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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.

Tuftsin (Thr-Lys-Pro-Arg) is a natural immunomodulating peptide. We have investigated for the presence of a specific tuftsin receptor on murine Kupffer cells using fluorescein-labeled tuftsin, which retains full biological activity. After incubation with fluorescein-labeled tuftsin, Kupffer cells displayed clear binding of this compound on the plasma membrane. Excess tuftsin inhibited this binding, indicating the presence of specific tuftsin receptors on the Kupffer cells. We then investigated the effect of tuftsin on the phagocytic activity of these cells. Phagocytosis assays were performed on 24-well plates between murine Kupffer cells and fluorescent microspheres. The greatest stimulatory effect of tuftsin on percent phagocytic cells over the control value was observed when the cells were incubated with particles at 1 microgram/ml tuftsin for 15 min at 37 degrees C with a particle-to-Kupffer cell ratio of 50:1. Tuftsin also markedly increased the number of particles engulfed by Kupffer cells under the same conditions. These results indicate that Kupffer cells have specific tuftsin receptors; thus tuftsin can stimulate phagocytic activity of Kupffer cells, which constitute the majority of macrophages in the host and are situated strategically in the liver for host defense.

Tuftsin (Thr-Lys-Pro-Arg), a natural immunomodulating peptide originally found to stimulate phagocytosis by polymorphonuclear leukocytes (PMNs), is now known to bind to both PMNs and monocyte-macrophages, affecting their phagocytosis and other functions. The potential roles of tuftsin in surgery-related infections have been documented using animal models. However, there have been some difficulties in demonstrating the phagocytosis-stimulating activity of tuftsin. In view of this, we have developed a suitable human PMN phagocytosis assay for tuftsin and performed preliminary kinetic studies. The assay was performed on 24-well plates between PMNs and fluorescent microspheres. The greatest effect of tuftsin over the control was observed under the following conditions: 15 min incubation at 37 degrees C with 5 micrograms/ml tuftsin and a 50:1 ratio of particle to PMN. Particles bound on the surface of PMNs were removed by washing and trypsin treatment, followed by centrifugation through fetal bovine serum. This allowed us to utilize flow cytometry in this study. A flow cytometric procedure was then successfully adapted to human PMN phagocytosis that established a high correlation between microscopic evaluation and flow cytometry of phagocytosis. In addition to the above determinations of the percentage of phagocytic cells, we evaluated the effect of tuftsin on the number of particles engulfed by PMNs. Under the above optimum conditions, tuftsin has greater impact on the number of particles engulfed than on the percentage of phagocytic cells.

Tuftsin, Thr-Lys-Pro-Arg, is a natural immunomodulating peptide originally found as a phagocytosis-stimulating factor for polymorphonuclear leukocytes. The peptide is now known to elicit various other activities including antimicrobial, antiviral and antitumor effects in vivo. Preliminary human studies revealed antitumor and anti-AIDS activities. In view of this clinical potential, we performed studies on the stability of sterile saline solutions of tuftsin stored at 25 degrees C, 5 degrees C, -20 degrees C or -70 degrees C utilizing a phagocytosis assay and analytical HPLC. Tuftsin stored at -20 degrees C or -70 degrees C gradually lost its phagocytosis-stimulating activity; tuftsin stored at 25 degrees C or 5 degrees C lost its phagocytosis activity rather rapidly. By 6 months, tuftsin stored at 25 degrees C lost all activity, and by 12 months, it significantly inhibited phagocytosis. In contrast, HPLC analysis indicated very minor changes. While tuftsin stored at 25 degrees C lost almost all activity by 6 months, we detected appearance of only 1% impurities by HPLC. This study indicates that if its activity is to be preserved, solutions of tuftsin must be frozen for storage.

1. [Leu1]tuftsin was reported to have greater phagocytosis-stimulating activity than tuftsin (Thr-Lys-Pro-Arg). 2. However, a study on inactivation of tuftsin by polymorphonuclear leukocytes (PMNs) demonstrated that leucine aminopeptidase, an ecto-enzyme, located on PMN surface was responsible for this mechanism. 3. Since leucine aminopeptidase is known to cleave Leu more easily than Thr at the N-terminal position of peptides, this suggested to us that [Leu1]tuftsin might then be inactivated by PMNs more easily than tuftsin, and thus this analog might be less active than tuftsin. 4. In addition, many tuftsin preparations used in earlier studies were not fully active, as high-performance liquid chromatography was not available to separate out many contaminating diastereomers. 5. In view of this, we have synthesized and purified [Leu1]tuftsin and compared its phagocytosis-stimulating activity with tuftsin. 6. Our results indicate that [Leu1]tuftsin is not as active as tuftsin in stimulating phagocytosis.

Tuftsin (Thr-Lys-Pro-Arg), a natural immunomodulating peptide originally found to stimulate phagocytosis by polymorphonuclear leukocytes (PMN), is now known to bind to both PMN and monocyte-macrophages, affecting many of their functions. Administration of tuftsin induces leukocytosis in vivo. We have recently observed that while tuftsin remains in the cytoplasm upon binding and internalization in human PMNs, it translocates into the human monocyte nucleus, suggesting that tuftsin may directly affect growth of monocytes. We have therefore examined the effect of tuftsin on [3H]thymidine incorporation in fractions of murine splenocytes to identify a cell population responding to tuftsin. Tuftsin showed the greatest effect in [3H]thymidine incorporation of splenocytes over controls at optimum conditions of 2% fetal bovine serum and 1 microgram/ml of tuftsin. Splenocyte fractionation by Lymphocyte Separation Medium indicated that tuftsin primarily affects the mononuclear cell fraction; further fractionation revealed that tuftsin affects mostly the monocytes that adhered to plastic. We subsequently further purified the splenic monocytes by repeated plastic adhesion and Percoll gradient separation, to show that tuftsin increases [3H]thymidine incorporation of these highly purified monocytes.