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.

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.

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.

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.

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.

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.

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.

Protease-activated receptors are potential regulators in the development of arterial endofibrosis in high-performance athletes.

OBJECTIVE: High-performance athletes can develop symptomatic arterial flow restriction during exercise caused by endofibrosis. The pathogenesis is poorly understood; however, coagulation enzymes, such as tissue factor (TF) and coagulation factor Xa, might contribute to the fibrotic process, which is mainly regulated through activation of protease-activated receptors (PARs). Therefore, the aim of this explorative study was to evaluate the presence of coagulation factors and PARs in endofibrotic tissue, which might be indicative of their potential role in the natural development of endofibrosis. METHODS: External iliac arterial specimens with endofibrosis (n = 19) were collected during surgical interventions. As control, arterial segments of the external iliac artery (n = 20) were collected post mortem from individuals with no medical history of cardiovascular disease who donated their body to medical science. Arteries were paraffinized and cut in tissue sections for immunohistochemical analysis. Positive staining within lesions was determined with ImageJ software (National Institutes of Health, Bethesda, Md). RESULTS: Endofibrotic segments contained a neointima, causing intraluminal stenosis, which was highly positive for collagen (+150%; P < .01) and elastin (+148%; P < .01) in comparison with controls. Intriguingly, endofibrosis was not limited to the intima because collagen (+213%) and elastin (+215%) were also significantly elevated in the media layer of endofibrotic segments. These findings were accompanied by significantly increased α-smooth muscle actin-positive cells, morphologically compatible with the presence of myofibroblasts. In addition, PAR1 and PAR4 and the membrane receptor TF were increased as well as coagulation factor X. CONCLUSIONS: We showed that myofibroblasts and the accompanying collagen and elastin synthesis might be key factors in the development of endofibrosis. The special association with increased presence of PARs, factor X, and TF suggests that protease-mediated cell signaling could be a contributing component in the mechanisms leading to endofibrosis.

Impaired mitochondrial activity explains platelet dysfunction in thrombocytopenic cancer patients undergoing chemotherapy.

Severe thrombocytopenia (≤50×109 platelets/L) due to hematological malignancy and intensive chemotherapy is associated with an increased risk of clinically significant bleeding. Since the bleeding risk is not linked to the platelet count only, other hemostatic factors must be involved. We studied platelet function in 77 patients with acute leukemia, multiple myeloma or malignant lymphoma, who experienced chemotherapy-induced thrombocytopenia. Platelets from all patients - independent of disease or treatment type - were to a variable extent compromised in Ca2+ flux, integrin a ß activation and P-selectin expression when stimulated with a panelIIbof3 agonists. The patients' platelets were also impaired in spreading on fibrinogen. Whereas the Ca2+ store content was unaffected, the patients' platelets showed ongoing phosphatidylserine exposure, which was not due to apoptotic caspase activity. Interestingly, mitochondrial function was markedly reduced in platelets from a representative subset of patients, as evidenced by a low mitochondrial membrane potential (P<0.001) and low oxygen consumption (P<0.05), while the mitochondrial content was normal. Moreover, the mitochondrial impairments coincided with elevated levels of reactive oxygen species (Spearman's rho=-0.459, P=0.012). Markedly, the impairment of platelet function only appeared after two days of chemotherapy, suggesting origination in the megakaryocytes. In patients with bone marrow recovery, platelet function improved. In conclusion, our findings disclose defective receptor signaling related to impaired mitochondrial bioenergetics, independent of apoptosis, in platelets from cancer patients treated with chemotherapy, explaining the low hemostatic potential of these patients.

Detecting clinically relevant rivaroxaban or dabigatran levels by routine coagulation tests or thromboelastography in a cohort of patients with atrial fibrillation.

BACKGROUND: Traditional coagulation tests are included in emergency guidelines for management of patients on direct oral anticoagulants (DOACs) who experience acute bleeding or require surgery. We determined the ability of traditional coagulation tests and fast whole blood thromboelastography (ROTEM®) to screen for anticoagulation activity of dabigatran and rivaroxaban as low as 30 ng/mL. METHODS: One hundred eighty-four citrated blood samples (75 dabigatran, 109 rivaroxaban) were collected from patients with non-valvular atrial fibrillation (NVAF), to perform screening tests from different manufacturers, (diluted, D) PT, aPTT, TT and ROTEM®. The activity of DOACs was quantitatively determined by clot detection assays: Hemoclot DTT and DiXaI test (Biophen), on CS2100 (Siemens). The clotting time (CT) of INTEM and EXTEM ROTEM® (Werfen) were used as test parameters. RESULTS: Dabigatran, ≥ 30 ng/mL, was accurately detected by five coagulation tests: APTT Actin FSL (93%), PT Neoplastin (93%), APTT Cephascreen, Thromboclotin, and Thrombin (all 100%), but not by PT Innovin (49%). CT-EXTEM (91%) was sufficiently sensitive, but not CT-INTEM (52%). APTT Cephascreen and Thrombin showed good linearity (R2 = 0.71,R2 = 0.72). For the other tests linearity was moderate to poor. Rivaroxaban was accurately detected by PT Neoplastin (98%) and less so by APTT Cephascreen (85%). In addition, rivaroxaban was also accurately detected by CT-INTEM (96%). PT Neoplastin showed good linearity (R2 = 0.81), all other tests had moderate to poor linearity. CONCLUSION: In patients with NVAF, the ability of routine coagulation tests to detect the presence of significant levels of DOACs is test and reagent dependent. CT-INTEM and CT-EXTEM may be fast whole blood alternatives. TRIAL REGISTRATION: The Institutional Review Board of the MUMC approved this study (December 2011, project number 114069).

Validation of a modified thromboelastometry approach to detect changes in fibrinolytic activity.

BACKGROUND: Thus far, validated whole blood assays used in in vitro fibrinolysis experiments using thromboelastometry (ROTEM) are lacking or have yet to be tested in humans. The objective was first, to establish a standardized modified ROTEM approach to detect both hypo- and hyperfibrinolysis. And second, to perform a technical and clinical validation of the assay. METHODS: Blood was used of healthy volunteers, patients with sepsis, patients after cardiothoracic surgery, pregnant women, and cirrhotic liver disease patients. A whole blood tissue factor (TF) activated ROTEM assay with and without the addition of recombinant tissue plasminogen activator (rTPA) was developed. Plasma fibrinolysis determinants were measured in all volunteers and patients. RESULTS: Thirty five pM TF and additions of 125 and 175 ng/ml rTPA resulted in full lysis within 60 min in healthy volunteers. Coefficients of variation were below 10 % without and below 20 % with rTPA addition. In sepsis the hypofibrinolytic ROTEM profiles with 175 ng/ml rTPA were in line with the plasma determinants (high PAI-1, high fibrinogen, low tPA activity, and high d-dimers). After cardiothoracic surgery, reduced fibrinogen and platelet levels accounted for the reduced maximum clot firmness. The hypofibrinolytic profile is attributed to tranexamic acid use and elevated PAI-1 levels. The lowest rTPA concentration in cirrhosis resulted in hyperfibrinolysis in only few of the patients. In pregnancy normal profiles were found. DISCUSSION: Our high rTPA concentration demonstrates hypofibrinolytic profiles adequately in sepsis and after cardiothoracic surgery. Our low rTPA concentration of 125 ng/ml seems too high for demonstrating hyperfibrinolysis in cirrhotic liver disease. CONCLUSIONS: We were able to present a validated whole blood ROTEM approach to fibrinolysis testing using added rTPA, which can be of added value next to classical plasma based fibrinolysis assays.

Early platelet recovery following cardiac surgery with cardiopulmonary bypass.

Coronary artery bypass grafting (CABG) with cardiopulmonary bypass (CPB) is frequently associated with low platelet count (PC) and disturbed platelet function (PF). While PC is easy to measure, PF is more difficult to assess. Moreover, the time-related platelet dysfunction and recovery after CPB is not fully elucidated. Platelet dysfunction could lead to bleeding but also to coronary graft failure. Laboratory tests could provide more insights into PF after CABG. The aim of the current study was to investigate the time-related PF induced by CPB. Blood samples of 20 patients with a preoperative PC of more than 250 × 109/L were collected before incision, after weaning from CPB, and 24 h postoperative. Platelet contribution to coagulation was quantified by PLTEM (calculated by means of EXTEM and FIBTEM results). PF was assessed by multiple electrode impedance aggregometry (MEIA) in whole blood and by light transmission aggregometry (LTA) in platelet-rich plasma after stimulation with arachidonic acid (AA), adenosine diphosphate, collagen, and thrombin-receptor-activating peptide. LTA and MEIA analysis demonstrated significant platelet dysfunction after CPB, with partial recovery within 24 h after surgery. AA-induced platelet aggregation increased to higher levels within 24 h after surgery compared to baseline values as measured by LTA. PLTEM maximum clot firmness remained unchanged throughout the study. Correlation analyses revealed that MEIA and rotational thromboelastometry (ROTEM), but not LTA, were dependent on PC and hematocrit. No correlations were found between LTA, MEIA, ROTEM, PC, and clinical outcome parameters. Our results demonstrate a reversible platelet dysfunction recovering within 24 h after CPB. Interestingly, AA-induced platelet aggregation increases to higher levels during the first 24 h postoperatively, which might be important for early initiation of antiplatelet therapy after CABG. MEIA as POC test is able to detect platelet dysfunction during cardiac surgery with a PC of ≥150 × 109/L.

Effects of ultrapure hemodialysis and low molecular weight heparin on the endothelial surface layer.

BACKGROUND: Chronic kidney disease patients show changes in the endothelial surface layer (ESL). Whether hemodialysis (HD) itself or low molecular weight heparins (LMWH) induce ESL alterations is unknown. METHODS: We studied the ESL in 20 HD patients with Sidestream Dark Field Imaging [measuring perfused boundary region (PBR)] and measurement of ESL constituents in plasma during HD in 2 studies. LMWH was administered at the start of HD in study A, and 120 min after the start of HD in study B. Mean platelet volume (MPV) and platelet large cell ratio (P-LCR) were also measured. RESULTS: Syndecan-1 increased significantly 30 min after LMWH administration. sP-Selectin increased 120 min after HD start, and MPV and P-LCR decreased significantly during HD. No significant changes of PBR, sE-Selectin, sICAM-1, or sVCAM-1 were perceived. CONCLUSIONS: HD caused a significant increase in Syndecan-1 without a change in PBR. The administration of LMWH appeared to precede the rise in Syndecan-1.

Comparative analysis of thrombin generation platforms for patients with coagulation factor deficiencies: A comprehensive assessment.

INTRODUCTION: Thrombin generation assays (TGAs) assess the overall functionality of the hemostatic system and thereby provide a reflection of the hemostatic capacity of patients with disorders in this system. Currently, four (semi-)automated TGA platforms are available: the Calibrated Automated Thrombogram, Nijmegen Hemostasis Assay, ST Genesia and Ceveron s100. In this study, we compared their performance for detecting patients with congenital single coagulation factor deficiencies. MATERIALS AND METHODS: Pooled patient samples, healthy control samples and normal pooled plasma were tested on all four platforms, using the available reagents that vary in tissue factor and phospholipid concentrations. The TGA parameters selected for analysis were peak height and thrombin potential. Results were normalized by using the calculated mean of healthy controls and a correction for between-run variation. Outcomes were presented as relative values, with the mean of healthy controls standardized to 100 %. RESULTS: Across all platforms and reagents used, thrombin potentials and peak heights of samples with coagulation factor deficiencies were lower than those of healthy controls. Reagents designed for bleeding tendencies yielded the lowest values on all platforms (relative median peak height 19-32 %, relative median thrombin potential 19-45 %). Samples representing more severe coagulation factor deficiencies generally exhibited lower relative peak heights and thrombin potentials. CONCLUSIONS: Thrombin generation assays prove effective in differentiating single coagulation factor deficient samples from healthy controls, with modest discrepancies observed between the platforms. Reagents designed for assessing bleeding tendencies, featuring the lowest tissue factor and phospholipid concentrations, emerged as the most suitable option for detecting coagulation factor deficiencies.

Increasing concentrations of prothrombin complex concentrate induce disseminated intravascular coagulation in a pig model of coagulopathy with blunt liver injury.

Despite increasing use of prothrombin complex concentrate (PCC) to treat hemorrhage-associated coagulopathy, few studies have investigated PCC in trauma, and there is a particular lack of safety data. This study was performed to evaluate PCC therapy in a porcine model of coagulopathy with blunt liver injury. Coagulopathy was induced in 27 anesthetized pigs by replacing approximately 70% blood volume with hydroxyethyl starch 130/0.4 and Ringer's lactate solution; erythrocytes were collected and retransfused. Ten minutes after trauma, animals randomly received PCC (35 or 50 IU/kg) or saline. Coagulation parameters including thromboelastometry, thrombin generation, and blood loss were monitored for 2 hours. Internal organs were examined macroscopically and histologically to determine the presence of emboli and assess liver injury. Total blood loss was significantly lower and survival was higher in both PCC groups versus the control group (P < .05). These outcomes appeared to be dose-independent. Thromboembolism was found in all animals treated with 50 IU/kg PCC; 44% also showed signs of disseminated intravascular coagulation. Liver injury was similar in all animals. In conclusion, 35 IU/kg PCC safely improved coagulation and attenuated blood loss. However, the higher dose of PCC (50 IU/kg) appeared to increase the risk of thromboembolism and disseminated intravascular coagulation.

Components of ambient air pollution affect thrombin generation in healthy humans: the RAPTES project.

OBJECTIVES: Increases in ambient particulate matter (PM) have been associated with an elevated risk of stroke, myocardial ischaemia and coronary heart disease, with activation of blood coagulation likely playing an important role. PM-mediated activation of two major activation pathways of coagulation provides a potential mechanism for the observed association between PM and cardiovascular disease. However, it remains unclear which specific characteristics and components of air pollution are responsible. METHODS: In order to investigate those characteristics and components, we semiexperimentally exposed healthy adult volunteers at five different locations with increased contrasts and reduced correlations among PM characteristics. Volunteers were exposed for 5 h, exercising intermittently, 3-7 times at different sites from March to October 2009. On site, we measured PM mass and number concentration, its oxidative potential (OP), content of elemental/organic carbon, trace metals, sulphate, nitrate and gaseous pollutants (ozone, nitrogen oxides). Before and 2 and 18 h after exposure we sampled blood from the participants and measured thrombin generation using the calibrated automated thrombogram. RESULTS: We found that thrombin generation increases in the intrinsic (FXII-mediated) blood coagulation pathway in relation to ambient air pollution exposure. The associations with NO2, nitrate and sulphate were consistent and robust, insensitive to adjustment for other pollutants. The associations with tissue factor-mediated thrombogenicity were not very consistent. CONCLUSIONS: Ex vivo thrombin generation was associated with exposure to NO2, nitrate and sulphate, but not PM mass, PM OP or other measured air pollutants.

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.

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.

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.