Chemical and pharmacological aspects of neutralization of heparins from different animal sources by protamine

Essentials

• Bovine (HBI) and porcine (HPI) heparins differ in structure and anticoagulant activity.
• Protamine‐neutralization was evaluated on a variety of physical‐chemical methods.
• HBI requires more protamine than HPI to fully neutralize its anticoagulant activity.
• Protamine preferentially removes higher‐sulfated chains of HBI while HPI is evenly precipitated.

Summary

Background

Protamine neutralization is an essential step for the safe use and inactivation of the unfractionated heparin (UFH) that is widely employed in surgical and non‐surgical procedures involving extracorporeal circulation.

Objective

To compare protamine neutralization of different pharmaceutical‐grade UFHs prepared from porcine or bovine intestine (HPI and HBI, respectively). HBI has approximately half the anticoagulant potency of HPI, mostly as consequence of its fraction enriched with N‐sulfated α‐glucosamine disaccharides.

Methods

Protamine neutralization of HPI and HBI was evaluated with in vitro, ex vivo and in vivo assays. We also performed in‐depth assessments of the complexation of protamine with these distinct UFHs by using nuclear magnetic resonance and mass spectroscopy.

Results

HPI and HBI interact similarly with protamine on a mass/mass basis; however, HBI requires more protamine than HPI to have its anticoagulant activity fully neutralized, because of its lower potency, which entails the use of higher doses. Nuclear magnetic resonance spectra revealed that HPI precipitates homogeneously with protamine. On the other hand, the low‐sulfated fraction of HBI, enriched with N‐sulfated α‐glucosamine, precipitates at higher concentrations of protamine than the fraction more like HPI, with a preponderance of N,6‐disulfated α‐glucosamine disaccharides. Finally, mass spectroscopy spectra showed that some of the different peptide components of protamine interact preferentially with the heparins, irrespective of their animal origin.

Conclusion

Our results have important medical implications, indicating that protamine neutralization of HBI, determined exclusively by point‐of‐care coagulation assessments, must fail because of its lower‐sulfated fraction with reduced anticoagulant activity that could remain in the circulation after the neutralization procedure.

     

Expression and functional characterization of two natural heparin‐binding site variants of antithrombin

Essentials

• Heparin‐binding site (HBS) variants of antithrombin (AT) are associated with thrombosis risk.
• HSB variants have, in general, normal progressive inhibitory activity but reduced heparin affinity.
• Thrombosis in HSB carriers has been primarily attributed to the loss of heparin cofactor activity.
• Results here demonstrate that HSB variants of AT also lack anti‐inflammatory signaling functions.

Summary

Background

Several heparin‐binding site (HBS) variants of antithrombin (AT) have been identified that predispose carriers to a higher incidence of thrombosis. Thrombosis in carriers of HBS variants has been primarily attributed to a loss in their heparin‐dependent anticoagulant function.

Objective

The objective of this study was to determine whether HSB mutations affect the anti‐inflammatory functions of variants.

Methods

Two HBS variants of AT (AT‐I7N and AT‐L99F), which are known to be associated with a higher incidence of thrombosis, were expressed in mammalian cells and purified to homogeneity. These variants were characterized by kinetic assays followed by analysis of their activities in established cellular and/or in vivo inflammatory models. The possible effects of mutations on AT structure were also evaluated by molecular modeling.

Results

The results indicated that, whereas progressive inhibitory activities of variants were minimally affected, their heparin affinity and inhibitory activity in the presence of heparin were markedly decreased. Unlike wild‐type AT, neither AT variant was capable of inhibiting activation of nuclear factor‐κB or downregulation of expression of cell adhesion molecules in response to lipopolysaccharide (LPS). Similarly, neither variant elicited barrier protective activity in response to LPS. Structural analysis suggested that the L99F substitution locally destabilizes AT structure.

Conclusions

It is concluded that the L99F mutation of AT is associated with destabilization of the serpin structure, and that the loss of anti‐inflammatory signaling function of the HBS variants may also contribute to enhanced thrombosis in carriers of HBS mutations.

     

Mortality due to bleeding,myocardial infarction and stroke in dialysis patients

Essentials

• Mortality due to bleeding vs. arterial thrombosis in dialysis patients is unknown.
• We compared death causes of 201 918 dialysis patients with the general population.
• Dialysis was associated with increased mortality risks of bleeding and arterial thrombosis.
• Clinicians should be aware of the increased bleeding and thrombosis risks.

Summary

Background

Dialysis has been associated with both bleeding and thrombotic events. However, there is limited information on bleeding as a cause of death versus arterial thrombosis as a cause of death.

Objectives

To investigate the occurrence of bleeding, myocardial infarction and stroke as causes of death in the dialysis population as compared with the general population.

Methods

We included 201 918 patients from 11 countries providing data to the ERA‐EDTA Registry who started dialysis treatment between 1994 and 2011, and followed them for 3 years. Age‐standardized and sex‐standardized mortality rate ratios for bleeding, myocardial infarction and stroke as causes of death were calculated in dialysis patients as compared with the European general population. Associations between potential risk factors and these causes of death in dialysis patients were investigated by calculating hazard ratios (HRs) with 95% confidence intervals (CIs) by the use of Cox proportional‐hazards regression.

Results

As compared with the general population, the age‐standardized and sex‐standardized mortality rate ratios in dialysis patients were 12.8 (95% CI 11.9–13.7) for bleeding as a cause of death (6.2 per 1000 person‐years among dialysis patients versus 0.3 per 1000 person‐years in the general population), 13.4 (95% CI 13.0–13.9) for myocardial infarction (22.5 versus 0.9 per 1000 person‐years), and 12.4 (95% CI 11.9–12.9) for stroke (14.3 versus 0.7 per 1000 person‐years).

Conclusion

Dialysis patients have highly increased risks of death caused by bleeding and arterial thrombosis as compared with the general population. Clinicians should be aware of the increased mortality risks caused by these conditions.

     

Preoperative platelet transfusions to reverse antiplatelet therapy for urgent non‐cardiac surgery: an observational cohort study

Essentials

• An increasing number of patients requiring surgery receive antiplatelet therapy (APT).
• We analyzed 181 patients receiving presurgery platelet transfusions to reverse APT.
• No coronary thrombosis occurred after platelet transfusion.
• This justifies a prospective trial to test preoperative platelet transfusions to reverse APT.

Summary

Background

Patients receiving antiplatelet therapy (APT) have an increased risk of perioperative bleeding and cardiac adverse events (CAE). Preoperative platelet transfusions may reduce the bleeding risk but may also increase the risk of CAE, particularly coronary thrombosis in patients after recent stent implantation.

Objectives

To analyze the incidence of perioperative CAE and bleeding in patients undergoing non‐cardiac surgery using a standardized management of transfusing two platelet concentrates preoperatively and restart of APT within 24–72 h after surgery.

Methods

A cohort of consecutive patients on APT treated with two platelet concentrates before non‐cardiac surgery between January 2012 and December 2014 was retrospectively identified. Patients were stratified by the risk of major adverse cardiac and cerebrovascular events (MACCE). The primary objective was the incidence of CAE (myocardial infarction, acute heart failure and cardiac troponine T increase). Secondary objectives were incidences of other thromboembolic events, bleedings, transfusions and mortality.

Results

Among 181 patients, 88 received aspirin, 21 clopidogrel and 72 dual APT. MACCE risk was high in 63, moderate in 103 and low in 15 patients; 67 had cardiac stents. Ten patients (5.5%; 95% CI, 3.0–9.9%) developed a CAE (three myocardial infarctions, four cardiac failures and three troponin T increases). None was caused by coronary thrombosis. Surgery‐related bleeding occurred in 22 patients (12.2%; 95% CI, 8.2–17.7%), making 12 re‐interventions necessary (6.6%; 95% CI, 3.8–11.2%).

Conclusion

Preoperative platelet transfusions and early restart of APT allowed urgent surgery and did not cause coronary thromboses, but non‐thrombotic CAEs and re‐bleeding occurred. Randomized trials are warranted to test platelet transfusion against other management strategies.

     

Abrogating fibrinolysis does not improve bleeding or rFVIIa/rFVIII treatment in a non‐mucosal venous injury model in haemophilic rodents

Essentials

• The efficacy of systemic antifibrinolytics for hemophilic non‐mucosal bleeding is undetermined.
• The effect of systemically inhibiting fibrinolysis in hemophilic mice and rats was explored.
• Neither bleeding nor the response to factor treatment was improved after inhibiting fibrinolysis.
• The non‐mucosal bleeding phenotype in hemophilia A appears largely unaffected by fibrinolysis.

Summary

Background

Fibrinolysis may exacerbate bleeding in patients with hemophilia A (HA). Accordingly, antifibrinolytics have been used to help maintain hemostatic control. Although antifibrinolytic drugs have been proven to be effective in the treatment of mucosal bleeds in the oral cavity, their efficacy in non‐mucosal tissues remain an open question of significant clinical interest.

Objective

To determine whether inhibiting fibrinolysis improves the outcome in non‐mucosal hemophilic tail vein transection (TVT) bleeding models, and to determine whether a standard ex vivo clotting/fibrinolysis assay can be used as a predictive surrogate for in vivo efficacy.

Methods

A highly sensitive TVT model was employed in hemophilic rodents with a suppressed fibrinolytic system to examine the effect of inhibiting fibrinolysis on bleeding in non‐mucosal tissue. In mice, induced and congenital hemophilia models were combined with fibrinolytic attenuation achieved either genetically or pharmacologically (tranexamic acid [TXA]). In hemophilic rats, tail bleeding was followed by whole blood rotational thromboelastometry evaluation of the same animals to gauge the predictive value of such assays.

Results

The beneficial effect of systemic TXA therapy observed ex vivo could not be confirmed in vivo in hemophilic rats. Furthermore, neither intravenously administered TXA nor congenital knockout of the fibrinolytic genes encoding plasminogen or tissue‐type plasminogen activator markedly improved the TVT bleeding phenotype or response to factor therapy in hemophilic mice.

Conclusions

The findings here suggest that inhibition of fibrinolysis is not effective in limiting the TVT bleeding phenotype of HA rodents in non‐mucosal tissues.

     

Rivaroxaban reversal with prothrombin complex concentrate or tranexamic acid in healthy volunteers

Essentials

• Specific reversal agents for managing severe factor Xa inhibitor‐associated bleeding are lacking.
• We assessed 4‐factor‐prothrombin complex concentrate (4F‐PCC) and tranexamic acid (TXA).
• 4F‐PCC, but not TXA, reduced the prothrombin time and increased endogenous thrombin potential.
• These agents may be viable options for reversal of therapeutic doses of rivaroxaban.

Summary

Background

Oral activated factor X inhibitors such as rivaroxaban are widely used, but specific reversal agents are lacking. Although four‐factor prothrombin complex concentrate (4F‐PCC) and tranexamic acid (TXA) are sometimes used to manage serious bleeding, their efficacy is unknown. Prior studies in healthy subjects taking rivaroxaban revealed that 4F‐PCC partially reverses the prolonged prothrombin time (PT), and fully restores the endogenous thrombin potential (ETP). The effect of TXA has not been evaluated.

Methods

In this double‐blind, parallel‐group study, 147 healthy volunteers given rivaroxaban 20 mg twice daily for 3 days were randomized after their morning dose on day 4 to receive intravenous 4F‐PCC (50 IU kg^?1), TXA (1.0 g), or saline. Standardized punch biopsies were performed at baseline and after 4F‐PCC, TXA or saline administration. Reversal was assessed by measuring bleeding duration and bleeding volume at biopsy sites, and by determining the PT and ETP.

Results

As compared with saline, 4F‐PCC partially reversed the PT and completely reversed the ETP, whereas TXA had no effect. Neither 4F‐PCC nor TXA reduced bleeding duration or volume. All treatments were well tolerated, with no recorded adverse events.

Conclusions

Although 4F‐PCC reduced the PT and increased the ETP in volunteers given supratherapeutic doses of rivaroxaban, neither 4F‐PCC nor TXA influenced punch biopsy bleeding.

     

The fibrinogen prothrombin time‐derived method is not useful in patients anticoagulated with low molecular weight heparins or rivaroxaban

Essentials

• Fibrinogen prothrombin time‐derived (FIBPT‐d) behavior in anticoagulated patients is under studied.
• FIBPT‐d method overestimates fibrinogen in rivaroxaban and low molecular weight heparin samples.
• Unfractionated heparin and dabigatran samples showed similar bias to the control group.
• Rabbit brain and human recombinant thromboplastin behavior was different in rivaroxaban samples.

Summary

Background

The fibrinogen prothrombin time‐derived (FIBPT‐d) method with photo‐optical coagulometers is easy and economical. However, there are few reports on the behavior of this test on samples from patients anticoagulated with direct oral anticoagulants or low molecular weight heparin (LMWH).

Objective

To compare fibrinogen results obtained with the Clauss (FIB C) method and the FIBPT‐d method with two thromboplastins in anticoagulated patients.

Population

The study population comprised 295 consecutive anticoagulated patients: 99 treated with vitamin K antagonists (VKAs), 49 treated with unfractionated heparin (UFH), 47 treated with LMWH, 50 treated with rivaroxaban, 50 treated with dabigatran, and 100 normal controls (NCs).

Methods

Dabigatran samples were analyzed by the use of FIB C with HemosIL Fibrinogen C or 100 NHI thrombin units mL^?1 reagents; rabbit brain and human recombinant thromboplastins with HemosIL PTFibrinogen HS plus (HS) and Recombiplastin 2G (RP) were used for FIBPT‐d method. Heparin and rivaroxaban levels were assessed with HemosIL Liq antiXa with specific calibrators; dabigatran levels were determined with the HemosIL Direct Thrombin Inhibitor Assay. All assays were performed on the ACL TOP platform in two laboratories. Percentage biases for the FIBPT‐d method versus the FIB C method were calculated by the use of Bland–Altman plots.

Results

Positive biases of the FIBPT‐d method versus the FIB C method with both thromboplastins were seen in NC samples (13.7% and 18.9% for HS and RP, respectively), but biases with HS in rivaroxaban and VKA patient samples were higher than that in NC samples, at 31.9% and 34.0%, respectively. LMWH patient samples showed higher bias than NC samples: 26.5% and 29.3.0% with HS and RP, respectively. UFH and dabigatran patient samples showed similar bias as NC samples.

Conclusion

The FIBPT‐d method should not be used in anticoagulated patients, because the FIBPT‐d mathematical algorithm has been validated only in normal subjects, so overestimation could occur in these patients.

     

Dabigatran versus vitamin k antagonist: an observational across‐cohort comparison in acute coronary syndrome patients with atrial fibrillation

Essentials

• Acute coronary syndrome (ACS) with atrial fibrillation (AF) is a therapeutic challenge.
• Dual and triple antithrombotic therapy showed a similar thrombotic risk in ACS patients with AF.
• The omission of aspirin during the first month did not increase the rate of ischemic events.
• Replacement of vitamin K antagonist by dabigatran leads to an increased thrombotic risk.

Summary

Background

Dual antithrombotic therapy comprising a vitamin K antagonist (VKA) plus clopidogrel reduces the incidence of major bleeding compared with triple therapy (VKA + clopidogrel + aspirin) in acute coronary syndrome (ACS) patients with atrial fibrillation (AF), with a similar thrombotic risk. The oral thrombin inhibitor dabigatran (150 mg twice a day) showed superiority over VKA in non‐valvular AF, but data supporting its use in AF patients presenting with ACS are limited.

Objective

We sought to evaluate the efficacy of dabigatran vs. VKA in the management of AF patients undergoing percutaneous coronary intervention for an ACS.

Methods

In this open‐label study, 133 consecutive patients received dabigatran plus clopidogrel. Another cohort of 133 patients treated with VKA plus clopidogrel was used as the control group.

Results

After propensity score adjustment, the cumulative incidence of major adverse cardiovascular events over 24 months was higher with dabigatran vs. VKA (adjusted hazard ratio, 2.28; 95% confidence interval, 1.46–3.56). Similar rates of major bleeding were found (adjusted hazard ratio, 1.17; 95% confidence interval, 0.46–2.96).

Conclusions

In AF patients presenting with ACS, replacement of VKA by dabigatran concurrently with clopidogrel is associated with an increased thrombotic risk, without a reduction in major bleeding.

     

Extended International Normalized Ratio testing intervals for warfarin‐treated patients

Essentials

• Warfarin typically requires International Normalized Ratio (INR) testing at least every 4 weeks.
• We implemented extended INR testing for stable warfarin patients in six anticoagulation clinics.
• Use of extended INR testing increased from 41.8% to 69.3% over the 3 year study.
• Use of extended INR testing appeared safe and effective.

Summary

Background

A previous single‐center randomized trial suggested that patients with stable International Normalized Ratio (INR) values could safely receive INR testing as infrequently as every 12 weeks.

Objective

To test the success of implementation of an extended INR testing interval for stable warfarin patients in a practice‐based, multicenter collaborative of anticoagulation clinics.

Methods

At six anticoagulation clinics, patients were identified as being eligible for extended INR testing on the basis of prior INR value stability and minimal warfarin dose changes between 2014 and 2016. We assessed the frequency with which anticoagulation clinic providers recommended an extended INR testing interval (> 5 weeks) to eligible patients. We also explored safety outcomes for eligible patients, including next INR values, bleeding events, and emergency department visits.

Results

At least one eligible period for extended INR testing was identified in 890 of 3362 (26.5%) warfarin‐treated patients. Overall, the use of extended INR testing in eligible patients increased from 41.8% in the first quarter of 2014 to 69.3% in the fourth quarter of 2016. The number of subsequent out‐of‐range next INR values were similar between eligible patients who did and did not have an extended INR testing interval (27.3% versus 28.4%, respectively). The numbers of major bleeding events were not different between the two groups, but rates of clinically relevant non‐major bleeding (0.02 per 100 patient‐years versus 0.09 per 100 patient‐years) and emergency department visits (0.07 per 100 patient‐years versus 0.19 per 100 patient‐years) were lower for eligible patients with extended INR testing intervals than for those with non‐extended INR testing intervals.

Conclusions

Extended INR testing for stable warfarin patients can be successfully and safely implemented in diverse, practice‐based anticoagulation clinic settings.

     

High and long‐term von Willebrand factor expression after Sleeping Beauty transposon‐mediated gene therapy in a mouse model of severe von Willebrand disease

Essentials

• von Willebrand disease (VWD) is the most common inherited bleeding disorder.
• Gene therapy for VWD offers long‐term therapy for VWD patients.
• Transposons efficiently integrate the large von Willebrand factor (VWF) cDNA in mice.
• Liver‐directed transposons support sustained VWF expression with suboptimal multimerization.

Summary

Background

Type 3 von Willebrand disease (VWD) is characterized by complete absence of von Willebrand factor (VWF). Current therapy is limited to treatment with exogenous VWF/FVIII products, which only provide a short‐term solution. Gene therapy offers the potential for a long‐term treatment for VWD.

Objectives

To develop an integrative Sleeping Beauty (SB) transposon‐mediated VWF gene transfer approach in a preclinical mouse model of severe VWD.

Methods

We established a robust platform for sustained transgene murine VWF (mVWF) expression in the liver of Vwf^?/? mice by combining a liver‐specific promoter with a sandwich transposon design and the SB100X transposase via hydrodynamic gene delivery.

Results

The sandwich SB transposon was suitable to deliver the full‐length mVWF cDNA (8.4 kb) and supported supra‐physiological expression that remained stable for up to 1.5 years after gene transfer. The sandwich vector stayed episomal (~60 weeks) or integrated in the host genome, respectively, in the absence or presence of the transposase. Transgene integration was confirmed using carbon tetrachloride‐induced liver regeneration. Analysis of integration sites by high‐throughput analysis revealed random integration of the sandwich vector. Although the SB vector supported long‐term expression of supra‐physiological VWF levels, the bleeding phenotype was not corrected in all mice. Long‐term expression of VWF by hepatocytes resulted in relatively reduced amounts of high‐molecular‐weight multimers, potentially limiting its hemostatic efficacy.

Conclusions

Although this integrative platform for VWF gene transfer is an important milestone of VWD gene therapy, cell type‐specific targeting is yet to be achieved.

     

Anti‐C1 domain antibodies that accelerate factor VIII clearance contribute to antibody pathogenicity in a murine hemophilia A model

Essentials

• Inhibitor formation remains a challenging complication of hemophilia A care.
• The Bethesda assay is the primary method used for determining bleeding risk and management.
• Antibodies that block factor VIII binding to von Willebrand factor can increase FVIII clearance.
• Antibodies that increase clearance contribute to antibody pathogenicity.

Summary

Background

The development of neutralizing anti‐factor VIII (FVIII) antibodies remains a challenging complication of modern hemophilia A care. In vitro assays are the primary method used for quantifying inhibitor titers, predicting bleeding risk, and determining bleeding management. However, other mechanisms of inhibition are not accounted for in these assays, which may result in discrepancies between the inhibitor titer and clinical bleeding symptoms.

Objectives

To evaluate FVIII clearance in vivo as a potential mechanism for antibody pathogenicity and to determine whether increased FVIII dosing regimens correct the associated bleeding phenotype.

Methods

FVIII^?/? or FVIII^?/?/von Willebrand factor (VWF)^?/? mice were infused with anti‐FVIII mAbs directed against the FVIII C1, C2 or A2 domains, followed by infusion of FVIII. Blood loss via the tail snip bleeding model, FVIII activity and FVIII antigen levels were subsequently measured.

Results

Pathogenic anti‐C1 mAbs that compete with VWF for FVIII binding increased the clearance of FVIII–mAb complexes in FVIII^?/? mice but not in FVIII^?/?/VWF^?/? mice. Additionally, pathogenic anti‐C2 mAbs that inhibit FVIII binding to VWF increased FVIII clearance in FVIII^?/? mice. Anti‐C1, anti‐C2 and anti‐A2 mAbs that do not inhibit VWF binding did not accelerate FVIII clearance. Infusion of increased doses of FVIII in the presence of anti‐C1 mAbs partially corrected blood loss in FVIII^?/? mice.

Conclusions

A subset of antibodies that inhibit VWF binding to FVIII increase the clearance of FVIII–mAb complexes, which contributes to antibody pathogenicity. This may explain differences in the bleeding phenotype observed despite factor replacement in some patients with hemophilia A and low‐titer inhibitors.

     

Reduced‐dose direct oral anticoagulants in the extended treatment of venous thromboembolism: a systematic review and meta‐analysis

Essentials

• In venous thromboembolism (VTE), benefits of extended treatment are balanced by bleeding risks.
• This is a meta‐analysis of reduced‐dose direct oral anticoagulants (DOACs) in extended treatment.
• Reduced‐dose DOACs are as effective as full anticoagulation with bleeding risks similar to placebo.
• Reduced‐dose DOACs are an attractive option for patients in the extended phase of VTE treatment.

Summary

Background

Extended‐duration anticoagulation is beneficial for preventing recurrent venous thromboembolism (VTE). Reduced‐dose direct oral anticoagulants (DOACs) may be preferable if they preserve efficacy and cause less bleeding. We conducted a systematic review and meta‐analysis of trials comparing reduced‐dose DOACs with full‐dose DOACs and aspirin or placebo in the extended phase of VTE treatment.

Methods

A literature search was conducted by use of the MEDLINE, EMBASE and CINAHL databases, supplemented by hand‐searching. One thousand three hundred and ninety‐nine titles were screened, with data from accepted studies being extracted by two independent reviewers. Major outcomes analyzed included recurrent VTE and major and clinically relevant non‐major bleeding events, presented as risk ratios (RRs) and 95% confidence intervals (CI).

Results

Two trials met the prespecified inclusion criteria. Data from 5847 patients were analyzed for efficacy outcomes, and from 5842 patients for safety outcomes. Reduced‐dose DOACs were as effective as full‐dose treatment in preventing recurrent VTE at 1 year (RR 1.12 [95% CI 0.67–1.87]), and more effective than aspirin or placebo (RR 0.26 [95% CI 0.14–0.46]). Rates of major or clinically relevant non‐major bleeding events were similar between patients receiving reduced‐dose DOACs and and those receiving aspirin or placebo (RR 1.19 [95% CI 0.81–1.77]). There was a trend towards less bleeding when reduced‐dose and full‐dose DOACs were compared (RR 0.74 [95% CI 0.52–1.05]).

Conclusions

Extended‐duration treatment of VTE with reduced‐dose DOACs may be as efficacious as full‐dose treatment, with rates of major bleeding being similar to those in patients receiving treatment with aspirin or placebo, but further long‐term studies are needed.

     

Polymorphism in dural arteriovenous fistula: matrix metalloproteinase‐2‐1306 C/T as a potential risk factor for sinus thrombosis

Essentials

• Sinus thrombosis may play a crucial role in development of dural arteriovenous fistula (DAVF).
• Little is known about the association between gene polymorphism and the development of DAVF.
• MMP‐2‐1306 C/T showed a higher prevalence rate in DAVF cases with sinus thrombosis.
• MMP‐2‐1306C/T polymorphism is likely a potential risk factor for sinus thrombosis in DAVF.

Summary

Background

Dural arteriovenous fistula (DAVF) is a rare but important cerebrovascular disorder in adults. Little is known about the molecular genetic pathogenesis underlying DAVF development.

Objectives

To investigate the associations of gene polymorphisms and DAVF.

Materials and Methods

By the use of real‐time PCR genotyping, seven single‐nucleotide polymorphisms (SNPs) of angiogenesis‐related genes were analyzed in 72 DAVF patients. Pertinent clinical and imaging data were subgrouped on the basis of location (cavernous sinus versus lateral sinus), lesions (single versus multiple), cerebral venous reflux (CVR) grading (Borden I versus Borden II/III), and sinus thrombosis (with versus without).

Results

We found that individuals carrying the polymorphic allele of matrix metalloproteinase (MMP)‐2‐1306 C/T (rs243865) had a significantly increased risk of sinus thrombosis in DAVF (odds ratio 6.2; 95% confidence interval 1.7–22.9). There was a weak difference in associations of tissue inhibitor of metalloproteinase (TIMP)‐2 (rs2277698) gene polymorphism and DAVF patients subgrouped by CVR grading.

Conclusions

These preliminary results indicate that MMP‐2‐1306 C/T, but not MMP‐9, TIMP‐1, TIMP‐2, and vascular endothelial growth factor A SNP variants, is a risk factor for the development of sinus thrombosis in DAVF patients.

     

Genes associated with venous thromboembolism in colorectal cancer patients

Essentials

• The underlying pathophysiological mechanisms behind cancer‐associated thrombosis are unknown.
• We compared expression profiles in tumor cells from patients with and without thrombosis.
• Tumors from patients with thrombosis showed significant differential gene expression profiles.
• Patients with thrombosis had a proinflammatory status and increased fibrin levels in the tumor.

Summary

Background

Venous thromboembolism (VTE) is a frequent complication in patients with cancer, and is associated with significant morbidity and mortality. However, the mechanisms behind cancer‐associated thrombosis are still incompletely understood.

Objectives

To identify novel genes that are associated with VTE in patients with colorectal cancer (CRC).

Methods

Twelve CRC patients with VTE were age‐matched and sex‐matched to 12 CRC patients without VTE. Tumor cells were isolated from surgical samples with laser capture microdissection approaches, and mRNA profiles were measured with next‐generation RNA sequencing.

Results

This approach led to the identification of new genes and pathways that might contribute to VTE in CRC patients. Application of ingenuity pathway analysis indicated significant links with inflammation, the methionine degradation pathway, and increased platelet function, which are all key processes in thrombus formation. Tumor samples of patients with VTE had a proinflammatory status and contained higher levels of fibrin and fibrin degradation products than samples of those without VTE.

Conclusion

This case–control study provides a proof‐of‐principle that tumor gene expression can discriminate between cancer patients with low and high risks of VTE. These findings may help to further unravel the pathogenesis of cancer‐related VTE. The identified genes could potentially be used as candidate biomarkers to select high‐risk CRC patients for thromboprophylaxis.

     

Metabolomic analysis of 92 pulmonary embolism patients from a nested case–control study identifies metabolites associated with adverse clinical outcomes

Essentials

• Risk‐stratification often fails to predict clinical deterioration in pulmonary embolism (PE).
• First‐ever high‐throughput metabolomics analysis of risk‐stratified PE patients.
• Changes in circulating metabolites reflect a compromised energy metabolism in PE.
• Metabolites play a key role in the pathophysiology and risk stratification of PE.

Summary

Background

Patients with acute pulmonary embolism (PE) exhibit wide variation in clinical presentation and outcomes. Our understanding of the pathophysiologic mechanisms differentiating low‐risk and high‐risk PE is limited, so current risk‐stratification efforts often fail to predict clinical deterioration and are insufficient to guide management.

Objectives

To improve our understanding of the physiology differentiating low‐risk from high‐risk PE, we conducted the first‐ever high‐throughput metabolomics analysis (843 named metabolites) comparing PE patients across risk strata within a nested case–control study.

Patients/methods

We enrolled 92 patients diagnosed with acute PE and collected plasma within 24 h of PE diagnosis. We used linear regression and pathway analysis to identify metabolites and pathways associated with PE risk‐strata.

Results

When we compared 46 low‐risk with 46 intermediate/high‐risk PEs, 50 metabolites were significantly different after multiple testing correction. These metabolites were enriched in the following pathways: tricarboxylic acid (TCA) cycle, fatty acid metabolism (acyl carnitine) and purine metabolism, (hypo)xanthine/inosine containing. Additionally, energy, nucleotide and amino acid pathways were downregulated in intermediate/high‐risk PE patients. When we compared 28 intermediate‐risk with 18 high‐risk PE patients, 41 metabolites differed at a nominal P‐value level. These metabolites were enriched in fatty acid metabolism (acyl cholines), and hemoglobin and porphyrin metabolism.

Conclusion

Our results suggest that high‐throughput metabolomics can provide insight into the pathophysiology of PE. Specifically, changes in circulating metabolites reflect compromised energy metabolism in intermediate/high‐risk PE patients. These findings demonstrate the important role metabolites play in the pathophysiology of PE and highlight metabolomics as a potential tool for risk stratification of PE.

     

Acute coronary syndrome remodels the antiplatelet aggregation properties of HDL particle subclasses

Essentials

• HDL subclasses were studied in acute coronary syndrome (ACS).
• HDL2 from ACS patients have better antiplatelet potency than HDL from non ACS subjects.
• ACS remodels the antiplatelet properties of HDL subclasses.
• Oxidized polyunsaturated fatty acids content of HDL is modified by ACS.

Summary

Background

Although HDLs have antithrombotic effects by reducing platelet activation, the relationship between HDL levels and the risk of acute coronary syndrome (ACS) is unclear, as HDL particles are heterogeneous in composition and biological properties.

Objective

To characterize the effects of HDL2 and HDL3 subclasses from ACS patients and non‐coronary artery disease (CAD) subjects on platelet activation.

Methods

We measured platelet aggregation and ex vivo thrombus formation, analyzed signaling pathways by flow cytometry, and performed a targeted lipidomics analysis on HDL subclasses.

Results

Analysis of human platelet aggregation in suspension, adhesion on von Willebrand factor and thrombus formation on collagen under arterial shear demonstrated that HDL2 from ACS patients had higher antiplatelet potency than HDL3 from ACS patients and HDL from non‐CAD subjects. HDL binding to scavenger receptor class B type I was essential for this effect. A lipidomics analysis revealed that HDL2 from ACS patients had more oxidized polyunsaturated fatty acids (PUFAs). An inverse correlation between the concentrations of 9‐hydroxyoctadecadienoic acid (9‐HODE), 13‐hydroxyoctadecadienoic acid (13‐HODE), the eicosapentaenoic acid metabolite 18‐hydroxyeicosapentaenoic acid (18‐HEPE) and hydroxyeicosatetraenoic acid isomers in HDL2 and platelet aggregation was observed. This relationship was further demonstrated by the direct inhibitory effects of 18‐HEPE, 9‐HODE, 13‐HODE, 17‐hydroxydocosahexaenoic acid and 14‐hydroxydocosahexaenoic acid on collagen‐related peptide‐induced platelet aggregation, indicating that oxidized PUFAs contribute to the antithrombotic effect of ACS HDL2.

Conclusions

Our data shed new light on the antiplatelet effects of HDL subclasses, and suggest physiological adaptation through the modulation of HDL properties in ACS patients that may limit their platelet‐dependent thrombotic risk.

     

Less abnormal uterine bleeding with dabigatran than warfarin in women treated for acute venous thromboembolism

Essentials

• Factor Xa inhibitors cause more abnormal menstrual bleeding (AUB) than vitamin‐K antagonists (VKA).
• We analyzed data of AUB in women, evaluating dabigatran versus VKA.
• We observed a 41% lower risk of AUB in women on dabigatran compared to those on VKA.
• Our findings of lower AUB risk on dabigatran should be corroborated in future studies.

Summary

Introduction

Although direct oral anticoagulants (DOACs) are associated with a better safety profile than warfarin in patients with acute venous thromboembolism (VTE), direct factor Xa inhibitors involve a higher risk of abnormal uterine bleeding (AUB). We aimed to determine the risk of AUB during anticoagulation with dabigatran compared with warfarin.

Methods

Post‐hoc analysis of the pooled RE‐COVER studies and the RE‐MEDY trial. Incidences of AUB, based on a defined preferred terms search for adverse events, in female patients aged 18–50 years treated with dabigatran, were compared with those in women treated with warfarin.

Results

Of the 2964 women included in the above‐mentioned trials, 1280 women were in the relevant age category (18–50 years) and included in the current analysis. A total of 643 patients were randomized to treatment with dabigatran and 637 to treatment with warfarin. The overall rate of AUB was 8.1%, 5.9% for the women treated with dabigatran and 9.6% in those treated with warfarin, for an odds ratio for dabigatran‐treated patients of 0.59 (95% confidence interval [CI], 0.39–0.90; P = 0.015). In the dabigatran‐treated patients, three (0.5%) suffered major bleeding (MB) vs. five (0.8%) in the warfarin‐treated patients (HR, 0.65; 95% CI, 0.15–2.72). MB or non‐major relevant bleeding occurred in 30 (4.7%) patients randomized to receive dabigatran and 57 (8.9%) randomized to receive warfarin (HR, 0.53; 95% CI, 0.34–0.83). None of the bleeding events was fatal.

Conclusion

Dabigatran treatment was associated with a significantly (41%) lower risk of AUB than warfarin. Future studies in daily practice are needed to corroborate these findings.

     

Evaluation of a microfluidic flow assay to screen for von Willebrand disease and low von Willebrand factor levels

Essentials

• von Willebrand factor (VWF) function is shear stress dependent.
• Platelet accumulation in a microfluidic assay correlates with VWF levels.
• The microfluidic assay discriminates type 1 von Willebrand disease from healthy controls.
• The microfluidic flow assay detects responses to therapeutic intervention (DDAVP).

Summary

Background

von Willebrand disease (VWD) is a mucocutaneous bleeding disorder with a reported prevalence of 1 in 10 000. von Willebrand factor (VWF) function and platelet adhesion are regulated by hemodynamic forces that are not integrated into most current clinical assays.

Objective

We evaluated whether a custom microfluidic flow assay (MFA) can screen for deficiencies in VWF in patients presenting with mucocutaneous bleeding.

Methods

Whole blood from individuals with mucocutaneous bleeding was assayed in a custom MFA.

Results

Thirty‐two patients with type 1 VWD (10/32) or reported mucocutaneous bleeding were enrolled. The platelet adhesion velocity (r = 0.5978 for 750 s^?1 and 0.6895 for 1500 s^?1) and the maximum platelet surface area coverage (r = 0.5719 for 750 s^?1 and 0.6633 for 1500 s^?1) in the MFA correlated with VWF levels. Furthermore, the platelet adhesion velocity at 750 s^?1 (type 1 VWD, mean 0.0009761, 95% confidence interval [CI] 0.0003404–0.001612; control, mean 0.003587, 95% CI 0.002455–0.004719) and at 1500 s^?1 (type 1 VWD, mean 0.0003585, 95% CI 0.00003914–0.0006778; control, mean 0.003132, 95% CI 0.001565–0.004699) differentiated type 1 VWD from controls. Maximum platelet surface area coverage at 750 s^?1 (type 1 VWD, mean 0.1831, 95% CI 0.03816–0.3281; control, mean 0.6755, 95% CI 0.471–0.88) and at 1500 s^?1 (type 1 VWD, mean 0.07873, 95% CI 0.01689–0.1406; control, mean 0.6432, 95% CI 0.3607–0.9257) also differentiated type 1 VWD from controls. We also observed an improvement in platelet accumulation after 1‐desamino‐8‐d ‐arginine vasopressin (DDAVP) treatment at 1500 s^?1 (pre‐DDAVP, mean 0.4784, 95% CI 0.1777–0.7791; post‐DDAVP, mean 0.8444, 95% CI 0.7162–0.9726).

Conclusions

These data suggest that this approach can be used as a screening tool for VWD.

     

Factor XI promotes hemostasis in factor IX‐deficient mice

Essentials

• Mice lacking factor IX (FIX) or factor XI (FXI) were tested in a saphenous vein bleeding model.
• FIX‐deficient mice displayed a hemostatic defect and FXI‐deficient mice were similar to wild type mice.
• Infusion of FXI or over‐expression of FXI in FIX‐deficient mice improved hemostasis.
• FXI may affect the phenotype of FIX‐deficiency (hemophilia B).

Summary

Background

In humans, deficiency of coagulation factor XI may be associated with a bleeding disorder, but, until recently, FXI‐deficient mice did not appear to have a hemostatic abnormality. A recent study, however, indicated that FXI‐deficient mice show a moderate hemostatic defect in a saphenous vein bleeding (SVB) model.

Objectives

To study the effect of FXI on bleeding in mice with normal levels of the FXI substrate FIX and in mice lacking FIX (a murine model of hemophilia B).

Methods

Wild‐type mice and mice lacking either FIX (F9^?) or FXI (F11^?/?) were tested in the SVB model. The plasma levels of FXI in F11^?/? mice were manipulated by infusion of FXI or its active form FXIa, or by overexpressing FXI by the use of hydrodynamic tail vein injection.

Results

F9^? mice showed a significant defect in the SVB model, whereas F11^?/? mice and wild‐type mice were indistinguishable. Intravenous infusion of FXI or FXIa into, or overexpression of FXI in, F9^? mice improved hemostasis in the SVB model. Overexpression of a FXI variant lacking a FIX‐binding site also improved hemostasis in F9^? mice.

Conclusions

Although we were unable to demonstrate a hemostatic defect in F11^?/? mice in the SVB model, our results support the premise that supraphysiological levels of FXI improve hemostasis in F9^? mice through FIX‐independent pathways.