Florian B. Mayr

Validation of rotation thrombelastography in a model of systemic activation of fibrinolysis and coagulation in humans

Summary.  Background: Thrombelastography (TEG) is a whole blood assay to evaluate the viscoelastic properties during blood clot formation and clot lysis. Rotation thrombelastography (e.g. ROTEM®) has overcome some of the limitations of classical TEG and is used as a point‐of‐care device in several clinical settings of coagulation disorders. Endotoxemia leads to systemic activation of the coagulation system and fibrinolysis in humans. Objectives: We validated whether ROTEM® is sensitive to endotoxin induced, tissue factor‐triggered coagulation and fibrinolysis and if its measures correlate with biohumoral markers of coagulation and fibrinolysis. Patients and methods: Twenty healthy male volunteers participated in this randomized placebo‐controlled trial. Volunteers received either 2 ng kg−1 National Reference Endotoxin or saline. Results: Endotoxemia significantly shortened ROTEM® clotting time (CT) by 36% (CI 0.26–0.46; P < 0.05) with a strong inverse correlation with the peak plasma levels of prothrombin fragments (F1 + 2) (r = −0.83, P < 0.05). Additionally, endotoxin infusion enhanced maximal lysis (ML) 3.9‐fold (CI: 2.5–5.2) compared with placebo or baseline after 2 h (P < 0.05). Peak ML and peak tissue plasminogen activator (t‐PA) values correlated excellently (r = 0.82, P < 0.05). ROTEM® parameters clot formation time and maximal clot firmness were not affected by LPS infusion, whereas platelet function analyzer (PFA‐100) closure times decreased. Conclusions: Rotation thrombelastography (ROTEM®) detects systemic changes of in vivo coagulation activation, and importantly it is a point of care device, which is sensitive to changes in fibrinolysis in humans. The ex vivo measures CT and ML correlate very well with established in vivo markers of coagulation activation (F1 + 2) and fibrinolysis (t‐PA), respectively.

Hemostasis in cardiac arrest patients treated with mild hypothermia initiated by cold fluids

AIM OF THE STUDY Application of mild hypothermia (32-33 degrees C) has been shown to improve neurological outcome in patients with cardiac arrest. However, hypothermia affects hemostasis, and even mild hypothermia is associated with bleeding and increased transfusion requirements in surgery patients. On the other hand, crystalloid hemodilution has been shown to induce a hypercoagulable state. The study aim was to elucidate in which way the induction of mild therapeutic hypothermia by a bolus infusion of cold crystalloids affects the coagulation system of patients with cardiac arrest. METHODS This was a prospective pilot study in 18 patients with cardiac arrest and return of spontaneous circulation (ROSC). Mild hypothermia was initiated by a bolus infusion of cold 0.9% saline fluid (4 degrees C; 30ml/kg/30min) and maintained for 24h. At 0h (before hypothermia), 1, 6 and 24h we assessed coagulation parameters (PT, APPT), platelet count and performed thrombelastography (ROTEM) after in vitro addition of heparinase. RESULTS A total amount of 2528 (+/-528)ml of 0.9% saline fluid was given. Hematocrit (p<0.01) and platelet count (-27%; p<0.05) declined, whereas APTT increased (2.7-fold; p<0.01) during the observation period. All ROTEM parameters besides clotting time (CT) after 1h (-20%; p<0.05) did not significantly change. CONCLUSION Mild hypothermia only slightly prolonged clotting time as measured by rotation thrombelastography. Therefore, therapeutic hypothermia initiated by cold crystalloid fluids has only minor overall effects on coagulation in patients with cardiac arrest.

Recombinant human antithrombin inhibits thrombin formation and interleukin 6 release in human endotoxemia

We hypothesized that infusion of recombinant human antithrombin without concomitant heparin would have dose‐dependent anticoagulant properties and potentially decrease endotoxin (lipopolysaccharide [LPS])–induced cytokine production. This was a randomized, double‐blind, placebo‐controlled study in parallel groups enrolling 30 healthy male volunteers. The active treatment groups received infusions of recombinant human antithrombin to increase antithrombin levels to 200% and 500% before infusion of 2 ng/kg endotoxin (LPS). Infusion of antithrombin dose‐dependently decreased coagulation (P<.01 by repeated‐measures ANOVA): peak levels of prothrombin fragment (1.8 nmol/L [95% confidence interval (CI), 1.3–2.3 nmol/L] in the 500% antithrombin group and 4.4 nmol/L [95% CI, 2.7–6.2 nmol/L] in the placebo group at 4 hours), thrombin antithrombin complexes (12 μg/L [95% CI, 8–16 μg/L] in the 500% antithrombin group and 34 μg/L [95% CI, 20–48 μg/L] in the placebo group at 4 hours), and D‐dimer (0.2 μg/L [95% CI, 0.1–0.2 μg/L] in the 500% antithrombin group and 0.5 μg/L [95% CI, 0.4–0.7 μg/L] in the placebo group). Recombinant human antithrombin decreased peak interleukin‐6 levels by 40% (222 pg/mL [95% CI, 148–295 pg/mL] and 216 pg/mL [95% CI, 112–320 pg/mL] in the 500% and 200% antithrombin groups, respectively, versus 357 pg/mL [95% CI, 241–474 pg/mL] in the placebo group; P<.001 by ANOVA). Finally, infusion of recombinant human antithrombin rapidly and transiently decreased neutrophil counts (by 19% [95% CI, 8%–30%] in the 500% antithrombin group versus 6% [95% CI, 1%–10%] in the placebo group, P = .002 by Kruskal‐Wallis ANOVA) and monocyte counts (by 30% [95% CI, 16%–44%] in the 500% antithrombin group and 18% [95% CI, 9%‐28%] in the 200% antithrombin group versus 8% [95% CI, 5%‐20%] in the placebo group, P = .04) before LPS challenge, indicating that recombinant human antithrombin directly interacts with these leukocyte subsets. In summary, recombinant human antithrombin dose‐dependently inhibited tissue factor‐triggered coagulation. Effects on leukocytes and inhibition of interleukin‐6 release seem to represent specific pharmacodynamic properties of recombinant human antithrombin.

The aptamer ARC1779 is a potent and specific inhibitor of von willebrand factor mediated ex vivo platelet function in acute myocardial infarction

ARC1779 is an aptamer, which blocks binding of the von Willebrand Factor (VWF) A1 domain to platelet GPIb receptors. VWF is increased in the elderly an in the setting of acute myocardial infarction (AMI), as reflected by increased shear-dependent platelet function. We hypothesized that ARC1779 concentration-dependently inhibits ex vivo platelet function, and that this concentration effect relationship may be shifted in patients with AMI. We studied ex vivo dose response curves for ARC1779 on VWF activity, shear-dependent platelet function, and agonist-induced platelet aggregation. We included patients with AMI on standard treatment (n = 40), young (n = 20) and elderly controls (n = 20) in this ex vivo dosing study. AMI patients displayed ∼2-fold increased plasma levels of VWF activity as compared to controls. ARC1779 inhibited VWF activity (IC90: ∼3–4 µg/mL) and shear dependent platelet function (Platelet Function Analyzer (PFA-100), IC50: ∼0.5–0.9 µg/mL and Cone and Plate Analyzer (CPA), IC50: ∼0.1–0.4 µg/mL in citrated blood) at comparable concentrations in all groups. In contrast to GPIIb/IIIa antagonists, ARC1779 did not inhibit platelet aggregation induced by ADP, collagen or arachidonic acid at concentrations (10 µg/mL) that fully inhibited VWF dependent platelet function. ARC1779 potently and specifically inhibits VWF activity and VWF dependent platelet function, even in the setting of AMI where VWF activity is increased.

Simvastatin and rosuvastatin mobilize Endothelial Progenitor Cells but do not prevent their acute decrease during systemic inflammation

INTRODUCTION Endothelial Progenitor Cells (EPCs) are a specific subtype of haematopoietic stem cells that contribute to the repair of injured endothelium. Treatment with atorvastatin has been shown to increase EPC counts in patients with stable coronary artery disease. Numbers of circulating EPCs decrease in various inflammatory diseases. Thus, we hypothesized that short term statin pre-treatment may alter the acute change in EPC levels during systemic inflammation. OBJECTIVES To explore the effect of statin pretreatment and low grade experimental endotoxemia on endothelial progenitor cells in humans. MATERIALS AND METHODS Randomized, double-blind, placebo-controlled three way cross-over trial in six healthy male volunteers. Each volunteer received three treatments consisting of 5 days of oral simvastatin (80 mg/day), rosuvastatin (40 mg/day) or placebo. On Day 5 of each study period, subjects received lipopolysaccharide (LPS; 2 ng/kg i.v.). This trial has been registered with Clinical.Trials.gov, trial number NCT00309374. RESULTS Statin pre-treatment led to a significant increase in circulating EPCs (1.9-3.5 fold, depending on statin and analytic method; P<0.05) but could not suppress the endotoxemia induced EPC decrease (approximately -75%; P<0.05) during the observation period. CONCLUSIONS Statin therapy significantly increases EPCs within 96 hours and this may be a class effect. However, statins could not counteract the acute decrease in circulating EPCs after LPS infusion.

Pharmacokinetics and Pharmacodynamics of the Dual FII/FX Inhibitor BIBT 986 in Endotoxin‐induced Coagulation

BIBT986 is a dual inhibitor of factors Xa and IIa. The aim of this study was to compare with placebo the effect of three doses of BIBT986 on coagulation, platelet activation, and inflammation. This was a prospective, randomized, double‐blind, placebo‐controlled, parallel‐group dose escalation trial in 48 healthy male volunteers. Participants received one of three doses of BIBT986 or placebo intravenously together with a bolus infusion of 2 ng/kg lipopolysaccharide (LPS). BIBT986 dose‐dependently changed global coagulation parameters and in vivo markers of thrombin generation and action: BIBT986 doses, which prolonged activated partial thromboplastin time by 100%, completely suppressed the LPS‐induced increases in prothrombin fragment, thrombin‐antithrombin complexes, and D‐dimer, which were 6.1‐, 14.5, and 3.5‐fold in the placebo group, respectively. BIBT986 did not influence inflammation, fibrinolysis, or platelet activation. Therefore, BIBT986 is a potent anticoagulant in the human endotoxemia model.

Endotoxemia enhances circulating endothelial cells in humans.

Catheter Cardiovasc Interv 2003; 59: 295–302. 3 Hulot JS, Bura A, Villard E, AziziM, Remones V, Goyenvalle C, Aiach M, Lechat P, Gaussem P. Cytochrome P450 2C19 loss-of-function polymorphism is a major determinant of clopidogrel responsiveness in healthy subjects. Blood 2006; 13: blood-2006-04-013052v1 [Epub ahead of print]. 4 Furata T, Shirai N, Xiao F, Ohashi K, Ishizaki T. Effect of high-dose lansoprazole on intragastric pH in subjects who are homozygous extensive metabolizers of cytochrome P4502C19. Clin Pharmacol Ther 2001; 70: 484–92.

Hypokalemic paralysis in a professional bodybuilder

Severe hypokalemia is a potentially life-threatening disorder and is associated with variable degrees of skeletal muscle weakness, even to the point of paralysis. On rare occasions, diaphragmatic paralysis from hypokalemia can lead to respiratory arrest. There may also be decreased motility of smooth muscle, manifesting with ileus or urinary retention. Rarely, severe hypokalemia may result in rhabdomyolysis. Other manifestations of severe hypokalemia include alteration of cardiac tissue excitability and conduction. Hypokalemia can produce electrocardiographic changes such as U waves, T-wave flattening, and arrhythmias, especially if the patient is taking digoxin. Common causes of hypokalemia include extrarenal potassium losses (vomiting and diarrhea) and renal potassium losses (eg, hyperaldosteronism, renal tubular acidosis, severe hyperglycemia, potassium-depleting diuretics) as well as hypokalemia due to potassium shifts (eg, insulin administration, catecholamine excess, familial periodic hypokalemic paralysis, thyrotoxic hypokalemic paralysis). Although the extent of diuretic misuse in professional bodybuilding is unknown, it may be regarded as substantial. Hence, diuretics must always be considered as a cause of hypokalemic paralysis in bodybuilders.

Current developments in anti-platelet therapy

ZusammenfassungThrombozyten spielen eine lebenswichtige Rolle in der Hämostase und der Blutgerinnung an Stellen von Gefäßverletzungen. Andererseits sind sie ebenso im pathologischen Gegenstück, der Thromboseentstehung, entscheidend involviert. Als Konsequenz hat sich die Hemmung der Thrombozytenfunktion als eine der Hauptstützen in der Behandlung sowie Prophylaxe von Erkrankungen wie Myokardinfarkt oder cerebrovaskulärem Insult etabliert. Acetylsalizylsäure (ASS, Aspirin®) ist das am häufigsten eingesetzte Medikament und wird generell als Protoyp eines Thrombozytenfunktionshemmers angesehen. Da die Plättchenaktivierung jedoch über unterschiedliche Signalwege abläuft, die zum Teil nicht durch ASS beeinflusst werden, wurden zusätzliche Thrombozytenfunktionshemmer entwickelt. Derzeit gibt es für den klinischen Gebrauch vier Hauptklassen von Thrombozytenfunktionshemmern: Acetylsalizylsäure (ASS), Phosphodiesterase (PDE) –Hemmer, Thienopyridine und intravenöse GPIIb/IIIa Antagonisten. Dieser Artikel gibt eine kurze Übersicht über die vier Hauptklassen von Thromboytenfunktionshemmern, jeweils mit Verweis auf ASS als Referenzstandard.SummaryPlatelets play a life-saving role in hemostasis and blood clotting at sites of vascular injury and consequently are similarly involved in the pathological counterpart, namely thrombosis. Thus, anti-platelet therapy has become a mainstay in treatment and/or prophylaxis of conditions like myocardial infarction, stroke and other cardiovascular diseases. Acetyl-salicylic acid (ASA, aspirin®) is still the most widely used agent and considered as the prototype antiplatelet drug. Since platelet activation occurs via several pathways that are not influenced by ASA, several other compounds have been developed to add to its beneficial effect. Currently four main classes of antiplatelet agents are available for clinical use: acetyl-salicylic acid (ASA), phosphodiesterase (PDE) inhibitors, thienopyridines and the intravenous GPIIb/IIIa antagonists. This article gives a concise review of these four classes of anti-platelet agents, using ASA as the reference standard.

Upregulation of cytokine mRNA in circulating leukocytes during human endotoxemia

BackgroundEndotoxemia induces pronounced changes in leukocyte count and enhances the release of many cytokines. However, the molecular regulation of this cytokine release is poorly characterized in humans.MethodsThe time course of mRNA expression of 24 cytokines in circulating leukocytes was studied in a wellstandardized model of human endotoxemia (2 ng/kg). Real-time polymerase chain reaction (RT-PCR) was used to quantify the lipopolysaccharide (LPS)-inducible mRNA levels of leukocytes from 16 healthy volunteers in a randomized, placebo-controlled trial.ResultsBaseline mRNA levels of interleukins including IL-1α, IL-3, IL-5, IL-6, IL-12p40, IL-13, IL-15, IL-17, granulocyte colony-stimulating factor (G-CSF) and granulocyte monocyte CSF (GM-CSF) were below detectable levels in normal blood of the healthy participants. After 2 h, LPS infusion increased median mRNA levels of IL-1α by >1100-fold and IL-1β and IL-8 by 33-fold and 46-fold, respectively. In contrast, levels of tumor necrosis factor (TNF-α) and IL-10 mRNA increased by only 7-fold, whereas changes in mRNA expression of other cytokines showed either a more than two fold increase or were undetectable. In vitro incubation of whole blood with 50 pg/mL LPS for 2 h enhanced transcription levels of IL-1α mRNA by >10,000-fold, IL-6 and IL-12p40 by >1000-fold, IL-1α by 400-fold, TNF-α by 100-fold, IL-8, IL-18, interferon γ (IFN-γ) and G-CSF by >10-25-fold, and IL-10, IL-12p35, TNF-β, and IL-13 by 10-25-fold.ConclusionOnly half of the 24 evaluated cytokines were expressed at the mRNA level in circulating leukocytes under basal conditions and after an LPS challenge. Only IL-1α, IL-1β, IL-10, IL-8, and TNF-α were upregulated in the circulating leukocytes, whereas several other cytokines (including IL-6 and G-CSF), were expressed on the mRNA level following in vitro incubation of blood with LPS. In addition, IL-1α and IL-1β might be potential diagnostic targets in inflammatory diseases.