyme immunoassay; vWF-Ag was measured using a STA clot analyzer. Plasma levels of these adhesion molecules and endothelial cell activation markers were not affected despite a 30-fold increase in insulin levels. Hyperinsulinemia has no adverse effect on circulating ICAM-1, vascular cell adhesion molecule-1, E-selectin, vWF, or sTM and therefore does not directly induce endothelial activation.

Adult↗
PubMed2000

Monitoring of aspirin (ASA) pharmacodynamics with the platelet function analyzer PFA-100.

BACKGROUND: Anti-platelet drug therapy is currently performed without monitoring, because the established method of platelet aggregometry is cumbersome. The recently developed platelet function analyzer PFA-100 measures shear stress dependent, collagen epinephrine (CEPI) and collagen adenosine diphosphate (CADP) induced platelet plug formation. As the PFA-100 provides a valuable tool to detect patients with platelet dysfunction more efficiently and cost-effectively than aggregometry, we investigated its potential to monitor the efficacy of aspirin treatment. METHODS: All healthy volunteers (n = 10) received a fractionated infusion of L-aspirin to establish individual dose-response curves. Further, in a randomized, double-blind, placebo controlled two-way cross over study the same volunteers received either 50 or 100 mg aspirin/day p.o. for a period of 11 days to determine the day-to-day variability CEPI induced closure time (CT) under constant intake of low dose aspirin, and to compare the efficacy of those two doses. RESULTS: Intra- and intersubject variability of CEPI-CT averaged 9% and 22%, respectively. Seven volunteers exceeded the maximum of CEPI-CT (>300 s) already after infusion of 100 mg L-aspirin. Intake of 100 mg of aspirin elicited a more rapid onset of effect than 50 mg, which was only significant on days 3 and 4 of aspirin intake. The aspirin induced CEPI-CT prolongation correlated positively with basal CEPI-CT values (r = 0.86; p = 0.001) and were strongly dependent on von Willebrand Factor levels (r = -0.9; p = 0.001). CONCLUSION: Thus, the PFA-100 system appears suitable to demonstrate an aspirin-induced platelet effect in a longitudinal study, and may be adequate to monitor a patient's compliance. However, prospective trials have to be conducted to demonstrate whether the EPI-CT achieved under ASA-intake has predictive value for cardiovascular outcome.

Adenosine Diphosphate↗