Axel Schlagenhauf

Ethanol Causes Protein Precipitation—New Safety Issues for Catheter Locking Techniques

Objective The ethanol lock technique has shown great potential to eradicate organisms in biofilms and to treat or prevent central venous catheter related infections. Following instillation of ethanol lock solution, however, the inherent density gradient between blood and ethanol causes gravity induced seepage of ethanol out of the catheter and blood influx into the catheter. Plasma proteins so are exposed to highly concentrated ethanol, which is a classic agent for protein precipitation. We aimed to investigate the precipitating effect of ethanol locks on plasma proteins as a possible cause for reported catheter occlusions. Methods Plasma samples were exposed in-vitro to ethanol (concentrations ranging from 7 to 70 v/v%) and heparin lock solutions. In catheter studies designed to mimic different in-vivo situations, the catheter tip was placed in a plasma reservoir and the material contained within the catheter was analyzed after ethanol lock instillation. The samples underwent standardized investigation for protein precipitation. Results Protein precipitation was observed in plasma samples containing ethanol solutions above a concentration of 28%, as well as in material retrieved from vertically positioned femoral catheters and jugular (subclavian) catheters simulating recumbent or head down tilt body positions. Precipitates could not be re-dissolved by dilution with plasma, urokinase or alteplase. Plasma samples containing heparin lock solutions showed no signs of precipitation. Conclusions Our in-vitro results demonstrate that ethanol locks may be associated with plasma protein precipitation in central venous catheters. This phenomenon could be related to occlusion of vascular access devices locked with ethanol, as has been reported. Concerns should be raised regarding possible complications upon injection or spontaneous gravity induced leakage of such irreversibly precipitated protein particles into the systemic circulation. We suggest limiting the maximum advisable concentration of ethanol to 28 v/v% in catheter lock solutions.

Coagulation Changes during Presyncope and Recovery

Orthostatic stress activates the coagulation system. The extent of coagulation activation with full orthostatic load leading to presyncope is unknown. We examined in 7 healthy males whether presyncope, using a combination of head up tilt (HUT) and lower body negative pressure (LBNP), leads to coagulation changes as well as in the return to baseline during recovery. Coagulation responses (whole blood thrombelastometry, whole blood platelet aggregation, endogenous thrombin potential, markers of endothelial activation and thrombin generation), blood cell counts and plasma mass density (for volume changes) were measured before, during, and 20 min after the orthostatic stress. Maximum orthostatic load led to a 25% plasma volume loss. Blood cell counts, prothrombin levels, thrombin peak, endogenous thrombin potential, and tissue factor pathway inhibitor levels increased during the protocol, commensurable with hemoconcentration. The markers of endothelial activation (tissue factor, tissue plasminogen activator), and thrombin generation (F1+2, prothrombin fragments 1 and 2, and TAT, thrombin-antithrombin complex) increased to an extent far beyond the hemoconcentration effect. During recovery, the markers of endothelial activation returned to initial supine values, but F1+2 and TAT remained elevated, suggestive of increased coagulability. Our findings of increased coagulability at 20 min of recovery from presyncope may have greater clinical significance than short-term procoagulant changes observed during standing. While our experiments were conducted in healthy subjects, the observed hypercoagulability during graded orthostatic challenge, at presyncope and in recovery may be an important risk factor particularly for patients already at high risk for thromboembolic events (e.g. those with coronary heart disease, atherosclerosis or hypertensives).

Increased procoagulant function of microparticles in pediatric inflammatory bowel disease: role in increased thrombin generation.

Objectives: Patients with inflammatory bowel disease (IBD) have a higher risk for venous thromboembolism compared with non-IBD subjects. The pathogenic mechanisms of the thrombotic events are not fully understood. We investigated levels of circulating microparticles and their influence on thrombin generation in pediatric patients with IBD during active and quiescent disease compared with healthy controls. Methods: Plasma samples were collected from 33 pediatric patients with Crohn disease (CD), 20 pediatric patients with ulcerative colitis (UC), and 60 healthy controls. Microparticles’ procoagulant activity was measured by enzyme-linked immunosorbent assay, and the dependency of thrombin generation on microparticles-derived tissue factor was determined by means of calibrated automated thrombography. Results: The procoagulant function of microparticles was significantly increased in patients with active and inactive CD, and active UC compared with controls. Endogenous thrombin potential was significantly higher in patients with CD and UC compared with controls. A minor influence of microparticles on thrombin generation was only observed for patients with active UC. Conclusions: Our study shows increased procoagulant function of microparticles in pediatric patients with active and quiescent CD and active UC compared with controls, but demonstrates that they are not a major cause for the higher thrombin generation in pediatric patients with IBD.

Urinary Total Globotriaosylceramide and Isoforms to Identify Women With Fabry Disease: A Diagnostic Test Study

BACKGROUND Fabry disease is a treatable X-linked lysosomal storage disorder caused by alterations in the structural gene (GLA) of α-galactosidase A (AGAL), manifesting with cardiovascular and/or kidney disease and decreased life span. Although males as well as females can be affected, females cannot be identified using AGAL activity. We evaluated urinary total globotriaosylceramide (Gb3) and single N-acyl isoforms for the detection of Fabry disease in female patients with and without chronic kidney disease (CKD). STUDY DESIGN Diagnostic accuracy study. SETTING & PARTICIPANTS 28 untreated women with Fabry disease and 335 female outpatients without Fabry disease with (n = 213) and without CKD (n = 122). INDEX TEST Assessment of urinary Gb3 using electrospray ionization tandem mass spectrometry, including 6 N-acyl isoforms, total Gb3 related to urinary creatinine, and ratios of Gb3-24 to Gb3-18 and Gb3-24 to urinary AGAL. REFERENCE TEST Fabry disease, diagnosed by identification of known pathogenic GLA mutations in patients or their male relatives. RESULTS 6 parameters (ratio of Gb3-24 to urinary AGAL activity; Gb3-24; ratio of Gb3-24 to Gb3-18; Gb3-22; Gb3-16; and total Gb3) were highly informative for the diagnosis of Fabry disease independent of the presence or absence of CKD (area under the receiver operating characteristic curve, 0.876-0.927; all P < 0.001). LIMITATIONS Because of low signal-to-noise ratios, 15.8% of samples had to be excluded. CONCLUSION Total urinary Gb3 and Gb3 isoforms can be used for the diagnosis of Fabry disease in women.

Thrombin receptor levels in platelet concentrates during storage and their impact on platelet functionality

BACKGROUND: Quality control of platelet (PLT) concentrates is challenging, due to PLT lesions, which are difficult to detect with routine methods. The search for reliable PLT lesion biomarkers is focused on the role of PLTs in primary hemostasis. PLT transfusions also have a significant impact on secondary hemostasis. In this phase, responsiveness of PLTs to small amounts of thrombin is crucial. PAR1 and PAR4 are protease‐activated receptors and are responsible for thrombin reactivity of human PLTs. This study should elucidate if levels of those two receptors are changing in PLT concentrates during storage and if those changes have an impact on PLT aggregation and support of thrombin generation.

Newborn platelets: lower levels of protease-activated receptors cause hypoaggregability to thrombin.

Newborn platelets show in vitro hypoaggregability to thrombin. Sensitivity of platelets to such a potent agonist is crucial for a functional clot formation. Nevertheless, newborns have an excellent hemostasis. We wanted to investigate the reason for this impairment by comparatively analysing levels of receptors known to be involved in thrombin signaling in newborn and adult platelets. Platelets of adult and cord blood were isolated, washed, and lysed. Resulting protein samples were separated by SDS-PAGE and blotted on nitrocellulose membranes. Receptors were visualized using immunodetection and evaluated densitometrically. Thrombin receptor activating peptide induced platelet aggregation was measured in citrated whole blood on a Multiplate analyzer. Statistical analysis was performed using SPSS 16.0. Significantly lower levels of protease-activated receptors (PAR1, PAR4) and higher levels of glycoprotein Ibα (GPIbα) were found in newborn platelets as compared to adult platelets. Platelet aggregation was lower in newborn samples than in adult controls and values correlated with the corresponding PAR levels. Our results suggest that lower levels of protease-activated receptors contribute to the poor thrombin induced aggregation observed with newborn platelets, which can not be compensated by higher levels of GPIbα.

Low endogenous thrombin potential in trained subjects

INTRODUCTION A paradox seems to exist: exercising leads to clotting activation in conventional clotting tests, but exercising persons have a low risk of thrombosis. In this study we tried to evaluate the effect of exercise performance status on in vitro plasma thrombin generation, which represents an overall function test of hemostasis. MATERIALS AND METHODS We compared 56 trained subjects to 98 healthy age matched sedentary volunteers. Blood samples were analyzed for thrombin generation using calibrated automated thrombography. Microparticles were quantified using ELISA. Additionally prothrombin fragments 1 + 2, thrombin-antithrombin complex, tissue factor pathway inhibitor, antithrombin and prothrombin were measured. The group of the trained subjects performed an incremental cycle-ergometer exercise test after taking the blood sample. RESULTS A significantly lower endogenous thrombin potential was observed in the group of the trained subjects compared to the sedentary individuals (p = 0.007). Microparticles (ELISA) were significantly lower in the trained subjects compared to the sedentary subjects (p = 0.001). Prothrombin fragments 1 + 2 (p < 0.001) and thrombin-antithrombin complex (p = 0.01) were significant higher in the trained subjects and antithrombin (p = 0.02) as well as prothrombin (p < 0.0001) were significantly lower in this group, whereas tissue factor pathway inhibitor values did not show significant differences. Both maximal and submaximal power output was significantly negatively related to endogenous thrombin potential (r = -0.43, r = -0.45) and thrombin peak (r = -0.44, r = -0.42). CONCLUSIONS Trained subjects have a lower endogenous thrombin potential than sedentary subjects possibly explaining the lower incidence of thrombosis in this group despite a higher acute clotting activation during strenuous exercise.

Effects of a single bout of walking exercise on blood coagulation parameters in obese women.

Obesity is associated with increased prevalence of thromboembolic events. We aimed to investigate whether obese women might benefit from vigorous aerobic exercise. Forty-two overweight and obese women performed a 30-min walking exercise test (treadmill ergometer) at an intensity of 70% of individual peak oxygen uptake. Blood samples were collected before and immediately after exercise. Thrombelastometry and platelet function measurements were performed on whole blood. Standard coagulation times, thrombin generation curves, markers of thrombin generation, fibrinolytic parameters, plasma levels of pro- and anticoagulatory factors, and microparticle procoagulant activity were determined in platelet-poor plasma samples. Thrombelastometry revealed a significant prolongation of clot formation time (P = 0.037) and a significant deceleration of fibrin build up (alpha angle, P = 0.034) after exercise. Calibrated automated thrombography revealed a significant exercise-induced decrease in endogenous thrombin potential (P = 0.039). Moreover, thrombin formation stopped earlier postexercise, reflected in shortened StartTail (P = 0.046). Significantly elevated tissue-plasminogen activator levels (P = 0.001) indicate an exercise-induced activation of the fibrinolytic system. White blood cell count increased significantly from pre- to postexercise (P = 0.045), indicating a mild exercise-induced leukocytosis. The results of this study demonstrate that vigorous aerobic exercise might be a suitable tool to protect obese women from thrombotic events. We show that a single bout of vigorous aerobic exercise is clearly associated with an activation of the fibrinolytic system and a decreased readiness of the postexercise samples to form a clot and to generate thrombin, the pivotal enzyme of hemostasis.

Bed rest does not induce hypercoagulability

Although there is no direct evidence, it is generally believed that bed rest shifts the haemostatic system towards hypercoagulability; thus, immobilized patients are commonly treated with anticoagulants. We therefore aimed to investigate whether long‐term bed rest actually leads to an elevated risk for thromboembolic events.

Effect of rivaroxaban, in contrast to heparin, is similar in neonatal and adult plasma.

Neonates have lower levels of clotting factors as well as inhibitors. Effects of heparin in neonatal plasma differ from those in adult plasma, and dosage recommendations cannot be extrapolated from adult trials. Riveroxaban is an oral direct factor Xa inhibitor that can achieve an anticoagulant effect without dependence on anti-thrombin. We performed comparative thrombin generation measurements in neonatal cord and adult plasma with different concentrations of unfractionated heparin and rivaroxaban to evaluate the potential of rivaroxaban in neonatal anticoagulation. The impact of heparin or rivaroxaban on the neonatal and adult hemostatic system was determined measuring calibrated automated thrombin generation and activated partial thromboplastin time in platelet-poor plasma pools of 15 adult samples or 15 neonatal cord samples and addition of seven increasing concentrations of heparin or rivaroxaban, respectively, to the pooled samples. Lag time, time to peak and peak height of thrombin generation in neonatal cord samples were significantly less affected by different heparin concentrations than in adult samples, whereas the impact on reduction of endogenous thrombin potential was higher in neonatal cord samples. The impact of rivaroxaban on thrombin generation parameters showed better comparability between neonatal cord and adult samples. Both anticoagulants showed the same differences in activated partial thromboplastin time between adult and neonatal plasma at each concentration. Rivaroxaban shows a very similar pattern in neonatal cord and adult plasma in suppressing thrombin generation and prolonging activated partial thromboplastin time values, suggesting that dose finding may be easier with rivaroxaban in neonates.