Associations between hemostatic markers and mortality in COVID-19 - Compounding effects of D-dimer, antithrombin and PAP complex.

In this single-center cohort study, we applied a panel of laboratory markers to characterize hemostatic function in 217 consecutive patients that underwent testing for COVID-19 as they were admitted to Linköping University Hospital between April and June 2020. In the 96 patients that tested positive for SARS-CoV-2 (COVID-19+), the cumulative incidences of death and venous thromboembolism were 24.0% and 19.8% as compared to 12.4% (p = 0.031) and 11.6% (p = 0.13) in the 121 patients that tested negative (COVID-19-). In COVID-19+ patients, we found pronounced increases in plasma levels of von Willebrand factor (vWF) and fibrinogen. Excess mortality was observed in COVID-19+ patients with the following aberrations in hemostatic markers: high D-dimer, low antithrombin or low plasmin-antiplasmin complex (PAP) formation, with Odds Ratios (OR) for death of 4.7 (95% confidence interval (CI95) 1.7-12.9; p = 0.003) for D-dimer >0.5 mg/L, 5.9 (CI95 1.8-19.7; p = 0.004) for antithrombin (AT) ˂0.85 kIU/l and 4.9 (CI95 1.3-18.3; p = 0.019) for PAP < 1000 µg/L. Compounding increases in mortality was observed in COVID-19+ patients with combined defects in markers of fibrinolysis and coagulation, with ORs for death of 15.7 (CI95 4.3-57; p < 0.001) for patients with PAP <1000 µg/L and D-dimer >0.5 mg/L and 15.5 (CI95 2.8-87, p = 0.002) for patients with PAP <1000 µg/L and AT ˂0.85 kIU/L. We observed an elevated fraction of incompletely degraded D-dimer fragments in COVID-19+ patients with low PAP, indicating impaired fibrinolytic breakdown of cross-linked fibrin.

Sex differences in platelet reactivity in patients with myocardial infarction treated with triple antiplatelet therapy - results from assessing platelet activity in coronary heart disease (APACHE).

)Several earlier studies have reported increased risk of bleeding in women with myocardial infarction, (MI) compared to men. The reasons for the observed difference are incompletely understood, but one suggested explanation has been excess dosing of antithrombotic drugs in women. The aim of this prospective observational study was to assess sex differences in platelet activity in patients treated with three different platelet inhibitors. We recruited 125 patients (37 women and 88 men) with MI, scheduled for coronary angiography. All patients received clopidogrel and aspirin. A subgroup of patients received glycoprotein (GP) IIb/IIIa-inhibitor. Platelet aggregation in whole blood was assessed at several time points, using impedance aggregometry. Soluble P-selectin was measured 3 days after admission. There were no significant differences between women and men in baseline features or comorbidities except higher frequency of diabetes, lower hemoglobin value, and lower estimated glomerular filtration rate, in women on admission. We observed significantly more in-hospital bleeding events in women compared to men (18.9% vs. 6.8%, p = .04). There were no differences in platelet aggregation using three different agonists, reflecting treatment effect of GPIIb/IIIa-inhibitors, clopidogrel, and aspirin, 6-8 hours, 3 days, 7-9 days, or 6 months after loading dose. Moreover, there was no significant difference in soluble P-selectin. The main finding of this study was a consistent lack of difference between the sexes in platelet aggregation, using three different agonists at several time-points. Our results do not support excess dosing of anti-platelet drugs as a major explanation for increased bleeding risk in women.

Unveiling the complex effects of direct oral anticoagulants on dilute Russell's viper venom time assays.

INTRODUCTION: Dilute Russell viper venom time (dRVVT) assays can be affected by direct oral anticoagulants (DOACs), which may cause false-positive results. However, there are conflicting results indicating significant differences between different reagents and DOACs. OBJECTIVES: To evaluate the effect of DOACs on dRVVT assays. MATERIAL AND METHODS: Samples were prepared by adding DOAC (dabigatran, rivaroxaban, apixaban, or edoxaban) to pooled normal plasma in the concentration range 0 to 800 µg/L. Six integrated dRVVT reagents were used, all composed of a screen assay (low phospholipid content) and a confirm assay (high phospholipid content). The screen/confirm dRVVT results were expressed as normalized ratios. To further evaluate the observed differences between tests and DOACs, addition of synthetic phospholipids was used. RESULTS: The dRVVT ratios increased dose dependently for all DOACs, with four of the six tests and the DOAC rivaroxaban having the greatest effect. With one test, the ratios were almost unaffected with increasing DOAC concentration, whereas another test revealed a negative dose dependency for all DOACs. Variable DOAC effects can be explained by different effects on dRVVT screen and confirm clotting time. Adding synthetic phospholipids to samples containing rivaroxaban resulted in greatly reduced screen clotting times and thereby lower calculated dRVVT ratios. CONCLUSIONS: There is a great variability in the dRVVT test result with different DOACs. The dRVVT ratios are unaffected for some reagents and this can be explained by an equal dose-dependent effect on both screen and confirm assays. The phospholipid type and content of the different reagents may contribute to the observed differences.

Individual long-term variation of platelet reactivity in patients with dual antiplatelet therapy after myocardial infarction.

There is a large inter-individual variation in response to clopidogrel treatment, and previous studies have indicated higher risk of thrombotic events in those with high residual platelet reactivity (HPR). Less is known about individual variation over time. The aim of this prospective cohort study was to investigate intra-individual variation in platelet reactivity. Platelet aggregation in whole blood was assessed in 77 patients, at 3 days, 8 days and 6 months after admission for acute myocardial infarction and loading dose of clopidogrel. All patients were treated with aspirin and clopidogrel through 6-month follow-up. We found a significant increase in median ADP-stimulated aggregation from third to eighth day (195 vs. 250 AU*min, p-value = 0.001) but not from day 8 to 6 months (250 vs. 223 AU*min, p-value = 0.666). There was no significant change in the overall rate of HPR (15.6% vs 20.8%, p-value 0.503) or low platelet reactivity (LPR) (37.7% vs 33.8%, p-value = 0.609) from day 8 to 6-month follow-up. In contrast, more than one in four changed HPR status, 15.6% from non-HPR to HPR and 10.4% HPR to non-HPR. A shift in LPR status appeared even more frequent, occurring in about one of three patients. In spite of similar median aggregation and rate of HPR during 6-month follow-up, about one in four of the patients changed HPR status and one in three changed LPR status. This may be important information for a concept of risk stratification based on a single aggregation value early after an acute coronary syndromes.

Effects of the oral, direct factor Xa inhibitor edoxaban on routine coagulation assays, lupus anticoagulant and anti-Xa assays.

Edoxaban is an oral direct factor Xa inhibitor for prophylaxis and treatment of thromboembolic disorders. The effects on common coagulation assays are clinically valuable information and in certain clinical situations a quick assessment of the anticoagulant is wanted. Our aim was to investigate the effect of edoxaban on routine coagulation methods and evaluate anti-Xa assays, commonly used for other direct factor Xa inhibitors, for estimation of the drug concentration. Edoxaban was spiked to plasma samples from healthy subjects in the concentration range 0-742 µg/L and analyzed using different reagents for activated partial thromboplastin time (APTT) and prothrombin time (PT). Assays for antithrombin, activated protein C resistance, lupus anticoagulant (LA) and chromogenic anti-Xa assays were also included. Edoxaban displayed similar effects in vitro to other oral direct Xa inhibitors. The concentration needed to double the coagulation time varied between assays and reagents; 539-758 µg/L for the APTT and between 329 and 2505 µg/L for the PT. Edoxaban gave false high antithrombin activities in assays based on Xa-inhibition. Two integrated assays for LA, both based on activation with dilute Russell's viper venom, displayed different results. Chromogenic anti-Xa assays displayed linear dose-response curves with edoxaban up to approximately 500 µg/L. In conclusion, therapeutic concentrations of edoxaban variably affect different coagulation assays, and even different reagents within an assay group. In comparison with other oral Xa-inhibitors, the in vitro effects of edoxaban were more similar to rivaroxaban than apixaban. For measurement of edoxaban concentration in plasma, it is possible to use the chromogenic anti-Xa assays.

Pretreatment with ticagrelor may offset additional inhibition of platelet and coagulation activation with bivalirudin compared to heparin during primary percutaneous coronary intervention.

BACKGROUND: It remains unknown if bivalirudin compared to heparin confers any additional inhibition of platelet and coagulation activation during primary percutaneous coronary intervention (PPCI) after pretreatment with ticagrelor. METHODS: In this substudy of VALIDATE-SWEDEHEART trial, 103 patients pretreated with ticagrelor were randomized before PPCI to heparin or bivalirudin. Blood samples were collected before and 1 and 12 h after PPCI. We measured platelet reactivity (PR) using Multiplate, soluble P-selectin, thrombin-antithrombin complexes (TAT) and prothrombin fragments 1 + 2 (F1 + 2) as markers of platelet and coagulation activation. RESULTS: The median (IQR) time from ticagrelor administration to randomization was 63 (29) vs 60 (24) minutes, p = 0.28. ADP-induced PR did not significantly differ between groups over time (heparin vs bivalirudin, AUC 73 (62) vs 74 (68), p = 0.74, 32 (42) vs 43 (51), p = 0.38, 15 (15) vs 19 (15), p = 0.29, before, 1 and 12 h after PPCI). Soluble P-selectin did not significantly differ between groups. At 1 h TAT significantly increased with bivalirudin (3.0 (1.3) to 4.3 (4.2) ug/L; p < 0.01), but not with UFH (3.1 (2.1) to 3.5 (1.6) ug/L, p = 0.24). F1 + 2 increased in both groups but the rise was numerically higher with bivalirudin (170 (85) to 213 (126) pmol/L vs 168 (118) to 191 (103) pmol/L). At 12 h, a comparable significant increase in thrombin generation was observed in both groups. CONCLUSION: In patients treated with ticagrelor, we found no major differences between bivalirudin and heparin in platelet aggregation or coagulation markers, which is in agreement with the neutral clinical results of the VALIDATE-SWEDEHEART study.

Hexabromocyclododecane affects benthic-pelagic coupling in an experimental ecosystem.

Hexabromocyclododecane (HBCDD) is an additive brominated flame retardant and a recognized PBT chemical. However, little is known about its effects on coastal species, and even less on ecosystem effects. We investigated the dose-response effects of HBCDD over 8 months in 1000 L experimental mesocosms assembled from coastal Baltic Sea ecosystem components. HBCDD was added via spiked plankton material and a range of structural and functional endpoints were measured during the experiment. Increasing HBCDD concentration decreased the biomass of large Macoma balthica, resulting in a decreased recirculation of nutrients to the water. Changes in plankton communities were also observed, either due to direct toxic HBCDD effects or indirect via changes in benthic-pelagic coupling of nutrients. Such complex ecosystem responses can only be quantified and understood by using realistic experimental set-ups, and including knowledge of system-specific ecological interactions. This is the first study of HBCDD effects on ecosystem level.

Large early variation of residual platelet reactivity in Acute Coronary Syndrome patients treated with clopidogrel: results from Assessing Platelet Activity in Coronary Heart Disease (APACHE).

INTRODUCTION: There is a large inter-individual variation in response to clopidogrel treatment and previous studies have indicated higher risk of thrombotic events in patients with high residual platelet reactivity (HRPR), but the optimal time-point for testing is not established. The aim of this study was to investigate the optimal time-point for aggregometry testing and the risk of major adverse cardiac events associated with HRPR. METHOD AND RESULTS: We included 125 patients with ACS (73 with STEMI, and 71 received abciximab). The prevalence of HRPR varied substantially over time. The rate of HRPR in patients treated and not treated with abciximab were 43% vs 67% (p=0.01) before, 2% vs 23% (p=0.001) 6-8h after, 8% vs 9% (p=0.749) 3days after, and 23% vs 12% (p=0.138) 7-9 days after loading dose of clopidogrel. We found HRPR in 18% of the patients but only four ischemic events during 6months follow-up, with no significant difference between HRPR patients compared to the rest of the population. There were 3 TIMI major bleedings, all of which occurred in the low residual platelet reactivity (LRPR) group. CONCLUSION: There is a large variation in platelet reactivity over time, also depending on adjunctive therapy, which has a large impact on optimal time-point for assessment. We found HRPR in almost 1 in 5 patients, but very few MACE, and not significantly higher in HRPR patients. In a contemporary ACS population, with low risk for stent thrombosis, the predictive value of HRPR for ischemic events will probably be low.

Effects of benthos, temperature, and dose on the fate of hexabromocyclododecane in experimental coastal ecosystems.

The authors studied the fate of the brominated flame retardant hexabromocyclododecane (HBCDD) added in a particulate suspension to experimental ecosystems assembled from brackish (Baltic Sea) coastal bays. Two experiments examined how benthic macrofauna (over 21 d) and increased temperature (14 d) affected HBCDD concentrations and fractionation of α, ß, and γ diastereomers in the water, sediment, and biota. A third experiment run over 3 seasons (231 d), studied the effect of HBCDD dose on the same endpoints. In all treatments of the 3 experiments, HBCDD partitioned mainly to the sediment, and this proportion increased with time. Presence of macrofauna tended to increase the HBCDD concentration in the sediment and decreased its concentration in the water. Increased temperature (+ 5°C) decreased the amount of HBCDD in sediment and water but not in the filter- and deposit-feeding infaunal bivalves (Macoma balthica). The partitioning between water, sediment, and biota was not concentration dependent. In all treatments, sediment became enriched in γ-HBCDD, M. balthica in α-HBCDD, and water in α- and ß-HBCDD. Bioaccumulation of HBCDD in M. balthica was high in all experiments (log biota-sediment accumulation factor [BSAF] > 1.25), the α diastereomer contributing the most (log BSAF 2.1-5.2). There is a risk of trophic transfer of HBCDD from benthic to pelagic food webs, as well as secondary poisoning of marine consumers.

More efficient reversal of dabigatran inhibition of coagulation by activated prothrombin complex concentrate or recombinant factor VIIa than by four-factor prothrombin complex concentrate.

The number of patients on antithrombotic treatment due to atrial fibrillation and venous thromboembolism is increasing fast due to an aging population. A growing proportion will be treated with novel oral anticoagulants, the first in clinical use was the direct oral thrombin inhibitor dabigatran (Pradaxa®). A small percentage of the patients on dabigatran will experience serious bleeding or be in need of urgent surgery. The aim of this study was to test the effects of different hemostatic agents in potentially reversing the anticoagulant effects in vitro in blood or platelet-rich plasma (PRP) spiked with dabigatran. Whole blood or PRP was spiked with the active substance dabigatran, 200 µg/L. We measured clotting time being induced by 1.4 pmol/L tissue factor using the instrument ReoRox2™ and initial clot growth velocity from a tissue factor covered surface using the instrument Thrombodynamics Analyzer T-2™. Dabigatran prolonged clotting time 5-fold but reduced clot growth velocity only slightly. The reversing effects of prothrombin complex concentrates (PCC), activated PCC (APCC) and recombinant activated factor VII (rFVIIa) were then tested. APCC (1.8 U/mL) reduced the prolonged clotting time by 1/3, rFVIIa (2 µg/L) only slightly (n = 10-20). The reduction was not significant using Mann-Whitney test but significant using t-test with Bonferronis' correction for multiple comparisons, whereas PCC (0.56 U/mL) had no effect on clotting time. APCC doubled initial clot growth velocity, although even more in the absence of dabigatran. In conclusion, APCC and rFVIIa, but not PCC, seem to reverse, at least partially, some effects of dabigatran on coagulation parameters. Systematic evaluation of case reports, registries and, ultimately, randomized clinical trials are needed to elucidate potential benefit for patients.

Fluorescence-based blood coagulation assay device for measuring activated partial thromboplastin time.

The measurement of blood clotting time is important in a range of clinical applications such as assessing coagulation disorders and controlling the effect of various anticoagulant drug therapies. Clotting time tests essentially measure the onset of clot formation which results from the formation of fibrin fibers in the blood sample. However, such assays are inherently imprecise due to the highly variable nature of the clot formation process and the sample matrix. This work describes a clotting time measurement assay which uses a fluorescent probe to very precisely detect the onset of fibrin clot formation. It uses a microstructured surface which enhances the formation of multiple localized clot loci and which results in the abrupt redistribution of the fluorescent label at the onset of clot formation in both whole blood and plasma. This methodology was applied to the development of an activated partial thromboplastin time (aPTT) test in a lateral flow microfluidic platform and used to monitor the effect of heparin dosage where it showed linearity from 0 to 2 U/mL in spiked plasma samples (R(2)=0.996, n = 3), correlation against gold standard coagulometry of 0.9986, and correlation against standard hospital aPTT in 32 patient samples of 0.78.

Effects of the oral, direct thrombin inhibitor dabigatran on five common coagulation assays.

Dabigatran is an oral, reversible thrombin inhibitor that has shown promising results in large clinical trials. Laboratory monitoring is not needed but the effects on common coagulation assays are incompletely known. Dabigatran was added to plasma from healthy subjects in the concentration range 0-1,000 µg/l and analysed using several reagents for activated thromboplastin time (APTT), prothrombin time (PT), fibrinogen, antithrombin, and activated protein C resistance. Typical trough concentrations are about 50 µg/l, peak concentrations 100-300 µg/l. At 100 µg/l all APTT-results were prolonged. The concentration required to double APTT ranged between 227 and 286 µg/l, the responses for all five reagents were similar. PT-reagents were much less affected with almost no samples above INR 1.2 at 100 µg/l. The effect was sample dilution dependent with PT Quick type more sensitive than PT Owren type methods. If a patient on dabigatran has prolonged APTT, >90 seconds, and Quick PT INR>2 or Owren PT INR>1.5 over-dosing or accumulation of dabigatran should be considered. Two of four fibrinogen reagents underestimated the fibrinogen concentration considerably at expected peak concentration. Methods based on inhibition of thrombin over-estimated the antithrombin concentration, but not Xa-based. The APC-resistance methods over-estimated the APC-ratio, which may lead to miss-classification of factor V Leiden patients as being normal. Different coagulation assays, and even different reagents within an assay group, display variable effects at therapeutic concentrations of dabigatran. Some of these assay variations are of clinical importance, thus knowledge is needed for a correct interpretation of results.

Direct and indirect effects of the fungicide azoxystrobin in outdoor brackish water microcosms.

The effects of the strobilurin fungicide azoxystrobin were studied in brackish water microcosms, with natural plankton communities and sediment. Two experiments were conducted: Experiment 1 (nominal conc. 0, 15 and 60 microg/L, 24-L outdoor microcosms for 21 days) and a second, follow-up, Experiment 2 (nominal conc. 0, 3, 7.5, 15 microg/L, 4-L indoor microcosms for 12 days). The microcosms represent a simplified brackish water community found in shallow semi-enclosed coastal areas in agricultural districts in the Baltic Sea region. Measured water concentrations of the fungicide (Experiment 1) were, on average, 83 and 62% of nominal concentrations directly after application, and 25 and 30% after 21 days, for the low and high dose treatments, respectively, corresponding to mean DT50-values of 15.1 and 25.8 days, for low and high dose treatments, respectively. In Experiment 1, direct toxic effects on calanoid copepods at both test concentrations were observed. Similarly, in Experiment 2, the copepod abundance was significantly reduced at all tested concentrations. There were also significant secondary effects on zooplankton and phytoplankton community structure, standing stocks and primary production. Very few ecotoxicological studies have investigated effects of plant protection products on Baltic organisms in general and effects on community structure and function specifically. Our results show that azoxystrobin is toxic to brackish water copepods at considerably lower concentrations than previously reported from single species tests on freshwater crustaceans, and that direct toxic effects on this ecologically important group may lead to cascade effects altering lower food webs and ecosystem functioning.

Older women's perceptions of independence versus dependence in food-related work.

This qualitative study aims to explore the cultural meaning of accomplishing food-related work by older women, when disease has diminished their abilities and threatens to make them dependent. Seventy-two women with stroke, rheumatoid arthritis, and Parkinson's disease, as well as women without those diseases, were interviewed. All were living at home. Results showed that older women valued independence and feared dependence when declining ability threatened performance of food-related work. They also had strong beliefs about living a "normal life," managing by oneself as long as possible, and becoming their own masters again. To remain independent, participants used three kinds of strategies: Public Health Service Support, self-managing, and adaptation. Their beliefs about dependence included not becoming a burden, retaining self-determination, and maintaining order in life. Implications for nursing include supporting independent cooking, developing care plans with the care recipient, and demonstrating respect for the women's self-determination.

Food-related health perceptions and food habits among older women.

AIM: The aim of the present study was to explore food-related health perceptions and food habits among older women. BACKGROUND: Food-related health promotion has its main focus on disease prevention. With a holistic perspective on health, social and psychological aspects also need to be considered for total wellbeing. METHODS: Qualitative interviews, representing an ethnographic approach, were carried out with 18 women, aged 65-88, living alone or cohabiting, who independently managed shopping and cooking. Interviews were conducted at the women's homes, and analysed for coherent themes. The women also gave data on three days' eating and drinking in a food diary. FINDINGS; Two themes were found: 'A healthy slimming meal or the usual' and 'Meals -- a pleasure or an obligation'. The first theme summarized the women's health perceptions related to food, where the dominating view was fear of fat. Some also had a bad conscience about not eating according to recommendations. Use of low-fat products was not a predominant habit among these women. In the second theme, meals in fellowship were perceived as a pleasure, while women living alone tended to simplify cooking and eating. This was also reflected in their food habits, with fewer cooked meals, as well as events with coffee with cakes, compared with cohabiting women. CONCLUSIONS: Food-related health promotion must pay more attention to women living alone. Women who have lost their partners, may be at risk for poor nutritional intake as they often simplified the entire meal situation, while cohabiting women perceived food and cooking as a central task in their lives. One responsibility for nurses working in the community is to recognize and assess older women's deficient eating habits, assess them and plan eating together with the woman to prevent poor nutritional intake. Relatives and community staff could also be involved in this work.