Rob Wagenvoord

Calibrated automated thrombin generation measurement in clotting plasma

Calibrated automated thrombography displays the concentration of thrombin in clotting plasma with or without platelets (platelet-rich plasma/platelet-poor plasma, PRP/PPP) in up to 48 samples by monitoring the splitting of a fluorogenic substrate and comparing it to a constant known thrombin activity in a parallel, non-clotting sample. Thus, the non-linearity of the reaction rate with thrombin concentration is compensated for, and adding an excess of substrate can be avoided. Standard conditions were established at which acceptable experimental variation accompanies sensitivity to pathological changes. The coefficients of variation of the surface under the curve (endogenous thrombin potential) are: within experiment ∼3%; intra-individual: <5% in PPP, <8% in PRP; interindividual 15% in PPP and 19% in PRP. In PPP, calibrated automated thrombography shows all clotting factor deficiencies (except factor XIII) and the effect of all anticoagulants [AVK, heparin(-likes), direct inhibitors]. In PRP, it is diminished in von Willebrand’s disease, but it also shows the effect of platelet inhibitors (e.g. aspirin and abciximab). Addition of activated protein C (APC) or thrombomodulin inhibits thrombin generation and reflects disorders of the APC system (congenital and acquired resistance, deficiencies and lupus antibodies) independent of concomitant inhibition of the procoagulant pathway as for example by anticoagulants.

The technique of measuring thrombin generation with fluorogenic substrates: 3. The effects of sample dilution

Assessing the clotting function inevitably brings about dilution of plasma. With the existing techniques of thrombin generation (TG) measurement, dilution ranges from 2:3 to 1:8. However, the possibility that dilution alters procoagulant and anticoagulant pathways differently has not been examined. We investigated the effects of dilution on the thrombin generation process and found that the anticoagulant pathways are far more affected by dilution than the procoagulant pathways. That is, when prothrombin and antithrombin concentrations are kept constant, dilution of plasma does not significantly affect tissue factor (TF)-driven thrombin generation. We demonstrate that dilution of plasma slows down the inhibitory activity of tissue factor pathway inhibitor (TFPI) to a greater extent when compared with the down regulation by diluting procoagulant factors. Dilution of plasma has also a negative effect on the participation of the antihaemophiliac factors VIII and IX in TG driven by contact activation or low TF concentration. We also investigated the effect of dilution on the participation of the anticoagulant system that consists of thrombomodulin, protein C and protein S (APC system). We found that plasma dilution causes a loss of sensitivity towards TM and APC. Furthermore, at high dilutions (> 1:12) a second wave of prothrombinase-activity was observed that could be attributed to the suppression of protein S-dependent inhibition. In conclusion, the mechanism of TG is profoundly disturbed by plasma dilution. As a consequence, the less a plasma sample is diluted, the better a TG experiment represents the physiological process.

Fluorogenic peptide-based substrates for monitoring thrombin activity

The synthesis of a series of peptides containing C‐terminal 7‐amino‐4‐methylcoumarin (AMC) for use in the thrombin generation test (TGT) is described. The lead structure in this project was H‐Gly‐Gly‐Arg‐AMC, of which the water solubility and kinetic parameters (KM and kcat) are greatly improved over those of the substrate in current use in the TGT: Cbz‐Gly‐Gly‐Arg‐AMC. A series of N‐terminally substituted Gly‐Gly‐Arg‐AMC derivatives were synthesized, as well as implementation of structural changes at either the P2 or P3 position of the peptide backbone. Furthermore, two substrates were synthesized that have structural similarities to the chromogenic thrombin substrate SQ68 or that contain a 1,2,3‐triazole moiety in the peptide chain, mimicking an amide bond. To determine the applicability of newly synthesized fluorogenic substrates for monitoring continuous thrombin generation, the KM and kcat values of the conversion of these fluorogenic substrates by thrombin (FIIa) and factoru2005Xa (FXa) were quantified. An initial selection was made on basis of these data, and suitable substrates were further evaluated as substrates in the thrombin generation assay. Assessment of the acquired data showed that several substrates, including the SQ68 derivative Et‐malonate‐Gly‐Arg‐AMC and N‐functionalized Gly‐Gly‐Arg‐AMC derivatives, are suitable candidates for replacement of the substrate currently in use.

Thrombin generation in mesalazine refractory ulcerative colitis and the influence of low molecular weight heparin

BackgroundIn ulcerative colitis (UC), a state of hypercoagulation has frequently been observed. Low molecular weight heparin (LMWH) has shown beneficial effects as an adjuvant treatment of steroid refractory UC in open trials. We assessed potential therapeutic effects of the LMWH reviparin in hospitalised patients with mesalazine refractory UC, as well as its influence on haemostasis factors.MethodsTwenty-nine patients with mild-to-moderately active UC were included in a double-blind placebo controlled trial. All patients had a flare-up of disease under mesalazine treatment. Reviparin (Clivarin®) 3,436 IU anti-Xa/0.6 ml or placebo s.c. was added, and self-administered twice daily for 8 weeks. Patients were monitored for possible adverse events and changes in clinical symptoms. Endoscopical, histological, biochemical and haemostasis parameters were analysed.ResultsTolerability and compliance were excellent and no serious adverse events occurred. No significant differences were observed on the clinical, endoscopical and histological outcome, as compared to placebo. A high intrinsic and extrinsic thrombin potential was found before LMWH therapy. However, the significant reduction in the thrombin generation by LMWH was not related to the reduction in disease activity.ConclusionThe LMWH reviparine reduces thrombin generation in patients with mild-to-moderately active, mesalazine refractory UC, but is not associated with a reduction in disease activity.

Low paediatric thrombin generation is caused by an attenuation of prothrombin conversion

Thrombin generation (TG) is decreased in children. TG is determined by two underlying processes: the conversion of prothrombin to thrombin and the inactivation of thrombin. Therefore, lower TG capacity in children can either be caused by a reduction of prothrombin conversion, an increase of thrombin inactivation, or both. In 36 children and 8 adults, TG and the factors that determine thrombin inactivation (antithrombin, α2Macroglobulin (α2M) and fibrinogen) were measured. Prothrombin conversion, thrombin inhibitor complex formation, and the overall thrombin decay capacity were determined. In silico modelling was performed to determine the contribution prothrombin conversion and thrombin inactivation to deviant paediatric TG. Both the amount of prothrombin converted and the maximal prothrombin conversion rate are significantly reduced in children as compared to adults. This is partly due to the prothrombin levels being lower and partly to a lower prothrombin conversion rate. The overall thrombin decay capacity is not significantly different in children, but α2Macroglobulin plays a more important role than it does in adults. In silico experiments demonstrate that reduced prothrombin conversion and to a lesser extent elevated α2M levels provide an explanation for low TG in children. Young age has a dual effect on prothrombin conversion. Lower plasma prothrombin levels result in decreased prothrombin conversion but the rate of prothrombin conversion is also decreased, i.u2009e. the development of prothrombinase is lower than in adults.

Decreased prothrombin conversion and reduced thrombin inactivation explain rebalanced thrombin generation in liver cirrhosis

Impaired coagulation factor synthesis in cirrhosis causes a reduction of most pro- and anticoagulant factors. Cirrhosis patients show no clear bleeding or thrombotic phenotype, although they are at risk for both types of hemostatic event. Thrombin generation (TG) is a global coagulation test and its outcome depends on underlying pro- and anticoagulant processes (prothrombin conversion and thrombin inactivation). We quantified the prothrombin conversion and thrombin inactivation during TG in 30 healthy subjects and 52 Child-Pugh (CP-) A, 15 CP-B and 6 CP-C cirrhosis patients to test the hypothesis that coagulation is rebalanced in liver cirrhosis patients. Both prothrombin conversion and thrombin inactivation are reduced in cirrhosis patients. The effect on pro- and anticoagulant processes partially cancel each other out and as a result TG is comparable at 5 pM tissue factor between healthy subjects and patients. This supports the hypothesis of rebalanced hemostasis, as TG in cirrhosis patients remains within the normal range, despite large changes in prothrombin conversion and thrombin inactivation. Nevertheless, in silico analysis shows that normalization of either prothrombin conversion or thrombin inactivation to physiological levels, by for example the administration of prothrombin complex concentrates would cause an elevation of TG, whereas the normalization of both simultaneously maintains a balanced TG. Therefore, cirrhosis patients might require adapted hemostatic treatment.

Inhibition of Thrombin-Catalyzed Reactions in Blood Coagulation and Platelet Activation by Heparin Fractions in the Absence of Antithrombin III

The antithrombin-III-independent effect of heparin was studied in the following thrombin-catalyzed reactions: activation of purified plasma factor V and partially purified plasma factor VIII:C, generation of factor Va from the platelets and, in the presence of collagen, of the platelet procoagulant activity. Five heparin fractions and a heparinoid were compared to crude heparin. Crude heparin was a more potent inhibitor of these reactions than the fractions or the heparinoid. The inhibitory action of heparin (fractions) appeared to be the result of the formation of a complex between heparin and thrombin that alters the specificity of thrombin towards high molecular weight substrates. The inhibition of these thrombin-dependent feedback reactions in blood coagulation might be of importance in the mechanisms for the dissociation between the antithrombotic and hemorrhagic properties of low molecular weight heparins.