New insights into the role of erythrocytes in thrombus formation.

The role of erythrocytes in thrombus formation has previously been regarded as passive by their influence on rheology. Erythrocytes are known, due to their abundance and size, to push platelets to the vascular wall (laminar shearing). This results in an increased platelet delivery at the vascular wall enabling platelets to seal off a vascular damage preventing excessive blood loss. Recently, there is new evidence for erythrocytes to influence thrombus formation in multiple ways besides their effect on rheology. Several groups have shown that besides platelets, erythrocytes are the main suppliers of phosphatidylserine-exposing membranes needed for coagulation resulting in fibrin formation. In addition, our group has found that the intercellular adhesion molecule 4-αIIbß3 interaction mediates erythrocyte-platelet interaction in flowing blood. By inhibiting this interaction, we found decreased thrombin formation and decreased incorporation of erythrocytes into a thrombus. This review will provide more in-detail information of existing and new hypotheses regarding the role of erythrocytes in thrombus formation.

Large inter-individual variation of the pharmacodynamic effect of anticoagulant drugs on thrombin generation.

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.

Access and quality of biomarker testing for precision oncology in Europe.

BACKGROUND: Predictive biomarkers are essential for selecting the best therapeutic strategy in patients with cancer. The International Quality Network for Pathology, the European Cancer Patient Coalition and the European Federation of Pharmaceuticals Industries and Associations evaluated the access to and quality of biomarker testing across Europe. METHODS: Data sources included surveys of 141 laboratory managers and 1.665 patients, and 58 in-depth interviews with laboratory managers, physicians and payers. Four access metrics (laboratory access, test availability, test reimbursement, test order rate) and three quality metrics (quality scheme participation, laboratory accreditation, test turnaround time) were applied to rank the results. RESULTS: The access to precision medicines is higher in countries with public national reimbursement processes in place. Lack of diagnostic laboratory infrastructure, inefficient organization and/or insufficient public reimbursement narrow the access to single biomarker tests in many European countries. In countries with limited public reimbursement, pharma and patients' out of pocket were the primary funding sources for testing. Uptake of multi-biomarker next generation sequencing (NGS) is highly varied, ranging from 0% to >50%. Financial constraints, a lack of NGS testing capabilities and the failure to include NGS testing in the guidelines represent the main barriers to NGS implementation. The quality of biomarker testing is highest in Western and Northern Europe, with more than 90% of laboratories participating in quality assurance schemes. CONCLUSIONS: Our data clearly indicate the need for a call to action to ensure the clinical implementation of precision medicine in Europe.

Tumor mutation burden testing: a survey of the International Quality Network for Pathology (IQN Path).

While tumour mutation burden (TMB) is emerging as a possible biomarker for immune-checkpoint inhibitors (ICI), methods for testing have not been standardised as yet. In April 2019, the International Quality Network for Pathology (IQN Path) launched a survey to assess the current practice of TMB testing. Of the 127 laboratories that replied, 69 (54.3%) had already introduced TMB analysis for research purposes and/or clinical applications. Fifty laboratories (72.5%) used targeted sequencing, although a number of different panels were employed. Most laboratories tested formalin-fixed paraffin-embedded material (94.2%), while 18/69 (26%) tested also cell-free DNA. Fifty-five laboratories used both single nucleotide variants and indels for TMB calculation; 20 centers included only non-synonymous variants. In conclusion, the data from this survey indicate that multiple global laboratories were capable of rapidly introducing routine clinical TMB testing. However, the variability of testing methods raises concerns about the reproducibility of results among centers.

e-Learning for Instruction and to Improve Reproducibility of Scoring Tumor-Stroma Ratio in Colon Carcinoma: Performance and Reproducibility Assessment in the UNITED Study.

BACKGROUND: The amount of stroma in the primary tumor is an important prognostic parameter. The tumor-stroma ratio (TSR) was previously validated by international research groups as a robust parameter with good interobserver agreement. OBJECTIVE: The Uniform Noting for International Application of the Tumor-Stroma Ratio as an Easy Diagnostic Tool (UNITED) study was developed to bring the TSR to clinical implementation. As part of the study, an e-Learning module was constructed to confirm the reproducibility of scoring the TSR after proper instruction. METHODS: The e-Learning module consists of an autoinstruction for TSR determination (instruction video or written protocol) and three sets of 40 cases (training, test, and repetition sets). Scoring the TSR is performed on hematoxylin and eosin-stained sections and takes only 1-2 minutes. Cases are considered stroma-low if the amount of stroma is ≤50%, whereas a stroma-high case is defined as >50% stroma. Inter- and intraobserver agreements were determined based on the Cohen κ score after each set to evaluate the reproducibility. RESULTS: Pathologists and pathology residents (N=63) with special interest in colorectal cancer participated in the e-Learning. Forty-nine participants started the e-Learning and 31 (63%) finished the whole cycle (3 sets). A significant improvement was observed from the training set to the test set; the median κ score improved from 0.72 to 0.77 (P=.002). CONCLUSIONS: e-Learning is an effective method to instruct pathologists and pathology residents for scoring the TSR. The reliability of scoring improved from the training to the test set and did not fall back with the repetition set, confirming the reproducibility of the TSR scoring method. TRIAL REGISTRATION: The Netherlands Trial Registry NTR7270; https://www.trialregister.nl/trial/7072. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): RR2-10.2196/13464.

The technique of measuring thrombin generation with fluorescent substrates: 4. The H-transform, a mathematical procedure to obtain thrombin concentrations without external calibration.

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.

Heparins: A Shift of Paradigm.

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 < 25. Clinical experience and epidemiological evidence show a direct relationship between the ETP and the risk of thrombosis and bleeding. Consequently, the therapeutic potency of a heparin is determined by its aIIa activity, i.e., the concentration of a domain in which 12 sugar flank the high affinity antithrombin-binding pentasaccharide (HA5) at one side. The response of individual plasmas to a fixed dose of any heparin is highly variable. This suggests that individualization of heparin dosage, on basis of the ETP, might reduce bleeding or re-thrombosis. There exist simple laboratory methods for both the ETP and the concentration of the active domain. These methods can be used both for unequivocally characterization of a heparin preparation and for controlling heparin therapy and allow arbitrary units relative to a standard to be abandoned. These tests are as robust as any hematological routine test but not yet routinely available, which severely encumbers progress in the field.

The technique of measuring thrombin generation with fluorogenic substrates: 1. Necessity of adequate calibration.

In 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 = 5 x 12), 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.

Linear diffusion of thrombin and factor Xa along the heparin molecule explains the effects of extended heparin chain lengths.

QUESTION: How does the size of the heparin moiety in the anti-thrombin (AT)-heparin complex influence its anticoagulant properties? APPROACH: 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. RESULTS: 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 Mr<10,000 but decreases at higher Mr. Thrombin inactivation is constant per pentasaccharide with twelve adjacent monosaccharides (C-domain). CONCLUSION: 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.

Thrombin generation, a function test of the haemostatic-thrombotic 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.

A reduction of prothrombin conversion by cardiac surgery with cardiopulmonary bypass shifts the haemostatic balance towards bleeding.

Cardiac 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.

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 Generating Capacity and Phenotypic Association in ABO Blood Groups.

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 α2Macroglobulin (α2M) 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 α2M 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, α2M 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 α2M 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.

Differences in the mechanism of blood clot formation and nanostructure in infants and children compared with adults.

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.

The thrombogram in rare inherited coagulation disorders: its relation to clinical bleeding.

We 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 some-what 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.

[The Choay domain -- the structure responsible for the anticoagulant action of heparins]. / Le domaine Choay--la structure responsable de l'activité anticoagulante des héparines.

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.

Measurement of thrombin generation intra-operatively and its association with bleeding tendency after cardiac surgery.

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<0.01) after heparinization in the presence and absence of platelets (37% and 50%) compared to baseline. After the start of CPB, TG elevated and persisted till the end of surgery but remained lower than preoperatively. Activated clotting time increased after heparinization and after the start of bypass compared to baseline (400% and 500%). Anti-FXa activity reduced on the start of CPB compared to the level after heparinization, to almost the baseline value following protamine reversal of heparin. The plasma levels of total and free TFPI elevated 9 and 14 fold during bypass and remained after protamine administration higher than preoperatively. Plasma D-dimer levels reduced (p<0.01) when bypass started. However, a marked elevation was observed in the following time points. TG in platelet-rich plasma measured after heparinization and after the start of CPB associated (p<0.05) with postoperative blood loss. 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.

Preoperative thrombin generation is predictive for the risk of blood loss after cardiac surgery: a research article.

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 <930 ml; group 2: patients with median blood loss ≥930 ml). RESULTS: On T1, patients of group 2 had a significantly lower endogenous thrombin potential and peak thrombin (p<0.001 and p=0.004 respectively) in platelet-rich plasma, a significantly lower endogenous thrombin potential (p=0.004) and peak thrombin (p=0.014) in platelet-poor plasma, and a lower platelet count (p=0.002). On T2 both endogenous thrombin potential and peak thrombin remain significantly lower (p=0.011 and p=0.010) in group 2, measured in platelet-rich plasma but not in platelet-poor plasma. In addition, platelet number remains lower in group 2 after protamine administration (p=0.002). CONCLUSIONS: The key finding is that the Calibrated Automated Thrombography assay, performed preoperatively, provides information predictive for blood loss after cardiac surgery.

Thrombin generation: what have we learned?

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.