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Background: Life-long vitamin K antagonist (VKA) therapy is recommended as a standard of care in antiphospholipid syndrome (APS) patients with thrombosis. Concerns have been raised about the validity of international normalized ratio (INR) measurements in lupus anticoagulant (LA)-positive APS patients because LA may interfere with phospholipid-dependent coagulation tests and could elevate INR measurements., Objectives: Here, we aimed to determine the interference of antigen-specific monoclonal and isolated patient antibodies with LA activity on INR measurements., Methods: Pooled normal plasma and control plasma from patients on VKA (without LA) were incubated with monoclonal and isolated patient immunoglobulin G antiprothrombin and anti-beta-2-glycoprotein I antibodies that express LA activity. INR was determined before and after addition using 3 laboratory assays (Owren STA-Hepato Prest, Quick STA-NeoPTimal, and Quick STA-Neoplastine R) and 1 point-of-care test device (CoaguChek Pro II)., Results: Antiprothrombin and anti-beta-2-glycoprotein I antibodies with LA activity interfered with recombinant human thromboplastin reagents (Quick STA-Neoplastine R and CoaguChek Pro II), particularly when added to plasma of VKA-treated controls. This effect was most evident on point-of-care test INR measurements, while the recombinant Quick reagent exhibited a lesser degree of interference. In contrast, tissue-derived thromboplastin reagents (Owren STA-Hepato Prest and Quick STA-NeoPTimal) remained largely unaffected by these antibodies, both in pooled normal plasma and VKA anticoagulated control plasma. Among these reagents, the Owren INR reagent exhibited the lowest sensitivity to the influence of LA antibodies. This observed difference in sensitivity is independent of the plasma dilution factor or the presence of factor V or fibrinogen in Owren reagent., Conclusion: INR reagents that utilize recombinant human thromboplastin are more sensitive to the presence of monoclonal and patient-derived antibodies with LA activity. Consequently, APS patients positive for LA should be monitored using tissue-derived thromboplastin reagents, given its reduced susceptibility to interference by LA-causing antibodies., (© 2024 The Author(s).)

Objectives: Extensive consumption of alcohol during pregnancy can lead to severe complications for the unborn child. Carbohydrate-deficient transferrin (CDT) levels in serum have become a common biomarker for excessive alcohol intake. However, during pregnancy CDT levels can rise to levels above commonly used cut-off values, for reasons unrelated to alcohol intake. The aim of this study is to investigate the changes in CDT values during pregnancy and to determine accurate, trimester dependent reference intervals., Methods: 439 serum samples of 147 healthy pregnant women were obtained for trimester 1, 2, 3, and post-partum and were analysed by high-performance liquid chromatography (HPLC) and an N-Latex immunonephelometric assay. New trimester-specific reference intervals were established., Results: This study demonstrates there is a trimester-dependent increase of %CDT, as up to 39.4% of the population exceeded the previously established upper reference limit of 1.7%. In our study the estimated upper reference limit for %DST/%CDT were 1.55%, 1.96%, 2.05% and 1.35% for trimester 1, 2, 3 and post-partum for the HPLC-method and 2.02%, 2.19%, 2.19% and 1.96% for the N-Latex immunoassay., Conclusions: We demonstrate that CDT levels rise during pregnancy. The magnitude of the increase is method-dependent and needs to be taken into account. We have established method- and trimester-specific reference intervals to prevent false-positive results in alcohol abuse screening tests during pregnancy., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Multiple myeloma (MM) is associated with a high prevalence of bleeding and an increased risk of thrombo-embolism. MM patients have reduced platelet- and red blood cell (RBC) numbers in blood, which may indicate that the paradoxical hemostasis profile is a consequence of a disturbed platelet and RBC homeostasis., Objectives: To get better insight in the disbalanced hemostasis of MM patients., Methods: We conducted a case-control study on the whole blood (WB) coagulation profiles of 21 MM patients and 21 controls. We measured thrombin generation (TG) in WB and platelet poor plasma (PPP) of MM patients and controls., Results: In WB-TG, we observed that the median time to the thrombin Peak was 52% longer in MM patients than in controls, while the median endogenous thrombin potential until the Peak (ETPp) was 39% higher in MM-patients than in controls. In line with these findings, the levels of platelets, RBCs, white blood cells and agonist induced platelet activation were decreased in MM patients compared to controls. The plasma TG experiments showed no differences between MM-patients and controls., Conclusion: Patients with MM have a disturbed blood cell metabolism and a disbalanced WB-TG profile. This disbalance may explain the paradoxically high prevalence of bleeding symptoms in MM patients vs. an increased thrombosis risk. There was no disturbance observed in plasma TG, indicating that blood cells are the major determinants for the disbalanced hemostasis in MM patients., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Li, Roest, Sang, Remijn, Fijnheer, Smit, Huskens, Wan, de Laat and Konings.)

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is associated with thrombosis. We conducted a cohort study of consecutive patients, suspected of SARS-CoV-2 infection presented to the emergency department. We investigated haemostatic differences between SARS-CoV-2 PCR positive and negative patients, with dedicated coagulation analysis. The 519 included patients had a median age of 66 years, and 52.5% of the patients were male. Twenty-six percent of the patients were PCR-positive for SARS-CoV-2.PCR positive patients had increased levels of fibrinogen and (active) von Willebrand Factor (VWF) and decreased levels of protein C and α2-macroglobulin compared to the PCR negative patients. In addition, we found acquired activated protein C resistance in PCR positive patients. Furthermore, we found that elevated levels of factor VIII and VWF and decreased levels of ADAMTS-13 were associated with an increased incidence of thrombosis in PCR positive patients. In conclusion, we found that PCR positive patients had a pronounced prothrombotic phenotype, mainly due to an increase of endothelial activation upon admission to the hospital. These findings show that coagulation tests may be considered useful to discriminate severe cases of COVID-19 at risk for thrombosis., Competing Interests: BdL, RMWDL-K, MN, DH, AM and MR are employees of Synapse Research Institute, a not-for-profit organization holding patents on thrombin generation and plasmin generation. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Background: Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), can manifest with varying disease severity and mortality. Genetic predisposition influences the clinical course of infectious diseases. We investigated whether genetic polymorphisms in candidate genes ACE2, TIRAP, and factor X are associated with clinical outcomes in COVID-19., Methods: We conducted a single-centre retrospective cohort study. All patients who visited the emergency department with SARS-CoV-2 infection proven by polymerase chain reaction were included. Single nucleotide polymorphisms in ACE2 (rs2285666), TIRAP (rs8177374) and factor X (rs3211783) were assessed. The outcomes were mortality, respiratory failure and venous thromboembolism. Respiratory failure was defined as the necessity of >5 litres/minute oxygen, high flow nasal oxygen suppletion or mechanical ventilation., Results: Between March and April 2020, 116 patients (35% female, median age 65 [inter quartile range 55-75] years) were included and treated according to the then applicable guidelines. Sixteen patients (14%) died, 44 patients (38%) had respiratory failure of whom 23 required endotracheal intubation for mechanical ventilation, and 20 patients (17%) developed venous thromboembolism. The percentage of TIRAP polymorphism carriers in the survivor group was 28% as compared to 0% in the non-survivor group (p = 0.01, Bonferroni corrected p = 0.02). Genotype distribution of ACE2 and factor X did not differ between survivors and non-survivors., Conclusion: This study shows that carriage of TIRAP polymorphism rs8177374 could be associated with a significantly lower mortality in COVID-19. This TIRAP polymorphism may be an important predictor in the outcome of COVID-19., Competing Interests: The authors have declared that no competing interests exist.

Background: Primary and secondary implant stability is of high importance for survival and success of dental implants in the short and long term. Measurements of implant stability during healing provide the opportunity to monitor the course of the osseointegration process., Purpose: To compare implant stability quotient (ISQ) by resonance frequency analysis (RFA), recorded with two different devices after implant placement., Materials and Methods: Patients with the need of single tooth extraction in posterior sites of the maxilla and the mandible were treated in a surgical center. All patients received additional augmentation with a bovine bone substitute and platelet-rich fibrin (PRF) after atraumatic tooth extraction. After a healing period of 10 weeks, 28 self-tapping titanium-implants were placed. Implant stability was recorded with two different devices (Osstell and Penguin) at the time of implant insertion (T0), 10 days later (T1), and after 7 (T2), or 17 weeks (T3)., Results: No implant was lost, and no postoperative complication occurred during follow-up. Patient cohort comprised 9 female (32.1%) and 19 male patients (67.9%), with a mean age of 52.8 years, 64.3 years, respectively. Mean overall insertion torque was 43.6 Ncm at implant placement with no significant difference between implant location, age, or gender. No patient dropped out. During observation period, a significant increase in mean ISQ was recorded with both devices. Significant positive correlations between insertion torque and ISQ were recorded with both devices at T0, T2, and T3. No significant differences were observed in ISQ-values between both devices, and measuring directions at any point of measurement., Conclusions: Within the limitations of this cohort study, both devices were suitable for RFA-measurement and revealed comparable results. Due to the cordless design, handling of the Penquin device was more comfortable. Reusability of the Penguin MultiPeg-transducers may offer an additional benefit with regard on ecological aspects., (© 2021 Wiley Periodicals LLC.)

Correct and reliable identification of SARS-CoV-2 in COVID-19 suspected patients is essential for diagnosis. Respiratory samples should always be tested with real-time PCR for SARS-CoV-2. In addition, blood samples have been tested, but without consistent results and therefore the added value of this sample type is unknown. The aim of this study was to determine the prevalence of SARS-CoV-2 by real-time PCR in blood samples obtained from PCR-proven COVID-19 patients and in addition to elaborate on the potential use of blood for diagnostics. In this single center study, blood samples drawn from patients at the emergency department with proven COVID-19 infection based on a positive SARS-CoV-2 PCR in respiratory samples were tested for the presence of SARS-CoV-2. Samples from 118 patients were selected, of which 102 could be included in the study (median age was 65 (IQR 10), 65.7 % men). In six (5.9 %) of the tested samples, SARS-CoV-2 was identified by real-time PCR. In conclusion, SARS-CoV-2 can be detected by real-time PCR in plasma samples from patients with proven COVID-19, but only in a minority of the patients. Plasma should therefore not be used as primary sample in an acute phase setting to identify SARS-CoV-2 infection. These findings are important to complete the knowledge on possible sample types to test to diagnose COVID-19., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Introduction: Patients with chronic obstructive pulmonary disease (COPD) are at increased risk for cardiovascular events, particularly following an acute exacerbation (AE-COPD). Exacerbations are associated with increased systemic inflammation, which may drive coagulation. This prospective cohort study aimed to determine how an AE-COPD affects platelet activation, the endothelium, plasmatic coagulation and fibrinolysis, and its association with systemic inflammation., Materials and Methods: Fifty-two patients with an AE-COPD were included. Blood samples at admission, at day 3 of treatment and at convalescence were available for 32 patients. Platelet-monocyte complex (PMC) formation, monocyte Mac-1 expression and platelet (re)activity (P-selectin expression, αIIbβ3 activation) were measured by flow cytometry. Von Willebrand Factor (VWF), thrombin generation (TG) and clot lysis time (CLT) were determined as measures of endothelial activation, plasmatic coagulation and fibrinolysis, respectively., Results: Exacerbations were associated with increased PMCs (MFI 31.3 vs 23.8, p = 0.004) and Mac-1 (MFI 38.2 vs 34.8, p = 0.006) compared to convalescence, but not with changes in platelet (re)activity. VWF (antigen, activity, active fraction) and TG (peak, ETP and velocity index) were all significantly higher during AE-COPD compared to convalescence. PMCs, Mac-1, VWF and TG were positively associated with systemic inflammation (CRP). CLT was prolonged in AE-COPD patients with systemic inflammation. Moreover, platelet hyperreactivity on admission was associated with an increased risk for exacerbation relapse., Conclusions: Acute exacerbations are associated with an inflammation-associated prothrombotic state, characterized by increased PMCs, endothelial activation and plasmatic coagulation. Our findings provide direction for future studies on biomarkers predicting the risk of exacerbation relapse and cardiovascular events., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Aims: The aim of the present study was to assess the association between dental implant stability and peripheral blood cell composition and levels of coagulation factors in patients treated with alveolar ridge preservation with platelet-rich fibrin (PRF) and bovine bone substitute., Materials and Methods: Fifty patients were included between 2015 and 2017. PRF was prepared from autologous blood, in which blood cells and coagulation factor levels were measured. PRF and bovine bone were placed in the socket, followed by closure with PRF membrane. Implants were placed 14 (±2.5) weeks postextraction. The implant stability quotient was measured at t = 0, t = 10 days, t = 7 weeks, and t = 17 weeks by resonance frequency analysis., Results: Erythrocyte count was inversely associated with PRF membrane length, but not with implant stability. Conversely, platelet count did not correlate with membrane size but inversely correlated with implant stability at 7 and 17 weeks. In addition, implant stability was directly correlated with levels FXIII (t = 0, p < .01), active von Willebrand factor (VWF; t = 0 and 7 weeks, p < .05), and total VWF (t = 7 weeks, p = .012)., Conclusion: Implant stability following alveolar ridge preservation with PRF and bovine bone substitute is associated with circulating blood cells and coagulation factors. In particular, fibrin structure, VWF, and FXIII may be important modulators of implant stability., (© 2019 The Authors. Clinical and Experimental Dental Research published by John Wiley & Sons Ltd.)

Introduction  Although physical exercise is protective against cardiovascular disease, it can also provoke sudden cardiac death (exercise paradox). Epidemiological studies suggest that systemic hypoxia at high altitude is a risk factor for venous thromboembolism. Forthcoming, this study investigated the effect of repeated exercise at high altitude on blood coagulation, platelet function, and fibrinolysis. Methods  Six trained male volunteers were recruited. Participants ascended from sea level to 3,375 m altitude. They performed four exercise tests at 65 to 80% of their heart-rate reserve during 2 hours: one time at sea level and three times on consecutive days at 3,375 m altitude. Thrombin generation (TG) was measured in whole blood (WB) and platelet-rich and platelet-poor plasma. Coagulation factor levels were measured. Platelet activation was measured as αIIbβ3 activation and P-selectin expression. Fibrinolysis was studied using a clot-lysis assay. Results  Normoxic exercise increased plasma peak TG through increased factor VIII (FVIII), and increased von Willebrand factor (VWF) and active VWF levels. Platelet granule release potential was slightly decreased. After repetitive hypoxic exercise, the increase in (active) VWF tapered, and there was no more distinct exercise-related increase in peak. Platelet aggregation potential and platelet-dependent TG decreased at high altitude. There were no effects on fibrinolysis upon exercise and/or hypoxia. Conclusion  Strenuous exercise induces a procoagulant state that is mediated by the endothelium, by increasing VWF and secondarily raising FVIII levels. After repetitive exercise, the amplitude of the endothelial response to exercise diminishes. A hypoxic environment appears to further attenuate the procoagulant changes by decreasing platelet activation and platelet-dependent TG.

Background: Interaction of von Willebrand factor (VWF) with platelets requires a conformational change that exposes an epitope within the VWF A1 domain, enabling platelet glycoprotein Ibα binding. Quantification of this ''active" conformation of VWF has been shown to provide pathophysiological insight into conditions characterized by excessive VWF-platelet interaction., Methods: We developed an immunosorbent assay based on a variable heavy chain antibody fragment against the VWF A1 domain as a capture antibody. Assay performance in terms of specificity (binding to active R1306W- and sheared VWF), precision, accuracy, linearity, limits of detection and stability were determined. Active VWF, VWF antigen, VWF ristocetin cofactor activity, VWF:GP1bM and VWF propeptide were measured in citrated plasma and platelet-VWF binding in whole blood from 120 healthy individuals to establish a reference interval for active VWF and to assess associations with other VWF parameters., Results: Intra- and inter-assay CVs were between 2.4-7.2% and 4.1-9.4%, depending on the level. Mean recovery of spiked recombinant R1306W VWF was 103±3%. The assay was linear in the range of 90.1-424.5% and had a limit of quantification of 101%. The reference interval for active VWF was 91.6-154.8% of NPP. Significant, positive correlations between active VWF and all other VWF parameters were found, with the strongest correlation with VWF:GP1bM binding., Conclusions: We developed and validated an immunosorbent assay for the accurate detection of active VWF levels in plasma. The assay fulfilled all analytical criteria in this study and a reference interval was established, allowing its use to quantify active VWF in pathological conditions for future research., Competing Interests: The authors have declared that no competing interests exist.

Physical activity is beneficial for health, for example, by lowering the risk of cardiovascular events. However, vigorous exercise is associated with the occurrence of thromboembolic events and sudden cardiac death, in particular in untrained individuals. Whereas acute exercise is known to cause a hypercoagulable state, repeated exposure to (strenuous) exercise by means of training may actually condition the hemostatic response to exercise. To date, the effects of exercise training on blood coagulability and the underlying mechanisms have yet to be fully discerned. In this review, the authors provide an overview of existing literature on how training programs and training status influence hemostasis in healthy individuals. Furthermore, they present data of a pilot study in which we studied the effects of repetitive submaximal intensity cycling on procoagulant and anticoagulant processes. It is known that factor VIII (FVIII) and von Willebrand factor (VWF) increase after exercise, but we found that this increase in FVIII and VWF (antigen, propeptide, and VWF in active conformation) was smaller on each of three subsequent days, suggesting either adaptation of endothelial activation or exhaustion of endothelial VWF supplies. With respect to thrombin generation, elevated FVIII significantly increased the thrombin generation peak but not the endogenous thrombin potential. In contrast, platelet activation in terms of P-selectin expression after stimulation with protease-activated receptor-1 and glycoprotein VI agonists decreased after exercise and did not recover, indicating exhaustion of the platelet response to repetitive exercise., Competing Interests: None., (Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.)

Background: Upon tooth extraction, extravascular tissue factor (TF) initiates coagulation to arrest bleeding. Additionally, saliva is in constant contact with the wound and contains extracellular vesicle-derived procoagulant TF. Since the duration of postextraction bleeding is highly variable between patients, we hypothesized this may be caused by variation in saliva-derived TF-induced clotting activity., Objectives: We aimed to assess the variability of saliva-induced thrombin generation (TG) in healthy individuals., Methods: TG was performed according to the calibrated automated thrombinography (CAT) method. Diluted saliva was added (instead of recombinant TF and phospholipids [PL]) to normal pooled plasma (NPP) in the absence/presence of anti-TF antibodies. Saliva was collected from healthy individuals in the morning, afternoon and evening., Results: Addition of saliva to NPP induced TG curves similar to those induced by r-TF and PL. Moreover, addition of anti-TF antibodies abolished saliva-induced TG, indicating TF-dependence. A large inter-individual variability (peak CV 31%, range 73-220 nmol/L thrombin) in saliva-induced TG was observed. Interestingly, within subjects, saliva-induced TG was significantly ( P  =   0.009) increased in the morning (167 ± 40 nmol/L thrombin) compared to the afternoon (124 ± 39 nmol/L thrombin) and evening (123 ± 38 nmol/L thrombin). This diurnal variation was not attributable to gingival stimulation or damage induced by tooth brushing., Conclusions: We identified a diurnal rhythm in salivary TF activity that may have implications for tooth extraction and dental surgery, as performing invasive procedures in the morning may be beneficial for rapid coagulation. Future studies should correlate salivary TF to clinical outcome (ie, postextraction bleeding) and assess a possible relation with bacterial status in the oral cavity.

Plasmin is the major fibrinolytic protease responsible for dissolving thrombi by cleavage of its primary substrate fibrin. In addition, emerging evidence points to other roles of plasmin: (1) as a back-up for ADAMTS13 in proteolysis of ultra-large von Willebrand factor (VWF) multimers and (2) as an activator of platelets. Although the molecular mechanisms of fibrinolysis are well defined, insights on the effects of plasmin on VWF and platelets are relatively scarce and sometimes conflicting. Hence, this review provides an overview of the literature on the effects of plasmin on VWF multimeric structures, on VWF binding to platelets, and on platelet activation. This information is placed in the context of possible applications of thrombolytic therapy for the condition thrombotic thrombocytopenic purpura.

Introduction: Heavy menstrual bleeding (HMB) is a condition that affects 20%-30% of women of reproductive age. HMB has a multifactorial pathophysiology, which is incompletely understood. HMB symptoms are very common in patients with established haemostasis defects, likewise, women with heavy menstrual bleeding have a higher prevalence of impaired Von Willebrand factor (VWF) levels and function, thrombocytopenia, impaired platelet function and impaired coagulation. The aim of this study was to quantify the prevalence of impaired platelet function, impaired coagulation and reduced VWF activity in patients with HMB., Methods: We have used thrombin generation (TG), a flow cytometry-based platelet function test and a flow cytometry-based VWF function test to study haemostasis in 58 women (median age: 48.4 years, range 40-60 years) with HMB. In addition, we determined VWF antigen levels and VWF ristocetin co-factor activity in platelet-poor plasma. Reference ranges of platelet function were measured in whole blood of 123 healthy volunteers, while reference ranges of TG were determined in platelet-poor plasma (PPP) of 126 healthy volunteers., Results: Fourteen (24%) patients with HMB had impaired platelet function and 17 (29.3%) patients had impaired coagulation. Five patients (8.6%) had both impaired platelet function and impaired coagulation. Only 2 (3.4%) patients had an impaired VWF function or levels; one of them was in combination with impaired coagulation., Conclusion: Our approach in women with HMB using a high precision platelet function test in combination with thrombin generation showed impaired coagulation or impaired platelet function in more than 40% of the patients., (© 2018 John Wiley & Sons Ltd.)

Introduction: Epidemiological studies suggest that hypobaric hypoxia at high altitude poses a risk for developing venous thromboembolism. The cause of this observed hypercoagulability remains unclear. Therefore, this study aimed to investigate the effect of hypobaric hypoxia at 3,883 m above sea level on thrombin generation and platelet activation., Methods: After complying with medical ethical procedures, 18 participants were recruited, of whom 1 had to leave the study prematurely due to mild acute mountain sickness. Blood was drawn first at 50 m above sea level and second at 3,883 m altitude after gradual acclimatization for 6 days. Thrombin generation was measured in whole blood, platelet-rich plasma and platelet-poor plasma. Platelet activation was assessed using a whole blood flow-cytometric assay. Coagulation factor levels, D-dimer levels and markers of dehydration and inflammation were measured., Results: Hypobaric hypoxia at 3,883 m altitude caused increased thrombin generation, measured as peak height and endogenous thrombin potential, in whole blood, platelet-rich and platelet-poor plasma without or at low tissue factor concentration. The elevated thrombin generation was mediated by increased factor VIII levels and not caused by dehydration or inflammation. In contrast, spontaneous and agonist-induced platelet activation was decreased at high altitude., Conclusion: Hypobaric hypoxia causes increased factor VIII-mediated thrombin generation. The hypercoagulability was balanced by decreased platelet activation. These findings may explain why venous, and not arterial thrombotic events occur more frequently at high altitude., Competing Interests: None., (Schattauer GmbH Stuttgart.)

Physical exercise is recommended for a healthy lifestyle. Strenuous exercise, however, may trigger the haemostatic system, increasing the risk of vascular thrombotic events and the incidence of primary cardiac arrest. Our goal was to study the effects of strenuous exercise on risk factors of cardiovascular disease. Blood was collected from 92 healthy volunteers who participated in the amateur version of the pro-tour Amstel Gold cycling race, before and directly after the race. Thrombin generation showed a shortening of the lag time and time to peak and an increase of the velocity index. Interestingly, the endogenous thrombin potential measured in plasma decreased due to reduced prothrombin conversion. Platelet reactivity increased and this effect was stronger in men than in women. Lower fibrinogen and higher D-dimer levels after exercise indicated higher fibrin formation. On the other hand, fibrinolysis was also elevated as indicated by a shortening of the clot lysis time. Exercise activated the endothelium (von Willebrand factor (VWF) and active VWF levels were elevated) and the immune system (concentrations IL-6, IL-8, MCP-1, RANTES and PDGF increased). Additionally, an increased cardiac troponin T level was measured post-exercise. Strenuous exercise induces a temporary hyperreactive state in the body with enhanced pro- and anticoagulant responses. As strenuous exercise has a more pronounced effect on platelet function in male subjects, this gives a possible explanation for the higher incidence of sudden cardiac death during exercise compared to women. This trial is registered at www.clinicaltrials.gov as NCT02048462.

Background: Risk factor burden and clinical characteristics of patients with coronary artery disease (CAD) differ among ethnic groups. We related biomarkers to CAD severity in Caucasians, Chinese, Indians and Malays., Methods: In the Dutch-Singaporean UNICORN coronary angiography cohort (n = 2033) we compared levels of five cardiovascular biomarkers: N-terminal pro-brain natriuretic peptide (NTproBNP), high-sensitivity C-reactive protein (hsCRP), cystatin C (CysC), myeloperoxidase (MPO) and high-sensitivity troponin I (hsTnI). We assessed ethnicity-specific associations of biomarkers with CAD severity, quantified by the SYNTAX score., Results: Adjusted for baseline differences, NTproBNP levels were significantly higher in Malays than in Chinese and Caucasians (72.1 vs. 34.4 and 41.1 pmol/l, p < 0.001 and p = 0.005, respectively). MPO levels were higher in Caucasians than in Indians (32.8 vs. 27.2 ng/ml, p = 0.026), hsTnI levels were higher in Malays than in Caucasians and Indians (33.3 vs. 16.4 and 17.8 ng/l, p < 0.001 and p = 0.029) and hsTnI levels were higher in Chinese than in Caucasians (23.3 vs. 16.4, p = 0.031). We found modifying effects of ethnicity on the association of biomarkers with SYNTAX score. NTproBNP associated more strongly with the SYNTAX score in Malays than Caucasians (β 0.132 vs. β 0.020 per 100 pmol/l increase in NTproBNP, p = 0.032). For MPO levels the association was stronger in Malays than Caucasians (β 1.146 vs. β 0.016 per 10 ng/ml increase, p = 0.017). Differing biomarker cut-off levels were found for the ethnic groups., Conclusion: When corrected for possible confounders we observe ethnicity-specific differences in biomarker levels. Moreover, biomarkers associated differently with CAD severity, suggesting that ethnicity-specific cut-off values should be considered.

Objectives: To evaluate current clinical practice for octogenarians with iron-deficiency anemia (IDA) by assessing referral patterns, diagnostic choices, clinical consequences of omission of endoscopy, and risks and benefits of IDA-related surgery., Design: Chart review., Setting: A regional hospital-based laboratory in the Netherlands between January 2008 and December 2010., Participants: All individuals aged 80 and older with newly ascertained IDA., Measurements: IDA was defined as a hemoglobin level of 11.1 g/dL or less and a ferritin level of 25 μg/L or less., Results: Four hundred seventy-one participants were newly diagnosed with IDA during the study period (median age 85.4), 276 of whom (59%) did not undergo any diagnostic procedures for IDA. A cause of anemia was identified during the initial examination in 50% of the 205 investigated participants, including nine (4%) upper and 37 (18%) lower gastrointestinal malignancies. Another 24 malignancies were identified during follow-up, of which 16 were in the gastrointestinal tract, primarily in participants for whom the initial diagnostic examination was limited or omitted. Perioperative mortality was 15% in individuals with colon cancer. Median survival for participants with colon cancer was 2.2 years, and the survival benefit of surgery over supportive care was not apparent until 1.3 years after ascertainment of IDA., Conclusion: The omission of endoscopy for IDA and the omission of surgery for colon cancer occur frequently in octogenarians and seem appropriate in the presence of significant comorbidity and in cases in which there is limited life expectancy. Further research is needed to determine which baseline factors should guide decision-making to optimize treatment outcomes and quality of life., (© 2013, Copyright the Authors Journal compilation © 2013, The American Geriatrics Society.)

Background: Platelet concentrate transfusion is the standard treatment for hemato-oncology patients to compensate for thrombocytopenia. We have developed a novel platelet activation test in anticoagulated unprocessed blood (pac-t-UB) to determine platelet function in platelet concentrates and in blood of thrombocytopenic patients., Methods: We have measured platelet activity in a platelet concentrate and in anticoagulated unprocessed blood of a post-transfusion thrombocytopenic patient., Results: Our data show time-dependent platelet activation by GPVI agonist (collagen related peptide; CRP), PAR-1 agonist (SFLLRN), P2Y12 agonist (ADP), and thromboxane receptor agonist (U46619) in a platelet concentrate. Furthermore, pac-t-UB showed time-dependent platelet activation in unprocessed blood of a post-transfusion patient with thrombocytopenia. Testing platelet function by different agonists in relation to storage show that 3-day-old platelet concentrates are still reactive to the studied agonists. This reactivity rapidly drops for each agonists during longer storage., Discussion: Pac-t-UB is a novel tool to estimate platelet function by different agonists in platelet concentrates and in unprocessed blood of thrombocytopenic patients. In the near future, we will validate whether pac-t-UB is an adequate test to monitor the quality of platelet concentrates and whether pac-t-UB predicts the bleeding risk of transfused thrombocytopenic patients.

We report 2 cases of visceral leishmaniasis in Dutch patients after a stay in Greece and the former Yugoslavia, respectively. Patient A, a 69-year-old woman, was referred to our department with abdominal pain. Additional examinations were suggestive of chronic liver disease. After a liver biopsy, which demonstrated hepatic granulomas, we admitted the patient due to a sudden onset of cyclic fever. Patient B, a 50-year-old woman, was admitted with cyclic fever and abdominal pain. We treated the patient with IV antibiotics and discontinued the methotrexate treatment for her rheumatoid arthritis. Both patients were diagnosed with visceral leishmaniasis and treated with liposomal amphotericin-B. Patient A, an immunocompetent patient, had stayed in Greece for prolonged periods. Patient B had lived in the former Yugoslavia until 1999, and her methotrexate use had likely activated an asymptomatic Leishmania infection. Visceral leishmaniasis, a potentially lethal protozoan disease, should be considered in patients who have travelled in Southern Europe.

Background: Iron and (stainless) steel are potent platelet aggregation activators, and may be involved in stent thrombosis, a serious complication after intracoronary stenting. Current platelet function tests are suboptimal, because of inappropriate agonists and/or lack of reproducibility. We tested the feasibility and reproducibility of a novel platelet function test using stainless steel as an agonist and compared it with other platelet function tests., Materials and Methods: In 111 patients with acute ST segment elevation myocardial infarction (STEMI), duplo measurements of iron (Fe)-induced platelet aggregation (FIPA) were performed after clopidogrel, acetylsalicylic acid and/or tirofiban treatment. Within 1 h, citrated blood samples drawn from the femoral sheath before primary percutaneous coronary intervention were added to 100 mg of low carbon steel and after 5 s mixing with vortex, the samples were incubated for 15 min. The ratio between the non-aggregated platelets in the agonist sample and platelets in a reference sample was calculated as the platelet aggregation inhibition., Results: FIPA measurement was highly reproducible (correlation coefficient (R)=0.942, P<0.001 between duplo samples). FIPA correlated well with adenosine diphosphate-induced platelet aggregation (R=0.83, P<0.001) but weakly with platelet function analyser-100 bleeding time (R=0.56, P<0.001). FIPA could be measured in patients in which platelet aggregation could not be measured by platelet function analyser-100 or after adenosine diphosphate., Conclusion: This study showed good reproducibility of a novel platelet function test using stainless steel as an agonist and showed correlation with validated platelet function tests. We found that the novel platelet function test is a suitable test for measurement of platelet aggregation inhibition in patients undergoing stenting for STEMI, even when they are taking multiple antiplatelet regimens.

Background: The platelet adenosine 5'-diphosphate (ADP) receptor P2Y(12) plays a crucial role in haemostasis. Only a few patients with haemorrhagic diathesis due to molecular defects in the P2Y(12) receptor have been described so far. We report a novel molecular defect in the gene coding for P2Y(12) in a patient with a history of epistaxis, easy bruising and excessive posttraumatic blood loss., Methods: Platelet aggregation studies, perfusion studies, in which patient blood was perfused over collagen surfaces at arterial shear rates, and PCR and sequencing were used., Results: Platelet aggregation studies showed impaired ADP and collagen-induced aggregation for patient G.S. Perfusion of patient blood over collagen surfaces showed small thrombi consisting of spread platelets overlayered with non-spread platelets. These thrombi were identical to control thrombi formed in the presence of a P2Y(12) antagonist. DNA analysis of the P2Y(12) gene revealed a novel heterozygous base pair C-->A substitution in exon 3, changing codon 258 from proline to threonine in the third extracellular loop of the P2Y(12) receptor., Conclusions: We conclude that perfusion studies with patient blood are of added value in the diagnostic process, which resulted in identification of a novel molecular defect in the P2Y(12) gene of a patient with haemorrhagic diathesis.

Vessel wall injury induces the formation of a haemostatic plug. Restoration of vascular integrity should involve cessation of further platelet and fibrin deposition and subsequent removal of these thrombi by both the fibrinolytic system and proteases delivered by infiltrating inflammatory cells. We hypothesized that adhesion of platelets and inflammatory cells [polymorphonuclear leucocyte (PMN)] to fibrin is differently supported after exposure of fibrin during fibrinolysis. Fibrin surfaces were exposed to fibrinolytic agents, and platelet and PMN adhesion was studied under conditions of flow. Specific adhesion of platelets to preformed fibrin was reduced by fibrinolytic treatment of the fibrin. PMN adhesion to fibrin was only slightly affected even after 180 min exposure to plasmin. With fibrin still present after fibrinolytic treatment, the impaired platelet adhesion seems explained by loss of the primary platelet adhesion site gamma400-411 on fibrin. PMN binding to fibrin clearly depends on other sites that are less degraded by fibrinolysis. We have shown that PMN adhesion in flowing blood to lysed fibrin was still present, whereas platelet adhesion was impaired due to the loss of the primary platelet adhesion site gamma400-411. Based on our in-vitro perfusion model, we conclude that fibrinolysis specifically interferes with the thrombogenicity of fibrin in the haemostatic plug, whereas the inflammatory response is preserved. The latter may participate in the long-term removal and restructuration of the plug.

A 16-year-old girl presented at the emergency unit with myalgia following a flu-like episode. Laboratory tests indicated severe rhabdomyolysis and nephritis. Autoimmune-induced myositis was excluded on the basis of negative tests for antinuclear antibodies; prednisolone treatment was discontinued 1 week later. The patient recovered gradually and was discharged with physiotherapy 2 weeks later. High positive titres of complement-binding antibody against influenza B virus were found, i.e. 1:125 and 1:250 on days to and 25 of illness, respectively. Viral myositis is an uncommon disease entity that occurs following a viral infection, especially with influenza virus, that has been experienced for the first time. It usually runs a benign course: children often present with calf tenderness that resolves within a few days. There are cases, however, with a more serious course involving severe rhabdomyolysis and acute renal failure that can be sometimes fatal.

Congenital afibrinogenaemia is a rare autosomal recessive disorder characterized by complete absence or trace amounts of fibrinogen. Here we report the identification of the molecular defect underlying afibrinogenaemia in a Dutch patient. DNA sequence analysis of the fibrinogen Aalpha, Bbeta and gamma-genes revealed a homozygous deletion of two adenines between nucleotides 3120 and 3122 in exon 4 of the gene coding for the Aalpha-chain. This deletion results in a frameshift with a predicted premature end of translation at codon 140. This is the first report of a patient homozygous for this rare mutation associated with afibrinogenaemia.

The interaction of platelets with fibrinogen is a key event in the maintenance of a haemostatic response. It has been shown that the 12-carboxy-terminal residues of the gamma-chain of fibrinogen mediate platelet adhesion to immobilized fibrinogen. These studies, however, did not exclude the possibility that other domains of fibrinogen are involved in interactions with platelets. To obtain more insight into the involvement of other domains of fibrinogen in platelet adhesion, we studied platelet adhesion in flowing blood to patient dysfibrinogen Vlissingen/Frankfurt IV (V/FIV), to several variant recombinant fibrinogens with abnormalities in the gamma-chain segments gamma318-320 and gamma408-411. Perfusion studies at physiological shear rates showed that platelet adhesion was absent to gammaDelta408-411, slightly reduced to the heterozygous patient dysfibrinogen V/FIV and strongly reduced to the homozygous recombinant fibrinogens: gammaDelta319-320, gamma318Asp-->Ala and gamma320Asp-->Ala. Furthermore, antibodies raised against the sequences gamma308-322 and gamma316-333 inhibited platelet adhesion under shear conditions. These experiments indicated that the overlapping segment gamma316-322 contains amino acids that could be involved in platelet adhesion to immobilized fibrinogen under flow conditions. In soluble fibrinogen, this sequence is buried inside the fibrinogen molecule and becomes exposed after polymerization. In addition, we have shown that this fibrin-specific sequence also becomes exposed when fibrinogen is immobilized on a surface.

ADP plays a central role in regulating platelet function. It induces platelet aggregation via the activation of 2 major ADP receptors, P2Y(1) and P2Y(12). We have investigated the role of P2Y(12) in platelet adhesion and thrombus formation under physiological flow by using blood from a patient with a defect in the gene encoding P2Y(12). Anticoagulated blood from the patient and from healthy volunteers was perfused over collagen-coated coverslips. The patient's thrombi were smaller and consisted of spread platelets overlying platelets that were not spread, whereas control thrombi were large and densely packed. Identical platelet surface coverage, aggregate size, and morphology were found when a P2Y(12) antagonist, N(6)-(2-methylthioethyl)-2-(3,3,3-trifluoropropylthio)-beta,gamma-dichloromethylene ATP (also known as AR-C69931 MX), was added to control blood. The addition of a P2Y(1) antagonist (adenosine-3',5'-diphospate) to control blood resulted in small, but normally structured, thrombi. Thus, the ADP-P2Y(12) interaction is essential for normal thrombus buildup on collagen. The patient's blood also showed reduced platelet adhesion on fibrinogen, which was not due to changes in morphology. Comparable results were found by using control blood with AR-C69931 MX and also with adenosine-3',5'-diphospate. This suggested that P2Y(12) and P2Y(1) were both involved in platelet adhesion on immobilized fibrinogen, thereby revealing it as ADP dependent. This was confirmed by complete inhibition on the addition of creatine phosphate/creatine phosphokinase.

We studied the role of fibrinogen in platelet thrombus formation under flow on adhesive proteins using afibrinogenemic blood (LMWH anticoagulated) in a perfusion system. Perfusions with afibrinogenemic blood showed strong increased surface coverage and thrombus volume that normalized upon addition of fibrinogen. Similar studies using citrate anticoagulated blood showed that this was due to fibrinogen and not fibrin. Morphological analysis showed that afibrinogenemic thrombi were loosely packed and consisted mainly of dendritic platelets that contacted one another through filopodia. However, in the presence of fibrinogen, platelets formed lamellipodia and spread out on top of one another. Studies with radiolabeled platelets showed similar numbers of platelets in both conditions demonstrating that the difference is one of packing and the larger size is due to absence of lamellipodia formation and spreading. The found increased thrombus size and loosely packed platelets might help to understand thrombotic complications sometimes seen in afibrinogenemia patients.

In this paper we report on studies of platelet adhesion to several fibrinogen gamma chain variants under physiological flow conditions. Reduced platelet adhesion was found to patient dysfibrinogen Vlissingen and its recombinant form (deletion of gamma 319-320). Furthermore, substitutions of the amino acids 318, 320, or both in the recombinant fibrinogen gamma chain showed a strong decrease in platelet adhesion under flow conditions in our perfusion system. Antibodies raised against peptides covering these sequences inhibited platelet adhesion completely, which suggested that the gamma 316-322 sequence could be involved in platelet adhesion in flowing blood.