Maastricht, Netherlands
Maastricht, Netherlands

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Hemker H.C.,Synapse BV
Nederlands tijdschrift voor geneeskunde | Year: 2011

A test is needed of which the score is low in case of haemorrhagic tendency and high in case of thrombotic tendency. Emerging evidence shows that thrombin generation is a promising candidate. Recently it became possible to measure the course in thrombin concentration in clotting blood or blood plasma in an simple way suitable for application in a routine clinical setting. It appears that the amount of thrombin developing in clotting blood or blood plasma is a better measure for thrombotic- or haemorrhagic tendency than any variant of the clotting time and that thrombin generation is a universal test for the effect of antithrombotic drugs.


Devreese K.M.J.,Ghent University | de Laat B.,Maastricht University | de Laat B.,Synapse B.V.
Journal of Thrombosis and Haemostasis | Year: 2015

Background: According to the ISTH guidelines for lupus anticoagulant (LAC) testing, the second step in the three-step procedure (screening, mixing, and confirmation) is the mixing test, which improves the discrimination between the presence of an inhibitor and coagulation factor deficiencies such as those occurring in patients receiving vitamin K antagonists (VKAs). Objectives: From a retrospective analysis of dilute Russell viper venom (dRVVT) results, we evaluated the impact of the mixing test result on the interpretation of LAC positivity. Methods: We interpreted the dRVVT clotting times with and without taking into account the results of the mixing test in a patient population with prolonged screening test (n = 267) with special attention to the patients receiving VKAs. Results and conclusions: The number of samples classified as 'LAC positive' differed substantially depending on the method of interpretation; 170 and 235 of 267 samples were classified as LAC positive with the three- and two-step procedure, respectively. Discrepancy between the two-step (without mixing step) and the three-step procedure was due to not including a mixing test and was more pronounced in the VKA patient population. Screen/confirm ratios carried out on a 1:1 mix of patient and normal pooled plasma (NPP) gave a lower incidence of 59 of 267. We advise continuing to perform mixing test to avoid false-positives. In patients with discrepant results between the two- and three-step dRVVT interpretation, mainly observed in VKA-treated patients, we advise retesting of the patients preferable beyond the period of anticoagulant therapy and additional testing for anti-beta2GPI and/or anti-cardiolipin antibodies. © 2015 International Society on Thrombosis and Haemostasis.


Hemker H.C.,Synapse BV. | Kremers R.,Synapse BV.
Thrombosis Research | Year: 2013

To obtain a thrombin generation (TG) curve from the conversion of added fluorogenic substrate, thrombin concentrations are to be derived from the observed velocity of increase of fluorescence (dF/dt). The relation between velocity and thrombin concentration varies during the experiment because substrate is consumed and because fluorescence is not linear with the concentration of product. Here we review the techniques that we developed to:A: Transform the fluorescence trace into the "ideal" trace that would be seen if substrate consumption and non-linearity of fluorescence would not play a role.B: Subtract the contribution of α2M-thrombin so as to obtain the course of free thrombin.C: Calculate the velocity of prothrombin conversion from the course of free thrombin.D: Fit a smooth curve through the points obtained in a TG experiment © 2012 Elsevier Ltd. All rights reserved.


Wagenvoord R.J.,Synapse BV | Deinum J.,Astrazeneca | Elg M.,Astrazeneca | Hemker H.C.,Synapse BV
Journal of Thrombosis and Haemostasis | Year: 2010

Background. Thrombin generation (TG) in plasma can be monitored continuously with a fluorogenic thrombin substrate using calibrated automated thrombinography (CAT). In the presence of low concentrations of a reversible direct thrombin inhibitor (DTI), CAT shows an unexpected effect: the endogenous thrombin potential (ETP) increases at low concentrations of the inhibitor to subsequently decrease concentration dependently at higher concentrations (> approximately 100 nm). Objectives. To find an explanation for this phenomenon, we measured the concentrations of free thrombin and α2-macroglobulin-thrombin complex (α2MT) with a sub-sampling technique in the presence of AR-H067637, a selective DTI. Results. At all concentrations of the DTI there was a gradual dose-dependent decrease in the concentration of free, not-inhibited thrombin but a transient increase in free α2MT due to competition of thrombin and α2MT for the inhibitor. Because the CAT technique uses an algorithm to subtract α2MT activity from the total amidolytic activity, this transient increase in α2MT activity is not subtracted and erroneously attributed to thrombin itself. Conclusions. This study explains the spurious increase in ETP observed at low DTI concentrations. The results obtained in plasma were corroborated by observations in a thrombin generating system reconstituted with purified factors. In practise, the effect of DTIs on TG can be reliably evaluated from the area under the curve till time-to-peak. © 2010 International Society on Thrombosis and Haemostasis.


Ninivaggi M.,Synapse BV | Apitz-Castro R.,Synapse BV | Dargaud Y.,Unite dHemostase Clinique | De Laat B.,Synapse BV | And 2 more authors.
Clinical Chemistry | Year: 2012

BACKGROUND: The calibrated automated thrombogram (CAT) assay in plasma is a versatile tool to investigate patients with hypo- or hypercoagulable phenotypes. The objective was to make this method applicable for whole blood measurements. METHODS: Thin-layer technology and the use of a rhodamine 110-based thrombin substrate appear to be essential for a reliable thrombin generation (TG) assay in whole blood. Using this knowledge we developed a whole blood CAT-based assay. RESULTS: We demonstrated that the whole blood CAT-based assay is a sensitive and rapid screening test to assess function of the hemostatic system under more nearly physiological conditions than the TG assay in plasma. Under conditions of low tissue factor concentration (0.5 pmol/L) and 50% diluted blood, the intraassay CV of the thrombogram parameters, endogenous thrombin potential and thrombin peak height, were 6.7% and 6.5%, respectively. The respective interassay CVs were 12% and 11%. The mean interindividual variation (SD) of 40 healthy volunteers was 633 (146) nmol · min/L for the endogenous thrombin potential and 128 (23) nmol/L for the thrombin peak. Surprisingly, erythrocytes contributed more than platelets to the procoagulant blood cell membranes necessary for optimal TG. Statistically significant (P <0.001) and potentially clinically significant correlations were observed between circulating factor-VIII concentrations in blood of hemophilia A patients and endogenous thrombin potential (r = 0.62) and thrombin peak height (r = 0.58). CONCLUSIONS: We have developed a reliable method to measure TG in whole blood. The assay can be performed with a drop of blood and may provide a useful measurement of TG under more physiological conditions than plasma. © 2012 American Association for Clinical Chemistry.


De Laat B.,University Utrecht | De Laat B.,Sanquin Research | De Laat B.,Maastricht University | De Laat B.,Synapse BV | And 7 more authors.
Arthritis and Rheumatism | Year: 2011

Objective The presence of autoantibodies against a cryptic epitope in domain I of β 2-glycoprotein I (β 2GPI) is strongly associated with thrombotic events in patients with the antiphospholipid syndrome. We hypothesized that a conformational change could be a trigger for the formation of antibodies against domain I of β 2GPI. Therefore, we investigated whether immune responses against β 2GPI are related to its conformation. Methods Conformational changes in β 2GPI were studied using various techniques, either upon binding to cardiolipin or after disruption of the internal disulfide bonds. The immunogenicity of β 2GPI in different conformations as well as the individual domains of β 2GPI were studied in vivo by monitoring the generation of antibodies after intravenous administration of β 2GPI to mice. Furthermore, plasma samples from these mice were assessed for lupus anticoagulant activity and thrombin-antithrombin complex levels. Results We observed that the interaction of β 2GPI with cardiolipin induced a conformational change in β 2GPI: electron microscopy revealed that β 2GPI assembled into polymeric meshworks. We next investigated the immunogenicity of both human and murine β 2GPI in mice. Both human and murine β 2GPI combined with cardiolipin and misfolded β 2GPI triggered antibody formation against the native protein as well as against domain I of β 2GPI, while native β 2GPI was not immunogenic. In addition, we observed that anti-domain I antibodies developed in mice injected with domain I of β 2GPI, and that antibodies did not develop in mice injected with domains II-V. The induced anti-domain I antibodies prolonged the dilute Russell's viper venom plasma clotting time. The plasma of mice with anti-domain I antibodies had increased levels of circulating thrombin-antithrombin complexes. Conclusion The results of our studies indicate that the exposure of cryptic epitopes due to conformational changes in β 2GPI can induce autoantibody formation. © 2011 by the American College of Rheumatology.


The present invention relates to compounds and methods for inhibition of binding of ICAM-4 to platelet integrin _(IIb)_(3). The present invention also relates to a screening method and to a kit to detect or monitor in vitro the effect of a substance, drug or pharmaceutical agent on the ICAM-4/_(IIb)_(3) interaction in a biological sample, while simulating blood flow conditions existing in vivo. It concerns in particular an anti-ICAM-4 antibody or a fragment thereof capable of blocking ICAM-4 binding to platelet integrin _(IIb)_(3), or an ICAM-4 mimetic peptide blocking ICAM-4 binding to platelet integrin _(IIb)_(3), or an anti-CD61 antibody or a fragment thereof capable of blocking ICAM-4 binding to platelet integrin _(IIb)_(3), for use in the treatment of a thrombotic disease, and for the preparation of pharmaceutical compositions for such treatment.


The present invention relates to the field of blood clotting. Specifically, the invention relates to particular inhibitors of artificial activation of the blood clotting process through contact with foreign surfaces.


The present invention relates to the simultaneous measurement of proteolylic enzyme generation and clot strength in plasma or whole blood or any appropriate biological sample derived from blood. The measurement method encompasses the use of a detectable substrate which comprises a moiety that can be released upon reaction with the targeted proteolytic enzyme, and means for measurement of an increase in viscosity of clot strength.


A method for the simultaneous measurement of proteolylic enzyme generation and clot strength in plasma or whole blood or any appropriate biological sample derived from blood. The measurement method encompasses the use of a detectable substrate which includes a moiety that can be released upon reaction with the targeted proteolytic enzyme, and elements for measurement of an increase in viscosity of clot strength.

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