M6229 Protects against Extracellular-Histone-Induced Liver Injury, Kidney Dysfunction, and Mortality in a Rat Model of Acute Hyperinflammation.

Extracellular histones have been shown to act as DAMPs in a variety of inflammatory diseases. Moreover, they have the ability to induce cell death. In this study, we show that M6229, a low-anticoagulant fraction of unfractionated heparin (UFH), rescues rats that were challenged by continuous infusion of calf thymus histones at a rate of 25 mg histones/kg/h. Histone infusion by itself induced hepatic and homeostatic dysfunction characterized by elevated activity of hepatic enzymes (ASAT and ALAT) and serum lactate levels as well as by a renal dysfunction, which contributed to the significantly increased mortality rate. M6229 was able to restore normal levels of both hepatic and renal parameters at 3 and 9 mg M6229/kg/h and prevented mortality of the animals. We conclude that M6229 is a promising therapeutic agent to treat histone-mediated disease.

Tissue factor expression in monocyte subsets during human immunothrombosis, endotoxemia and sepsis.

INTRODUCTION: Tissue factor expression on monocytes is implicated in the pathophysiology of sepsis-induced coagulopathy. How tissue factor is expressed by monocyte subsets (classical, intermediate and non-classical) is unknown. METHODS: Monocytic tissue factor surface expression was investigated during three conditions. Primary human monocytes and microvascular endothelial cell co-cultures were used for in vitro studies. Volunteers received a bolus of lipopolysaccharide (2 ng/kg) to induce endotoxemia. Patients with sepsis, or controls with critical illness unrelated to sepsis, were recruited from four intensive care units. RESULTS: Contact with endothelium and stimulation with lipopolysaccharide reduced the proportion of intermediate monocytes. Lipopolysaccharide increased tissue factor surface expression on classical and non-classical monocytes. Endotoxemia induced profound, transient monocytopenia, along with activation of coagulation pathways. In the remaining circulating monocytes, tissue factor was up-regulated in intermediate monocytes, though approximately 60 % of individuals (responders) up-regulated tissue factor across all monocyte subsets. In critically ill patients, tissue factor expression on intermediate and non-classical monocytes was significantly higher in patients with established sepsis than among non-septic patients. Upon recovery of sepsis, expression of tissue factor increased significantly in classical monocytes. CONCLUSION: Tissue factor expression in monocyte subsets varies significantly during health, endotoxemia and sepsis.

Effectiveness and costs of a stepwise versus an all-in-one approach to diagnose mild bleeding disorders.

The diagnostic work-up of patients referred to the haematologist for bleeding evaluation is performed in a stepwise way: bleeding history and results of screening laboratory tests guide further diagnostic evaluation. This can be ineffective, time-consuming and burdensome for patients. To improve this strategy, the initial laboratory investigation can be extended. In a model-based approach, effectiveness and costs of a conventional stepwise versus a newly proposed all-in-one diagnostic approach for bleeding evaluation were evaluated and compared, using data from an observational patient cohort study, including adult patients referred for bleeding evaluation. In the all-in-one approach, specialized platelet function tests, coagulation factors, and fibrinolysis tests were included in the initial investigation. Final diagnosis, hospital resource use and costs and patient burden were compared. A total of 150 patients were included. Compared to the stepwise approach, in the all-in-one approach, 19 additional patients reached a diagnosis and patient burden was lower, but total costs per patient were higher [€359, 95% bootstrapped confidence interval (BCI) 283-518, p = 0.001]. For bleeding evaluation of patients referred to the haematologist, an all-in-one diagnostic approach has a higher diagnostic yield and reduces patient burden, at a higher cost. This raises the question what costs justify the diagnosis of a bleeding disorder and a less burdensome diagnostic strategy.

Lower levels of vWF are associated with lower risk of cardiovascular disease.

Objective: The current study was undertaken to prospectively explore whether having low levels of von Willebrand factor (vWF) antigen and vWF activity reduce the risk for cardiovascular disease and death. Methods: VWF antigen and vWF activity were measured by enzyme-linked immunosorbent assay and an immunological-based assay, respectively, in a subsample of 4857 individuals aged between 35 and 74 years old, enrolled between April 2007 and October 2008 in the population-based Gutenberg Health Study. VWF antigen and activity below the 20th percentile was set as a measure of "low vWF." Adjusted robust Poisson regression models were used to analyze the relation between low vWF and the incidence of cardiovascular disease (CVD). Consequent adjusted cox regression models as well as cumulative incidence plots were calculated to explore the relation between all-cause and cardiovascular mortality and low vWF. Results: VWF activity levels <20th percentile (i.e., <76.2%) were associated with a decreased relative risk for CVD (RR: 0.59, 95% CI: 0.37-0.95), despite adjusting for age and sex. After adjusting for levels of F-VIII, the association persisted (RR: 0.60, 95% CI: 0.36-0.99). The cumulative incidence plots demonstrated that vWF antigen <20th percentile significantly correlated with decreased cardiovascular mortality. VWF antigen<20th percentile (i.e., <83%) was significantly associated with lower risk of all-cause mortality, despite adjusting for clinical factors (RR: 61, 95% CI: 0.41-0.91). Conclusion: The study demonstrated that having low vWF activity levels were associated with a lower risk for CVD. Additionally, it revealed a decreased risk of cardiovascular and all-cause mortality in individuals with low levels of vWF antigen, shining new light on vWF as a potential target for novel therapies.

Serial thrombin generation and exploration of alternative anticoagulants in critically ill COVID-19 patients: Observations from Maastricht Intensive Care COVID Cohort.

Background: COVID-19 associated coagulopathy (CAC) is associated with an increase in thromboembolic events. Current guidelines recommend prophylactic heparins in the management of CAC. However, the efficacy of this strategy in the intensive care population remains uncertain. Objective: We aimed to measure thrombin generation (TG) to assess CAC in intensive care unit (ICU) patients receiving thromboprophylaxis with low molecular weight heparin (LMWH) or unfractionated heparin (UFH). In addition, we performed statistical modeling to link TG parameters to patient characteristics and clinical parameters. Lastly, we studied the potency of different anticoagulants as an alternative to LMWH treatment in ex vivo COVID-19 plasma. Patients/Methods: We included 33 patients with confirmed COVID-19 admitted at the ICU. TG was measured at least twice over the course of 6 weeks after admission. Thrombin generation parameters peak height and endogenous thrombin potential (ETP) were compared to healthy controls. Results were subsequently correlated with a patient characteristics and laboratory measurements. In vitro spiking in TG with rivaroxaban, dabigatran, argatroban and orgaran was performed and compared to LMWH. Results: Anti-Xa levels of all patients remained within the therapeutic range throughout follow-up. At baseline, the mean (SE) endogenous thrombin potential (ETP) was 1,727 (170) nM min and 1,620 (460) nM min for ellagic acid (EA) and tissue factor (TF), respectively. In line with this we found a mean (SE) peak height of 353 (45) nM and 264 (96) nM for EA and TF. Although fluctuating across the weeks of follow-up, TG parameters remained elevated despite thromboprophylaxis. In vitro comparison of LMWHs and direct thrombin inhibitors (e.g., agratroban, dabigatran) revealed a higher efficacy in reducing coagulation potential for direct thrombin inhibition in both ellagic acid (EA) and tissue factor (TF) triggered TG. Conclusion: In a sub-group of mechanically ventilated, critically ill COVID-19 patients, despite apparent adequate anti-coagulation doses evaluated by anti-Xa levels, thrombin generation potential remained high during ICU admission independent of age, sex, body mass index, APACHE II score, cardiovascular disease, and smoking status. These observations could, only partially, be explained by (anti)coagulation and thrombosis, inflammation, and multi-organ failure. Our in vitro data suggested that direct thrombin inhibition compared with LMWH might offer an alternate, more effective anticoagulant strategy in COVID-19.

Relationships between coagulation factors and thrombin generation in a general population with arterial and venous disease background.

BACKGROUND: The current study aims to identify the relationships between coagulation factors and plasma thrombin generation in a large population-based study by comparing individuals with a history of arterial or venous thrombosis to cardiovascular healthy individuals. METHODS: This study comprised 502 individuals with a history of arterial disease, 195 with history of venous thrombosis and 1402 cardiovascular healthy individuals (reference group) from the population-based Gutenberg Health Study (GHS). Calibrated Automated Thrombography was assessed and coagulation factors were measured by means of BCS XP Systems. To assess the biochemical determinants of TG variables, a multiple linear regression analysis, adjusted for age, sex and antithrombotic therapy, was conducted. RESULTS: The lag time, the time to form the first thrombin, was mainly positively associated with the natural coagulant and anti-coagulant factors in the reference group, i.e. higher factors result in a longer lag time. The same determinants were negative for individuals with a history of arterial or venous thrombosis, with a 10 times higher effect size. Endogenous thrombin potential, or area under the curve, was predominantly positively determined by factor II, VIII, X and IX in all groups. However, the effect sizes of the reported associations were 4 times higher for the arterial and venous disease groups in comparison to the reference group. CONCLUSION: This large-scale analysis demonstrated a stronger effect of the coagulant and natural anti-coagulant factors on the thrombin potential in individuals with a history of arterial or venous thrombosis as compared to healthy individuals, which implicates sustained alterations in the plasma coagulome in subjects with a history of thrombotic vascular disease, despite intake of antithrombotic therapy.

Protein C or Protein S deficiency associates with paradoxically impaired platelet-dependent thrombus and fibrin formation under flow.

Background: Low plasma levels of protein C or protein S are associated with venous thromboembolism rather than myocardial infarction. The high coagulant activity in patients with thrombophilia with a (familial) defect in protein C or S is explained by defective protein C activation, involving thrombomodulin and protein S. This causes increased plasmatic thrombin generation. Objective: Assess the role of platelets in the thrombus- and fibrin-forming potential in patients with familial protein C or protein S deficiency under high-shear flow conditions. Patients/Methods: Whole blood from 23 patients and 15 control subjects was perfused over six glycoprotein VI-dependent microspot surfaces. By real-time multicolor microscopic imaging, kinetics of platelet thrombus and fibrin formation were characterized in 49 parameters. Results and Conclusion: Whole-blood flow perfusion over collagen, collagen-like peptide, and fibrin surfaces with low or high GPVI dependency indicated an unexpected impairment of platelet activation, thrombus phenotype, and fibrin formation but unchanged platelet adhesion, observed in patients with protein C deficiency and to a lesser extent protein S deficiency, when compared to controls. The defect extended from diminished phosphatidylserine exposure and thrombus contraction to delayed and suppressed fibrin formation. The mechanism was thrombomodulin independent, and may involve negative platelet priming by plasma components.

Thrombin generation by calibrated automated thrombography in goat plasma: Optimization of an assay.

The goat model of atrial fibrillation (AF) allows investigation of the effect of AF on coagulation. However, assays for goat plasma are not available from commercial sources. Calibrated automated thrombography (CAT) provides a global view of the coagulation profile by assessing in vitro thrombin generation (TG). We describe the customization of the CAT assay in goat platelet-poor plasma (PPP) and in factor Xa (FXa)-inhibitor-anticoagulated PPP. TG was initiated in the presence of phospholipids and either (a) PPP reagent, reagent low, or reagent high; (b) goat brain protein extraction (GBP); or (c) Russell's viper venom-factor X activator (RVV-X). Contact activation was assessed by adding corn trypsin inhibitor. Different concentrations of prothrombin complex concentrate (PCC) were used to determine the sensitivity of both the GBP and RVV-X method. To obtain FXa-inhibitor anticoagulated plasma, rivaroxaban was added to plasma. TG settings with human reagents were not suitable for goat plasma. TG triggered with GBP increased peak height and ETP values. Similarly, the RVV-X method produced comparable TG curves and was more sensitive to PCC titration. Finally, both methods were able to detect the decrease in clotting potential induced by FXa inhibition. This is the first study that reports the customization of the CAT assay for goats. The GBP and RVV-X methods were comparable in triggering TG in goat plasma. The RVV-X method seemed to better discriminate changes in TG curves due to increases in clotting potential as well as to FXa inhibition by rivaroxaban in goat plasma.

Potential value of the calibrated automated thrombogram in patients after a cerebral venous sinus thrombosis; an exploratory study.

BACKGROUND: Cerebral venous sinus thrombosis (CVST) is a relatively rare, but potentially lethal condition. In approximately 15% of the patients, the cause of CVST remains unclear. Conventional clotting tests such as prothrombin time and activated partial thromboplastin time are not sensitive enough to detect prothrombotic conditions nor mild haemostatic abnormalities. The calibrated automated thrombogram (CAT) is a physiological function test that might be able to detect minor aberrations in haemostasis. Therefore, we aimed to detect the presence of a prothrombotic state in patients who endured idiopathic CVST with the CAT assay. METHODS: Five adult patients with an idiopathic, radiologically proven CVST that had been admitted during the past 3 years were included in this study. The control group consisted of five age/gender matched healthy volunteers. Exclusion criteria were known haematological disorders, malignancy (current/past) or hormonal and anticoagulant therapy recipients. We obtained venous blood samples from all participants following cessation of anticoagulation. Using the CAT assay, we determined lag time, normalized endogenous thrombin potential (ETP), ETP reduction and normalized peak height. In addition, prothrombin concentrations were determined. RESULTS: We found no significant differences in lag time (4.7 min [4.5-4.9] vs 5.3 min [3.7-5.7], p = 0.691), normalized ETP (142% [124-148] vs 124% [88-138], p = 0.222), ETP reduction (29% [26-35] vs 28% [24-58], p > 0.999), and normalized peak height (155% [153-175] vs 137 [94-154], p = 0.056) between patients and their age/gender matched controls. In addition, prothrombin concentrations did not significantly differ between patients and controls (120% [105-132] vs 127% [87-139], p > 0.999). CONCLUSION: Reasons for absent overt hypercoagulability within this study population may be the small patient sample, long time since the event (e.g. 3 years) and avoidance of acquired risk factors like oral contraception. Given the fact that CVST is a serious condition with a more than negligible risk of venous thrombosis event recurrence, exclusion of clinically relevant hypercoagulability remains a challenging topic to further study at the acute and later time points, particularly in patients with idiopathic CVST.

Activated factor XI-antithrombin complex presenting as an independent predictor of 30-days mortality in out-of-hospital cardiac arrest patients.

BACKGROUND: Cardiac arrest and cardiopulmonary resuscitation (CPR) are associated with activated coagulation and microvascular fibrin deposition with subsequent multiorgan failure and adverse outcome. OBJECTIVES: Activated Factor XI-antithrombin (FXIa-AT) complex, activated Factor IX-antithrombin (FIXa-AT) complex and thrombin-antithrombin (TAT) complex were measured as markers of coagulation activation, and evaluated as independent prognostic indicators in out-of-hospital cardiac arrest (OHCA) patients. METHODS: From February 2007 until December 2010 blood samples were collected in close approximation to CPR from patients with OHCA of assumed cardiac origin. Follow-up samples in survivors were drawn 8-12 h and 24-48 h after hospital admission. All measurements were determined by ELISA. RESULTS: Thirty-seven patients presented with asystole and 77 with ventricular fibrillation as first recorded heart rhythm. At 30-days follow-up, 70 patients (61.4%) had died. All patients had elevated levels of FXIa-AT complex, FIXa-AT complex and TAT. Initial levels were significantly higher in non-survivors compared to 30-days survivors. A significant increase in risk of 30-days all-cause mortality was observed through increasing quartiles of all three biomarkers in univariate Cox regression analysis. Compared to the lowest quartile (Q1), only FXIa-AT complex levels in Q3 (HR 3.17, p = 0.011) and Q2 (HR 3.02, p = 0.016) were independently associated with all-cause mortality in the multivariable analysis. FIXa-AT complex and TAT-complex did not behave as independent predictors. CONCLUSIONS: Complexes of FXIa-AT were independently associated with 30-days survival in OHCA-patients. CLINICAL TRIAL REGISTRATION: ClinicalTrials. gov, NCT02886273.

Evaluation of the analytical performance of the PC100 platelet counter.

INTRODUCTION: Platelet count can be altered in various diseases and treatments and measuring it may provide better insight into the expected outcome. So far, quantification of platelet count is done within laboratory conditions by using established hematology analyzers, whereas a point-of-care device could be used for this purpose outside of the clinical laboratories. AIM: Our aim was to assess the closeness of agreement between a newly developed point-of-care PC100 platelet counter and two reference methods (Sysmex® XP-300, Sysmex® XN-9000) in measuring platelet counts in whole blood and platelet-rich-plasma (PRP). METHOD: Whole blood was obtained from 119 individuals, of which 74 were used to prepare PRP samples. Whole blood platelet count was measured by the two reference methods and the PC100 platelet counter. PRP was prepared from the whole blood and platelet count was adjusted to the range of 250-3600 × 103/µl and measured with the PC100 platelet counter and Sysmex® XP-300. RESULTS: A median difference of - 1.35% and - 2.98% occurred in whole blood platelet count between the PC100 platelet counter and the Sysmex® XP-300 and Sysmex® XN-9000, respectively. A strong linear correlation (r ≥ 0.98) was seen in both cases and regression equations indicated neither a constant nor a proportional bias between the methods. Direct comparison of the two reference methods revealed a median difference of - 1.15% and a strongly linear relationship (r = 0.99). Platelet count in PRP resulted in a median difference of 1.42% between the PC100 platelet counter and the reference method, Sysmex® XP-300. While the difference between two methods increased with concentration of platelets in PRP, a strong linear relationship remained throughout the whole measuring interval indicated by the high correlation coefficient (r = 0.99). Assessment of the predicted bias at predefined platelet counts showed that the bias in platelet counts falls within the acceptance criterion for both whole blood and PRP measurements. CONCLUSIONS: Our results show that the PC100 platelet counter can be used interchangeably with the reference methods for determining platelet counts.

Potent Cyclic Peptide Inhibitors of FXIIa Discovered by mRNA Display with Genetic Code Reprogramming.

The contact system comprises a series of serine proteases that mediate procoagulant and proinflammatory activities via the intrinsic pathway of coagulation and the kallikrein-kinin system, respectively. Inhibition of Factor XIIa (FXIIa), an initiator of the contact system, has been demonstrated to lead to thrombo-protection and anti-inflammatory effects in animal models and serves as a potentially safer target for the development of antithrombotics. Herein, we describe the use of the Randomised Nonstandard Peptide Integrated Discovery (RaPID) mRNA display technology to identify a series of potent and selective cyclic peptide inhibitors of FXIIa. Cyclic peptides were evaluated in vitro, and three lead compounds exhibited significant prolongation of aPTT, a reduction in thrombin generation, and an inhibition of bradykinin formation. We also describe our efforts to identify the critical residues for binding FXIIa through alanine scanning, analogue generation, and via in silico methods to predict the binding mode of our lead cyclic peptide inhibitors.

Multiparameter microfluidics assay of thrombus formation reveals increased sensitivity to contraction and antiplatelet agents at physiological temperature.

INTRODUCTION: Current developments to assess qualitative and quantitative platelet traits in flowed whole-blood are based on microfluidic devices that mostly operate at room temperature. However, operation at physiological temperature (37 °C) may increase the assay's sensitivity, and facilitates the comparison to other platelet function tests of the diagnostic laboratory. MATERIALS AND METHODS: We adapted the conventional microspot-based microfluidic device with a simple thermo-coupled pre-heating module. Automated analysis of microscopic images assisted in obtaining five time-dependent parameters of thrombus formation over collagen microspots (shear rate 1000 s-1). These modifications allowed rapid testing of control and patient blood samples at physiological temperature. RESULTS AND CONCLUSION: The higher temperature enhanced platelet adhesion and aggregation as well as late thrombus characteristics such as size and contraction, when compared to room temperature. Moreover, assessment at 37 °C indicated a time-dependent impairment of the thrombus parameters in blood from patients taking common antiplatelet medication, i.e. aspirin and/or clopidogrel. This pointed to increased contribution of the autocrine platelet agonists thromboxane A2 and ADP in the buildup of contracted thrombi under flow. Overall, this study underlined the advantage of multiparameter assessment of microfluidic thrombus formation in detecting an acquired platelet dysfunction, when operating at physiological temperature. This work may bring microfluidics tests closer to the diagnostic laboratory.

Suggestions for global coagulation assays for the assessment of COVID-19 associated hypercoagulability.

INTRODUCTION: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) infection is associated with a clear prothrombotic phenotype. Although the exact pathophysiological mechanisms are not yet fully understood, thrombosis is clearly a highly important in the prognosis and outcome of COVID-19. As such, there is a need for diagnostic analysis and quantification of the coagulation potential in these patients, both at diagnosis and follow-up. Global coagulation assays like thrombin generation (TG) and rotational thromboelastometry (ROTEM) might be suitable in estimating COVID-19 associated coagulopathy and thrombosis risk. Therefore, we aimed at validating both assays for samples with high levels of fibrinogen and in the presence of anticoagulant heparins, such as commonly observed for COVID-19 ICU patients. MATERIALS AND METHODS: Calibrated Automated Thrombography (CAT) was optimized to assess plasma thrombin generation in the presence of heparins. The final conditions with either 10 µg/mL Ellagic acid (EA) or PPP Reagent HIGH (high tissue factor; HPPH) were validated according to the EP5 protocol for within-run and between-run variability. Overall variability was well below 10%. To estimate the influences of heparins and high fibrinogen levels, CAT was performed on spiked plasma aliquots from 13 healthy volunteers. Comparable to the CAT method, tPA-ROTEM was used to validate the effect of high fibrinogen and heparins on clotting time, clot firmness and clot lysis parameters. RESULTS: Our adjusted COVID-19 assay showed a heparin dose dependent decrease in peak height and endogenous thrombin potential (ETP) for both EA and HPPH triggered variants. High fibrinogen did not alter the inhibitory effect of either LMWH or UFH, nor did it influence the peak height or ETP in any of the conditions. The tPA-ROTEM showed a significant prolongation in clotting time with the additions of heparin, which normalized with the addition of high fibrinogen. MCF was markedly increased in all hyperfibrinogenemic conditions. A trend towards increased lysis time and, thus, decreased fibrinolysis was observed. CONCLUSION: Thrombin generation and tPA-ROTEM protocols for measurements in the COVID-19 populations were adjusted and validated. The adjusted thrombin generation assay shows good sensitivity for measurements in heparin spiked plasma. High levels of fibrinogen did not alter the assay or the effectiveness of heparins as measured in this assay. t-PA ROTEM was effective in measurement of both high fibrinogen and heparins spiked samples and was sensitive to the expected relevant coagulant changes by these conditions. No clear fibrinolytic effect was observed in different conditions.

Relation between Tissue Factor Pathway Inhibitor Activity and Cardiovascular Risk Factors and Diseases in a Large Population Sample.

OBJECTIVE: Tissue factor pathway inhibitor (TFPI) is a potent anticoagulant protein in the extrinsic coagulation pathway. In the present study, we aim to identify the cardiovascular determinants for total TFPI activity and its association with cardiovascular disease (CVD) and total mortality. METHODS: Total TFPI activity was assessed in a selection of the population-based Gutenberg Health Study (n = 5,000). Statistical analysis was performed to identify the determinants for total TFPI activity as well as the associations with CVD and mortality. RESULTS: Multivariable linear regression analysis identified smoking (ß 0.095 [0.054-0.136]) as a positive determinant for total TFPI activity, while diabetes (ß -0.072 [-0.134 to -0.009]), obesity (ß -0.063 [-0.101 to -0.024]), and history of coronary artery disease (CAD) were negatively associated with total TFPI activity, independent of age, sex, and the remaining cardiovascular risk factors. After adjustment for lipoprotein levels, the association between total TFPI activity levels and obesity and CAD was lost. The analysis additionally revealed a strong positive association between total TFPI activity levels and low-density lipoprotein (ß 0.221 [0.204-0.237]). The Cox regression models revealed that a higher total TFPI activity, above 97.5th percentile of the reference group, was associated with an increased mortality risk (hazard ratio = 2.58 [95% confidence interval: 1.49-4.47]), independent of age, sex, and cardiovascular risk profile. CONCLUSION: In the Gutenberg Health Study population-based cohort, the highest percentage of total TFPI correlated with an increased mortality risk. While elevated TFPI may reflect endothelial cell activation, the associations between total TFPI activity and obesity and CAD, points to additional mechanistic interactions.

Thrombin generation in cardiovascular disease and mortality - results from the Gutenberg Health Study.

Thrombin generation may be a potential tool to improve risk stratification for cardiovascular diseases. This study aims to explore the relation between thrombin generation and cardiovascular risk factors, cardiovascular diseases, and total mortality. For this study, N=5000 subjects from the population-based Gutenberg Health Study were analysed in a highly standardized setting. Thrombin generation was assessed by the Calibrated Automated Thrombogram method at 1 and 5 pM tissue factors trigger in platelet poor plasma. Lag time, endogenous thrombin potential, and peak height were derived from the thrombin generation curve. Sex-specific multivariable linear regression analysis adjusted for age, cardiovascular risk factors, cardiovascular diseases and therapy, was used to assess clinical determinants of thrombin generation. Cox regression models adjusted for age, sex, cardiovascular risk factors and vitamin K antagonists investigated the association between thrombin generation parameters and total mortality. Lag time was positively associated with obesity and dyslipidaemia for both sexes (p<0.0001). Obesity was also positively associated with endogenous thrombin potential in both sexes (p<0.0001) and peak height in males (1 pM tissue factor, p=0.0048) and females (p<0.0001). Cox regression models showed an increased mortality in individuals with lag time (1 pM tissue factor, hazard ratio=1.46, [95% CI: 1.07; 2.00], p=0.018) and endogenous thrombin potential (5 pM tissue factor, hazard ratio = 1.50, [1.06; 2.13], p=0.023) above the 95th percentile of the reference group, independent of the cardiovascular risk profile. This large-scale study demonstrates traditional cardiovascular risk factors, particularly obesity, as relevant determinants of thrombin generation. Lag time and endogenous thrombin potential were found as potentially relevant predictors of increased total mortality, which deserves further investigation.

Platelet-primed interactions of coagulation and anticoagulation pathways in flow-dependent thrombus formation.

In haemostasis and thrombosis, platelet, coagulation and anticoagulation pathways act together to produce fibrin-containing thrombi. We developed a microspot-based technique, in which we assessed platelet adhesion, platelet activation, thrombus structure and fibrin clot formation in real time using flowing whole blood. Microspots were made from distinct platelet-adhesive surfaces in the absence or presence of tissue factor, thrombomodulin or activated protein C. Kinetics of platelet activation, thrombus structure and fibrin formation were assessed by fluorescence microscopy. This work revealed: (1) a priming role of platelet adhesion in thrombus contraction and subsequent fibrin formation; (2) a surface-independent role of tissue factor, independent of the shear rate; (3) a mechanism of tissue factor-enhanced activation of the intrinsic coagulation pathway; (4) a local, suppressive role of the anticoagulant thrombomodulin/protein C pathway under flow. Multiparameter analysis using blood samples from patients with (anti)coagulation disorders indicated characteristic defects in thrombus formation, in cases of factor V, XI or XII deficiency; and in contrast, thrombogenic effects in patients with factor V-Leiden. Taken together, this integrative phenotyping approach of platelet-fibrin thrombus formation has revealed interaction mechanisms of platelet-primed key haemostatic pathways with alterations in patients with (anti)coagulation defects. It can help as an important functional add-on whole-blood phenotyping.

Plasma Kallikrein Contributes to Coagulation in the Absence of Factor XI by Activating Factor IX.

OBJECTIVES: FXIa (factor XIa) induces clot formation, and human congenital FXI deficiency protects against venous thromboembolism and stroke. In contrast, the role of FXI in hemostasis is rather small, especially compared with FIX deficiency. Little is known about the cause of the difference in phenotypes associated with FIX deficiency and FXI deficiency. We speculated that activation of FIX via the intrinsic coagulation is not solely dependent on FXI(a; activated FXI) and aimed at identifying an FXI-independent FIX activation pathway. Approach and Results: We observed that ellagic acid and long-chain polyphosphates activated the coagulation system in FXI-deficient plasma, as could be demonstrated by measurement of thrombin generation, FIXa-AT (antithrombin), and FXa-AT complex levels, suggesting an FXI bypass route of FIX activation. Addition of a specific PKa (plasma kallikrein) inhibitor to FXI-deficient plasma decreased thrombin generation, prolonged activated partial thromboplastin time, and diminished FIXa-AT and FXa-AT complex formation, indicating that PKa plays a role in the FXI bypass route of FIX activation. In addition, FIXa-AT complex formation was significantly increased in F11-/- mice treated with ellagic acid or long-chain polyphosphates compared with controls and this increase was significantly reduced by inhibition of PKa. CONCLUSIONS: We demonstrated that activation of FXII leads to thrombin generation via FIX activation by PKa in the absence of FXI. These findings may, in part, explain the different phenotypes associated with FIX and FXI deficiencies.

The daily practice of direct oral anticoagulant use in patients with atrial fibrillation; an observational cohort study.

BACKGROUND: Direct oral anticoagulants (DOACs) are administered in fixed doses without monitoring. There is still little published data on the impact of the absence of monitoring on adherence to medication and stability of DOAC plasma levels over time. OBJECTIVES: To explore adherence and stability of DOAC plasma levels over time in patients with atrial fibrillation (NVAF) recently started on DOAC therapy. PATIENTS AND METHODS: A prospective observational cohort study with structured follow up including assessment of adherence to medication, plasma levels at baseline, 3,6 and 12 months and adverse events. RESULTS: We included 164 patients; 89% were previous users of a vitamin K antagonist (VKA). One-year adherence was reasonably good: Morisky adherence measurement scores of 6-8 in 92%. The majority of DOAC plasma levels were within reported on-therapy ranges; dabigatran (median 104.4 ng/ml, IQR 110.2), rivaroxaban (median 185.2 ng/ml, IQR 216.1) and on average levels were not different for full and adjusted doses. There was significant variation between patients, but no significant differences over time within individuals. A substantial proportion of patients starting in the upper-or lower 20th percentiles remained there during the entire follow up. Seventeen bleedings (16 minor, 1 major) were reported, no ischemic events and bleeding or thrombotic events were not associated with DOAC plasma levels. CONCLUSIONS: Adherence was reasonably good in the majority of patients. Our data confirm the stability of DOAC plasma levels over time. Knowledge of such data may, in the individual patient, contribute to optimal drug and dose selection.