Martin Engström

Calponin reduces shortening velocity in skinned taenia coli smooth muscle fibres

Calponin (4.1–5.9 μM, pig stomach) inhibited maximal shortening velocity (V max) by 20–25% with only minor influence on force in skinned smooth muscle from guinea‐pig taenia coli activated at different Ca2+ levels and with thiophosphorylation. Similar results were obtained with a fragment of the N‐terminal 1–228 amino acids engineered using a mouse cDNA construct (5.4 μM). Both the native calponin and the fragment inhibited actin filament sliding in a graded manner in an in vitro motility assay. We conclude that calponin influences the kinetics of the actin‐myosin interaction in the organised smooth muscle contractile system and that engineered fragments of calponin can be used to probe its action in muscle fibres. The effects can be due to an introduction of an internal load during filament sliding, possibly by decreasing the detachment rates and increasing the cross‐bridge time spent in the attached state.

Increased Lactate Levels Impair the Coagulation System-A Potential Contributing Factor to Progressive Hemorrhage After Traumatic Brain Injury.

Progressive intracerebral contusions are a major problem in the management of patients with severe traumatic brain injury that is also linked to worse outcome. Microdialysis studies have revealed that lactate levels are very high inside contusions, corresponding to significant acidosis. The current study was performed in an effort to investigate whether the lactate accumulation inside cerebral contusions may be a contributing factor to the prolonged bleeding inside contusions. We have investigated the effects of lactic acidosis on the coagulation system with rotational thromboelastometry. It was a laboratory study involving 6 healthy volunteers. Blood was drawn and the pH was adjusted by addition of lactic acid in vitro. The pH levels studied were 7.4, 7.2, 7.0, and 6.8. The pH was also readjusted to 7.4 by addition the buffer THAM to blood initially adjusted to a pH of 6.8 to study the reversibility of potential adverse effects induced by the lactic acidosis. We found the coagulation to be significantly impaired by lactic acidosis (P=0.000l). The impairment found was reversible after correction of the acidosis by a buffer. In conclusion, we found that lactic acidosis impaired the coagulation system. The impairment caused by lactic acidosis may be one factor causing the progressive hemorrhage in posttraumatic cerebral contusions, known to have high levels of lactate and correspondingly low pH. It may also be important to consider in bleeding trauma patients.

Bactericidal/permeability‐increasing protein inhibits endotoxin‐induced vascular nitric oxide synthesis

Background: Endotoxin (lipopolysaccharide, LPS) up‐regulates inducible nitric oxide synthase (iNOS) in blood vessels during septic shock. This promotes the production of nitric oxide (NO), leading to dilation of the vessels. The aim of the study was to investigate the effects of the LPS‐binding endogenous antibiotic bactericidal/permeability‐increasing protein (BPI) on the action of LPS on the blood vessels wall and to identify possible influence on underlying NO‐related mechanisms.

Monitoring fibrinolysis in whole blood by viscoelastic instruments: A comparison of ROTEM and ReoRox.

Abstract Objective. Increased fibrinolysis with the risk of bleeding is a consequence of thrombolytic therapy and can also be seen in clinical situations such as acute trauma. Thrombelastography and thrombelastometry are viscoelastic coagulation instruments that can detect higher degrees of fibrinolysis; hyperfibrinolysis. A newer viscoelastic instrument is the ReoRox, which uses free oscillation rheometry to detect clot formation, strength and fibrinolysis. The ReoRox has a new test for detection of fibrinolysis, called ReoLyse. The aim of this study was to compare ReoRox with its new ReoLyse test with rotational thrombelastometry (ROTEM) in the monitoring of in vitro-induced fibrinolysis. Methods. Whole blood from 10 healthy volunteers was mixed with tissue plasminogen activator (t-PA) to obtain seven different plasma concentrations (0, 0.25, 0.5, 0.75, 1, 3 and 5 μg/mL). Whole blood samples with the different t-PA plasma concentrations were analyzed with ROTEM EXTEM and FIBTEM tests, ReoRox standard test Fib1 (clot formation/strength) and ReoLyse (fibrinolysis) tests. Results. The fibrinolysis variables with the best dose-response effect were the ReoRox ReoLyse lysis variables and ROTEM EXTEM Time to complete lysis. However, these variables only detected high t-PA levels (> 1 μg/mL). Conclusions. The new ReoRox ReoLyse test provides more information on fibrinolysis compared to the ReoRox Fib1 program. Neither ReoRox nor ROTEM could detect lower degrees of fibrinolysis. ReoRox is a valuable alternative to ROTEM to study high degrees of fibrinolysis and should be evaluated in clinical situations with increased fibrinolysis and during therapeutic thrombolysis.

Ethanol impairs coagulation and fibrinolysis in whole blood: a study performed with rotational thromboelastometry.

The objective was to study the effects of ethanol on coagulation and fibrinolysis in whole blood. Blood samples from healthy volunteers were analyzed before and after in-vitro addition of ethanol in order to achieve ethanol concentrations of 0, 1, 2 and 4‰, respectively (0, 22, 44 and 88 mmol/l). Coagulation and fibrinolysis were then assessed using rotational thromboelastometry. We found that increasing ethanol levels increasingly impaired coagulation as evaluated with rotational thromboelastometry, with a maximum prolongation of the clot formation time of 118% at an ethanol level of 4‰ (P < 0.000001). We also found a very strong impairment of fibrinolysis already at an ethanol level of 1‰. This is the first study assessing the effects of ethanol on coagulation and fibrinolysis in a whole blood model. The impairment of coagulation is similar in nature to the impairment found in patients suffering from hypothermia. The impairment is at a level that may be of clinical importance (e.g. in patients suffering from trauma). The inhibition of fibrinolysis is obvious already at an ethanol level of 1‰ and it may be a contributing factor to the increased amount of coronary and cerebrovascular ischemic events after binge drinking.

An in vitro evaluation of standard rotational thromboelastography in monitoring of effects of recombinant factor VIIa on coagulopathy induced by hydroxy ethyl starch

BackgroundRotational thromboelastography (ROTEG) has been proposed as a monitoring tool that can be used to monitor treatment of hemophilia with recombinant factor VIIa (rFVIIa). In these studies special non-standard reagents were used as activators of the coagulation. The aim of this study was to evaluate if standard ROTEG analysis could be used for monitoring of effects of recombinant factor VIIa (rFVIIa) on Hydroxy Ethyl Starch-induced dilutional coagulopathy.MethodsThe study was performed in vitro on healthy volunteers. Prothrombin time (PT) and ROTEG analysis were performed after dilution with 33% hydroxy ethyl starch and also after addition of rFVIIa to the diluted blood.ResultsPT was impaired with INR changing from 0.9 before dilution to 1.2 after dilution while addition of rFVIIa to diluted blood lead to an overcorrection of the PT to an International Normalized Ratio (INR) value of 0.6 (p = 0.01). ROTEG activated with the contact activator ellagic acid was impaired by hemodilution (p = 0.01) while addition of rFVIIa had no further effects. ROTEG activated with tissue factor (TF) was also impaired by hemodilution (p = 0.01) while addition of rFVIIa lead to further impairment of the coagulation (p = 0.01).ConclusionsThe parameters affected in the ROTEG analysis were Clot Formation Time and Amplitude after 15 minutes while the Clotting Time was unaffected. We believe these effects to be due to methodological problems when using standard activators of the coagulation in the ROTEG analysis in combination with rFVIIa.

Monitoring fondaparinux with the Sonoclot

Fondaparinux is a new anticoagulant that interacts with antithrombin III and activated coagulation factor X resulting in an inhibition of the coagulation system. It has been successful in doses of 2.5 mg for thromboprophylaxis as well as in higher therapeutic doses of 5–7.5 mg. No optimal method for monitoring the effects of fondaparinux has been proposed. The aim of the present study was to investigate whether a viscoelastic coagulation analyzer, the Sonoclot (Sienco, Denver, Colorado, USA), could be used for in-vitro monitoring of fondaparinux. Different concentrations of fondaparinux were added in vitro to whole blood taken from eight volunteers. The blood samples mixed with the various amounts of fondaparinux were analyzed using the Sonoclot. The whole-blood activated partial thromboplastin time with the Hemochron Jr (ITC, Edison, New Jersey, USA) was used as the reference coagulation analysis. All analyses were started expeditiously, within 30 s from sampling, and were performed at 37°C. The values of the Sonoclot parameter clot rate, which measures the rate of fibrin formation, fibrin polymerization and platelet–fibrin interactions, were significantly correlated to increasing concentrations of fondaparinux (R = −0.90). The Sonoclot parameters of activated coagulation time, time to peak and clot retraction had weaker, but still significant, correlations to fondaparinux concentrations. At prophylactic doses (0.38 μg/ml blood) the clot rate decreased 15% compared with the initial unanticoagulated value, whereas at therapeutic doses (1.53 μg/ml blood) there was a 27% decrease. In conclusion, the Sonoclot parameter clot rate could be of clinical value to individualize the fondaparinux dosage, especially the higher, therapeutic, dosages.

Monitoring of low molecular weight heparin anticoagulation during haemodialysis with a Sonoclot Analyzer.

Objectives: Sonoclot was used in this study to monitor low molecular weight heparin (LMWH) during haemodialysis. Material and Methods: Two different intravenous doses (standard / half-dose) of dalteparin were studied in eight patients. Blood was sampled for coagulation analyses with Sonoclot, thrombin-antithrombin (TAT) and anti-Xa. A visual fibrin deposition score (VFS) in the venous drip chamber was also evaluated. Results: All patients completed their dialysis. There was a progressive increase in TAT levels, which correlated to the dalteparin dose. Significant differences (p<0.05) were found for TAT, VFS and Sonoclot celite-activated clotting time (SonACT) between the different LMWH dosages. TAT and Sonoclot correlated to each other, but not to the VFS. SonACT was significantly increased at two hours, with the high dalteparin dose compared to the lower dose. Conclusion: Both Sonoclot and TAT failed to predict the VFS. No patient had any clinical clotting events and all dialyses were completed successfully.

Dabigatran and its reversal with recombinant factor VIIa and prothrombin complex concentrate: A Sonoclot in vitro study

Abstract Objective. Dabigatran is a new oral direct thrombin inhibitor. No specific antidote exists in the event of hemorrhage, but prothrombin complex concentrate (PCC) and recombinant activated factor VII (rFVIIa) are suggested therapies. Sonoclot is a bedside viscoelastic instrument for monitoring the coagulation process in whole blood. The aim of this study was to investigate the effect of dabigatran and reversal with PCC and rFVIIa, as monitored by the Sonoclot. Methods. Citrated whole blood was drawn and mixed in vitro with dabigatran, dabigatran + PCC or dabigatran + rFVIIa and analyzed with three different Sonoclot cuvettes: Glassbead, kaolin and tissue factor (diluted) activated. Results. The Sonoclot detected in vitro-induced anticoagulation due to dabigatran with the glassbead- and kaolin-activated cuvettes. There was no reversing effect of PCC, probably due to the presence of heparin in the PCC we used. There was no certain reversing effect of rFVIIa. Conclusions. The Sonoclot can detect the anticoagulant effect of dabigatran. Our results do not support efficient reversal of dabigatran with PCC and rFVIIa, or alternatively do not support the ability of Sonoclot to detect a reversing effect of the PCC and rFVIIa in our study. Clinical studies of dabigatran-treated patients with severe bleeding are called for, as well as the continued development of specific antidotes and monitoring techniques.

Effects of recombinant human activated protein C on the coagulation system: a study with rotational thromboelastometry.

Background: Recombinant human activated protein C (rhAPC) is an anticoagulant that can be used for treatment of patients with severe sepsis. The use of rhAPC is accompanied by an increased risk of severe bleeding. Rotational thromboelastometry is a method for measuring the status of the coagulation. The aim of the study was to investigate whether rotational thromboelastometry could be used for monitoring the effects of rhAPC on the coagulation.