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 PATIENTSThe 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 MEASURESThe 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.

Summary. Background: Warfarin is commonly prescribed for prophylaxis and treatment of thromboembolism after orthopedic surgery. During warfarin initiation, out-of-range International Normalized Ratio (INR) values and adverse events are common. Methods: In orthopedic patients beginning warfarin therapy, we developed and prospectively validated pharmacogenetic and clinical dose refinement algorithms to revise the estimated therapeutic dose after 4 days of therapy. Results: The pharmacogenetic algorithm used the cytochrome P450 (CYP) 2C9 genotype, smoking status, peri-operative blood loss, liver disease, INR values and dose history to predict the therapeutic dose. The R 2 was 82% in a derivation cohort (n = 86) and 70% when used prospectively (n = 146). The R 2 of the clinical algorithm that used INR values and dose history to predict the therapeutic dose was 57% in a derivation cohort (n = 178) and 48% in a prospective validation cohort (n = 146). In 1 month of prospective follow-up, the percent time spent in the therapeutic range was 7% higher (95% CI: 2.7-11.7) in the pharmacogenetic cohort. The risk of a laboratory or clinical adverse event was also significantly reduced in the pharmacogenetic cohort (Hazard Ratio 0.54; 95% CI: 0.30-0.97). Conclusions: Warfarin dose adjustments that incorporate genotype and clinical variables available after four warfarin doses are accurate. In this non-randomized, prospective study, pharmacogenetic dose refinements were associated with more time spent in the therapeutic range and fewer laboratory or clinical adverse events. To facilitate gene-guided warfarin dosing we created a non-profit website, http://www.WarfarinDosing.org.

Background Warfarin demonstrates wide interindividual variability that is partly mediated by variants in CYP2C9 and VKORC1. Whether variants in CALU (vitamin K reductase regulator) influence warfarin dose is unknown. Methods and Results We resequenced CALU regions in a discovery cohort of dose-outliers: patients with high(>90th percentile, n=55) or low(<10th percentile, n=53) dose requirements(after accounting for known genetic and nongenetic variables). One CALU variant, rs339097, was associated with high-doses(p=0.01). We validated this variant as a predictor of higher warfarin doses in two replication cohorts: 1)496 patients of mixed ethnicity, 2)194 African-American patients. The G allele of rs339097(African-American and Caucasian allele frequency 0.14 and 0.002, respectively), was associated with a 14.5%(SD±7%) greater therapeutic dose(p=0.03) in the first replication cohort and a higher than predicted dose in the second replication cohort(allele frequency=0.14, one-sided p=0.03). Conclusions CALU rs339097 A>G is associated with higher warfarin dose requirements independent of known genetic and nongenetic predictors of warfarin dose in African-Americans.

Herein we describe a recombinant factor X (fX) with a single substitution at position 347 (fXR347N). Activated fXR347N had a reduced affinity for factor Va (fVa), although the catalytic impact of fVa binding remained intact. The mutation was selective as demonstrated by normal activation and inhibition, except in the presence of subsaturating heparin where the rate of inhibition by antithrombin III (ATIII) was 15% of normal. The reactivity of fXaR347N toward prothrombin was equivalent to wild-type fXa (fXaWT) in the absence of fVa and phospholipid. Addition (without phospholipid) of fVa dramatically increased the catalytic efficiency of fXaWT toward prothrombin but had a negligible effect on fXaR347N. On addition of phosphatidylcholine:phosphatidylserine (PC:PS, 3:1) vesicles, fXaR347Ndisplayed an increased catalytic activity in response to fVa, but the apparent affinity for fVa on the phospholipid surface was 5-20-fold lower than that of fXaWT. On an activated platelet surface, however, fXaWT and fXaR347N activated prothrombin similarly. In a competitive binding assay that measures the displacement of radiolabeled fXa from fVa on a phospholipid surface, fXaR347N was approximately 10-fold less effective than fXaWT. Substitution of fXa at position 347 selectively attenuates the interaction between fXa and fVa without affecting its catalytic activity.

We reported previously that residue 347 in activated fX (fXa) contributes to binding of the cofactor, factor Va (fVa) (Rudolph, A. E., Porche-Sorbet, R. and Miletich, J. P. (2000) Biochemistry 39, 2861-2867). Four additional residues that participate in fVa binding have now been identified by mutagenesis. All five resulting fX species, fX R306A , fX E310N , fX R347N , fX K351A , and fX K414A , are activated and inhibited normally. However, the rate of inhibition by antithrombin III in the presence of submaximal concentrations of heparin is reduced for all the enzymes. In the absence of fVa, all of the enzymes bind and activate prothrombin similarly except fXa E310N , which has a reduced apparent affinity (ϳ3-fold) for prothrombin compared with wild type fXa (fXa WT ). In the absence of phospholipid, fVa enhances the catalytic activity of fXa WT significantly, but the response of the variant enzymes was greatly diminished. On addition of 100 nm PC:PS (3:1) vesicles, fVa enhanced fXa WT , fXa R306A , and fXa E310N similarly, whereas fXa R347N , fXa K351A , and fXa K414A demonstrated near-normal catalytic activity but reduced apparent affinity for fVa under these conditions. All enzymes function similarly to fXa WT on activated platelets, which provide saturating fVa on an ideal surface. Loss of binding affinity for fVa as a result of the substitutions in residues Arg-347, Lys-351, and Lys-414 was verified by a competition binding assay. Thus, Arg-347, Lys-351, and Lys-414 are likely part of a core fVa binding site, whereas Arg-306 and Glu-310 serve a less critical role.