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Heparins inhibit the thrombin forming capacity of plasma, i. e., the endogenous thrombin potential (ETP), by their anti-thrombin (aIIa) activity, the anti-factor Xa (aXa) activity is of minimal importance. This holds for both unfractionated heparin (UFH) and low molecular weight heparin (LMWH) at aXa/aIIa ratios

Anticoagulation by a standard dosage of an inhibitor of thrombin generation presupposes predictable pharmacokinetics and pharmacodynamics of the anticoagulant. We determined the inter-individual variation of the effect on thrombin generation of a fixed concentration of direct and antithrombin-mediated inhibitors of thrombin and factor Xa. Thrombin generation was determined by calibrated automated thrombinography in platelet-poor plasma from 44 apparently healthy subjects which was spiked with fixed concentrations of otamixaban, melagatran, unfractionated heparin, dermatan sulfate and pentasaccharide. The variability of the inhibitory effect of the different anticoagulants within the population was determined using the coefficient of variation, i.e. the standard deviation expressed as a percentage of the mean. The inter-individual coefficients of variation of the endogenous thrombin potential and peak height before inhibition were 18% and 16%, respectively and became 20%-24% and 24%-43% after inhibition. The average inhibition of endogenous thrombin potential and peak height (ETP, peak) brought about by the anticoagulants was respectively: otamixaban (27%, 83%), melagatran (56%, 63%), unfractionated heparin (43%, 58%), dermatan sulfate (68%, 57%) and pentasaccharide (25%, 67%). This study demonstrates that the addition of a fixed concentration of any type of anticoagulant tested causes an inhibition that is highly variable from one individual to another. In this respect there is no difference between direct inhibitors of thrombin and factor Xa and heparin(-like) inhibitors acting on the same factors.

Thrombin is a pivotal player in the coagulation system. In clotting blood a transient wave of thrombin appears after a lag time. Clotting occurs at the start of the wave. The amount of thrombin formed reflects the function of the hemostatic system much better than the clotting time does: "The more thrombin the less bleeding but the more thrombosis, the less thrombin the more bleeding but the less thrombosis" has been shown to hold for congenital and acquired tendencies to venous thrombosis and bleeding and under all variants of antithrombotic treatment. The situation with arterial thrombosis is less clear. Calibrated automated thrombinography (CAT) allows quantitative assessment of the thrombin generation (TG) curve in platelet poor as well as in platelet rich plasma. Procedures to measure TG in whole blood and at the point of care are under development. TG measurement in platelet rich plasma underlines the close cooperation between platelets and the clotting system and challenges the traditional division between primary and secondary hemostases.

SummaryIn fluorogenic thrombin generation (TG) experiments, thrombin concentrations cannot be easily calculated from the rate of the fluorescent signal increase, because the calibration coefficient increases during the experiment, due to substrate consumption and quenching of the fluorescent signal by the product. Continuous, external calibration via an in a parallel sample therefore was hitherto required for an accurate calculation of the TG curve. A technique is presented that allows mathematical transformation of experimental fluorescence intensities into "ideal" data, i.e. in the data that would have been obtained if substrate consumption and quenching by the product would not play a role. The method applies to fluorescence intensities up to 90% of the maximal fluorescent signal corresponding to total substrate conversion and thereby covers the entire region of interest encountered in practice. The first derivative of the transformed signal can then be converted into thrombin concentrations via a conventional, fixed calibration factor. This calibration factor can be obtained from a separate experiment but also by measuring the amidolytic activity of the α2macroglobulin-thrombin complex present in the reaction mixture ("serum") after thrombin generation is over. This method halves the amount of sample required per experiment. Keywordscalibration, thrombin generation, fluorogenic methods

SummaryCardiac surgery with cardiopulmonary bypass (CPB) is associated with blood loss and post-surgery thrombotic complications. The process of thrombin generation is disturbed during surgery with CPB because of haemodilution, coagulation factor consumption and heparin administration. We aimed to investigate the changes in thrombin generation during cardiac surgery and its underlying pro- and anticoagulant processes, and to explore the clinical consequences of these changes using in silico experimentation. Plasma was obtained from 29 patients undergoing surgery with CPB before heparinisation, after heparinisation, after haemodilution, and after protamine administration. Thrombin generation was measured and prothrombin conversion and thrombin inactivation were quantified. In silico experimentation was used to investigate the reaction of patients to the administration of procoagulant factors and/or anticoagulant factors. Surgery with CPB causes significant coagulation factor consumption and a reduction of thrombin generation. The total amount of prothrombin converted and the rate of prothrombin conversion decreased during surgery. As the surgery progressed, the relative contribution of α2-macroglobulin-dependent thrombin inhibition increased, at the expense of antithrombin-dependent inhibition. In silico restoration of post-surgical prothrombin conversion to pre-surgical levels increased thrombin generation excessively, whereas co-administration of antithrombin resulted in the normalisation of post-surgical thrombin generation. Thrombin generation is reduced during surgery with cardiopulmonary bypass because of a balance shift between prothrombin conversion and thrombin inactivation. According to in silico predictions of thrombin generation, this new balance increases the risk of thrombotic complications with prothrombin complex concentrate administration, but not if antithrombin is co-administered.

Introduction Infants and children have a lower incidence of thrombosis compared with adults. Yet, the mechanism of blood clot formation and structure in infants and children, as the end product of coagulation, has not been studied. This study aimed to establish differences in the mechanism of thrombin generation, fibrin clot formation and response to thrombolysis in infants and children compared with adults. Materials and methods We studied thrombin generation, fibrin clot formation, structure and fibrinolysis in healthy infants, children and adults. Results Younger populations had a decreased potential to generate thrombin, at a slower velocity compared with adults, correlating positively with age. Clot formation at venous shear rate was decreased in infants and children compared with adults, with increased time for fibrin formation, decreased fibrin formation velocity, resulting in decreased tendency for fibrin formation in younger populations. These differences were less pronounced at arterial shear rate. Studies of the fibrin clot structure in paediatric age groups showed a significantly larger pore size compared with adults, suggestive of a clot that is less resistant to fibrinolysis. The presence of tissue plasminogen activator (tPA) resulted in a significant decrease in the pore size of infants and children, but not in adults. Conclusions This is the first study to suggest that the mechanism of blood clot formation and nanostructure, as well as response to thrombolytic therapy is different in infants and children compared with adults.

SummaryBy 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.

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 similar to3%; intra-individual

Individuals with blood group O have a higher bleeding risk than non-O blood groups. This could be explained by the lower levels of FVIII and von Willebrand Factor (VWF) levels in O individuals. We investigated the relationship between blood groups, thrombin generation (TG), prothrombin activation and thrombin inactivation. Plasma levels of VWF, FVIII, antithrombin, fibrinogen, prothrombin and alpha(2)Macroglobulin (alpha M-2) levels were determined. TG was measured in platelet rich (PRP) and platelet poor plasma (PPP) of 217 healthy donors and prothrombin conversion and thrombin inactivation were calculated. VWF and FVIII levels were lower (75% and 78%) and alpha M-2 levels were higher (125%) in the O group. TG is 10% lower in the O group in PPP and PRP. Less prothrombin was converted in the O group (86%) and the thrombin decay capacity was lower as well. In the O group, alpha M-2 plays a significantly larger role in the inhibition of thrombin (126%). In conclusion, TG is lower in the O group due to lower prothrombin conversion, and a larger contribution of alpha M-2 to thrombin inactivation. The former is unrelated to platelet function because it is similar in PRP and PPP, but can be explained by the lower levels of FVIII.

Thrombin generation in the presence of platelets is sensitive to the activation status of von Willebrand factor

Introduction: Patients undergoing cardiac surgery with cardiopulmonary bypass (CPB) are susceptible to haemostatic disturbances. Monitoring the haemostatic capacity by conventional clotting tests is challenging.Materials and Methods: Thrombin generation (TG) by Calibrated Automated Thrombography, clotting tests and tissue factor pathway inhibitor (TFPI) measurements were performed to describe the relationship between haemostatic changes and alterations in these tests. Blood samples were collected before, during and after CPB. Furthermore, it was investigated whether TG measured intraoperatively, is associated with increased risk of bleeding postoperatively.Results: TG diminished significantly (p Conclusions: TG can be determined during CPB despite the high heparinization level, it reflects the haemostatic capacity better than clotting-based assays and might better predict bleeding when performed intraoperatively. (C) 2013 Elsevier Ltd. All rights reserved.

Background: In this study the value of thrombin generation parameters measured by the Calibrated Automated Thrombography for prediction of blood loss after cardiac surgery with cardiopulmonary bypass was investigated. Methods: Thirty male patients undergoing first-time coronary artery bypass grafting were enrolled. Blood samples were taken pre-bypass before heparinisation (T1) and 5 min after protamine administration (T2). Thrombin generation was measured both in platelet-rich plasma and in platelet-poor plasma. Besides thrombin generation measurements, activated clotting time, haematocrit, haemoglobin, platelet number, fibrinogen, antithrombin, D-dimers, prothrombin time and activated partial thromboplastin time were determined. Blood loss was measured and the amount of transfusion products was recorded postoperatively until 20 hours after surgery. Patients were divided into two groups based on the median volume of postoperative blood loss (group 1: patients with median blood loss

PURPOSE. An increased mRNA expression of genes related to blood coagulation has been demonstrated in an experimental retinal detachment model but has not yet been confirmed in human clinical specimens. Tissue factor (TF), the initiating factor of blood coagulation, may be a determinant of the extent of tissue injury after rhegmatogenous retinal detachment (RRD). This study was conducted to determine whether subretinal fluid and vitreous fluid collected from patients with RRD have a procoagulant effect. METHODS. Calibrated thrombin generation (CAT) was used to investigate the thrombogenic properties of 28 subretinal fluids collected during scleral buckling surgery for RRD. Further, the thrombogenic properties of vitreous fluids from RRD (n 12), macular pucker (n 5), macular hole (n 6), and proliferative diabetic retinopathy (n 5) were compared with the properties of eye bank eyes (n 11), which served as control specimens. The procoagulant activity of TF was determined with Western blot analysis. RESULTS. The addition of subretinal fluid from all RRD patients (28/28, 100%) induced thrombin generation in normal and severely factor (F)XII-deficient plasma. Contrary to the subretinal fluid, the addition of vitreous fluids from various ocular disorders evoked very little thrombin generation in normal and severely FXII-deficient plasma (4/12, 33% RRD; 1/5, 20% macular pucker; 0/6, 0% macular hole; 0/5, 0% proliferative diabetic retinopathy; and 2/11, 18% eye bank eyes). The procoagulant activity in subretinal fluid was almost completely neutralized by antibodies against human TF. The presence of TF in subretinal fluid was confirmed by Western blot. CONCLUSIONS. Subretinal fluid of patients with RRD induces high procoagulant activity, determined by measuring the level of tissue factor. (Invest Ophthalmol Vis Sci. 2010;51: 5234‐5239) DOI:10.1167/iovs.10-5354 T issue factor (TF), a transmembrane glycoprotein, is the major initiating factor of blood coagulation in normal hemostasis in vivo. Upon the complexing of coagulation factor VII and TF, coagulation factor X and IX further activate downstream proteases. 1 Ultimately, this activation generates thrombin, the key enzyme in the conversion of fibrinogen into fibrin. 2 TF is ubiquitous throughout the human body and maintains hemostasis when blood vessels are injured. Therefore, TF is constitutively expressed by subendothelial cells and epithelia, to prevent blood loss into extravascular spaces. 3 In the human eye, TF is mostly expressed in the retinal vessel walls but only faintly in anterior segmental tissue. 4 Recently, it was shown that genes related to blood coagulation were upregulated in an experimental retinal detachment model in the pig. 5 In this study, TF gene expression showed a threefold change in expression level and was considered to be upregulated in detached and peridetached neural retinas and in retinal pigment epithelial (RPE) cells, compared with control retinas. It is postulated that TF may be a critical determinant of the extent of tissue injury mediated by an ongoing inflammatory response. 6 For instance, it was suggested that plasma TF levels reflect endothelial cell injury and/or destruction of TFcontaining cells in patients with diabetes mellitus with retinopathy or nephropathy. 3 Since the blood‐retinal barrier with its TF-containing RPE cells may be damaged during rhegmatogenous retinal detachment (RRD), it is conceivable that TF is released into the subretinal space. Moreover, blood coagulation components may play an essential role in the development of proliferative vitreoretinopathy (PVR) after RRD, as is shown in animal models. 7,8

In fluorogenic thrombin generation (TG) experiments, thrombin concentrations cannot be easily calculated from the rate of the fluorescent signal increase, because the calibration coefficient increases during the experiment, due to substrate consumption and quenching of the fluorescent signal by the product. Continuous, external calibration via an in a parallel sample therefore was hitherto required for an accurate calculation of the TG curve. A technique is presented that allows mathematical transformation of experimental fluorescence intensities into "ideal" data, i.e. in the data that would have been obtained if substrate consumption and quenching by the product would not play a role. The method applies to fluorescence intensities up to 90% of the maximal fluorescent signal corresponding to total substrate conversion and thereby covers the entire region of interest encountered in practice. The first derivative of the transformed signal can then be converted into thrombin concentrations via a conventional, fixed calibration factor. This calibration factor can be obtained from a separate experiment but also by measuring the amidolytic activity of the alpha(2)macroglobulin-thrombin complex present in the reaction mixture ("serum") after thrombin generation is over. This method halves the amount of sample required per experiment.

SummaryIn fluorogenic thrombin generation (TG) measurement the concentrations of thrombin are obtained from the course of fluorescence intensity. Because of fluorescence quenching, in one series of normal plasmas (n=60), the rate of fluorescence increase at fixed thrombin activity was 70 ± 13% of that in buffer, in another (n= 139) 75 ± 8%. Using a calibration factor (CF) measured in buffer therefore underestimates thrombin concentrations in plasma and introduces a source of error. A fixed CF also neglects the 25 – 35% increase of CF during the experiment and thus distorts the form of the TG curve so that the ETP cannot be determined. Continuous individual calibration (CIC), in which CF is determined continuously in a parallel sample, avoids such systematic errors but adds random error because the thrombin course is calculated from two different measurements. We determined the intra-individual coefficients of variation (CV) of the peak-height and ETP as obtained with CIC to those obtained with a fixed CF measured in buffer. With the fixed CF, the CVs varied between 18% and 49%; with CIC they lowered to 4–7% (n=5x12), i.e. in a range allowing clinical application. It is shown that CIC can be discarded for the measurement of peak thrombin values and replaced by comparison to a reference plasma only if quenching is not a systematic confounder. This was shown to be the case in the set of 139 normal plasmas but not in the set used for determining the intraindividual CVs, a difference that may depend upon preanalytical conditions.

How does the size of the heparin moiety in the anti-thrombin (AT)-heparin complex influence its anticoagulant properties?Of 52 heparin fractions of precise Mr between 2800 and 37,000 we determined the dissociation constant (Kd) of the binding of the enzyme to the AT-heparin complex and the decay constant (kdec) of thrombin and factor Xa at 1 microM of that complex.The Kd of thrombin or factor Xa is constant when expressed in terms of the concentration of sugar units, i.e. the enzymes bind the better the longer the heparin. Thrombin (Kd=1.86+/-0.13 microM) binds 11 times tighter than factor Xa (Kd=20.2 +/-1.5 microM). Factor Xa inactivation velocity is proportional to the concentration of pentasaccharide-bound AT if Mr10,000 but decreases at higher Mr. Thrombin inactivation is constant per pentasaccharide with twelve adjacent monosaccharides (C-domain).The data fit a model in which thrombin and factor Xa bind at a random site on the heparin chain and, via one-dimensional diffusion, reach the AT that is bound to its specific binding site on the heparin. Factor Xa, but not thrombin, can dissociate from heparin before reaching bound AT.

Introduction Cardiac surgery with cardiopulmonary bypass (CPB) causes hemodilution and coagulation factor consumption. The administration of unfractionated heparin accelerates thrombin inactivation and leads to elevated consumption of antithrombin (AT). The heparin-mediated release of tissue factor pathway inhibitor (TFPI) from the vessel wall reduces prothrombin activation. CPB surgery thus affects both pro- and anticoagulant processes, which are reflected in changes in a patient's thrombin generation potential. In this study we investigated how cardiac surgery changes thrombin generation (TG), prothrombin conversion and thrombin inactivation. In addition, we studied the consequences of these changes in pro- and anticoagulant processes for transfusion management using in silico experimentation. Methods Thirty patients undergoing cardiac surgery with CPB were included and samples were collected before and after surgery and protamine administration. AT and prothrombin levels were determined and TG was measured at 5 pM tissue factor by calibrated automated thrombinography. Prothrombin conversion and thrombin inactivation were quantified from each TG curve. The effect of transfusion of prothrombin complex concentrate (PCC) was simulated by an in silico increase of prothrombin conversion. The effect of PCC administration in combination with AT was modeled as an increase of prothrombin conversion and antithrombin levels. The effect of in silico transfusion of PCC on coagulation was quantified by TG parameters. Results CPB surgery causes a reduction of plasma coagulation factor levels: AT (47%, p We modeled the effect of the transfusion of prothrombin complex concentrate with or without added antithrombin on TG. If the prothrombin conversion capacity was increased to pre-surgery levels in the absence of antithrombin, TG peak height increases to 454 ± 72 nM (figure A). A slight increase of prothrombin conversion (up to 83% of the pre-surgery level) brings TG peak height into the normal range (386±61 nM), but the ETP remains elevated (3278 ± 1141 nM). The optimal way to restore thrombin generation is the simultaneous infusion of PCC and antithrombin. Our in silico experiments indicate that this restores both the peak height (362 ± 69 nM) and the ETP (1601 ± 369 nM∙min) to the pre-surgery level (figure B). Conclusions Cardiac surgery with CPB causes the hemostatic balance to shift toward bleeding due to a reduction of prothrombin conversion. This is only partially compensated by a reduction of thrombin inactivation, resulting in reduced TG after surgery. Post-surgery bleeding can be (and often is) remedied by the transfusion of PCC. However, full restoration of the prothrombin conversion capacity results in a TG profile that predicts a prothrombotic state, as an ETP value in the highest quartile is associated with a 5 times elevated thrombosis risk according to Winckers et al. Equilibrated restoration of the hemostatic capacity asks for the simultaneous supplementation of both pro- and anticoagulant factors, i.e. PCC containing AT or fresh frozen plasma. Figure 1. The effect of prothrombin complex concentrate (± antithrombin) transfusion on in silico thrombin generation. ■ Pre-surgery TG, ● Post-surgery TG, □ in silico PCC transfusion (left panel), and ○ in silico PCC and AT transfusion (right panel). Figure 1. The effect of prothrombin complex concentrate (± antithrombin) transfusion on in silico thrombin generation. ■ Pre-surgery TG, ● Post-surgery TG, □ in silico PCC transfusion (left panel), and ○ in silico PCC and AT transfusion (right panel). Disclosures Kremers: Synapse bv: Employment. Bloemen:Synapse bv: Employment. De Laat:Synapse bv: Employment. Wagenvoord:Synapse bv: Employment. Al Dieri:Synapse bv: Employment. Hemker:Synapse bv: Employment.

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.

SummaryIn 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.

Purpose of review After 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 findings The 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.Summary The 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.

We describe the common structural basis for the anticoagulant action of the many different heparins available to the clinician. From different types of heparin we prepared fractions of virtually single molecular weight. We determined the molar concentration of material (HAM) containing the antithrombin (AT) binding pentasaccharide (A-domain), the specific catalytic activity in thrombin- and factor Xa inactivation and the capacity to inhibit thrombin generation (TG). We also calculated the molar concentration of A-domain with 12 sugar units at its non-reducing end, i.e. the structure that carries anti-thrombin activity (Choay- or C-domain). The anti-thrombin activity and the effects on TG are determined by the concentration of C-domain and independent of the source material or Mr. High Mr fractions (15,000) are less active, probably through interaction with non-AT plasma proteins. Anti-factor Xa activity is not indicative of anticoagulant potency but is a sensitive indicator of Mr and therefore predicts favourable pharmacokinetic properties: long half-life in the circulation and high bioavailability.

Activated factor VII (FVIIa)/tissue factor enzyme complex is the initiator of the coagulation cascade in vivo. FVIIa is of particular interest because it has been found to induce haemostasis in various bleeding disorders. In order to evaluate the FVII threshold that is required to initiate the clotting cascade, we measured thrombin generation in FVII-depleted plasmas spiked with increasing amounts of normal pooled plasma and in inherited FVII-deficient plasmas. According to the literature, only trace amounts of FVII are sufficient to initiate blood coagulation in vitro. By contrast, results on inherited FVII-deficient plasmas showed a wide variety of the amounts of thrombin generated in plasmas with the same FVII coagulant activity levels. This suggests that the threshold of FVII required to initiate haemostasis in vivo depends on one or more, hitherto unknown, plasmatic or cellular factors.

Thromb Haemost 2006; 95: 756–7 The paper “Age dependency measured by calibrated automated thrombinography (CAT)” by Haidl et al. (1) is one in a series of investigations from the Graz group that significantly contributes to the understanding of paediatric haemostasis. More or less as a by-product of these studies they now found that adults of over 35 years of age have a significantly higher thrombin generation potential (ETP) than those aged from 18 – 35. The figure in this publication suggests an apparently linear increase with age that is as high as ~30% between the ages of 20 and 50. This is an important observation because it shows that, also in a group of apparently healthy volunteers without known prothrombotic traits, the age group that is more likely to get thrombosis is also the one that generates more thrombin. In a series of congenital conditions, such as antithrombin deficiency (2), congenital hyperprothrombinaemia (G20210A mutation) (3), and dysfunction of the protein C-system (unpublished observation), it is evident that the genetic defect primarily causes higher thrombin generation, and one concludes that higher thrombin generation leads to a higher risk of venous thrombosis. If we observeAPC-resistance in the presence of the lupus anti(!)-coagulant (4) or in women using oral contraceptives (5), it is not a long shot to surmise that in these cases, as in congenital conditions, higher thrombin generation is the cause of the thrombotic tendency. Taken together, the body of circumstantial evidence for high thrombin generation potential being the cause of a prothrombotic condition becomes rather impressive. It is reinforced again by the inverse observation, i.e. the fact that all antithrombotic drugs decrease thrombin generation [see further (6)]. The observation by Haidl et al. carries the subject a step further. It has been observed that thrombin generation is rather variable in a normal population. Where the intraindividual variation of the ETP is ~5%, the interindividual variation is >15% (7). This means that 99% of all normal individuals have a thrombin generating capacity of between 55 and 145 % of the mean. In other words: some people in the normal population generate twice as much thrombin as others. In congenital conditions such as antithrombin deficiency or the prothrombin A20210G mutation, such large variations can be attributed to one single cause. Apparently, in the normal population they can also be caused by a combination of circumstances. “Mr. Low” who, by chance, has a low normal set of procoagulant factors and a high normal set of anticoagulant factors may produce half as much thrombin when his plasma is triggered compared to “Mrs. High”, a person with high procoagulant and low anticoagulant factors. The question now is whether, as a consequence, he has a much smaller risk of developing thrombosis. Now that automated calibrated measurement of thrombin generation allows such variations to be measured, we feel that the time is ripe for larger epidemiological studies relating the parameters of thrombin generation to thrombotic risk in those populations where no recognised risk factors can be found. Before embarking upon such studies, epidemiologist may well ask why thrombin generation might do better than the time honoured clotting times, which have essentially failed as predictors of thrombotic disease. The answer can be given on different levels: i) Clotting times are insensitive to variations in clotting function in the region of interest, i.e. in a large area around the normal mean. If the thromboplastin time, as defined by Armand Quick, is invariably 12 seconds in the normal, this is because it does not distinguish between the clotting system of Mr. Low and Mrs. High. This again is caused by the fact that the clotting time already approaches its minimum when the concentration of prothrombin (or of any other clotting factor) exceeds the ~25% level (8). ii) At the moment that the clotting time is recorded there is only very little thrombin formed. The large majority of all prothrombin (> 98%) still has to be converted. So, not enough thrombin is formed for the protein C system to start, and the plasma antithrombins are not yet of significant influence. Contrary to the clotting time, thrombin generation is very sensitive to variations of prothrombin and antithrombins around the normal mean as well as to the activity of the APC system. iii) The conditions of a thrombin generation experiment can be obtained reproducibly at almost any concentration of tissue factor (TF). For clotting times we have essentially the choice between an unphysiologically large excess of TF, as in the thromboplastin time, or an equally unphysiological absence of TF as in the activated partial thromboplastin time (aPTT). Measuring clotting times at lower TF concentrations is possible and has been practiced in the

Introduction We tested whether the recently introduced measurement of thrombin generation (TG) in whole blood can be used to evaluate the clotting status of patients on vitamin K antagonist (VKA) prophylaxis. The prothrombin time, and hence the International Normalized Ratio (INR), only evaluates the vitamin K dependent factors II, VII and X but not the anticoagulant factors, protein C and S as well as factor IX. In TG all factors play their role and when thrombomodulin (TM) is added the function of proteins C and S is stressed. The thrombotic tendency in congenital protein C resistance proves the importance of this protein C pathway. Aim To compare the INR in samples from patients under VKA prophylaxis to TG in whole blood and in platelet rich and platelet poor plasma (PRP, PPP) both in the presence and in the absence of TM. Materials & Methods Blood samples were collected from 123 consenting patients on VKA. In two thirds (67%) the indication for prophylaxis was atrial fibrillation. Other indications included prosthetic valves, lung embolisms or thrombosis. The INR was determined in the PPP of the samples and the patients were stratified into 5 groups: INR of 1.0 to 1.5, 1.5 to 2.5, 2.5 to 3.5, 3.5 to 4.5 and higher than 4.5. Thrombin generation (TG) was measured via Calibrated Automated Thrombinography (CAT) in whole blood and in PRP and PPP, with and without 20 nM added TM. From the TG curve lag time and time to peak were obtained as well as the maximal thrombin concentration (peak) and the area under the curve (endogenous thrombin potential: ETP). Also red and white blood cells and platelets were counted. Results With increasing INR values, the ETP and peak height decrease and lag time and time to peak prolong. All TG parameters measured in whole blood were significantly correlated (p-values< 0.01) with the values determined in both PRP and PPP. INR was linearly correlated with lag time and time to peak (p-value< 0.01), whereas for the concentration dependent parameters (ETP and peak height) the correlation with the INR was hyperbolical (p-value< 0.01). In plasma, 20 nM TM causes a diminution of ETP and peak of 50-60 % in normals and in patients in the INR 1 – 1.5 group. At higher INR values inhibition is between 25 and 40%, independent of the INR value. In whole blood, on the contrary, the same concentration of TM causes around 30 % of inhibition in normals and in all patients alike. Conclusions Whole blood TG data correlate well with INR and reflect more of the coagulation mechanism than the INR does. Like the INR it does not reflect the function of the VKAs on the natural anticoagulant factors, however. In PPP and PRP addition of TM shows that VKA treatment induces TM resistance in patients with an INR value higher than 1.5. Disclosures: No relevant conflicts of interest to declare.

Introduction Age specific differences in the function and concentration of haemostatic proteins are well established and are thought to contribute to the lower incidence of thromboembolism in infants and children compared to adults. We hypothesized that there are age-specific differences in the fibrin clot nanostructure and that these differences impact on fibrinolytic and anticoagulant treatment. Aim To determine the differences in fibrin clot nanostructure and response to tissue Plasminogen Activator (tPA) and Unfractionated Heparin (UFH) in infants compared to adults. Materials and Methods Four plasma samples from infants (0.86±0.16 years) and four samples from adults (32.04±15.80 years) were investigated. Thrombin generation (TG) was performed on paper discs in a microplate with 300mmol/L rhodamine substrate (P2Rho), 4mmol/L phospholipids, 5x10-9 mmol/L tissue factor and 16.7 mmol/L CaCl2; with and without additional 50ng/ml tPA, and 0.01IU/ml UFH. Fluorescence signal was recorded with a Fluoroskan Ascent fluorometer, with samples tested in triplicate. The resulting fibrin clots were fixed with 2.5% glutaraldehyde; washed with PBS and fixed with osmium tetroxide OsO4. The clots were dehydrated in ethanol and hexamethyldisilazane and were placed on carbon adhesive tabs. Each clot was coated with gold and studied using Scanning Electron Microscopy (SEM) on a Phenom Pro desktop SEM. The age-specific differences in fibrin fibre thickness and pore size were quantified using the Fibermetric software, with 36 representative images for infants and the same number for adults at each baseline, tPA and UFH, at magnification 10.000, counting 500 fibres per image. Results We observed significant differences in fibrin clot nanostructure between infants and adults (Fig 1). Results are expressed as median and range. Fibrin fibers were thicker in infants compared to adults (343.3nm, 61.2nm to 997.1nm vs 352.3nm, 26.1nm to 997.5nm, p In the presence of tPA in the adult sample, the fibrin fibers were thicker (352.3nm, 26.1nm to 997.5nm vs 355.8nm, 70.0nm to 999.6nm, p0.05) and pore size (0.057pm2, 0.0041pm2 to 19.97pm2 vs 0.050pm2, 0.0041pm2 to 19.34pm2, p>0.05). In the presence of UFH in adults fibrin fibers were thicker (352.3nm, 26.1nm to 997.5nm vs 339.0, 66.9nm to 996.4nm, p Discussion and Conclusion Our study suggests that the fibrin clots in infants are weaker compared to those of adults and is consistent with the lower TG in infants. This is further supported by published evidence that clots produced with low thrombin concentrations are composed of thick fibrin fibres, whereas those generated with high thrombin concentrations are composed of a tight network of thin fibres. The effect of both tPA and UFH on fibrin clots nanostructure in adults is consistent with published literature and is investigated in children for the first time. The fact that tPA did not have an effect on the fibrin clots of infants is not surprising considering that the concentration of plasminogen is 39%, tPA is 40% and TAFI function is 60% of the adult value; while PAI function is 50% higher compared to adults. UFH had an effect of the fibrin clot nanostructure in both infants and adults, with the effect on pore size being more pronounced in adults. While developmental haemostasis has been understood for many years, this is the first study to provide definitive proof that the end product of coagulation is a different clot structure in children. This further enhances our understanding of why thrombosis are less common in children, and has significant implications for treatment. Disclosures: No relevant conflicts of interest to declare.

Introduction Developmental Haemostasis, the concept describing maturation of the haemostatic system is based on established age specific differences in the quantity and function of haemostatic proteins. Interestingly, infants and children have a lower incidence of thromboembolic events than adults. Understanding the precise mechanism of thromboprotection in infants and children may therefore provide novel prevention and treatment strategies for adults. We hypothesized that a significant component of the thromboprotective mechanism is contributed to by the age-specific differences in thrombin generation. Aim This study was designed to investigate differences in thrombin generation in different age groups from infants to adults, to obtain more insight into age related differences in thromboembolic rates. Materials and Methods Plasma samples from 86 infants, children and adults were tested individually and the results were grouped according to age: 1 month to 1 year (N=13), 1-5 years (N=20), 6-10 years (N=19), 11-16 years (N=16) and adults (N=18). Thrombin generation (TG) was measured using Calibrated Automated Thrombinography (CAT) triggered with tissue factor (TF) at 5pM and 1pM final concentration. Parameters measured from the TG curve included: lag time, maximal thrombin concentration (peak), time to peak, as well as the area under the curve (endogenous thrombin potential, ETP). The raw data from TG was also used to calculate the overall, as well as alpha-2-macroglobulin (A2M) dependent decay constants. A2M function was measured with a thrombin inhibition assay developed in house. Results Both ETP and peak thrombin in infants and children was consistently significantly lower than in adults at both 1pM and 5pM tissue factor concentrations. The difference in ETP ranged from 27% to 35% (p Discussion and Conclusions This study confirms that TG in children (including adolescents) is lower than in adults and shows for the first time that this is predominantly caused by higher thrombin inhibition, largely due to an increase of A2M activity. Considering that the levels of A2M are increased by 50%, Antithrombin is decreased by approximately 37%, whilst the levels of Prothrombin, Fibrinogen and Heparin Cofactor II remain the same in infants and children compared to adults; therefore A2M is an important inhibitor of thrombin in early life and during childhood. Age-specific differences in thrombin generation as demonstrated by our study possibly play an important role in the thromboprotective mechanism functioning in infants and children. Disclosures: No relevant conflicts of interest to declare.

SummaryWe investigated the relation between clotting factor concentration, the parameters of the thrombin generation curve (the thrombogram) and the severity of clinically observed bleeding in patients with congenital deficiency of prothrombin (n = 21), factor V (n = 22), factor VII (n = 22), factor X (n = 10), factor XI (n = 7) and factor XII (n = 6). The parameters used were: area under the curve (endogenous thrombin potential, ETP), peak concentration of thrombin attained and lag time before manifest formation.Peak height and ETP varied linearly with the concentration of prothrombin. For the other factors these parameters hyperbolically approached to the 100% limit with increasing clotting factor concentration. Half normal ETP was seen at about the following concentrations: prothrombin (50%), factor V (1%), factor VII (2%), factor X (5%) and factor XI (1%). As a rule, the peak height was somewhat more sensitive to clotting factor decrease than the ETP was.In all the patients with severe bleeding symptoms the ETP was less than 20% of normal. Bleeding tendency was absent or mild in patients with an ETP of 30% or higher. This value (except for prothrombin) is already obtained at concentrations of clotting factor of 1%-2%, which corroborates the clinical observation that a severe bleeding tendency is only seen in severe clotting factor deficiencies (less than 1%). The one exception was a patient with factor VII deficiency and severe bleeding, who showed a normal ETP value, albeit with a decreased peak height and a prolonged lag-time.