Thrombin-Fibrin(ogen) Interactions, Host Defense and Risk of Thrombosis.

Fibrinogen is a well-known risk factor for arterial and venous thrombosis. Its function is not restricted to clot formation, however, as it partakes in a complex interplay between thrombin, soluble plasma fibrinogen, and deposited fibrin matrices. Fibrinogen, like thrombin, participates predominantly in hemostasis to maintain vascular integrity, but executes some important pleiotropic effects: firstly, as observed in thrombin generation experiments, fibrin removes thrombin from free solution by adsorption. The adsorbed thrombin is protected from antithrombins, notably α2-macroglobulin, and remains physiologically active as it can activate factors V, VIII, and platelets. Secondly, immobilized fibrinogen or fibrin matrices activate monocytes/macrophages and neutrophils via Mac-1 interactions. Immobilized fibrin(ogen) thereby elicits a pro-inflammatory response with a reciprocal stimulating effect of the immune system on coagulation. In contrast, soluble fibrinogen prohibits recruitment of these immune cells. Thus, while fibrin matrices elicit a procoagulant response, both directly by protecting thrombin and indirectly through the immune system, high soluble fibrinogen levels might protect patients due to its immune diminutive function. The in vivo influence of the 'protective' plasma fibrinogen versus the 'pro-thrombotic' fibrin matrices on thrombosis should be explored in future research.

The Anticoagulant and Nonanticoagulant Properties of Heparin.

Heparins represent one of the most frequently used pharmacotherapeutics. Discovered around 1926, routine clinical anticoagulant use of heparin was initiated only after the publication of several seminal papers in the early 1970s by the group of Kakkar. It was shown that heparin prevents venous thromboembolism and mortality from pulmonary embolism in patients after surgery. With the subsequent development of low-molecular-weight heparins and synthetic heparin derivatives, a family of related drugs was created that continues to prove its clinical value in thromboprophylaxis and in prevention of clotting in extracorporeal devices. Fundamental and applied research has revealed a complex pharmacodynamic profile of heparins that goes beyond its anticoagulant use. Recognition of the complex multifaceted beneficial effects of heparin underscores its therapeutic potential in various clinical situations. In this review we focus on the anticoagulant and nonanticoagulant activities of heparin and, where possible, discuss the underlying molecular mechanisms that explain the diversity of heparin's biological actions.

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.

Prothrombin conversion is accelerated in the antiphospholipid syndrome and insensitive to thrombomodulin.

Antiphospholipid syndrome (APS) is a condition in which the presence of antibodies against phospholipid-binding proteins is associated with thrombophilia and/or pregnancy morbidity. Although antiphospholipid antibodies have anticoagulant characteristics in vitro, they are associated with thromboembolic complications. Thrombin generation (TG) is a sensitive global test of coagulation, and elevated TG is associated with thrombosis. Increased TG can be caused by increased prothrombin conversion, decreased thrombin inactivation, or a combination of both. In this study, we measured TG in APS patients and healthy controls with and without vitamin K antagonist (VKA) treatment at 1 and 5 pM tissue factor and with thrombomodulin. Prothrombin conversion and thrombin inactivation were determined by thrombin dynamics analysis. The TG peak was increased in nontreated APS patients at 1 pM TF compared with nontreated controls. Prothrombin conversion was significantly increased in nontreated APS patients. In contrast, prothrombin conversion did not differ in controls and patients that were on VKA therapy. Thrombin inactivation was comparable between controls and APS patients in the presence and absence of VKAs. Both TG (peak and ETP) and prothrombin conversion were significantly higher in APS patients with prior thrombosis compared with patients without a history of thrombosis. In this study, we demonstrate that in APS, the hemostatic balance shifts toward a more prothrombotic phenotype due to elevated prothrombin conversion but unchanged thrombin inactivation rates. Within the group of APS patients, increased TG and prothrombin conversion are associated with a history of thrombosis.

Thrombin generation in low plasma volumes.

Accurate thrombin generation determination by calibrated automated thrombinography can be sustained when reducing the plasma and reagent volumes up to half, but not for higher reductions or plasma dilutions.

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.

Sex hormone-binding globulin and thrombin generation in women using hormonal contraception.

OBJECTIVES: We investigated the impact of serum sex hormone-binding globulin (SHBG) on thrombin generation (TG) in women according to hormonal contraception. PATIENTS AND METHODS: A cross-sectional study of SHBG and TG measured via calibrated automated thrombography was conducted in 150 healthy women, including 75 users of combined oral contraceptives (COC), 22 users of progestin-only contraceptives (POC) and 53 nonusers. RESULTS: COC but not POC-users had significantly higher SHBG levels compared with nonusers. In hormonal contraceptive users, SHBG was positively associated with both activated protein C (APC) resistance and baseline TG, and protein S and prothrombin were important mediators. CONCLUSION: These data provide further evidence that SHBG may be used as a biomarker in assessing prothrombotic profile of hormonal contraception.

Simultaneous measurement of thrombin generation and fibrin formation in whole blood under flow conditions.

Assays based on the formation of thrombin and fibrin are frequently used, and results are considered exchangeable in research/clinical settings. However, thrombin generation and fibrin formation do not always go hand in hand and flow profoundly influences thrombus formation. We describe the technical/clinical evaluation of an assay to simultaneously measure thrombin generation and fibrin formation under conditions of flow. Introduction of a fluorometer into a 'cone and base principle'-based rheometer allowed the measurement of thrombin generation (using a thrombin-sensitive substrate) and fibrin formation (changes in viscosity), while applying a linear shear flow. Increasing shear rates inversely related with thrombin generation and fibrin formation. Increasing fibrinogen concentrations in defibrinated plasma resulted in increased thrombin generation and fibrin formation. In pre-operative samples of 70 patients undergoing cardiothoracic surgery, fibrin formation and thrombin generation parameters correlated with fibrinogen content, rotational thromboelastometry (ROTEM) and whole blood Calibrated Automated Thrombinography (CAT) parameters, respectively. Upon dividing patients into two groups based on the median clot strength, a significant difference in perioperative/total blood loss was established. In conclusion, we clinically evaluated a method capable of simultaneously measuring thrombin generation and fibrin formation in plasma/whole blood under continuous flow, rendering our method one step closer to physiology. Importantly, our test proved to be indicative for the amount of blood loss during/after cardiothoracic surgery.

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.

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. e. the development of prothrombinase is lower than in adults.

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.

Thrombin generation: biochemical possibilities and clinical reality.

In this issue of Blood, under the unassuming title "Sample conditions determine the ability of thrombin generation parameters to identify bleeding phenotype in FXI deficiency," Pike et al publish observations on a very rare condition, but the results validate the real-life importance of a scheme of thrombin generation that has been emerging from biochemical research over the last decades and that challenges such stereotypes as the "clotting cascade" and "primary and secondary hemostasis." Moreover, this article shows how a bleeding phenotype is best recognized in the laboratory.

Characterization of an autosomal dominant bleeding disorder caused by a thrombomodulin mutation.

We describe a family with an autosomal dominant disorder characterized by severe trauma- and surgery-related bleeding. The proband, who experienced life-threatening bleeding during a routine operation, had normal clotting times, but markedly reduced prothrombin consumption. Plasma levels of all coagulation factors and of the main coagulation inhibitors were normal. Thrombin generation at low triggers was severely impaired and mixing experiments suggested the presence of a coagulation inhibitor. Using whole exome sequencing, the underlying genetic defect was identified as the THBD c.1611C>A mutation (p.Cys537Stop), which predicts a truncated form of thrombomodulin that is shed from the vascular endothelium. The patient had decreased expression of endothelium-bound thrombomodulin, but extremely elevated levels of soluble thrombomodulin in plasma, impairing the propagation phase of coagulation via rapid activation of protein C and consequent inactivation of factors Va and VIIIa. The same thrombomodulin mutation has been recently described in an unrelated British family with strikingly similar features.

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.

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.

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:

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.