Rate Of Prothrombin Conversion Research Articles

Abacavir use is associated with increased prothrombin conversion.

There is ongoing debate as to whether abacavir (ABC) increases the risk for cardiovascular disease(CVD) in people living with HIV (PLHIV) and the mechanisms underlying this possible association. We recently showed that the use of an ABC-containing regimen was independently associated with increased thrombin generation (TG). In the present study, we aim to explore these findings further, by studying the mechanistical processes that underly the global thrombin generation test via thrombin dynamics analysis. Thrombin dynamics analysis can pinpoint the cause of increased thrombin generation associated with ABC-use either to the procoagulant prothrombin conversion pathway or the anticoagulant thrombin inactivation pathway. In this cross-sectional study, 208 virally suppressed PLHIV were included, of whom 94 were on a ABC-containing regimen, 92 on a tenofovir disoproxil fumarate (TDF)-containing regimen, and the remainder on other regimens. We used Calibrated Automated Thrombinography to measure thrombin generation and perform thrombin dynamics analysis. The total amount of prothrombin conversion, as well as the maximum rate of prothrombin conversion were significantly increased in PLHIV on an ABC containing regimen compared to other treatment regimens. The levels of pro- and anticoagulant factors were comparable, indicating that the ABC-induced changes affect the kinetics of prothrombin conversion rather than procoagulant factor levels. Moreover, Von Willebrand Factor (VWF), active VWF and VWF pro-peptide levels were significantly higher in PLHIV than controls without HIV. However, they did not differ between ABC and non-ABC treated participants.

Neural Network Diagnoses the Antiphospholipid Syndrome without Interrupting Anticoagulant Therapy

Background The antiphospholipid syndrome (APS) is characterized by the presence of antiphospholipid antibodies (aPL) and is associated with an increased risk of thrombosis and pregnancy morbidity. The Laboratory diagnosis of APS is difficult because most patients already use anticoagulants due to a thrombotic event, resulting in interference with the clotting assays used for lupus anticoagulant (LAC) testing. Nevertheless, the aPL profile defines patient management, making aPL testing warranted during anticoagulant therapy. Sometimes, cessation of anticoagulant treatment is necessary to perform the lupus anticoagulant (LAC) test, which is unfavorable. The thrombin generation (TG) test is a functional and global hemostatic test. Clinical studies have shown that increased TG is associated with thrombosis, also in thrombotic APS patients. We have previously shown that the artificial intelligence method neural networks can be used to diagnose untreated APS patients from a group of healthy, auto-immune disease, and thrombosis controls with high accuracy. At the moment of aPL testing, many patients are already on anticoagulants, hampering the detection of LAC. Therefore, we developed a neural net (NN) for diagnoses APS in a cohort of anticoagulated subjects, based on the TG test. Methods Antiphospholipid antibodies positivity was diagnosed according to the Sapporo-Sydney guidelines. TG was measured using the calibrated automated thrombogram assay (CAT) on platelet poor plasma with PPP reagent (intermediate concentration of tissue factor) in 48 APS patients and 64 control non-APS patients, both treated with vitamin K antagonists (VKAs) TG was explored in more depth by quantifying parameters of prothrombin conversion and thrombin inactivation, using thrombin dynamics (TD) analysis, and assessing the sensitivity of the activated protein C pathway through the addition of thrombomodulin (TM). Five NNs were developed and the most accurate NN was selected for clinical validation based on its sensitivity and specificity. Input parameters for the NN were TG parameters lag time, peak, endogenous thrombin potential, time-to-peak, and velocity index, and TD parameters total prothrombin conversion, maximum prothrombin conversion rate, thrombin-antithrombin complex formation, and thrombin-α2-macroglobulin complex formation, and the inhibition of TG and TD by TM. The top performing NN was clinically validated using the TG and TD data acquired in the validation cohort, consisting of 33 APS patients and 62 controls, both on VKA therapy, 49 auto-immune disease controls, 38 thrombosis controls, 92 patients visiting the hospital for other indications and 37 healthy subjects. All control subjects were negative for aPL. Results TG was slightly increased in APS patients compared to controls. VKA therapy abolished the typical lag time prolongation seen in non-anticoagulated APS patients due to the LAC effect. The rate of prothrombin conversion was significantly increased in anticoagulated APS patients compared to controls, indicating a more prothrombotic phenotype. The selected NN classified APS patients with a sensitivity of 92% and a specificity of 95% in the developmental cohort consisting of APS patients and controls on VKA antagonists (ROCAUC = 0.9805; 0.9542-1.000; p<0.0001). On average, controls and APS patients were assigned an APS likelihood score of 12% and 89%, respectively. The NN was clinically validated in a separate validation cohort of 311 individuals, including 33 anticoagulated APS patients, 62 anticoagulated controls, 38 thrombosis patients, 49 auto-immune disease patients, 92 hospital controls, and 37 healthy controls. The sensitivity of the NN for the detection of APS was 85%. The overall specificity of the NN was 93%, and 100%, 96%, 96%, 92% and 76% respectively in healthy controls, auto-immune disease controls, hospital controls, anticoagulated controls, and thrombosis controls. Conclusions We developed a NN that accurately classifies APS under anticoagulant treatment. Therefore, this NN could be an alternative for diagnosing APS patients already treated with VKAs. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Applying Neural Networking Identifies Antiphospholipid Syndrome Patients with an Accuracy of 98.7%

Background: The antiphospholipid syndrome (APS) is characterized by the presence of antiphospholipid antibodies predominantly directed against β2-glycoprotein I. The syndrome is associated with an increased risk of thrombosis and pregnancy morbidity. In APS patients an altered thrombin generation (TG) profile has recently been described; however, it was not possible to differentiate between patients and healthy controls based on the value of a single TG parameter.Therefore, we used a novel machine learning approach called ‘neural networking’ to look for hidden patterns in patient and healthy subject data. A neural network is a computing system inspired by the human brain and consists of a collection of artificial neurons. These systems can learn to classify samples (healthy or APS patient) using examples from a database containing many input variables (i.e. training). Once the network has been trained and tested it can be used to classify new samples as healthy or APS patient.Aim: To develop a neural network to predict APS in subjects based on TG results.Materials and methods: 31antiphospholipid syndrome patients and 44 age- and sex-matched healthy subjects were enrolled in the study. Patients on anticoagulant therapy were excluded from the study. Thrombin generation was measured at 1 pM tissue factor (TF) and thrombin dynamics analysis was performed to study prothrombin conversion and thrombin inactivation. The sensitivity of the activated protein C system was tested by measuring TG in the presence and absence of 20 nM thrombomodulin (TM).A neural network was trained and tested to predict whether a subject is a patient or a healthy subject using Matlab software. The input of the neural network consisted of TG data (lag time, time-to-peak, peak height, ETP and TM effect) and thrombin dynamics data (total and maximal prothrombin conversion and thrombin inactivation).Results: Thrombin generation was measured in platelet poor plasma at 1 pM TF and prothrombin conversion parameters were determined computationally (Figure 1). The lag time was significantly prolonged in APS patients and the peak height was elevated. Time-to-peak and ETP were comparable between healthy subjects and APS patients, but the inhibitory effect of TM was significantly less in APS patients than healthy controls (p<0.001). The total amount of prothrombin converted during thrombin generation (PCtot) did not differ between healthy subjects and patients, but the maximum rate of prothrombin conversion (PCmax) was significantly elevated in APS patients.The neural networking tool was used to develop a classification network. The network consists of an input layer (thrombin generation and thrombin dynamics data, 1 layer of hidden processing neurons and two outputs (either APS patient or healthy subject) (Figure 2). After a training and testing phase, we obtained a network that accurately predicts a subject's class (APS patient or healthy) based on the given input values with an accuracy of 98.7%.Discussion:APS is a complex condition associated with thrombosis, pregnancy morbidity and alterations of diagnostic variables, for example in the thrombin generation profile. Single TG parameters are unable to predict whether or not a subject has APS, but a neural network based on TG and thrombin dynamics data correctly classifies APS patients in 98.7% of the cases. We have found a novel approach that is helpful in correctly categorizing APS patients and therefore might be included to improve treatment strategies. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Reference values for thrombin dynamics in platelet rich plasma

Thrombin generation (TG) is a better determinant of the overall function of the hemostatic system than routinely used clotting time-based assays and can be studied more in detail by thrombin dynamics analysis. Platelet poor plasma is often used to measure TG, however, measuring the contribution of the platelets is also important as patients with a low platelet count or with dysfunctional platelets have an increased risk of developing bleeding. In this study, platelet rich plasma (PRP) was collected from 117 healthy individuals. PRP was measured undiluted and diluted to a varying platelet concentration of 10*109/L to 400*109/L. Prothrombin conversion and thrombin inactivation were calculated from the data obtained by the TG parameters and coagulation factor levels (antithrombin, α2Macroglobulin (α2M) and fibrinogen). Reference ranges of TG and thrombin dynamics in PRP of 117 healthy individuals were established. Peak, velocity index and the maximum rate of prothrombin conversion increased linearly with platelet count, but endogenous thrombin potential reached a maximum at 150*109/L as seen in a subset population (n = 20). More extensive analysis revealed that a platelet count below 50*109/L did not affect TG parameters (except for the ETP). Correlation analysis indicated that the platelet count mainly affected the rate of prothrombin conversion. Inhibition of thrombin by antithrombin and α2M increased with increasing TG, but the ratio of inhibition by antithrombin or α2M remained the same independently of the total thrombin formed. In conclusion, TG and thrombin dynamics were assessed in PRP of healthy donors to provide reference values for future TG studies in PRP. Increasing the platelet count mainly affected the rate of prothrombin conversion and TG, rather than the total amount of thrombin formed.

Thrombin dynamics in children with liver disease or extrahepatic portal vein obstruction or shunt

IntroductionChanges in the coagulation profile in children with liver disease and extrahepatic portal vein obstruction or shunt result in both bleeding and thrombosis. Routine coagulation tests do not accurately predict bleeding risk, as they are not sensitive to changes in anticoagulant factors. The thrombin generation assay could be suitable for describing the overall balance of coagulation in children with liver disease. This study aims to characterise the mechanism of thrombin generation in this population, focusing on prothrombin conversion and thrombin inhibition. MethodsPatients were categorised as: severe (paediatric end stage liver disease score > 15) and mild disease, or portal vein obstruction or shunt. Age and gender matched healthy controls were used. The thrombin generation assay was performed in plasma samples from patients and controls with and without exogenous thrombomodulin and the results were further analysed with the computational thrombin dynamics method. ResultsA total of 42 patients (severe, n = 5; mild, n = 29, obstruction/shunt, n = 8) and 20 controls were included in this study. The total prothrombin conversion, thrombin-antithrombin formation and the thrombin decay capacity, in the presence and absence of thrombomodulin were reduced in children with severe liver disease. The rate of prothrombin conversion was increased and thrombin decay capacity was decreased in patients with portal vein obstruction or shunt compared to controls. ConclusionThis study demonstrates changes in the mechanism in thrombin generation seen in severe chronic liver disease. The changes vary in parenchymal versus non parenchymal liver disease and further study assessing the clinical significance of these variations in mechanism is required.

The Rate of Prothrombin Conversion Is Increased in Antiphospholipid Syndrome and Is Insensitive to Thrombomodulin

Background: The antiphospholipid syndrome (APS) is characterized by the presence of antiphospholipid antibodies directed mainly against prothrombin and β2-glycoprotein I. The syndrome is associated with an increased risk of thrombosis. The global hemostatic state in a patient can be tested by measuring thrombin generation (TG). Recently, we developed a method to study the main pro- and anticoagulant processes at the basis of TG, called the thrombin dynamics method.Aim: In this study we investigated the dynamics of thrombin generation in healthy subjects and APS patients.Materials and methods: Healthy subjects (n=129) and antiphospholipid syndrome (APS) patients (n=31) were included in the study. Sixty-eight percent of the APS patients were lupus anticoagulant positive, anti-cardiolipin antibodies were detected in 84% of the patients, and 52% presented with anti-β2-glycoprotein I antibodies. Patients on anticoagulant therapy were excluded from the study. Thrombin generation was measured at 1 pM tissue factor (TF) and activated protein C (APC) system sensitivity was tested by measuring TG in the presence and absence of 20 nM thrombomodulin (TM).Results: Thrombin generation was measured in platelet poor plasma at 1 pM tissue factor. The lag time and time-to-peak were significantly prolonged in APS patients compared to healthy subjects (lag time: 3.30 ± 0.59 vs. 6.69 ± 4.26 min, p<0.001; time-to-peak: 8.33 ± 1.29 vs. 10.76 ± 4.51 min, p<0.001). The peak height was significantly higher in APS patients (240 ± 84 vs. 214 ± 58 nM, p<0.05) and the velocity index was elevated in APS patients (134 ± 66 vs. 70 ± 32 nM/min, p<0.001) compared to healthy subjects. The ETP values were comparable between healthy subjects and APS patients (1260 ± 235 vs. 1176 ± 362 nM*min).The pro- and anticoagulant processes underlying thrombin generation were studied separately. The total amount of prothrombin converted during thrombin generation (PCtot) did not differ between healthy subjects and patients (1234 vs. 1165 nM). However, the maximum rate of prothrombin conversion (PCmax) was significantly elevated in APS patients (291 vs 425 nM/min; p<0.001). The amount of thrombin-antithrombin (T-AT) complexes formed was comparable between patients and controls (1169 vs. 1098 nM), and the thrombin decay capacity (TDC) was comparable as well (0.675 vs. 0.674 min-1). These results are in line with the finding that the plasma levels of the main thrombin inhibitors are unchanged in APS patients. Antithrombin levels are on average 2.31 ± 0.44 μM in healthy subjects and 2.36 ± 0.56 μM in APS patients, and the mean α2-macroglobulin levels were 3.22 ± 0.77 μM in healthy subjects and 3.23 ± 1.11 μM in patients.Thrombomodulin reduced the ETP by 45% in healthy subjects, but had significantly less effect in APS patients (10%). The addition of TM decreased total prothrombin conversion by 40% and the maximum prothrombin conversion rate by 50% in healthy subjects. In patients, TM only slightly reduced total prothrombin conversion (8%) and the maximum prothrombin conversion rate (7%).Discussion: The thrombin generation results indicate a predisposition to thrombosis in APS patients, as the TG parameters peak height and the velocity index are increased. Examination of the underlying pro- and anticoagulant processes of prothrombin conversion and thrombin inactivation revealed that although the same amount of prothrombin is converted in patients, the maximum activity of the prothrombinase complex is higher, indicating that patients can generate thrombin faster. In addition, APS patients have a dysfunctional APC system, as prothrombin conversion and thrombin generation could be only slightly inhibited by the addition of thrombomodulin. DisclosuresNo relevant conflicts of interest to declare.

Elevated Fibrinogen Levels in Patients Treated with Vitamin K Antagonists Attenuate the Inactivation of Thrombin

Introduction: Thrombin generation (TG) assesses the hemostatic capacity of an individual and is indicative of a person's bleeding and thrombosis risk. TG dependson the balance between the pro- and anticoagulant processes in clotting plasma. Recently, we developed a method to study the main pro- and anticoagulant processes at the basis of TG, called the thrombin dynamics method. Vitamin K antagonist (VKA) treatment is known to reduce TG and that the reduction of TG correlates with the international normalized ratio (INR). In this study, we aim to investigate the effect of VKA therapy on the dynamics of TG by examining prothrombin conversion and thrombin inactivation.Materials and methods: TG was measured in platelet poor plasma at 1 pM tissue factor in 129 healthy subjects and 129 patients treated with VKA. The patients were classified according to the INR value: INR<2 (n=17), 2<INR<3 (n=55), 3<INR<4 (n=38) and INR>4 (n=19). Thrombin dynamics was calculated from TG curves and plasma antithrombin (AT), α2-macroglobulin (α2M) and fibrinogen levels. Three thrombin dynamics parameters were determined: the total amount of prothrombin conversion (PCtot), the maximum rate of prothrombin conversion (PCmax) and the thrombin decay capacity (TDC) of each plasma sample.Results: Vitamin K antagonist treatment reduced the ETP and peak height significantly at all INR values (p<0.001), and the INR is negatively correlated with the TEP and the peak height (R2=0.47, p<0.001). The lag time was increased in patients compared to controls (3.3 vs. 16.3 min, P<0.001). VKA reduce prothrombin conversion by decreasing both the total amount of prothrombin converted and the maximum rate of the prothrombinase (figure 1A-C). Surprisingly, the rate of thrombin inactivation was attenuated in patients treated with VKA, and this effect was independent of the INR (0.67 vs. 0.58 min-1, p<0.001, figure 1D).As previously reported, the thrombin decay capacity is mainly dependent on the plasma antithrombin and fibrinogen level. Plasma antithrombin levels were comparable between healthy subjects and patients on VKA (figure 2A) and showed a clear positive correlation with the thrombin decay constant in the patient group (R2=0.72, p<0.001, figure 2C). As antithrombin levels did not differ between healthy subjects and patients, the difference in TDC could not be attributed to antithrombin. In contrast, fibrinogen levels were elevated in all patients, regardless of the INR (figure 2B), and were were negatively correlated with the TDC (R2=0.13, p<0.001, figure 2D). Therefore we performed in silico testing to investigate whether thrombin decay is attenuated in patients by elevated levels of fibrinogen, by calculating prothrombin conversion as if fibrinogen levels were physiological (the average fibrinogen level found in the group of healthy subjects). Figure 2E-F shows that indeed, the thrombin decay capacity in patients is reduced compared to controls due to increased fibrinogen levels. If in an in silico model of the thrombin decay capacity the measured fibrinogen levels are substituted by the average healthy subject fibrinogen levels, the thrombin decay constant restores to normal (0.67 vs. 0.63 min-1, p=0.238) in the VKA-treated patients.Conclusion: Thrombin dynamics analysis shows that prothrombin conversion is reduced in patients treated with VKA and that thrombin inactivation is significantly attenuated. We demonstrate that the latter effect can be attributed to the elevated fibrinogen levels that accompany VKA treatment. [Display omitted] [Display omitted] DisclosuresHemker:Diagnostica Stago: Consultancy.

Asthma is associated with enhanced thrombin formation and impaired fibrinolysis.

There is evidence that altered blood coagulation and fibrinolysis are involved in the pathogenesis of asthma. Increased thromboembolic risk has been reported in asthmatics. To investigate whether enhanced thrombin generation and impaired fibrinolysis occur in asthmatics. Plasma thrombin generation profile together with a computational assessment of thrombin dynamics and fibrinolytic capacity expressed as clot lysis time (CLT) were determined in 164 consecutive patients with stable asthma and 72 controls matched for age, gender, weight and smoking. Asthma patients had 20.2% increased endogenous thrombin potential (ETP), 41.4% higher peak thrombin concentration, 61% higher maximal prothrombin conversion rate, 15.5% faster rate of thrombin formation (all, P < 0.0001) and 10% lower thrombin decay capacity (P = 0.0004) compared with controls. Asthmatics had also 14.4% longer CLT (P = 0.001) associated with 21.3% higher plasminogen activator inhibitor-1 (PAI-1) (P < 0.0001), and 13% higher plasma α2 -macroglobulin (P = 0.0002). Using ETP and CLT above 75th percentile of the control values as the cut-off levels, we found increased risks of enhanced thrombin generation and hypofibrinolysis in asthmatics, also after correction for potential confounders. ETP and CLT were associated inversely with forced expiratory volume in 1 s/vital capacity (FEV1 /VC) index, after adjustment for age and body mass index. Non-allergic asthma (n = 70, 42.6%) was characterized by 17.5% longer CLT (P = 0.02), which positively associated with PAI-1. Thrombin generation profile was not affected by allergy. Asthma is associated with enhanced thrombin generation and impaired fibrinolysis, which might contribute to thromboembolic events in this disease.

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.

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.

Engineered Factor Xa Variants Retain Procoagulant Activity Independent of Direct Factor Xa-Inhibitors

The venom of the Australian Elapid snake Pseudonaja textilis contains a prothrombin-activating complex that consists of factor Xa (FXa) and factor Va (FVa) homologs which are evolutionary adapted to derail the hemostatic system of its prey, leading to runaway coagulation. These adaptations include functional resistance to inactivation by the main inhibitors of coagulation, antithrombin and activated protein C. Further studies revealed that venom FXa, unlike other FXa species, is also resistant to inhibition by direct oral FXa-inhibitors such as rivaroxaban and apixaban (Ki >1000 nM for venom FXa vs. 1 nM for human FXa). Accordingly, venom FXa is able to support thrombin generation (TG) in FX-depleted plasma spiked with pharmacological concentrations (0.4-2 μM) of these FXa-inhibitors. While human FXa-initiated TG resulted in a 8-fold prolonged lag time and a 70% reduced thrombin peak, those parameters were within normal range in venom FXa-triggered TG.Venom FX homologs produced by Elapid snakes comprise a heterogeneous insertion between His91-Tyr99 (chymotrypsin numbering) in the serine protease domain. A recent crystal structure of one of these homologs shows that this insertion is in close proximity of the active site pocket. In contrast, P. textilis liver-derived plasma FX, which, when activated, is fully inhibited by the FXa-inhibitors (Ki10 nM), lacks this structural feature. We investigated whether the His91-Tyr99 insertion is at the basis of the reduced sensitivity of venom FXa towards FXa-inhibitors. To do so, we constructed and stably expressed human-snake FX chimeras (FX-A, -B, -C) that incorporate His91-Tyr99 insertions from three venom FXa homologs. The chimeric FX variants were purified by successive ion-exchange and hydrophobic interaction chromatography steps, and FXa was generated following RVV-X-activation and size-exclusion chromatography.Evaluation of the kinetic parameters of prothrombin conversion in the presence of saturating amounts of FVa and anionic phospholipids revealed that the chimeric FXa variants exhibit an up to ~4-fold enhanced affinity for prothrombin (Km 0.11-0.29 μM) as compared to recombinant human FXa (rhFXa; Km 0.41 μM). The rate of prothrombin activation was 3-10-fold reduced (kcat 118-370 min-1 vs. 1243 min-1 for rhFXa), which may be indicative of a modified active site conformation. Consistent with this, the rate of chimeric FXa inhibition by antithrombin was impaired (kapp 0.12-0.95 x 103 M-1 s-1 vs. 4.07 x 103 M-1 s-1 for rhFXa). Furthermore, the variant that was most poorly inhibited by antithrombin (variant A) also exhibited the lowest catalytic rate of prothrombin conversion and vice versa (variant C). Conversely, apixaban or edoxaban inhibition of the FXa variants assembled into prothrombinase led to the highest Ki for chimeric variant C (2.3 or 0.3 µM), followed by variants B (1.4 or 0.2 µM), and A (0.2 or 0.006 µM) compared to rhFXa (0.004 or 0.0005 µM). Evaluation of the inhibition of uncomplexed FXa variants employing peptidyl substrate conversion revealed a similar decrease in sensitivity to the FXa-inhibitors (≤500-fold). These data suggest that insertion of the snake venom His91-Tyr99 regions indeed results in impaired engagement of the FXa active site pocket.We next assessed whether chimeric variant C, which is most resistant to inhibition by the direct FXa-inhibitors, is able to restore thrombin generation in a plasma system in the presence of apixaban or edoxaban. While rhFXa-triggered (5 nM) thrombin formation in FX-depleted plasma was inhibited by 2 μM apixaban, initiation with FXa-C (5 nM) resulted in normal thrombin generation parameters (peak thrombin 98%). In addition, the zymogen form of variant C also supported tissue factor-initiated (2 pM) thrombin generation in FX-depleted plasma with inhibitor concentrations up to 6 μM (apixaban) or 2 μM (edoxaban). Under these conditions, little if any thrombin was formed with rhFX present (peak thrombin 5%). We obtained similar results when performing these experiments in normal pooled plasma.Taken together, these results show that chimeric FX is able to restore hemostasis in plasma inhibited by the direct FXa-inhibitors, both in the zymogen as well as protease state. As such, these variants have the potential to serve as rescue therapeutic agents to overcome the effect of FXa-inhibitors in case of potential life-threatening bleeding events or emergency surgical interventions. DisclosuresBos:Bayer Hemophilia Awards: Research Funding.

The balance of pro‐ and anticoagulant processes underlying thrombin generation

The generation of thrombin in time is the combined effect of the processes of prothrombin conversion and thrombin inactivation. Measurement of prothrombin consumption used to provide valuable information on hemostatic disorders, but is no longer used, due to its elaborate nature. Because thrombin generation (TG) curves are easily obtained with modern techniques, we developed a method to extract the prothrombin conversion curve from the TG curve, using a computational model for thrombin inactivation. Thrombin inactivation was modelled computationally by a reaction scheme with antithrombin, α(2) Macroglobulin and fibrinogen, taking into account the presence of the thrombin substrate ZGGR-AMC used to obtain the experimental data. The model was validated by comparison with data obtained from plasma as well as from a reaction mixture containing the same reactants as plasma. The computational model fitted experimental data within the limits of experimental error. Thrombin inactivation curves were predicted within 2 SD in 96% of healthy subjects. Prothrombin conversion was calculated in 24 healthy subjects and validated by comparison with the experimental consumption of prothrombin during TG. The endogenous thrombin potential (ETP) mainly depends on the total amount of prothrombin converted and the thrombin decay capacity, and the peak height is determined by the maximum prothrombin conversion rate and the thrombin decay capacity. Thrombin inactivation can be accurately predicted by the proposed computational model and prothrombin conversion can be extracted from a TG curve using this computational prediction. This additional computational analysis of TG facilitates the analysis of the process of disturbed TG.

Prothrombin Saint‐Denis: a natural variant with a point mutation resulting in Asp to Glu substitution at position 552 in prothrombin

A new prothrombin variant, with a point mutation at nucleotide 20 029 resulting in Asp 552 to Glu substitution (prothrombin numbering), has been identified in a male newborn. Plasma prothrombin level was <3%, 16% and 60% when measured by clotting, chromogenic and immunological assays respectively. The substitution did not affect the rate of prothrombin conversion to thrombin but altered thrombin activity. Amino acid 552 has been reported to be involved in the allosteric transition, which is induced by sodium binding to thrombin. This is the first known amino acid substitution at this site to result in dysprothrombinaemia.

Complement-induced vesiculation and exposure of membrane prothrombinase sites in platelets of paroxysmal nocturnal hemoglobinuria

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired stem-cell disorder in which the glycolipid-anchored membrane proteins, including the cell-surface complement inhibitors, CD55 and CD59, are partially or completely deleted from the plasma membranes of mature blood cells. To gain insight into the pathogenesis of thrombosis that is frequently observed in this disorder, the procoagulant responses of PNH platelets exposed to the human terminal complement proteins C5b-9 were investigated. C5b-9 complexes were assembled on gel-filtered platelets by incubation with purified C5b6, C7, C9, and limiting amounts of C8. Platelet microparticle formation and exposure of plasma membrane- binding sites for coagulation factor Va were then analyzed by flow cytometry. PNH platelets exhibiting undetectable levels of surface CD59 antigen showed an approximately 10-fold increase in sensitivity to C5b- 9-stimulated expression of membrane-binding sites for factor Va when compared with platelets from normal controls. Expression of catalytic surface for the prothrombinase complex (VaXa) paralleled the exposure of factor Va-binding sites; the rate of prothrombin conversion by C5b-9- treated PNH platelets exceeded that of C5b-9-treated normal controls by approximately 10-fold at the maximal input of C8 tested (500 ng/mL). These data indicate that PNH platelets deficient in plasma membrane CD59 antigen are exquisitely sensitive to C5b-9-induced expression of prothrombinase activity, and suggest that the tendency toward thrombosis in these patients may be due, at least in part, to the deletion of this complement inhibitor from the platelet plasma membrane.

Complement-Induced Vesiculation and Exposure of Membrane Prothrombinase Sites in Platelets of Paroxysmal Nocturnal Hemoglobinuria

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired stem-cell disorder in which the glycolipid-anchored membrane proteins, including the cell-surface complement inhibitors, CD55 and CD59, are partially or completely deleted from the plasma membranes of mature blood cells. To gain insight into the pathogenesis of thrombosis that is frequently observed in this disorder, the procoagulant responses of PNH platelets exposed to the human terminal complement proteins C5b-9 were investigated. C5b-9 complexes were assembled on gel-filtered platelets by incubation with purified C5b6, C7, C9, and limiting amounts of C8. Platelet microparticle formation and exposure of plasma membrane-binding sites for coagulation factor Va were then analyzed by flow cytometry. PNH platelets exhibiting undetectable levels of surface CD59 antigen showed an approximately 10-fold increase in sensitivity to C5b-9-stimulated expression of membrane-binding sites for factor Va when compared with platelets from normal controls. Expression of catalytic surface for the prothrombinase complex (VaXa) paralleled the exposure of factor Va-binding sites; the rate of prothrombin conversion by C5b-9-treated PNH platelets exceeded that of C5b-9-treated normal controls by approximately 10-fold at the maximal input of C8 tested (500 ng/mL). These data indicate that PNH platelets deficient in plasma membrane CD59 antigen are exquisitely sensitive to C5b-9-induced expression of prothrombinase activity, and suggest that the tendency toward thrombosis in these patients may be due, at least in part, to the deletion of this complement inhibitor from the platelet plasma membrane.

Activation of Bovine Prothrombin as Monitored by the Clotting Assay

The activation of bovine prothrombin was studied with highly purified clotting factors and using a coagulation assay developed to look at the initial rate of prothrombin conversion as well as the conversion rate over a time course of 75 min. Activation of prothrombin by factor Xa alone was slow. The rate of prothrombin conversion increased markedly with the addition of each of the accessory components Ca2+, phospholipid and bovine factor V, respectively. With the complete prothrombinase complex comprising factor Xa, Ca2+, phospholipid and factor V, the rate increase was about 22,000-fold higher compared to the action of factor Xa and Ca2+ on prothrombin alone. The rates of thrombin formation obtained with activated factor X1 were only about 70% the values obtained with factor X2. The rate of prothrombin activation and the difference between the activities of the activated factors X1 and X2 are discussed.

Inhibition by human thrombomodulin of factor Xa-mediated cleavage of prothrombin.

Human thrombomodulin significantly inhibited the rate of prothrombin conversion to thrombin by Factor Xa in the presence of phospholipid or platelets, calcium, and Factor Va. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of 125I-prothrombin activation revealed that thrombomodulin reduced the rate of prothrombin activation but did not alter the cleavage pattern. The inhibition was reversed by the inclusion of a highly specific rabbit antithrombomodulin antibody. If thrombomodulin was replaced by hirudin, the rate of thrombin generation was not decreased excluding the possibility that the inhibition by thrombomodulin was secondary to the binding of small amounts of thrombin formed early in the reaction and the prevention of feedback breakdown of prothrombin by thrombin. The inhibitory activity of thrombomodulin was overcome by increasing the concentration of Factor Xa and specific, saturable binding of thrombomodulin to Factor Xa was demonstrated. These results indicate that thrombomodulin binds to Factor Xa and thereby inhibits the activity of the prothrombinase complex.

Determinants of the Formation and Activity of Factor V-phospholipid Complexes II. Molecular Properties of the Complexes

The stability, buoyancy, and intrinsic activity for factor V-phospholipid complexes were investigated. The decay of factor V activity in the absence of phospholipid followed first order kinetics; however, in the presence of several phospholipids a biphasic decay curve was observed. The addition of phosphatidylethanolamine to factor V produced only a small loss of activity in the first 2 minutes but decreased the subsequent rate of inactivation fourfold. A PE-factor V complex with a low bouyant density was separated from uncomplexed factor V by sucrose density ultracentrifugation. The association constant for this complex was 5 X 10(6) M-1 with approximately 2 moles of factor V bound per mole of lipid micelle. The isolated complex was capable of increasing prothrombin conversion 10-fold without additional phospholipid. A still lighter complex increased the rate of prothrombin conversion 18-fold. Phosphatidyl serine produced a concentration-dependent loss of up to 95% of the factor V activity in the first 2 minutes. After ultracentrifugation on a sucrose density gradient, a PS-factor V complex of increased density was detected. This complex failed to accelerate prothrombin conversion in the intrinsic two-stage assay. Except at very high concentrations, phosphatidylcholine did not alter the kinetics of inactivation of factor V. A factor V-phosphatidylcholine complex could not be detected after ultracentrifugation. When added to factor V, cardiolipin (200 mug/ml), produced a rapid 50% decline in activity with a subsequent three-fold increase in the rate of inactivation. No activity was recovered after ultracentrifugation of factor V in the presence of cardiolipin. Saturated phosphatidylethanolamine produced a concentration dependent initial loss of activity, but only a minimal increase in the subsequent rate of inactivation. At 200 mug/ml almost no light complex was detected after ultracentrifugation, but at 800 mug/ml a light complex was observed. This behavior corresponds to the ability of saturated phosphatidylethanolamine to accelerate prothrombin conversion only at very high concentrations. Thus, phospholipids combine with factor V to form complexes which differ in their ability to accelerate prothrombin conversion. The most active species are stable lipoprotein complexes of lower buoyant density than factor V.

The Conversion of Prothrombin to Thrombin: V. THE ACTIVATION OF PROTHROMBIN BY FACTOR Xa IN THE PRESENCE OF PHOSPHOLIPID

Abstract As shown previously for activated Factor X and Ca2+ alone, (Esmon, C. T., and Jackson, C. M. (1974) J. Biol. Chem. 249, 7782–7790) prothrombin activation by activated Factor X and Ca2+ in the presence of phospholipid also proceeds via Intermediate 2 and Fragment 1·2, i.e. [see PDF for equation] Unless rapidly inactivated, thrombin cleaves Fragment 1·2 into two smaller activation products, i.e. Fragment 1·2 (Thrombin)/→ Fragment 1 + Fragment 2 2) The direct and concomitant formation of Intermediate 2 and Fragment 1·2 was demonstrated by activating prothrombin in the presence of iPr2P-F. In the absence of this inhibitor, thrombin, in addition to catalyzing the reaction of Equation 2, cleaves Fragment 1 from prothrombin and in so doing eliminates the possibility of observing phospholipid catalysis from all but the earliest stages of reaction (Gitel, S. N., Owen, W. G., Esmon, C. T., and Jackson, C. M. (1973) Proc. Nat. Acad. Sci. U. S. A. 70, 1344–1348). The reaction of Equation 1 was shown to occur independent of any special effect of diisopropyl fluorophosphate (iPr2P-F) by a direct demonstration of Intermediate 2 and Fragment 1·2 formation from tritium-labeled prothrombin. Both the formation of Intermediate 2 and its conversion to thrombin are accelerated by phospholipid. However, in the presence of phospholipid, Intermediate 2 was found to satisfy the necessary condition for an intermediate on a sequential activation path, i.e. that its rate of conversion to thrombin be greater than or equal to the rate of prothrombin conversion to thrombin, if and only if, Fragment 1·2 were also present in the activation mixture. In view of this observation, the true activation intermediate of the phospholipid-catalyzed pathway must consist of both Intermediate 2 and Fragment 1·2. Fragment 1 plus Fragment 2 was not able to substitute for Fragment 1·2. Intermediate 2 and Fragment 1·2 were demonstrated to associate noncovalently to form a product with gel electrophoretic properties indistinguishable from those of prothrombin. This association of Fragment 1·2 with Intermediate 2 apparently provides the mechanism by which Intermediate 2 can be retained on the phospholipid surface and thus be readily accessible to the activated Factor X for the final proteolysis which is required to form thrombin. An alternative prothrombin activation pathway which maintains the integrity of the Intermediate 2 and Fragment 1·2 portions of the prothrombin molecule during the two-step activation process was proposed previously (Esmon, C. T., Owen, W. G., and Jackson, C. M. (1974) J. Biol. Chem. 249, 606–611). No unambiguous evidence for the existence of the alternative activation route could be demonstrated and thus the reactions of Equation 1 appear to describe the principal, if not exclusive, pathway of prothrombin activation by Factor Xa, phospholipid, and Ca2+.

Interaction of Soybean Trypsin Inhibitor with Thrombin and Its Effect on Prothrombin Activation

Abstract Because of its importance to an understanding of the mechanisms involved in the conversion of prothrombin to thrombin, the inhibitory properties of soybean trypsin inhibitor on this system were investigated. Kinetic studies showed that the inhibitor participates in a bimolecular reversible reaction with one of the components of the prothrombin-thrombin conversion system and to have an association constant dependent upon the rate of prothrombin conversion. With the use of three different systems for the activation of prothrombin, soybean trypsin inhibitor was found to block the conversion of the zymogen at two stages in the activation sequence. The first stage occurs during the initial peptide bond-breaking step which produces a polymorphic prothrombin intermediate plus a peptide fragment. The second point of inhibition occurs when this intermediate is split into smaller molecules, one of which has thrombin activity. Indirect evidence suggests that the proteolytic activity of thrombin is responsible for these initial events and that some of the bonds normally broken at these two stages are similar to bonds which have been implicated with the reactive site of soybean trypsin inhibitor. By the use of gel filtration columns equilibrated with buffers containing soybean trypsin inhibitor, it is possible to show that human or bovine thrombin (mol wt approximately 30,000 g) combines with soybean trypsin inhibitor (mol wt approximately 20,000 g) to form a mole for mole dissociable enzyme-inhibitor complex (mol wt approximately 50,000 g). This was confirmed by ultracentrifuge analysis. The complex thus formed is enzymatically active against fibrinogen (Arg-Gly bonds) and synthetic ester substrates but its formation is not affected when these catalytic properties are inhibited by sulfonation or alkylation of the residues at the active site of thrombin. Because of this, it is suggested that the mechanism of inhibition may be associated with alterations of secondary binding sites on the enzyme or with steric effects. From other experiments and for reasons which are discussed, these results cannot be explained on the basis of Factor X contamination of the thrombin preparations used. Although these studies do not dispute the possible interaction of soybean trypsin inhibitor with preparations having Factor X activity, they do not support the view that the inhibitor combines with intermediates or derivatives of the prothrombin-thrombin transition. Rather, these studies indicate that soybean trypsin inhibitor interacts with thrombin to block its specific proteolytic capacity to initiate prothrombin activation.