Factor IX assay discrepancies in the setting of liver gene therapy using a hyperfunctional variant factor IX-Padua.

BACKGROUND: Limited information exists regarding the factor IX (FIX) coagulant activity (FIX:C) measured by different assays following FIX-Padua gene therapy. OBJECTIVE: Assess for the first time FIX:C in five commonly used coagulation assays in plasma samples from hemophilia B subjects receiving FIX-Padua gene transfer. METHODS: FIX:C was compared between central (n = 1) and local laboratories (n = 5) in the study, and across four commonly used FIX:C one-stage assays and one FIX:C chromogenic assay. For comparison, samples of pooled congenital FIX-deficient plasma spiked with purified recombinant human FIX (rHFIX)-Padua protein or rHFIX (nonacog alfa) to obtain FIX:C concentrations from ~20% to ~40% were tested. RESULTS: FIX:C results at local laboratories strongly correlated with central laboratory results. However, absolute values at the central laboratory were consistently lower than those at local laboratories. Across five different FIX:C assays, a consistent pattern of FIX:C was observed for subjects receiving fidanacogene elaparvovec-expressed gene transfer. Use of Actin FSL activated partial thromboplastin time (APTT) reagent in the central laboratory resulted in lower FIX:C values compared with other APTT reagents tested. The chromogenic assay determined lower FIX:C than any of the one-stage assays. The rHFIX-Padua protein-spiked samples showed similar results. In contrast, FIX:C results for rHFIX-nonacog alfa measured within 25% of expected for all one-stage assays and below 25% in the chromogenic assay. CONCLUSIONS: Assay-based differences in FIX:C were observed for fidanacogene elaparvovec transgene product and rHFIX-Padua protein, suggesting the variable FIX:C determined with different assay reagents is inherent to the FIX-Padua protein and is not specific to gene therapy-derived FIX-Padua.

Once-weekly prophylaxis regimen of nonacog alfa in patients with hemophilia B: an analysis of timing of bleeding event onset.

In a pivotal, multicenter, open-label study, 25 patients aged 12-54 years with moderately severe/severe hemophilia B received on-demand nonacog alfa (6 months; dose at investigator's discretion) followed by once-weekly prophylaxis with nonacog alfa 100 IU/kg (12 months). During prophylaxis, patients had a median spontaneous annualized bleeding rate (sABR) of 1.0 and significant reductions in ABR (P < 0.0001). This post hoc analysis examined the time of onset of spontaneous bleeding events (sBEs) and spontaneous target joint bleeding events (sTJBEs). The postdosing day (D) of onset of sBEs observed during prophylaxis and steady-state FIX activity data (FIX:C) between 144 and 196 h postdose were collected at weeks 26 and 78. Twelve patients (48%) had no sBEs; the remaining 13 (52%) had the following onset of sBEs: less than 1 D (0%), 1 to less than 2D (5%), 2 to less than 3 D (22%), 3 to less than 4 D (9%), 4 to less than 5D (22%), 5 to less than 6D (23%), 6 to less than 7D (11%), and at least 7D (8%). Reductions in sBEs and sTJBEs during on-demand versus prophylaxis treatment were experienced by all 13 patients. Target joint sABR during prophylaxis was 0 for 5/13 patients. ABR reduction ranged from 66.1% (27.2→9.2) to 97.8% (46.2→1.0); sTJBE reductions ranged from 6.2% (2.1→2.0) to 100% (from 40.1, 19.1, 3.9, 9.0, 6.1--0). During prophylaxis, 47% (8/17) of trough FIX activity samples were more than 2%. In sBE patients, ABR and number of TJBEs were reduced with once-weekly nonacog alfa. When sBEs occurred, they followed no apparent pattern for day of occurrence. Clinicaltrials.gov identifier: NCT01335061.

Hemophilia B Gene Therapy with a High-Specific-Activity Factor IX Variant.

BACKGROUND: The prevention of bleeding with adequately sustained levels of clotting factor, after a single therapeutic intervention and without the need for further medical intervention, represents an important goal in the treatment of hemophilia. METHODS: We infused a single-stranded adeno-associated viral (AAV) vector consisting of a bioengineered capsid, liver-specific promoter and factor IX Padua (factor IX-R338L) transgene at a dose of 5×1011 vector genomes per kilogram of body weight in 10 men with hemophilia B who had factor IX coagulant activity of 2% or less of the normal value. Laboratory values, bleeding frequency, and consumption of factor IX concentrate were prospectively evaluated after vector infusion and were compared with baseline values. RESULTS: No serious adverse events occurred during or after vector infusion. Vector-derived factor IX coagulant activity was sustained in all the participants, with a mean (±SD) steady-state factor IX coagulant activity of 33.7±18.5% (range, 14 to 81). On cumulative follow-up of 492 weeks among all the participants (range of follow-up in individual participants, 28 to 78 weeks), the annualized bleeding rate was significantly reduced (mean rate, 11.1 events per year [range, 0 to 48] before vector administration vs. 0.4 events per year [range, 0 to 4] after administration; P=0.02), as was factor use (mean dose, 2908 IU per kilogram [range, 0 to 8090] before vector administration vs. 49.3 IU per kilogram [range, 0 to 376] after administration; P=0.004). A total of 8 of 10 participants did not use factor, and 9 of 10 did not have bleeds after vector administration. An asymptomatic increase in liver-enzyme levels developed in 2 participants and resolved with short-term prednisone treatment. One participant, who had substantial, advanced arthropathy at baseline, administered factor for bleeding but overall used 91% less factor than before vector infusion. CONCLUSIONS: We found sustained therapeutic expression of factor IX coagulant activity after gene transfer in 10 participants with hemophilia who received the same vector dose. Transgene-derived factor IX coagulant activity enabled the termination of baseline prophylaxis and the near elimination of bleeding and factor use. (Funded by Spark Therapeutics and Pfizer; ClinicalTrials.gov number, NCT02484092 .).

Emerging and future therapies for hemophilia.

The evolution of care in hemophilia is a remarkable story. Over the last 60 years, advances in protein purification, protein chemistry, donor screening, viral inactivation, gene sequencing, gene cloning, and recombinant protein production have dramatically enhanced the treatment and lives of patients with hemophilia. Recent efforts have produced enhanced half-life (EHL) clotting factors to better support prophylaxis and decrease the frequency of infusions. Medical needs remain in the areas of alternate modes of administration to decrease the need for venous access, better treatment, and prophylaxis for patients who form antibodies to clotting factors, and ultimately a cure of the underlying genetic defect. In this brief review, the authors summarize data on EHL clotting factors, introduce agents whose mode of action is not clotting factor replacement, and list current gene therapy efforts.

Life, limb or off-label recombinant VIIa use in the setting of limited blood assets: a case study.

Due to the lack of adequate controlled trials, the off-label use of recombinant factor VIIa (rFVIIa) to control hemorrhage in trauma patients remains controversial. The decision regarding when to initiate rFVIIa therapy is particularly problematic. Whereas most reports and trials have delayed use until significant bleeding has occurred, there is some evidence that coagulopathy develops early in some trauma patients, raising the possibility that early rFVIIa use may be more clinically efficacious. Herein, we report the case of a hemodynamically unstable patient with massive blood loss from multiple gunshot wounds and who had a potentially salvageable upper extremity. Rapid hemorrhage despite efforts to surgically control the bleeding resulted in virtual exhaustion of the facilities' limited blood component supply. Hemorrhage was controlled when rFVIIa was added to hypotensive resuscitation allowing salvage of the arm and significant conservation of blood products. This case raises the question as to whether earlier off-label use of this agent should be considered when amputation for hemorrhage control is being considered and/or conservation of limited blood assets is needed.

Reversal of clopidogrel-induced bleeding with rFVIIa in healthy subjects: a randomized, placebo-controlled, double-blind, exploratory study.

BACKGROUND: Clopidogrel (Plavix®) therapy, although effective for minimizing risk of thrombotic events, is also associated with potential bleeding risk. Recombinant activated FVII (rFVIIa, NovoSeven®) induces hemostasis in hemophilia patients with inhibitors (alloantibodies) and has been proposed as potential treatment for mitigating clopidogrel therapy-mediated bleeding. METHODS: In this single-center, randomized, placebo-controlled, double-blind, dose-escalation, exploratory phase I trial, we assessed the safety and effects of rFVIIa in reversing clopidogrel-enhanced bleeding in an experimentally induced punch biopsy in healthy subjects. Efficacy assessments included the reversal of bleeding characteristics (bleed duration [BD], the primary end point and blood loss volume [BV] induced by punch biopsy, and thromboelastograph [TEG®] parameters) with rFVIIa or placebo after clopidogrel treatment. RESULTS: A significant number of subjects (56%) had limited response to clopidogrel (defined as ≤30% platelet aggregation inhibition) and were discontinued from study. The remaining subjects continued and had 4 biopsies. Of 40 subjects randomized, 37 were evaluated for efficacy. Clopidogrel treatment increased BD and BV compared with the baseline biopsy. Recombinant FVIIa (10 and 20 µg/kg) significantly mitigated the clopidogrel-induced effects on BV (P = 0.007 and P = 0.001, respectively). Early trial termination limited the evaluation of effects of higher rFVIIa doses. Subgroup analyses of subjects biopsied by the same physician demonstrated significant reduction of clopidogrel-induced BD with 20 µg/kg rFVIIa (P = 0.048). Ex vivo analysis of rFVIIa demonstrated clotting dynamics presented by parameters time to clot onset (TEG®-R) and clot angle (TEG®-A) (P < 0.005). CONCLUSIONS: In this clinical study, rFVIIa (10 and 20 µg/kg) reversed the effect of clopidogrel on blood loss.

Treatment of massive pulmonary embolism in a soldier in Kosovo: the potential value of cardiopulmonary resuscitation and fibrinolytic therapy.

After an 18-hour bus ride, a 29-year-old soldier complained of leg pain. Ten days later, he collapsed. After cardiopulmonary resuscitation (CPR), he revived but complained of chest pain and shortness of breath. Computed tomography revealed massive thrombus in the right pulmonary artery, emboli in the left pulmonary artery, and right ventricle ballooning. Adequate anticoagulation required repeated boluses and continuous infusion (1,600 units/hour) of heparin. Vena caval filter was not available, and possible additional clot in the legs could not be completely assessed. After no improvement in 24 hours, alteplase was given (10 mg IV bolus and 90 mg over 2 hours). At 12 hours, tachycardia, tachypnea, and dyspnea resolved and computed tomography revealed marked resolution. This case illustrates both the value of CPR and aggressive fibrinolytic therapy in patients who suddenly collapse from massive pulmonary embolism. The collapse was likely due to a saddle embolus. Chest compressions probably fractured the large clot. Although not completely reestablished, enough flow occurred for successful resuscitation. Even though delayed, fibrinolytic therapy was effective and should be considered even in patients where vena caval filter placement is not feasible and/or complete evaluation of the extremity deep venous system is not possible.

Exploratory study on the reversal of warfarin with rFVIIa in healthy subjects.

The use of warfarin has a well-known bleeding risk. Recombinant activated factor VII (rFVIIa) is a non-plasma-derived, rapid-acting, and rapidly infused potential treatment. This randomized, single-center, placebo-controlled, double-blinded, dose-escalation, exploratory phase 1 trial assessed safety and effects of rFVIIa in reversing warfarin-induced changes in bleeding and coagulation parameters, using a punch biopsy-induced bleeding model in healthy subjects. The effects of warfarin (experiment 1) and rFVIIa (5-80 microg/kg; experiment 2) were evaluated. Outcomes were bleeding duration, blood loss, coagulation parameters, and safety. Warfarin treatment significantly increased bleeding duration and blood loss from pretreatment (experiment 1, 12 subjects). However, these parameters after rFVIIa treatment were not significantly different from placebo (experiment 2, 85 subjects). Mean activated partial thromboplastin time, prothrombin time, and international normalized ratio were reduced from warfarin-elevated levels. rFVIIa (80 microg/kg) significantly reversed warfarin effects on all thromboelastography parameters, compared with placebo (P < .05), and returned the thrombin generation speed to baseline. There were no thromboembolic or serious adverse events. In this exploratory trial, the reversal of warfarin effects was observed in the thromboelastography, thrombin generation, and clotting assays. However, this reversal did not translate to improvements in the bleeding model parameters evaluated in the punch biopsy model. Trial registration is exempt (phase 1).

Future directions in hemostasis: normalizing the lives of patients with hemophilia.

The ultimate goal of hemophilia research is to cure the disease by permanently replacing the abnormal or deficient clotting factor. The most promising current approach is to insert a functional gene into the patient that will lead to the production of either the native protein or a compatible non-immunogenic equivalent version. This protein will circulate at adequate levels to prevent spontaneous bleeds and to treat traumatic hemorrhage. While there continues to be progress in this area, there remain significant hurdles to allow the process to be done at minimal risk to the patient. In the short term the goals are: 1) to prevent spontaneous bleeding whenever possible through the provision of effective prophylactic therapy, and 2) to rapidly establish hemostasis when bleeding occurs via effective, safe and widely available treatment modalities. In addition, all therapy should be convenient and simple to use so as to allow the vast majority of hemophilia patients to routinely treat themselves. Optimally, such treatment should not require highly trained medical personnel or vascular access. Prophylactic therapy will be facilitated via the development and introduction of clotting factors with prolonged circulating times. Long acting versions of rFVIIa, rFVIII and rFIX are either already in or are about to enter clinical trials. Rapid treatment of bleeding could be facilitated by bio-engineered molecules with increased activity and/or altered binding characteristics. At least one such agent is in phase II clinical trials. Ease of administration may be accomplished by the introduction of room temperature stable products and the development of subcutaneous and even orally administered therapeutics. It is conceivable that a broad spectrum hemostatic agent capable of normalizing thrombin generation in a variety of deficiencies could be developed. If this is accomplished, the steps to a room temperature stable agent, contained within a pre-filled pen, capable of self-delivery by subcutaneous injection are already within the realm of current developmental capabilities.

Creatine kinase BB isoenzyme blood levels in trauma patients with suspected mild traumatic brain injury.

This pilot study sought to determine if creatine kinase isoenzyme BB (CK-BB) levels might serve as a biological marker of injury severity in mild TBI (mTBI). This was a retrospective study of 64 soldiers with suspected mTBI seen in a combat support hospital in Mosul between March and August of 2007. Four of the 64 total samples were positive for CK-BB. One major trauma patient had a negative CK-BB. This yields a sensitivity of 11% and a specificity of 97%. Military Acute Concussion Evaluation (MACE) scores collected also did not appear to reflect extent of injury. Although the low sensitivity of CK-BB from this study does not support its use as an early marker of suspected mTBI, the result is not conclusive given the small sample and the possibility of isoenzyme degradation during transport. Although limited, the data collected on MACE scores warrant additional evaluation of whether this measure is clinically relevant.

The effect of uremia on platelet contractile force, clot elastic modulus and bleeding time in hemodialysis patients.

INTRODUCTION: Uremic bleeding frequently occurs in dialysis patients. Although its mechanism is not well characterized, acquired platelet dysfunction has been implicated in its pathogenesis. Skin bleeding time has been used to characterize platelet dysfunction in this population. However, the bleeding time is prone to error. The goal of this study was to compare the bleeding time to the novel platelet function parameters platelet contractile force and clot elastic modulus as well as platelet aggregation studies in controls and patients receiving maintenance hemodialysis. MATERIALS AND METHODS: Forty-five subjects completed this study (25 controls, 20 dialysis). All subjects had the Ivy skin bleeding time procedure performed, as well as the collection of whole blood samples for the determination of platelet contractile force, clot elastic modulus, % von Willebrand Factor antigen, and platelet aggregation studies. Pearson's correlation determined the relationships between skin bleeding time and platelet function and clot structure parameters and markers of renal dysfunction. RESULTS: Bleeding time was significantly prolonged in the dialysis group relative to controls. The platelet function parameters were not significantly different between groups. There was a significant relationship between bleeding time and creatinine concentration, however, no relationship existed between bleeding time and platelet function parameters. CONCLUSIONS: Skin bleeding time poorly correlates with measurements of platelet function. There were no significant differences noted in platelet function between the groups despite the prolongations in bleeding time in the dialysis group. These data may suggest that the bleeding time reflects perturbations in platelet adhesion or secretion, and not aggregation. Further study is needed to characterize platelet function in dialysis patients.

The pharmacokinetics of enoxaparin do not correlate with its pharmacodynamic effect in patients receiving dialysis therapies.

The pharmacokinetics and pharmacodynamics of enoxaparin were studied in healthy volunteers and hemodialysis and peritoneal dialysis subjects. Antifactor Xa activity estimated the pharmacokinetics, whereas thrombin generation time (TGT) estimated the pharmacodynamics. Enoxaparin 1 mg/kg was given subcutaneously to all subjects. Antifactor Xa Amax and AUC(0-12) were similar between groups, but the TGTmax was significantly greater in the dialysis groups (P = .001). The thrombin generation time remained significantly more prolonged throughout the 12-hour study period, and there was a trend toward greater TGT AUEC(0-12) for both dialysis groups (P = .07). Patients receiving hemodialysis had greater sensitivity to enoxaparin compared to the other groups. These results suggest that in dialysis patients, there may be accumulation of active heparin metabolites that are undetected by the antifactor Xa assay. Therefore, these subjects exhibit greater thrombin generation time prolongation despite similar antifactor Xa exposure. Further large-scale studies are needed to corroborate the results of this exploratory pilot study.

Glucose-6-phosphate dehydrogenase deficiency in two returning Operation Iraqi Freedom soldiers who developed hemolytic anemia while receiving primaquine prophylaxis for malaria.

Use of antimalarial prophylaxis continues to be routine practice among military personnel returning from areas where malaria is endemic. Primaquine may be used for terminal prophylaxis against Plasmodium ovale and Plasmodium vivax. Serious complications of this regimen are infrequent. We report the occurrence of significant hemolytic anemia for two soldiers returning from Operation Iraqi Freedom. They presented with dark urine, headaches, and classic laboratory findings of hemolysis. Both soldiers were subsequently found to have glucose-6-phosphate dehydrogenase deficiency, and both responded to conservative treatment and cessation of medication. Although this complication is unusual, medical personnel involved in the care of recently returned deployed service members should be alert to its potential occurrence among patients who are receiving antimalarial prophylaxis. This complication could be completely avoided with prescreening of personnel for glucose-6-phosphate dehydrogenase deficiency, as is currently done in the Air Force and Navy, before the use of primaquine.

A hypothesis-generating study to evaluate platelet activity in diabetics with chronic kidney disease.

BACKGROUND: It is well described that diabetes mellitus is a hypercoagulable state. It is also known that patients with renal dysfunction have impaired platelet aggregation and function. It is not well described how renal dysfunction affects the hypercoagulability associated with diabetes. This post-hoc sub-group analysis compares platelet function, clot structure and thrombin generation time at baseline, and following enoxaparin exposure in three groups of subjects. METHODS: 30 total subjects were evaluated in the three groups: Group I: normal controls (n = 10), Group II: subjects with renal dysfunction but without diabetes (n = 13), and Group III: subjects with concomitant diabetes and renal dysfunction (n = 7). For each subject, platelet contractile force (PCF), clot elastic modulus (CEM) and thrombin generation time (TGT) were simultaneously measured in whole blood at baseline, and following increasing enoxaparin antifactor Xa activity exposure. The group means for each parameter were determined and compared using one-way analysis of variance, with post-hoc Tukey-Kramer test. RESULTS: At baseline, subjects in Group III (diabetics with concomitant renal dysfunction) display significantly enhanced platelet activity, as measured by PCF (p = 0.003) and CEM (p = 0.03), relative to the non-diabetic Groups I and II. Subjects in Group II (renal dysfunction without diabetes) had significantly prolonged TGT values relative to controls when the antifactor Xa activity concentration reached 0.5 (p = 0.007), 1.0 (p = 0.005) and 3.0 IU/mL (p < 0.0001), respectively. There were no differences between Group II and Group III with respect to TGT at these antifactor Xa activity concentrations. When the antifactor Xa activity concentration reached 3.0 IU/mL, Groups II and III formed significantly less rigid blood clots (CEM p = 0.003) and also trended toward reduced PCF (p = 0.06) relative to Group I. CONCLUSION: This hypothesis-generating sub-group analysis suggests that at baseline, patients with concomitant diabetes and renal dysfunction have significantly enhanced platelet activity (PCF), and form more rigid blood clots (CEM) compared to controls and subjects with renal dysfunction but no diabetes. This may suggest that the presence of renal dysfunction does not ameliorate the hypercoagulable state associated with diabetes. Secondly, it appears that subjects with renal dysfunction but without diabetes have an enhanced response to enoxaparin relative to controls.

Monitoring of hemostatic status in four patients being treated with recombinant factor VIIa.

Recombinant Factor VIIa (rVIIa) is a potent hemostatic agent for the management of refractory bleeding in patients with Factor VII deficiency or Factor VIII inhibitors. While the current recommended dose is usually effective, the most appropriate dose remains a subject of debate. Since factor VII levels and shortening of the pro-thrombin time do not appear to correlate with response, an appropriate laboratory marker of clinical response has not been identified. In this article we report changes noted in thrombin generation, platelet function and clot structure in blood from patients treated with rVIIa. Thrombin generation was assessed via a thrombin generation time (TGT) assay using a Hemodyne HAS instrument. Changes in clot structure were assessed as changes in clot elastic modulus in the HAS, changes in maximum amplitude in the TEG and changes in maximum clot firmness in the ROTEG. The cases presented confirmed improvement in thrombin generation with administration of rVIIa. The cases also illustrate that: a) in the factor VII deficient patient, 25% of the 90 microg/kg dose is sufficient to totally correct the defect, b) patients with high level factor VIII inhibitors may require significantly more than the recommended dose of 90 microg/kg, c) thrombin generation may not be completely corrected despite dramatic shortening of the prothrombin time, and d) increasing rVIIa doses does not by itself ensure improved thrombin generation.

Evolving techniques for monitoring clotting in plasma and whole blood samples.

New models of hemostatic function emphasize the importance of clotting factor interactions with cells and highlight the central event of thrombin production. As the coagulation cascade has evolved into a scheme of overlapping phases of initiation, amplification and propagation, the relevance of plasma based coagulation assays are being brought into question. Since platelets are critical to both amplification and propagation of the thrombin signal, assays performed in the absence of platelets would appear to completely miss these events. The lack of sensitivity to platelet influences may explain the inability of the prothrombin time (PT) and the partial thromboplastin time (PTT) to detect/reflect the therapeutic and clinical effects of agents such as recombinant FVIIa. This article reviews several evolving technologies for measuring thrombin generation and hemostatic function in samples of plasma and whole blood. Such assays may better reflect global hemostasis and hold potential for detecting hypo- and hyper-hemostatic states as well as monitoring both hemostatic and anticoagulant agents.

Recombinant Factor VIIa: clinical applications for an intravenous hemostatic agent with broad-spectrum potential.

Recombinant Factor VIIa (NovoSeven, Novo Nordisk) is a unique hemostatic agent with potential for broad-spectrum applications in bleeding patients with congenital and acquired bleeding abnormalities. At present, recombinant Factor VIIa is only approved for the treatment of hemophilia A and B patients who have acquired antibodies to Factors VIII and IX. However, the literature is rapidly expanding indicating that rFVIIa could benefit patients with bleeding due to a variety of etiologies. Unfortunately, the vast majority of these reports are case studies or small-series summaries, and are neither prospective nor controlled. Controlled trials are currently underway in several potential areas of application. While the possibility of thrombotic complications has been a clinical concern, safety data on patients treated to date have not revealed a significant problem. Expansion of the clinical applications for this interesting and important hemostatic agent are hampered by its expense, the limited scope of the US Food and Drug Administration approval for its use, and the absence of an appropriate laboratory monitoring assay.

Hand-foot syndrome in a patient with multiple fire ant stings.

The case is reported of an elderly patient with known previous exposure to fire ant stings, and who presented with hand-foot syndrome (HFS) in the setting of multiple fire ant stings to the lower extremities. Both hands and both feet were red, swollen, and mildly tender. Treatment was with fluocinonide cream, and all symptoms resolved as the classic fire ant skin lesions regressed. HFS was initially reported in association with acute crisis in sickle cell anemia and thalassemia and more recently as a common toxicity of chemotherapy administration. This is the first report of its occurrence in the setting of fire ant envenomization. Although recent literature may indicate a potential therapeutic benefit from COX-2 antagonists, the process appears to be self-limited, and requires only conservative treatment.

Enhanced anticoagulant activity of enoxaparin in patients with ESRD as measured by thrombin generation time.

BACKGROUND: Patients with renal dysfunction who undergo systemic anticoagulation with enoxaparin are at increased risk for bleeding. Although there is decreased renal clearance of enoxaparin in this population, the clinical utility of monitoring antifactor Xa activity is controversial because it is weakly correlated to bleeding. The goal of this study was to investigate the role of other novel anticoagulation markers, such as thrombin generation time, platelet contractile force, and clot elastic modulus, while controlling for antifactor Xa activity in patients with and without renal dysfunction. METHODS: Thirty anticoagulant- and antiplatelet-naive subjects completed this trial (10 controls, 10 patients with chronic kidney disease, and 10 patients with end-stage renal disease [ESRD]). Blood samples were obtained and spiked ex vivo with increasing concentrations of enoxaparin antifactor Xa activity (0.25, 0.5, 1.0, and 3.0 IU/mL). Thrombin generation time, platelet contractile force, and clot elastic modulus were measured in each group at each antifactor Xa activity concentration. RESULTS: Subjects with ESRD had an approximately 50% greater anticoagulant effect, determined by thrombin generation time prolongation, than controls at antifactor Xa activity concentrations of 0.5 to 3.0 IU/mL. This may explain why subjects with ESRD with seemingly therapeutic antifactor Xa levels still experience adverse bleeding. There were no intergroup differences in platelet function, determined by platelet contractile force and clot elastic modulus. CONCLUSION: Antifactor Xa poorly predicts the degree of anticoagulation in patients with ESRD administered low-molecular-weight heparin (LMWH). Thrombin generation time may be a clinically useful anticoagulation monitoring tool to monitor LMWH therapy, especially in patients with renal dysfunction. Additional randomized prospective studies are needed to corroborate these findings.