Pall T. Onundarson

Apolipoprotein(a) genetic sequence variants associated with systemic atherosclerosis and coronary atherosclerotic burden but not with venous thromboembolism.

OBJECTIVES The purpose of this study is investigate the effects of variants in the apolipoprotein(a) gene (LPA) on vascular diseases with different atherosclerotic and thrombotic components. BACKGROUND It is unclear whether the LPA variants rs10455872 and rs3798220, which correlate with lipoprotein(a) levels and coronary artery disease (CAD), confer susceptibility predominantly via atherosclerosis or thrombosis. METHODS The 2 LPA variants were combined and examined as LPA scores for the association with ischemic stroke (and TOAST [Trial of Org 10172 in Acute Stroke Treatment] subtypes) (effective sample size [n(e)] = 9,396); peripheral arterial disease (n(e) = 5,215); abdominal aortic aneurysm (n(e) = 4,572); venous thromboembolism (n(e) = 4,607); intracranial aneurysm (n(e) = 1,328); CAD (n(e) = 12,716), carotid intima-media thickness (n = 3,714), and angiographic CAD severity (n = 5,588). RESULTS LPA score was associated with ischemic stroke subtype large artery atherosclerosis (odds ratio [OR]: 1.27; p = 6.7 × 10(-4)), peripheral artery disease (OR: 1.47; p = 2.9 × 10(-14)), and abdominal aortic aneurysm (OR: 1.23; p = 6.0 × 10(-5)), but not with the ischemic stroke subtypes cardioembolism (OR: 1.03; p = 0.69) or small vessel disease (OR: 1.06; p = 0.52). Although the LPA variants were not associated with carotid intima-media thickness, they were associated with the number of obstructed coronary vessels (p = 4.8 × 10(-12)). Furthermore, CAD cases carrying LPA risk variants had increased susceptibility to atherosclerotic manifestations outside of the coronary tree (OR: 1.26; p = 0.0010) and had earlier onset of CAD (-1.58 years/allele; p = 8.2 × 10(-8)) than CAD cases not carrying the risk variants. There was no association of LPA score with venous thromboembolism (OR: 0.97; p = 0.63) or intracranial aneurysm (OR: 0.85; p = 0.15). CONCLUSIONS LPA sequence variants were associated with atherosclerotic burden, but not with primarily thrombotic phenotypes.

Activated and Total Coagulation Factor VII, and Fibrinogen in Coronary Artery Disease

Fibrinogen (FBG) and total coagulation factor VII (FVIIc) concentrations are higher in those patients with coronary artery disease who are at increased future risk of acute ischemic events. The relationship between activated factor VII (FVIIa) and cardiovascular events, however, has not been intensively studied. Data were collected from 401 consecutive patients who underwent coronary angiography because of suspected coronary artery disease. Conventional risk factors FVIIc, FVIIa and FBG were assessed in relation to the severity of coronary artery disease, left ventricular ejection fraction, and previous clinical events. A strong positive correlation was found between FVIIa and FVIIc (p < 0.001), but neither FVIIa nor FVIIc correlated with FBG. No correlation was found between FVIIa, FVIIc or FBG levels and stenosis score for the severity of coronary artery disease, and all were similar in patients with stable or unstable angina pectoris. Multivariate regression analysis showed FVIIc to be higher in women (p = 0.004), and positively related to triglycerides (p = 0.001) and HDL cholesterol (p = 0.006), but not to a previous myocardial infarction or total cholesterol. FVIIa, on the other hand, was lower in patients with a previous myocardial infarction (p = 0.004), higher in women (p = 0.001) and those that previously had undergone percutaneous transluminal coronary angioplasty (p = 0.039), and positively related to total cholesterol (p = 0.011), duration of coronary artery disease (p = 0.032), and smoking (p = 0.008). FBG was positively associated with a previous myocardial infarction (p = 0.013), hypertension (p = 0.016), smoking (p = 0.005), and the thrombocyte count (p < 0.001). Finally, stepwise logistic regression analysis verified a previous myocardial infarction to be negatively associated with FVIIa (p = 0.03), and positively with FBG (p = 0.03), total cholesterol (p = 0.02), and the severity of coronary artery disease (p < 0.001). In conclusion, in patients suspected of coronary artery disease undergoing cardiac catheterization, FVIIa was decreased and FBG increased in those who had a previous myocardial infarction. FVIIa, FVIIc, or FBG levels were not, however, related to the severity of coronary artery disease, and they were similar in patients with stable or unstable angina pectoris.

Warfarin anticoagulation intensity in specialist-based and in computer-assisted dosing practice

The efficacy and safety of oral anticoagulation (OA) with vitamin K antagonists depends on maintaining anticoagulation intensity, measured as international normalized ratio (INR), within defined target ranges. We assessed the quality of our current software‐assisted warfarin dosing in the year 2006 in 941 unselected consecutive patients on stable OA with atrial fibrillation (AF), venous thromboembolism (VTE) and prosthetic heart valves (PHV) by comparing it to our previous cardiologist‐based dosing practice in 1992 when a study was carried out on 241 comparable patients. Over 14 years, the time within target range increased in all three anticoagulated groups, i.e. in AF patients from 46% to 81%, in VTE patients from 62% to 84% and in patients with PHV from 40% to 64%. Only 1% of the treatment time is now spent at INR < 1.5 compared to 7% previously (P < 0.0001) and 0.4% of the treatment time at INR > 4.0 presently compared to 2.8% in the past (P < 0.0001). INR‐targets are better achieved with the current software‐assisted dosing practice and extreme low and high values are less common than previously. The results provide indirect evidence suggesting that both efficacy and safety has improved with the current practice.

The combination of recombinant factor VIIa and fibrinogen correct clotting ex vivo in patient samples obtained following cardiopulmonary bypass surgery.

Cardiac surgery involving cardio pulmonary bypass (CPB) may be associated with development of a coagulopathy that increases risk of bleeding. In the present ex vivo study we investigated the influence of fibrinogen and rFVIIa, alone or in combination, on whole blood coagulation thromboelastometry using pre- and postoperative blood samples from 18 consecutive adult patients undergoing CPB surgery. Dynamic thromboelastometric clotting profiles were recorded using citrated whole blood activated with trace amounts of tissue factor (Innovin, final dilution 1:17000). Blood samples were collected before surgery (control) and postoperative samples were obtained following in vivo neutralization of heparin with protamine sulphate. All blood samples were treated with heparinase to ensure neutralization of possible residual heparin effect. The post-operative blood samples were spiked with buffer, rFVIIa (2 microg/mL), fibrinogen (1 mg/mL), or the combination of rFVIIa and fibrinogen. Despite neutralization of heparin, CPB surgery left a measurable coagulopathy that was thromboelastometrically characterized by prolonged onset of clotting, reduced maximum velocity of clot formation (MaxVel), and decreased maximum clot firmness (MCF). Ex vivo spiking of the postoperative samples with rFVIIa shortened the clotting time. Fibrinogen also shortened the clotting time and, in addition, improved the MaxVel, and MCF. Finally, adding the combination of rFVIIa and fibrinogen to the postoperative samples corrected all thromboelastometric parameters to the preoperative range. In conclusion, the correction of whole blood clotting abnormalities that occurs with rFVIIa and/or fibrinogen suggests that future clinical trials on treatment of bleeding during CPB surgery should study the haemostatic effect of fibrinogen or possibly the combination of rFVIIa and fibrinogen.

A rare IL33 loss-of-function mutation reduces blood eosinophil counts and protects from asthma

IL-33 is a tissue-derived cytokine that induces and amplifies eosinophilic inflammation and has emerged as a promising new drug target for asthma and allergic disease. Common variants at IL33 and IL1RL1, encoding the IL-33 receptor ST2, associate with eosinophil counts and asthma. Through whole-genome sequencing and imputation into the Icelandic population, we found a rare variant in IL33 (NM_001199640:exon7:c.487-1G>C (rs146597587-C), allele frequency = 0.65%) that disrupts a canonical splice acceptor site before the last coding exon. It is also found at low frequency in European populations. rs146597587-C associates with lower eosinophil counts (β = -0.21 SD, P = 2.5×10–16, N = 103,104), and reduced risk of asthma in Europeans (OR = 0.47; 95%CI: 0.32, 0.70, P = 1.8×10–4, N cases = 6,465, N controls = 302,977). Heterozygotes have about 40% lower total IL33 mRNA expression than non-carriers and allele-specific analysis based on RNA sequencing and phased genotypes shows that only 20% of the total expression is from the mutated chromosome. In half of those transcripts the mutation causes retention of the last intron, predicted to result in a premature stop codon that leads to truncation of 66 amino acids. The truncated IL-33 has normal intracellular localization but neither binds IL-33R/ST2 nor activates ST2-expressing cells. Together these data demonstrate that rs146597587-C is a loss of function mutation and support the hypothesis that IL-33 haploinsufficiency protects against asthma.

Risk of excessive bleeding associated with marginally low von Willebrand factor and mild platelet dysfunction

Summary.  Background: Bleeding symptoms are so commonly reported that it is not known whether they associate causally or coincidentally with mild but measurable primary hemostatic defects. Objectives/patients/methods: In order to evaluate if the mild primary hemostatic defects are truly causative of increased bleeding symptoms, we surveyed a population of healthy teenagers for bleeding symptoms. Using a case–control approach, we then estimated the risk of excessive bleeding associated with low von Willebrand factor (defined as VWF below the 5th percentile of a normal reference population), and with mild platelet dysfunction [PD, defined as concurrent reduced platelet aggregation responses to two agonists (adenosine diphosphate and epinephrine)]. Results: Excessive bleeding was present in 63 out of 809 teenagers (7.8%). Among the 49 cases who were tested for VWF, low values by three measures were more commonly present than in 166 controls, specifically, ristocetin cofactor (RCo) activity [20.4% vs. 5.4%, odds ratio (OR) 4.5], collagen binding (14.3% vs. 4.2%, OR 3.8), and antigen level (20.4% vs. 6.0%, OR 4.0). The low RCo values ranged from 35 to 45 U dL−1 except for a single case with 26 U dL−1. Of the 47 teenagers with excessive bleeding who underwent platelet aggregation studies, reduced responses were more common than in controls (12.8% vs. 4.4%, OR 3.2). Twenty‐nine per cent of cases with excessive bleeding had either low RCo or PD. Conclusion: Almost one in three teenagers who report excessive bleeding is likely to have a measurable hemostatic disturbance manifested either by marginally low VWF (by three measures) or by mild PD.

Critical role of factors II and X during coumarin anticoagulation and their combined measurement with a new Fiix-prothrombin time

Vitamin K antagonists (VKA) are monitored with prothrombin time (PT) based assays that are equally sensitive to reductions in factors II, VII or X. We compared the effect of vitamin K dependent (VKD) coagulation factors on PT and also on rotational thromboelastometric (ROTEM) parameters. The PT was equally sensitive to reductions in factors II, VII or X but ROTEM parameters correlated poorly with the PT in anticoagulated patients´ plasmas. ROTEM parameters were more affected by mild and moderate reductions in FII or FX than by FVII or FIX which had little influence except at very low coagulant activity. We developed a modified PT that was sensitive only to reductions in factors II and X. The Fiix-PT (Fiix-INR) correlated well with PT (INR) but the Fiix-INR fluctuated less than the INR in an anticoagulated patient reflecting its insensitivity to FVII. The ROTEM results suggest that mild to moderate reductions in factors II or X are more important during clot formation than factors VII or IX. Reductions in FII and X may better reflect anticoagulation with VKA than FVII or IX. The new Fiix-PT may more accurately reflect the degree of therapeutic anticoagulation in patients treated with VKA than the current PT which is subject to a confounding variation caused by FVII.

Fiix-prothrombin time versus standard prothrombin time for monitoring of warfarin anticoagulation: a single centre, double-blind, randomised, non-inferiority trial

BACKGROUND Rapid fluctuations in factor VII during warfarin anticoagulation change the international normalised ratio (INR) but contribute little to the antithrombotic effect. We aimed to assess non-inferiority of anticoagulation stabilisation with a warfarin monitoring method affected only by factors II and X (Fiix-prothrombin time [Fiix-PT]) compared with standard PT-INR monitoring that includes factor VII measurement as well. METHODS The Fiix trial was a single centre, double-blind, prospective, non-inferiority, randomised controlled clinical trial. Ambulatory adults on warfarin with an INR target of 2-3 managed by an anticoagulation dosing service using software-assisted dosing at the National University Hospital of Iceland, Reykjavik, Iceland, were eligible for inclusion in this study. We excluded patients undergoing electroconversion and nursing home residents. Patients were randomly assigned (1:1) to either the Fiix-PT monitoring group or the PT monitoring group by block randomisation. A blinded research INR (R-INR) based on results of the respective test was reported to the dosing staff. Participants were contacted by a study nurse at 4-week intervals to elicit information about thromboembolism or bleeding otherwise unknown to the anticoagulation management centre. The primary efficacy outcome was a composite of objectively diagnosed non-fatal and fatal arterial or venous thromboembolism, including myocardial infarction and transient ischaemic attacks, assessed in all eligible patients who were randomised (intention-to-monitor population). The safety endpoint was major bleeding or other clinically relevant bleeding, assessed in the per-protocol population. We assumed a 3% annual thromboembolism incidence and a non-inferiority margin of 2·5%. This trial is registered with ClinicalTrials.gov, number NCT01565239. FINDINGS Between March 1, 2012, and Feb 28, 2014, we enrolled 1156 patients. 573 patients were assigned to Fiix-PT and 575 to PT-INR monitoring after exclusion of four patients from each group for various reasons. Median follow-up was 1·7 years (IQR 1·1-1·9). During days 1-720, ten (1·2% per patient year) thromboembolic events occurred in the Fiix-PT group versus 19 (2·3% per patient year) in the PT group (relative risk [RR] 0·52, 95% CI 0·25-1·13; pnon-inferiority<0·0001). Major bleeding occurred in 17 of 571 patients in the Fiix group (2·2% per patient year) versus 20 of 573 patients in the PT group (2·5% per patient year; RR 0·85, 0·45-1·61; pnon-inferiority=0·0034). Anticoagulation stability was improved with Fiix-PT monitoring as manifested by fewer tests, fewer dose adjustments, increased time in range and less INR variability than reported with standard PT monitoring. INTERPRETATION Monitoring of warfarin with Fiix-PT improved anticoagulation and dosing stability and was clinically non-inferior to PT monitoring. Results from this trial suggest that during vitamin K antagonist treatment INR monitoring could be replaced by Fiix-PT and that this would lead to at least a non-inferior clinical outcome compared with monitoring with PT-INR. FUNDING Innovation Center Iceland, University of Iceland Science Fund, Landspitali Science Fund and Actavis.

Quantification of menstrual flow by weighing protective pads in women with normal, decreased or increased menstruation

Objective. To investigate the association between menstrual flow weight measured from modern sanitary pads (converting liquid to non‐evaporating gel) and clinically assessed normal, increased or decreased menstrual flow. Design. Objective method development study. Setting. Outpatient clinic, University Hospital, Reykjavik. Population. One hundred and thirteen volunteers included 26 normally menstruating adult women and 52 normally menstruating teenagers not using oral or intrauterine contraception, seven normally menstruating women using oral contraception, 17 women with clinically diagnosed menorrhagia, five women using oral contraception for clinical menorrhagia, and six teenage girls claiming heavy menstrual flow. Methods. Menstruation length, menstrual flow weight and history of iron deficiency were assessed. During the menstruation following recruitment, all women collected their used protective pads in a hygienic manner and returned them to the laboratory for accurate weighing. Main outcome measures. Menstrual flow total weight measured in grams. Results. Mean menstrual flow total weight in the 78 asymptomatic women was 51 g (median 44, range 5–144). The mean flow in 17 women clinically diagnosed with menorrhagia was 217 g (median 207, range 63 – 402) (p<0.0001 compared to healthy women). The seven healthy women using oral contraceptives discharged 13 g (13–19) (p = 0.0004 compared with normals). Menstruation lasted

Clinical phenotype in heterozygote and biallelic Bernard‐Soulier syndrome—A case control study

Bernard‐Soulier syndrome (BSS) is a rare severe autosomal recessive bleeding disorder. To date heterozygous carriers of BSS mutations have not been shown to have bleeding symptoms. We assessed bleeding using a semi‐quantitative questionnaire, platelet parameters, PFA‐100 closure times, ristocetin response, GP Ib/IX expression and VWF antigen in 14 BSS patients, 30 heterozygote carriers for related mutations and 29 controls. Eight mutations in GP1BA, GP1BB or GP9 were identified including four previously unknown pathogenic mutations. Subjects with BSS reported markedly more mucocutaneous bleeding than controls. Increased bleeding was also observed in heterozygotes. Compared to controls, patients with BSS had lower optical platelet counts (P < 0.001), CD61‐platelet counts (P < 0.001) and higher mean platelet volume (17.7 vs. 7.8 fL, P < 0.001) and ristocetin response and closure times were unmeasurable. Heterozygotes had higher MPV (9.7 fL, P < 0.001) and lower platelet counts (P < 0.001) than controls but response to ristocetin and closure times were normal. The VWF was elevated in both BSS and in heterozygotes (P = 0.005). We conclude that heterozygotes for BSS mutations have lower platelet counts than controls and show a bleeding phenotype albeit much milder than in BSS. Both patients with BSS and heterozygote carriers of pathogenic mutations have raised VWF. Am. J. Hematol. 90:149–155, 2015.