Effect of Low-Intensity vs Standard-Intensity Warfarin Prophylaxis on Venous Thromboembolism or Death Among Patients Undergoing Hip or Knee Arthroplasty: A Randomized Clinical Trial.

Importance: The optimal international normalized ratio (INR) to prevent venous thromboembolism (VTE) in warfarin-treated patients with recent arthroplasty is unknown. Objective: To determine the safety and efficacy of a target INR of 1.8 vs 2.5 for VTE prophylaxis after orthopedic surgery. Design, Setting, and Participants: The randomized Genetic Informatics Trial (GIFT) of Warfarin to Prevent Deep Vein Thrombosis enrolled 1650 patients aged 65 years or older initiating warfarin for elective hip or knee arthroplasty at 6 US medical centers. Enrollment began in April 2011 and follow-up concluded in October 2016. Interventions: In a 2 × 2 factorial design, participants were randomized to a target INR of 1.8 (n = 823) or 2.5 (n = 827) and to either genotype-guided or clinically guided warfarin dosing. For the first 11 days of therapy, open-label warfarin dosing was guided by a web application. Main Outcomes and Measures: The primary outcome was the composite of VTE (within 60 days) or death (within 30 days). Participants underwent screening duplex ultrasound postoperatively. The hypothesis was that an INR target of 1.8 would be noninferior to an INR target of 2.5, using a noninferiority margin of 3% for the absolute risk of VTE. Secondary end points were bleeding and INR values of 4 or more. Results: Among 1650 patients who were randomized (mean age, 72.1 years; 1049 women [63.6%]; 1502 white [91.0%]), 1597 (96.8%) received at least 1 dose of warfarin and were included in the primary analysis. The rate of the primary composite outcome of VTE or death was 5.1% (41 of 804) in the low-intensity-warfarin group (INR target, 1.8) vs 3.8% (30 of 793) in the standard-treatment-warfarin group (INR target, 2.5), for a difference of 1.3% (1-sided 95% CI, -∞ to 3.05%, P = .06 for noninferiority). Major bleeding occurred in 0.4% of patients in the low-intensity group and 0.9% of patients in the standard-intensity group, for a difference of -0.5% (95% CI, -1.6% to 0.4%). The INR values of 4 or more occurred in 4.5% of patients in the low-intensity group and 12.2% of the standard-intensity group, for a difference of -7.8% (95% CI, -10.5% to -5.1%). Conclusions and Relevance: Among older patients undergoing hip or knee arthroplasty and receiving warfarin prophylaxis, an international normalized ratio goal of 1.8 compared with 2.5 did not meet the criterion for noninferiority for risk of the composite outcome of VTE or death. However, the trial may have been underpowered to meet this criterion and further research may be warranted. Trial Registration: ClinicalTrials.gov Identifier: NCT01006733.

Effect of Genotype-Guided Warfarin Dosing on Clinical Events and Anticoagulation Control Among Patients Undergoing Hip or Knee Arthroplasty: The GIFT Randomized Clinical Trial.

Importance: Warfarin use accounts for more medication-related emergency department visits among older patients than any other drug. Whether genotype-guided warfarin dosing can prevent these adverse events is unknown. Objective: To determine whether genotype-guided dosing improves the safety of warfarin initiation. Design, Setting, and Patients: The randomized clinical Genetic Informatics Trial (GIFT) of Warfarin to Prevent Deep Vein Thrombosis included patients aged 65 years or older initiating warfarin for elective hip or knee arthroplasty and was conducted at 6 US medical centers. Enrollment began in April 2011 and follow-up concluded in October 2016. Interventions: Patients were genotyped for the following polymorphisms: VKORC1-1639G>A, CYP2C9*2, CYP2C9*3, and CYP4F2 V433M. In a 2 × 2 factorial design, patients were randomized to genotype-guided (n = 831) or clinically guided (n = 819) warfarin dosing on days 1 through 11 of therapy and to a target international normalized ratio (INR) of either 1.8 or 2.5. The recommended doses of warfarin were open label, but the patients and clinicians were blinded to study group assignment. Main Outcomes and Measures: The primary end point was the composite of major bleeding, INR of 4 or greater, venous thromboembolism, or death. Patients underwent a screening lower-extremity duplex ultrasound approximately 1 month after arthroplasty. Results: Among 1650 randomized patients (mean age, 72.1 years [SD, 5.4 years]; 63.6% women; 91.0% white), 1597 (96.8%) received at least 1 dose of warfarin therapy and completed the trial (n = 808 in genotype-guided group vs n = 789 in clinically guided group). A total of 87 patients (10.8%) in the genotype-guided group vs 116 patients (14.7%) in the clinically guided warfarin dosing group met at least 1 of the end points (absolute difference, 3.9% [95% CI, 0.7%-7.2%], P = .02; relative rate [RR], 0.73 [95% CI, 0.56-0.95]). The numbers of individual events in the genotype-guided group vs the clinically guided group were 2 vs 8 for major bleeding (RR, 0.24; 95% CI, 0.05-1.15), 56 vs 77 for INR of 4 or greater (RR, 0.71; 95% CI, 0.51-0.99), 33 vs 38 for venous thromboembolism (RR, 0.85; 95% CI, 0.54-1.34), and there were no deaths. Conclusions and Relevance: Among patients undergoing elective hip or knee arthroplasty and treated with perioperative warfarin, genotype-guided warfarin dosing, compared with clinically guided dosing, reduced the combined risk of major bleeding, INR of 4 or greater, venous thromboembolism, or death. Further research is needed to determine the cost-effectiveness of personalized warfarin dosing. Trial Registration: clinicaltrials.gov Identifier: NCT01006733.

High-resolution HLA typing by long reads from the R10.3 Oxford nanopore flow cells.

Nanopore sequencing has been investigated as a rapid and cost-efficient option for HLA typing in recent years. Despite the lower raw read accuracy, encouraging typing accuracy has been reported, and long reads from the platform offer additional benefits of the improved phasing of distant variants. The newly released R10.3 flow cells are expected to provide higher read-level accuracy than previous chemistries. We examined the performance of R10.3 flow cells on the MinION device in HLA typing after enrichment of target genes by multiplexed PCR. We also aimed to mimic a 1-day workflow with 8-24 samples per sequencing run. A diverse collection of 102 unique samples were typed for HLA-A, -B, -C, -DPA1, -DPB1, -DQA1, -DQB1, -DRB1, -DRB3/4/5 loci. The concordance rates at 2-field and 3-field resolutions were 99.5% (1836 alleles) and 99.3% (1710 alleles). We also report important quality metrics from these sequencing runs. Continued research and independent validations are warranted to increase the robustness of nanopore-based HLA typing for broad clinical application.

Performance of commercial platforms for rapid genotyping of polymorphisms affecting warfarin dose.

Initiation of warfarin therapy is associated with bleeding owing to its narrow therapeutic window and unpredictable therapeutic dose. Pharmacogenetic-based dosing algorithms can improve accuracy of initial warfarin dosing but require rapid genotyping for cytochrome P-450 2C9 (CYP2C9) *2 and *3 single nucleotide polymorphisms (SNPs) and a vitamin K epoxide reductase (VKORC1) SNP. We evaluated 4 commercial systems: INFINITI analyzer (AutoGenomics, Carlsbad, CA), Invader assay (Third Wave Technologies, Madison, WI), Tag-It Mutation Detection assay (Luminex Molecular Diagnostics, formerly Tm Bioscience, Toronto, Canada), and Pyrosequencing (Biotage, Uppsala, Sweden). We genotyped 112 DNA samples and resolved any discrepancies with bidirectional sequencing. The INFINITI analyzer was 100% accurate for all SNPs and required 8 hours. Invader and Tag-It were 100% accurate for CYP2C9 SNPs, 99% accurate for VKORC1 -1639/3673 SNP, and required 3 hours and 8 hours, respectively. Pyrosequencing was 99% accurate for CYP2C9 *2, 100% accurate for CYP2C9 *3, and 100% accurate for VKORC1 and required 4 hours. Current commercial platforms provide accurate and rapid genotypes for pharmacogenetic dosing during initiation of warfarin therapy.

The role of the factor X activation peptide: a deletion mutagenesis approach.

To understand the role of the factor X (fX) activation peptide (AP), a deletion mutagenesis approach was employed. Two single-chain, variant enzymes were generated in which 41 residues were deleted from the AP: fX (des-137-183) and fX(des-137-183;N191A), which lacks a carbohydrate moiety at Asn191 due to an alanine substitution. Deletion of the fX AP did not impact fXa catalytic activity. Activation of the variant zymogens, however, was altered. Neither mutant enzyme was activated by the fX coagulant protein from Russell's viper venom (RVV-X(1)). Activation by factor VIIa (fVIIa) and fVIIa in the presence of cofactor, lipidated tissue factor (TF), occurred at an accelerated rate for both variants as compared to wild-type fX (WTfX). Similar to fVII, the mutants auto-activated in a cofactor-independent manner, which was characterized by a lag period and accelerated dose-dependently by plasma fXa (kcat/Km, 0.046 +/- 0.004 micro M(-1) s(-1)). Both mutants were also found to be activated by fVIIa (0.31 +/- 0.03 micro M(-1) s(-1)), fIXa (0.30 +/- 0.03 micro M(-1) s(-1)), and thrombin (0.00078 +/- 0.00015 micro M(-1) s(-1)). In all cases, the rate of activation was faster for fX(des-137-183;N191A) as compared to fX(des-137-183). We propose that the fX AP and Asn191 carbohydrate serve primarily as negative autoregulation mechanisms to prevent spurious activation of fX and secondarily in cofactor dependence and activator specificity.

Expression of the regenerating gene family in inflammatory bowel disease mucosa: Reg Ialpha upregulation, processing, and antiapoptotic activity.

BACKGROUND: The pathophysiology of inflammatory bowel disease (IBD) reflects a balance between mucosal injury related to an ongoing inflammatory process and mucosal reparative mechanisms. Proreparative mucosal factors may offer new therapeutic paradigms. Transcriptional profiling can be applied to identify candidate gene products involved in colonic mucosal regeneration. METHODS: Resection specimens from patients who underwent colonic resection for IBD or non-IBD indications were analyzed by performing Affymetrix GeneChip hybridization (Affymetrix, Inc., Santa Clara, Calif) and histopathologic scoring. Expression and physiologic processing of Reg Ialpha, the most highly expressed member of the regenerating (Reg) gene family, was further studied by performing specific immunohistochemistry, protein sequencing, and mass spectroscopy. RESULTS: Foregut-derived tissues normally express human Reg proteins with minimal expression in the colon. In the setting of tissue injury associated with IBD, Reg Ialpha Reg Ibeta, and Reg III mRNA were highly expressed in colonic mucosa. Paired histopathologic scoring demonstrated that Reg expression was not related to the presence or the degree of mucosal inflammation. Studies of the Reg Ialpha protein revealed evidence of proteolytic cleavage at the N-terminus. In IBD, intact Reg Ialpha protein was expressed by the metaplastic Paneth granular cell population. Whereas Reg Ialpha cleaved at the N-terminus, it was also deposited throughout the lamina propria. Reg Ialpha treatment was shown to reduce epithelial apoptosis that occurred in response to treatment with hydrogen peroxide. CONCLUSION: Ectopic expression, physiologic processing, and directed tissue deposition of Reg Ialpha are components of the colonic mucosal regenerative response in IBD. Reg Ialpha may serve to reduce epithelial apoptosis in inflammation.

Heparin and oversulfated heparin byproduct induce thrombin generation through contact system activation in plasma of patients with HIT.

Heparin-induced thrombocytopenia with thrombosis (HITT) is the most severe side effect of heparin administration. Patients with HITT may die or have permanent sequelae such as stroke or limb amputation. Contaminated heparin is associated with anaphylactic reactions and deaths by activating the contact system. It is also associated with high incidence of HIT via a yet unknown mechanism. This study showed that although oversulfated heparin byproduct induced thrombin activities in both normal and HIT patient plasmas through the contact system activation, authentic heparin induced thrombin activities only in HIT patient plasmas containing autoantibodies against protein/ heparin complex. These data suggest that the negatively charged immunoglobulin G (IgG)/platelet factor 4 (PF4)/heparin complex activate the contact system and produce thrombin in human plasma, and thrombin partially activates the platelets allowing subsequent platelet activation through IgG/Fc receptor II signaling. The newly discovered mechanism of heparin-induced thrombin activity could explain the increased incidence of HIT in patients exposed to contaminated heparin. Furthermore, the assays used in these studies would be valuable for HIT diagnosis, prevention, and treatment.

Gamma-glutamyl carboxylase and its influence on warfarin dose.

Via generation of vitamin K-dependent proteins, gamma-glutamyl carboxylase (GGCX) plays a critical role in the vitamin K cycle. Single nucleotide polymorphisms (SNPs) in GGCX, therefore, may affect dosing of the vitamin K antagonist, warfarin. In a multi-centered, cross-sectional study of 985 patients prescribed warfarin therapy, we genotyped for two GGCX SNPs (rs11676382 and rs12714145) and quantified their relationship to therapeutic dose. GGCX rs11676382 was a significant (p=0.03) predictor of residual dosing error and was associated with a 6.1% reduction in warfarin dose (95% CI: 0.6%-11.4%) per G allele. The prevalence was 14.1% in our predominantly (78%) Caucasian cohort, but the overall contribution to dosing accuracy was modest (partial R2 = 0.2%). GGCX rs12714145 was not a significant predictor of therapeutic dose (p = 0.26). GGCX rs11676382 is a statistically significant predictor of warfarin dose, but the clinical relevance is modest. Given the potentially low marginal cost of adding this SNP to existing genotyping platforms, we have modified our non-profit website (www.WarfarinDosing.org) to accommodate knowledge of this variant.