H. Coenraad Hemker

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.

Thrombin generation, a function test of the haemostaticthrombotic system

By the use of a fluorogenic thrombin substrate and continuous calibration of each individual sample, it is now possible to obtain a thrombin generation (TG) curve (or thrombogram) in plasma, with or without platelets, in an easy routine procedure at high throughput and with an acceptable experimental error (<5%). Evidence is growing that the parameters of the thrombogram, and notably the area under the curve (endogenous thrombin potential, ETP), are useful in assessing bleeding- or thrombotic risk and its modification by antithrombotic- or haemostatic treatment. Available data strongly suggest that conditions (congenital, acquired, drug-induced) that increase TG all cause a thrombotic tendency and that conditions that decrease TG prevent thrombosis but, beyond a limit, cause bleeding. Diminution of TG is a common denominator of all antithrombotic treatment, including anti-platelet drugs. The thrombogram can also be used as a tool in the search for new antithrombotics and reflects the haemorrhagic or thrombotic side effects of other drugs (e.g. oral contraceptives). The thrombogram thus is a promising new approach to clinical management of bleeding and thrombotic disease as well as a tool in drug research and epidemiology. Our experience at this moment is insufficient, however, to already clearly define its limits.

Evaluation of thrombin generating capacity in plasma from patients with haemophilia A and B

In haemophilia patients, a relationship is usually observed between the clinical expression of the disease and plasmatic factor VIII/factor IX (FVIII/FIX) activity. However, it is known from clinical experience, that some haemophilia patients, despite similar FVIII/FIX plasma levels, could exhibit different bleeding phenotype. After determining preanalytical test conditions, we evaluated the thrombin generation capacity from haemophilia plasma samples in various conditions and the potential usefulness of thrombin generation test (TGT) in haemophilia patients. In a series of 46 haemophilia patients (34 haemophilia A and 12 haemophilia B patients), we found a significant correlation between plasmatic FVIII/FIX levels and endogenous thrombin potential (ETP), peak and time to peak obtained by thrombin generation measurement. In addition, a correlation was found between severe clinical bleeding phenotype and ETP. Our results suggest that TGT could be a promising tool to evaluate haemostasis capacity in patients with haemophilia. Our ex vivo results, obtained 24 hours after FVIII concentrate administration, showed that in patients presenting similar plasmatic FVIIII levels, thrombin generation capacity may be significantly different. These results suggest that in patients with haemophilia, TGT could be useful for individually tailoring prophylactic regimens as well as for adapting clotting factors infusions in surgical situations, in addition to FVIII/FIX plasma clotting activities.

Thrombin generation assays: accruing clinical relevance

Purpose of reviewAfter decades of near oblivion, thrombin generation is being revived as an overall function test of the plasmatic coagulation system in platelet-poor plasma (PPP). In platelet-rich plasma (PRP) it assesses platelet procoagulant functions as well. Recent findingsThe recently developed use of special fluorogenic thrombin substrates allows monitoring of thrombin concentration in clotting PPP and PRP on line in up to 24 parallel samples. Studies in model systems stress the importance of cell-bound thrombin generation such as measured in PRP. SummaryThe method can be profitably applied to various hitherto unyielding problems such as the control of (low-molecular-weight) heparin therapy, the detection of lupus anticoagulant, and various forms of thrombomodulin and activated protein C resistance (including the use of oral contraceptives) as well as monitoring the treatment of hemophiliacs by factor VIII bypassing therapy. In PRP it reflects the abnormalities encountered in von Willebrand disease and Glanzmann and Bernard-Soulier thrombopathy as well as the action of antiplatelet drugs.

Nonanticoagulant heparin prevents histone-mediated cytotoxicity in vitro and improves survival in sepsis

Extracellular histones are considered to be major mediators of death in sepsis. Although sepsis is a condition that may benefit from low-dose heparin administration, medical doctors need to take into consideration the potential bleeding risk in sepsis patients who are already at increased risk of bleeding due to a consumption coagulopathy. Here, we show that mechanisms that are independent of the anticoagulant properties of heparin may contribute to the observed beneficial effects of heparin in the treatment of sepsis patients. We show that nonanticoagulant heparin, purified from clinical grade heparin, binds histones and prevents histone-mediated cytotoxicity in vitro and reduces mortality from sterile inflammation and sepsis in mouse models without increasing the risk of bleeding. Our results demonstrate that administration of nonanticoagulant heparin is a novel and promising approach that may be further developed to treat patients suffering from sepsis.

Thrombin generation for the control of heparin treatment, comparison with the activated partial thromboplastin time

Summary.  Heparin can be quantified with antifactor Xa and IIa tests (aXa, aIIa) but the anticoagulant power of heparin depends upon plasma properties as well as upon heparin concentrations and thus differs between subjects. Measuring the effect, as with the activated partial thromboplastin time (APTT) therefore is clinically more relevant. Here we investigate the use of the endogenous thrombin potential (ETP) for this purpose. In 12 volunteers 9000 IU of four heparins of different mol. wt distributions were injected. Samples were taken at 11 time points between 0 and 24 h. With the exception of the 0 and 24‐h time points, heparin could be demonstrated by its aIIa and aXa activity in virtually all samples. The APTT showed the effect of this heparin in 34% of the samples; the ETP in 80%. This is partly due to the wide margins of the normal values, caused by large interindividual variation [coefficient of variation (CV) approximately 12% for the APTT, approximately 17% for the ETP]. The intraindividual variation is much smaller (CV approximately 4% for the APTT, approximately 5% for the ETP). Relative to the baseline value of the individual, the heparin effect was recognized by the APTT in 55% of the cases and by the ETP in 98%. There were no large differences between the different types of heparin.

Acquired APC resistance and oral contraceptives : differences between two functional tests

Resistance to activated protein C (APC) is often associated with a mutation in factor V (factor VLeiden). Individuals without factor VLeiden who exhibit a response in functional APC‐resistance tests similar to that of carriers of factor VLeiden are considered to be acquired APC resistant. This phenomenon is particularly observed in women using oral contraceptives (OC).

Whole-Blood Thrombin Generation Monitored with a Calibrated Automated Thrombogram-Based Assay

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.

Factor XI–Dependent Reciprocal Thrombin Generation Consolidates Blood Coagulation when Tissue Factor Is Not Available

Objective—Feedback activation of factor XI by thrombin is a likely alternative for tissue factor-dependent propagation of thrombus formation. However, the hypothesis that thrombin can initiate and propagate its formation in a factor XI-dependent and platelet-dependent manner has not been tested in a plasma milieu. Methods and Results—We investigated thrombin generation in recalcified platelet-rich plasma activated with varying amounts of thrombin or factor VIIa. Thrombin initiates and propagates dose-dependently thrombin generation only when platelets and plasma factor XI are present. Incubation of thrombin-activated platelets with a tissue factor neutralizing antibody had no effect on thrombin formation, indicating that platelet-associated tissue factor, if present at all, is not involved. In the absence of factor VIII, thrombin could not initiate its own formation, whereas factor VIIa-induced thrombin generation was reduced. Collagen strongly stimulated both thrombin-initiated and factor VIIa-initiated thrombin generation. Conclusions—These findings support the notion that platelet-localized feedback activation of factor XI by thrombin plays an important role in maintaining normal hemostasis as well as in sustaining thrombus formation when the TF pathway is inhibited by tissue factor pathway inhibitor.

Data management in Thrombin Generation

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: