DNA Fragility and Repair: Some Personal Recollections.

In this autobiographical article, I reflect on my Swedish background. Then I discuss endogenous DNA alterations and the base excision repair pathway and alternative repair strategies for some unusual DNA lesions. Endogenous DNA damage, such as loss of purine bases and cytosine deamination, is proposed as a major source of cancer-causing mutations.

The world of DNA in glycol solution.

The properties of high-molecular-weight DNA are usually investigated in neutral aqueous solutions. Strong acids and strong alkaline solutions are obviously unsuitable, as are corrosive solvents, and DNA is insoluble in most organic solvents; precipitation of DNA from aqueous solution with ethanol or isopropanol is therefore frequently used as a purification step. An exception is the organic solvent glycol (ethylene glycol, 1,2-ethanediol, dihydroxyethane, HOCH2CH2OH) and the similar solvent glycerol. Double-stranded DNA remains soluble in salt-containing glycol, although it precipitates in polyethylene glycol. (DNA also remains soluble in formamide, but the double-helical structure of DNA is much less stable in this solvent than in glycol.) However, DNA in glycol has been little investigated during the last half-century.

The effect of tinzaparin on biomarkers in FIGO stages III-IV ovarian cancer patients undergoing neoadjuvant chemotherapy - the TABANETOC trial: study protocol for a randomized clinical multicenter trial.

BACKGROUND: Tinzaparin, a low-molecular weight heparin (LMWH), has shown anti-neoplastic properties in animal models and in in vitro studies of human cancer cell lines. The reduction of CA-125 levels during neoadjuvant chemotherapy (NACT) in patients with epithelial ovarian cancer (EOC) co-varies with the prognosis; the larger the decrease in CA-125, the better the prognosis. PURPOSE: This study aims to evaluate the potential anti-neoplastic effects of tinzaparin by investigating changes in serum CA-125 levels in advanced EOC patients who receive NACT. MATERIAL AND METHODS: This is an open randomized multicenter pilot trial. Forty patients with EOC selected to receive NACT will be randomized 1:1 to receive daily addition of tinzaparin or no tinzaparin. The processing and treatment of the patients will otherwise follow the recommendations in the Swedish National Guidelines for Ovarian Cancer. Before every cycle of chemotherapy, preoperatively, and 3 weeks after the last cycle of chemotherapy, a panel of biomarkers, including CA-125, will be measured. PATIENTS: Inclusion criteria are women aged 18 years or older, World Health Organization performance status 0-1, histologically confirmed high-grade serous, endometrioid or clear cell EOC, International Federation of Gynecology and Obstetrics (FIGO) stages III-IV. In addition, a CA-125 level of ≥ 250 kIE/L at diagnosis. Exclusion criteria are contraindications to LMWH, ongoing or recent treatment with unfractionated heparin, LMWH, warfarin or non-vitamin K antagonist oral anticoagulants. INTERPRETATION: This study will make an important contribution to the knowledge of the anti-neoplastic effects of tinzaparin in EOC patients and may thus guide the planning of a future study on the impact of tinzaparin on survival in EOC.

Thrombus remodelling by reversible and irreversible P2Y12 inhibitors.

Pharmacological inhibition of the platelet ADP-receptor P2Y12 is a cornerstone in the prevention of atherothrombotic events in adult patients with acute coronary syndrome (ACS). Thienopyridines such as clopidogrel and prasugrel exert their antithrombotic effect by means of active metabolites that irreversibly inhibit P2Y12. Due to the short half-life of these metabolites, a subpopulation of ADP-responsive platelets will form in between dosing. With increased platelet turnover rate or poor patient compliance, the fraction of ADP-responsive platelets will increase, potentially increasing the risk for new thrombotic events. In contrast, the reversible P2Y12 inhibition produced by direct-acting ADP blockers such as ticagrelor and cangrelor inhibit the entire platelet population. In this study, we evaluated the impact of these pharmacological differences on thrombus formation in an ex vivo flow chamber model. A customized image analysis pipeline was used for automatized, large-scale identification and tracking of single platelets incorporated into the thrombus, enabling quantitative analysis of the relative contribution of inhibited and uninhibited platelets to thrombus growth and consolidation. Comparative experiments were conducted using the irreversible and reversible P2Y12 inhibitors prasugrel active metabolite (PAM) and ticagrelor, respectively. Our results show that PAM inhibited thrombus platelet recruitment more gradually than ticagrelor, with a slower onset of inhibition. Further, we show that the presence of a small fraction (<10%) of uninhibited platelets did not abrogate the antithrombotic effect of PAM to any significant extent. Finally, we demonstrate a gradual enrichment of inhibited platelets in the thrombus shell due to selective recruitment of inhibited platelets to the thrombus periphery.

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.

Biomarker dynamics in cardiac surgery: a prospective observational study on MR-proADM, MR-proANP, hs-CRP and sP-selectin plasma levels in the perioperative period.

Background: For many biomarkers in cardiac surgery, there is a lack of knowledge regarding the normal dynamics of plasma levels during the perioperative course. The aim of this study was to investigate the perioperative dynamics of MR-proADM, MR-proANP, hs-CRP and sP-selectin in cardiac surgery.Method: A prospective observational pilot study with 20 patients scheduled for open cardiac surgery procedures with cardiopulmonary bypass (CPB). Plasma samples were taken for each patient and biomarker during the pre-, per- and postoperative period until Day 6 postoperatively.Results: MR-proADM increased significantly from 0.62 [IQR; 0.54-0.93] nmol/L preoperatively to 1.20 [1.04-1.80] nmol/L postoperative Day 1. MR-proANP increased significantly from 125 [77-152] pmol/L preoperatively to 198 [168-307] pmol/L on weaning from CPB. hs-CRP increased significantly from 2.5 mg/L [0.4-12] preoperatively to peak at 208 mg/L [186-239] postoperative Day 3. The preoperative level of sP-selectin at 23.0 [21.3-26.3] ng/mL initially fell at weaning from CPB, followed by a significant peak of 25.5 [22.7-27.7] ng/mL 8 h postoperatively.Conclusions: The findings in this study may help to understand the physiology of the biomarkers analysed and their response to cardiac surgical trauma including CPB. Furthermore, these findings will guide us in further research on the clinical usefulness of these biomarkers.

Gradient-dependent inhibition of stimulatory signaling from platelet G protein-coupled receptors.

As platelet activation is an irreversible and potentially harmful event, platelet stimulatory signaling must be tightly regulated to ensure the filtering-out of inconsequential fluctuations of agonist concentrations in the vascular milieu. Herein, we show that platelet activation via G protein-coupled receptors is gradient-dependent, i.e., determined not only by agonist concentrations per se but also by how rapidly concentrations change over time. We demonstrate that gradient-dependent inhibition is a common feature of all major platelet stimulatory G protein-coupled receptors, while platelet activation via the non-G protein-coupled receptor glycoprotein VI is strictly concentration-dependent. By systematically characterizing the effects of variations in temporal agonist concentration gradients on different aspects of platelet activation, we demonstrate that gradient-dependent inhibition of protease-activated receptors exhibits different kinetics, with platelet activation occurring at lower agonist gradients for protease-activated receptor 4 than for protease-activated receptor 1, but shares a characteristic bimodal effect distribution, as gradient-dependent inhibition increases over a narrow range of gradients, below which aggregation and granule secretion is effectively shut off. In contrast, the effects of gradient-dependent inhibition on platelet activation via adenosine diphosphate and thromboxane receptors increase incrementally over a large range of gradients. Furthermore, depending on the affected activation pathway, gradient-dependent inhibition results in different degrees of refractoriness to subsequent autologous agonist stimulation. Mechanistically, our study identifies an important role for the cyclic adenosine monophosphate-dependent pathway in gradient-dependent inhibition. Together, our findings suggest that gradient-dependent inhibition may represent a new general mechanism for hemostatic regulation in platelets.

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.

Flow cytometry-based platelet function testing is predictive of symptom burden in a cohort of bleeders.

Platelet function disorders (PFDs) are common in patients with mild bleeding disorders (MBDs), yet the significance of laboratory findings suggestive of a PFD remain unclear due to the lack of evidence for a clinical correlation between the test results and the patient phenotype. Herein, we present the results from a study evaluating the potential utility of platelet function testing using whole-blood flow cytometry in a cohort of 105 patients undergoing investigation for MBD. Subjects were evaluated with a test panel comprising two different activation markers (fibrinogen binding and P-selectin exposure) and four physiologically relevant platelet agonists (ADP, PAR1-AP, PAR4-AP, and CRP-XL). Abnormal test results were identified by comparison with reference ranges constructed from 24 healthy controls or with the fifth percentile of the entire patient cohort. We found that the abnormal test results are predictive of bleeding symptom severity, and that the greatest predictive strength was achieved using a subset of the panel, comparing measurements of fibrinogen binding after activation with all four agonists with the fifth percentile of the patient cohort (p = 0.00008, hazard ratio 8.7; 95% CI 2.5-40). Our results suggest that whole-blood flow cytometry-based platelet function testing could become a feasible alternative for the investigation of MBDs. We also show that platelet function testing using whole-blood flow cytometry could provide a clinically relevant quantitative assessment of platelet-related hemostasis.

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.

Copeptin Release in Cardiac Surgery-A New Biomarker to Identify Risk Patients?

OBJECTIVE: To describe the dynamics of copeptin in open cardiac surgery during the perioperative course. DESIGN: Prospective cohort study. SETTING: Single tertiary hospital. PARTICIPANTS: Twenty patients scheduled for open cardiac surgery procedures with cardiopulmonary bypass (CPB). INTERVENTIONS: No intervention. MEASUREMENTS AND MAIN RESULTS: Copeptin concentrations were measured pre-, peri-, and postoperatively until day 6 after surgery. Patients were analyzed as a whole cohort (n = 20) and in a restricted "normal cohort" consisting of patients with normal preoperative copeptin concentration (<10 pmol/L) and perioperative uneventful course (n = 11). In the whole cohort, preoperative copeptin concentration was 7.0 pmol/L (interquartile range: 3.1-11 pmol/L). All patients had an early rise of copeptin, with 80% having peak copeptin concentration at weaning from CPB or upon arrival in the intensive care unit. Patients in the "normal cohort" had copeptin concentration at weaning from CPB of 194 pmol/L (98-275), postoperative day 1, 27 pmol/L (18-31); and day 3, 8.9 pmol/L (6.3-12). CONCLUSIONS: Regardless of cardiac surgical procedure and perioperative course, all patients had an early significant rise of copeptin concentrations, generally peaking at weaning from CBP or upon arrival in the intensive care unit. Among patients with normal copeptin concentration preoperatively and uneventful course, the postoperative copeptin concentrations decreased to normal values within 3-to-4 days after cardiac surgery. Furthermore, the restricted "normal cohort" generally tended to display lower levels of copeptin concentration postoperatively. Further studies may evaluate whether copeptin can be a tool in identifying risk patients in cardiac surgery.

Biochemical Foundations of Health and Energy Conservation in Hibernating Free-ranging Subadult Brown Bear Ursus arctos.

Brown bears (Ursus arctos) hibernate for 5-7 months without eating, drinking, urinating, and defecating at a metabolic rate of only 25% of the summer activity rate. Nonetheless, they emerge healthy and alert in spring. We quantified the biochemical adaptations for hibernation by comparing the proteome, metabolome, and hematological features of blood from hibernating and active free-ranging subadult brown bears with a focus on conservation of health and energy. We found that total plasma protein concentration increased during hibernation, even though the concentrations of most individual plasma proteins decreased, as did the white blood cell types. Strikingly, antimicrobial defense proteins increased in concentration. Central functions in hibernation involving the coagulation response and protease inhibition, as well as lipid transport and metabolism, were upheld by increased levels of very few key or broad specificity proteins. The changes in coagulation factor levels matched the changes in activity measurements. A dramatic 45-fold increase in sex hormone-binding globulin levels during hibernation draws, for the first time, attention to its significant but unknown role in maintaining hibernation physiology. We propose that energy for the costly protein synthesis is reduced by three mechanisms as follows: (i) dehydration, which increases protein concentration without de novo synthesis; (ii) reduced protein degradation rates due to a 6 °C reduction in body temperature and decreased protease activity; and (iii) a marked redistribution of energy resources only increasing de novo synthesis of a few key proteins. The comprehensive global data identified novel biochemical strategies for bear adaptations to the extreme condition of hibernation and have implications for our understanding of physiology in general.

Caveats in studies of the physiological role of polyphosphates in coagulation.

Platelet-derived polyphosphates (polyP), stored in dense granule and released upon platelet activation, have been claimed to enhance thrombin activation of coagulation factor XI (FXI) and to activate FXII directly. The latter claim is controversial and principal results leading to these conclusions are probably influenced by methodological problems. It is important to consider that low-grade contact activation is initiated by all surfaces and is greatly amplified by the presence of phospholipids simulating the procoagulant membranes of activated platelets. Thus, proper use of inhibitors of the contact pathway and a careful choice of materials for plates and tubes is important to avoid artefacts. The use of phosphatases used to degrade polyP has an important drawback as it also degrades the secondary activators ADP and ATP, which are released from activated platelets. In addition, the use of positively charged inhibitors, such as polymyxin B, to inhibit polyP in platelet-rich plasma and blood is problematic, as polymyxin B also slows coagulation in the absence of polyP. In conclusion we hope awareness of the above caveats may improve research on the physiological roles of polyP in coagulation.

Platelet Function Determined by Flow Cytometry: New Perspectives?

Flow cytometry enables studies of several different aspects of platelet function in response to a variety of platelet agonists. This can be done using only a small volume of whole blood, and also in blood with low platelet counts. These properties, together with the increasing number of flow cytometers available in hospitals worldwide, make flow cytometry an interesting option for laboratories interested in studies of platelet function in different clinical settings. This review focuses on practical issues regarding the use of flow cytometry for platelet function testing. It provides an overview of available activation markers, platelet agonists, and experimental setup issues. The review summarizes previous experience and factors important to consider to perform high-quality platelet function testing by flow cytometry. It also discusses its current use and possibilities and challenges for future use of flow cytometry in clinical settings.

Inflammatory and endothelial markers during the menstrual cycle.

BACKGROUND: The menstrual cycle exhibits a pattern of repeated inflammatory activity. The present study aims to evaluate inflammatory and endothelial markers during the two phases of a menstrual cycle. METHODS: The study cohort consisted of 102 women with regular menstrual cycles. Inflammatory and endothelial markers (interleukin-6 [IL-6], pentraxin-3 [PTX-3], hs-C reactive protein [hs-CRP], sE-selectin, sP-selectin, intracellular and vascular cell adhesion molecules [ICAM-1 and VCAM-1] and cathepsins L, B and S) were measured during the early follicular and the late luteal phase of a normal menstrual cycle. RESULTS: Pentraxin-3 (PTX-3) and hs-CRP were significantly higher during the follicular phase compared to the luteal phase (p < 0.001 respectively p = 0.025). The other inflammatory and endothelial markers, with the exception of cathepsin B, were higher, albeit not significantly, during the follicular phase. CONCLUSIONS: Inflammatory activity, expressed mainly by members of the pentraxin family, is higher during the early follicular compared to the luteal phase. This could be associated to menstruation but the exact mechanisms behind this pattern are unclear and might involve the ovarian hormones or an effect on hepatocytes.

Increased Thrombopoiesis and Platelet Activation in Hantavirus-Infected Patients.

BACKGROUND: Thrombocytopenia is a common finding during viral hemorrhagic fever, which includes hemorrhagic fever with renal syndrome (HFRS). The 2 main causes for thrombocytopenia are impaired thrombopoiesis and/or increased peripheral destruction of platelets. In addition, there is an increased intravascular coagulation risk during HFRS, which could be due to platelet activation. METHODS: Thrombopoiesis was determined by quantification of platelet counts, thrombopoietin, immature platelet fraction, and mean platelet volume during HFRS. The in vivo platelet activation was determined by quantification of soluble P-selectin (sP-selectin) and glycoprotein VI (sGPVI). The function of circulating platelets was determined by ex vivo stimulation followed by flow cytometry analysis of platelet surface-bound fibrinogen and P-selectin exposure. Intravascular coagulation during disease was determined by scoring for disseminated intravascular coagulation (DIC) and recording thromboembolic complications. RESULTS: The levels of thrombopoietin, immature platelet fraction, and mean platelet volume all indicate increased thrombopoiesis during HFRS. Circulating platelets had reduced ex vivo function during disease compared to follow-up. Most interestingly, we observed significantly increased in vivo platelet activation in HFRS patients with intravascular coagulation (DIC and thromboembolic complications) as shown by sP-selectin and sGPVI levels. CONCLUSIONS: HFRS patients have increased thrombopoiesis and platelet activation, which contributes to intravascular coagulation.

The Intrinsic Fragility of DNA (Nobel Lecture).

Our cells contain common molecules, such as water or oxygen, that can damage DNA. In his studies Tomas Lindahl has shown how specific repair enzymes remove and replace damaged parts of DNA in a process of vital importance.

Putting polyphosphates to the test: evidence against platelet-induced activation of factor XII.

The recent claim that stimulated platelets activate the intrinsic pathway of coagulation by the release of polyphosphates has been considered a breakthrough in hemostasis research. In little more than 3 years, the original publication by Müller et al has been cited >100 times. However, none of the citing articles has sought to independently validate this potentially paradigm-shifting concept. To this end, we performed extensive experimentation in vitro and in vivo in an attempt to verify the claim that factor XII (FXII) is primarily activated by stimulated platelets. In contrast to the original assertion, platelet-derived polyphosphates were found to be weak activators of FXII, with a FXIIa-generating activity of <10% compared with equivalent concentrations of kaolin. Using different coagulation assays, it was shown that platelet contribution to whole blood coagulation was unrelated to the generation of activated FXII in vitro. Additionally, key results used to verify the hypothesis in the original study in vivo were found to be irreproducible. We conclude that platelet-derived polyphosphates are not physiologically relevant activators of FXII.

Assays of different aspects of haemostasis - what do they measure?

Haemostasis is a complex process affected by many factors including both cellular and plasma components. It is a multistep process starting with platelet adhesion to damaged endothelium and ending in clot fibrinolysis. There are several methods available to study different aspects of haemostasis including adhesion, aggregation, coagulation and fibrinolysis. This review describes the different methods, what aspects of haemostasis they measure and their limitations. Methods discussed include methods to study adhesion (e.g. PFA-100, cone and platelet(let) analyzer and perfusion chambers) and aggregation (e.g. Multiplate, VerifyNow and Plateletworks). Furthermore the principles behind viscoelastic haemostatic assays are presented as well as methods that can analyse aspects of haemostasis in plasma or platelet-rich-plasma samples (thrombin generation, overall haemostasis potential and Thrombodynamics Analyzer).