During warfarin induction, the Fiix-prothrombin time reflects the anticoagulation level better than the standard prothrombin time.

Essentials Fiix-prothrombin time (PT) monitoring of warfarin measuring factor (F) II and X, is effective. Plasma obtained during warfarin induction and stable phase in Fiix-trial was assayed. Fiix-PT stabilized anticoagulation earlier than monitoring with traditional PT-INR. FVII had little effect on thrombin generation that was mainly determined by FII and FX. SUMMARY: Background The prothrombin time (PT) is equally prolonged by reduction of each of the vitamin K-dependent (VKD) factors (F) II, VII and X. The Fiix-PT is only affected by FII and FX, the main contributors to thrombin generation (TG). Objective To test the hypothesis that variability in warfarin anticoagulation is reduced early during monitoring with the normalized PT-ratio calculated from Fiix-PT (Fiix-International Normalized Ratio [INR]) compared with traditional PT-INR monitoring. Also, that because of its insensitivity to FVII, Fiix-PT more accurately reflects TG when Fiix-INR and PT-INR are discrepant. Methods Samples from Fiix-trial participants monitored with either Fiix-PT or PT were used. VKD coagulation factors and TG were measured in samples from 40 patients during stable anticoagulation and in serial samples obtained from 26 patients during warfarin induction. TG was assessed in relation to selective reduction in single VKD factors. Results During Fiix-warfarin induction full anticoagulation measured as FII or FX activity was achieved at a similar rate to that with PT-warfarin but subsequently stabilized better. Fiix-INR but not PT-INR mirrored total TG during initiation. During induction, FII (R2 = 0.66) and FX (R2 = 0.52) correlated better with TG and with a steeper slope than did FIX (R2 = 0.37) and in particular FVII (R2 = 0.21). In vitro, FII and FX were the main determinants of TG at concentrations observed during VKA anticoagulation, whereas FVII and FIX had little influence. Conclusions Fiix-PT monitoring reduces anticoagulation variability, suggesting that monitoring FVII has a limited role during VKA management. TG is better reflected by Fiix-PT.

Fiix-prothrombin time monitoring improves warfarin anticoagulation outcome in atrial fibrillation: a systematic review of randomized trials comparing Fiix-warfarin or direct oral anticoagulants to standard PT-warfarin.

BACKGROUND: Monitoring warfarin with Fiix-prothrombin time (Fiix-PT), which is only affected by coagulation factors II and X, stabilizes anticoagulation and reduces thromboembolism compared to PT/INR monitoring. We compared outcome in nonvalvular atrial fibrillation (NVAF) patients treated with Fiix-warfarin, direct oral anticoagulants (DOACs), or PT-warfarin. METHODS: A systematic efficacy and safety assessment by retrieving data from the Fiix trial and the four major phase III DOAC trials in NVAF. Prespecified outcomes included stroke and systemic embolism (SSE), SSE and myocardial infarction (MI), major bleeding (MB), composite major vascular events (SSEMI and MB; CMVE), and deaths. We calculated relative risk, 95% CI, and 95% confidence limits (CL) for each outcome and performed meta-analysis using fixed- and random-effects modeling. RESULTS: There were 613 and 628 observation years with Fiix-warfarin and PT-warfarin in the Fiix trial, and 70 628 and 57 962 with DOACs and PT-warfarin in DOAC trials. Populations were comparable although death rates were lower in the Fiix trial. Compared to pooled PT-warfarin, Fiix-warfarin reduced SSE (RR 0.54;95% CI 0.26-1.10/95% CL <1.00), SSEMI (0.51;0.26-0.99/<0.90), MB (RR 0.63;0.37-1.07/<0.99), and CMVE (RR 0.66;0.43-1.00/<0.94). Vascular death was lower (RR 0.13;0.04-0.47/<0.42). Compared to pooled DOACs, Fiix-warfarin consistently had lower point estimates for the RR for efficacy and safety, but only significant for lower death rates (vascular death RR 0.14;0.04-0.49/<0.43). Meta-analysis comparing Fiix-warfarin and DOACs with PT-warfarin consistently found Fiix-warfarin to have the lowest point estimates for efficacy. CONCLUSION: Monitoring warfarin with Fiix-PT reduces risk of vascular events in NVAF patients as much as DOACs. Warfarin monitored with Fiix-PT is an improved anticoagulant.

A single test to assay warfarin, dabigatran, rivaroxaban, apixaban, unfractionated heparin, and enoxaparin in plasma.

UNLABELLED: Essentials Simple and fast assaying of different anticoagulants (ACs) is useful in emergent situations. We used highly diluted prothrombin time (dPT) or highly diluted Fiix-PT (dFiix-PT) to assay ACs. Both tests could quantify target specific anticoagulants and warfarin anticoagulation. Improved results were consistently observed with the dFiix-PT compared with the dPT. SUMMARY: Background Assaying anticoagulants is useful in emergency situations or before surgery. Different specific assays are currently needed depending on the anticoagulant. Objectives We hypothesized that levels of warfarin, dabigatran, rivaroxaban, apixaban, and heparins could be measured with use of the diluted prothrombin time (dPT) and diluted Fiix-PT (dFiix-PT), using highly diluted thromboplastin (TP). The latter test is affected only by reduced levels of active factors II and X but corrects test plasma for other deficiencies Methods Increasing TP dilutions were used to identify suitable dilutions to measure dabigatran, rivaroxaban, apixaban, unfractionated heparin (UFH), and enoxaparin. Calibrators containing known amounts of direct oral anticoagulants (DOACs) were used to make standard curves. Citrated plasma samples were obtained from patients taking warfarin or DOACs with known drug concentrations as determined by specific assays. Results The dFiix-PT at a TP dilution of 1:1156 could be used to measure all of the drugs tested at therapeutic concentrations except for fondaparinux. The dPT achieved the same but required two TP dilutions (1:750 and 1:300). The warfarin effect could be assessed by using dFiix-PT at 1:1156 with a PT ratio identical to the international normalized ratio. Six different TPs yielded similar results, but two were less sensitive. Dabigatran, rivaroxaban, and apixaban could be accurately measured in patient samples using both dilute PT assays, but a better correlation was consistently observed between the dFiix-PT and specific assays than with the dPT. Conclusion The dFiix-PT using a single dilution of TP may be suitable to assess the anticoagulant effects of warfarin, dabigatran, rivaroxaban, apixaban, heparin, and enoxaparin.

ABO-immune complex formation and impact on platelet function, red cell structural integrity and haemostasis: an in vitro model of ABO non-identical transfusion.

BACKGROUND: Transfusion of ABO non-identical platelets has been associated with fatal haemolytic reactions, increased red cell transfusion needs and other adverse effects, but the practice of ABO matching in platelet transfusion is controversial. Immune complexes can be formed from the anti-A and/or anti-B antibodies and ABO soluble antigen(s) present in donor and recipient plasma after ABO non-identical transfusions. We hypothesized that these immune complexes affect recipient red cell structural integrity, platelet function and haemostasis. STUDY DESIGN AND METHODS: Haemolysis, platelet function and haemostatic function were assessed before and after incubation of recipient red cells, platelets and whole blood with normal saline controls, ABO-identical plasma controls or in vitro-generated ABO-immune complexes. RESULTS: ABO-immune complexes caused significantly increased haemolysis (P < 0·001), inhibition of platelet function (P = 0·001) and disruption of clot formation kinetics (P < 0·005) in both group A and O recipient samples. CONCLUSIONS: Substantial changes in platelet function, red cell integrity and haemostasis occur after in vitro exposure to immune complexes. These in vitro findings may explain, in part, previously observed associations of ABO non-identical platelet transfusions with adverse effects including increased red cell transfusion needs, organ failure and mortality.

Treatment of venous thromboembolism in cancer patients with dalteparin for up to 12 months: the DALTECAN Study.

BACKGROUND: Treatment of venous thromboembolism (VTE) in patients with cancer has a high rate of recurrence and bleeding complications. Guidelines recommend low-molecular-weight heparin (LMWH) for at least 3-6 months and possibly indefinitely for patients with active malignancy. There are, however, few data supporting treatment with LMWH beyond 6 months. The primary aim of the DALTECAN study (NCT00942968) was to determine the safety of dalteparin between 6 and 12 months in cancer-associated VTE. METHODS: Patients with active cancer and newly diagnosed VTE were enrolled in a prospective, multicenter study and received subcutaneous dalteparin for 12 months. The rates of bleeding and recurrent VTE were evaluated at months 1, 2-6 and 7-12. FINDINGS: Of 334 patients enrolled, 185 and 109 completed 6 and 12 months of therapy; 49.1% had deep vein thrombosis (DVT); 38.9% had pulmonary embolism (PE); and 12.0% had both on presentation. The overall frequency of major bleeding was 10.2% (34/334). Major bleeding occurred in 3.6% (12/334) in the first month, and 1.1% (14/1237) and 0.7% (8/1086) per patient-month during months 2-6 and 7-12, respectively. Recurrent VTE occurred in 11.1% (37/334); the incidence rate was 5.7% (19/334) for month 1, 3.4% (10/296) during months 2-6, and 4.1% (8/194) during months 7-12. One hundred and sixteen patients died, four due to recurrent VTE and two due to bleeding. CONCLUSION: Major bleeding was less frequent during dalteparin therapy beyond 6 months. The risk of developing major bleeding complications or VTE recurrence was greatest in the first month of therapy and lower over the subsequent 11 months.

Challenges of rare disease research: limited patients and competing priorities.

Rare disease research is increasingly challenging. For those with haemophilia, this is an exciting time, with the promise of new therapies at the bench and in early phase clinical trials. Yet, it is also a time for critical assessment and planning to assure the success of the clinical research effort. As successes at the bench have enabled transition of novel peptides, longer-acting factor products and gene therapy to clinical trials, clinicians face the challenges of limited number of patients, competing priorities and strained resources. To solve these problems and assure the success of the clinical research effort, it is essential that the research process be enabling and the dialogue be global, involving academia with industry, and physicians with patients. This is a critical juncture in the process, especially with new national initiatives in clinical research at hand. Needs must be assessed and priorities must be set to assure that despite the challenges, exciting new therapies will ultimately translate into safe, effective therapies for patients. Finally, these challenges are by no means restricted only to rare disease research. With the evolution of genetic medicine, it is likely that the general medical disease research of the future will include small clinical trials of new agents for small subsets of patients with certain disease mutations. Thus, the milestones we achieve in this ongoing process will hopefully not only enable clinical trials research in a rare disease, but also in many medical genetic disease of the future.

Dabigatran versus enoxaparin for prevention of venous thromboembolism after hip or knee arthroplasty: a pooled analysis of three trials.

BACKGROUND: Three randomized, double-blind trials compared dabigatran, an oral direct thrombin inhibitor, with enoxaparin for the primary prevention of venous thromboembolism (VTE) in patients undergoing elective total hip and knee arthroplasty. OBJECTIVES AND METHODS: We conducted a pre-specified pooled analysis of these trials. 8,210 patients were randomized, of whom 8,135 were treated (evaluable for safety) with dabigatran 220 mg or 150 mg once-daily, or subcutaneous enoxaparin (40 mg once-daily or 30 mg twice-daily). Efficacy analyses were based on the modified intention-to-treat population of 6,200 patients with an evaluable outcome. The common risk difference (RD) of treatment effect between each dabigatran dose and enoxaparin was estimated using fixed-effects models, and statistical heterogeneity was estimated using the I2 statistic. RESULTS: The composite outcome of major VTE (proximal deep vein thrombosis and/or pulmonary embolism) and VTE-related mortality occurred in 3.3% of the enoxaparin group versus 3.0% of the dabigatran 220 mg group (RD vs. enoxaparin -0.2%, 95% CI -1.3% to 0.9%, I2=37%) and 3.8% of the 150 mg group (RD vs. enoxaparin 0.5%, -0.6% to 1.6%, I2=0%). Major bleeding occurred in 1.4% of the enoxaparin group versus 1.4% of the dabigatran 220 mg group (RD vs. enoxaparin -0.2%, -0.8% to 0.5%, I2=40%) and 1.1% of the 150 mg group (RD vs. enoxaparin -0.4%, -1.0% to 0.2%, I2=0%). CONCLUSIONS: Oral dabigatran was as effective as subcutaneous enoxaparin in reducing the risk of major VTE and VTE-related mortality after hip or knee arthroplasty and has a similar bleeding profile.

15-deoxy-Delta12,14 prostaglandin J2-induced heme oxygenase-1 in megakaryocytes regulates thrombopoiesis.

BACKGROUND: Platelet production is an intricate process that is poorly understood. Recently, we demonstrated that the natural peroxisome proliferator-activated receptor gamma (PPARgamma) ligand, 15-deoxy-Delta(12,14) prostaglandin J(2) (15d-PGJ(2)), augments platelet numbers by increasing platelet release from megakaryocytes through the induction of reactive oxygen species (ROS). 15d-PGJ(2) can exert effects independent of PPARgamma, such as increasing oxidative stress. Heme oxygenase-1 (HO-1) is a potent antioxidant and may influence platelet production. OBJECTIVES: To further investigate the influence of 15d-PGJ(2) on megakaryocytes and to understand whether HO-1 plays a role in platelet production. METHODS: Meg-01 cells (a primary megakaryoblastic cell line) and primary human megakaryocytes derived from cord blood were used to examine the effects of 15d-PGJ(2) on HO-1 expression in megakaryocytes and their daughter platelets. The role of HO-1 activity in thrombopoiesis was studied using established in vitro models of platelet production. RESULTS AND CONCLUSIONS: 15d-PGJ(2) potently induced HO-1 protein expression in Meg-01 cells and primary human megakaryocytes. The platelets produced from these megakaryocytes also expressed elevated levels of HO-1. 15d-PGJ(2)-induced HO-1 was independent of PPARgamma, but could be replicated using other electrophilic prostaglandins, suggesting that the electrophilic properties of 15d-PGJ(2) were important for HO-1 induction. Interestingly, inhibiting HO-1 activity enhanced ROS generation and augmented 15d-PGJ(2)-induced platelet production, which could be attenuated by antioxidants. These new data reveal that HO-1 negatively regulates thrombopoiesis by inhibiting ROS.

The PPAR-Platelet Connection: Modulators of Inflammation and Potential Cardiovascular Effects.

Historically, platelets were viewed as simple anucleate cells responsible for initiating thrombosis and maintaining hemostasis, but clearly they are also key mediators of inflammation and immune cell activation. An emerging body of evidence links platelet function and thrombosis to vascular inflammation. peroxisome proliferator-activated receptors (PPARs) play a major role in modulating inflammation and, interestingly, PPARs (PPARbeta/delta and PPARgamma) were recently identified in platelets. Additionally, PPAR agonists attenuate platelet activation; an important discovery for two reasons. First, activated platelets are formidable antagonists that initiate and prolong a cascade of events that contribute to cardiovascular disease (CVD) progression. Dampening platelet release of proinflammatory mediators, including CD40 ligand (CD40L, CD154), is essential to hinder this cascade. Second, understanding the biologic importance of platelet PPARs and the mechanism(s) by which PPARs regulate platelet activation will be imperative in designing therapeutic strategies lacking the deleterious or unwanted side effects of current treatment options.

Fibrinogen synthesized by cancer cells augments the proliferative effect of fibroblast growth factor-2 (FGF-2).

BACKGROUND: Fibroblast growth factor (FGF)-2 is a critical growth factor in normal and malignant cell proliferation and tumor-associated angiogenesis. Fibrinogen and fibrin bind to FGF-2 and modulate FGF-2 functions. Furthermore, we have shown that extrahepatic epithelial cells are capable of endogenous production of fibrinogen. OBJECTIVE: Herein we examined the role of fibrinogen and FGF-2 interactions on prostate and lung adenocarcinoma cell growth in vitro. METHODS: Cell proliferation was measured by (3)H-thymidine uptake and the specificity of FGF-2-fibrinogen interactions was measured using wild-type and mutant FGF-2s, fibrinogen gamma-chain (FGG) RNAi and co-immunoprecipitation. Metabolic labeling, immunopurification and fluorography demonstrated de novo fibrinogen production. RESULTS: FGF-2 stimulated DU-145 cell proliferation, whereas neither FGF-2 nor fibrinogen affected the growth of PC-3 or A549 cells. Fibrinogen augmented the proliferative effect of FGF-2 on DU-145 cells. The role of fibrinogen in FGF-2-enhanced DNA synthesis was confirmed using an FGF-2 mutant that exhibits no binding affinity for fibrinogen. FGG transcripts were present in PC-3, A549 and DU-145 cells, but only PC-3 and A549 cells produced detectable levels of intact protein. RNAi-mediated knockdown of FGG expression resulted in decreased production of fibrinogen protein and inhibited (3)H-thymidine uptake in A549 and PC-3 cells by 60%, which was restored by exogenously added fibrinogen. FGF-2 and fibrinogen secreted by the cells were present in the medium as a soluble complex, as determined by coimmunoprecipitation studies. CONCLUSIONS: These data indicate that endogenously synthesized fibrinogen promotes the growth of lung and prostate cancer cells through interaction with FGF-2.

Effects of pioglitazone on fasting and postprandial levels of lipid and hemostatic variables in overweight non-diabetic patients with coronary artery disease.

OBJECTIVES: To evaluate the effects of pioglitazone on insulin sensitivity and levels of biomarkers associated with thrombotic risk in overweight and obese, non-diabetic subjects with coronary artery disease. BACKGROUND: Little information is available regarding the effects of thiazolidinediones in the absence of diabetes. Further, although postprandial hyperlipemia is a risk factor for cardiovascular diseases, there is limited information about the postprandial effects. METHODS: Twenty overweight and obese, non-diabetic patients with coronary artery disease were enrolled in a randomized, placebo-controlled, double-blind study. Subjects were on atorvastatin for the duration of the study and received either placebo or pioglitazone (45 mg day(-1)) for 12 weeks and then crossed over to the alternative therapy for an additional 12 weeks. Insulin sensitivity, fasting and postprandial levels of lipid, hemostatic, and inflammatory variables were measured, and endothelial function was assessed. RESULTS: Insulin sensitivity improved from 0.03 micromol kg(-1) x min pM(-1) on placebo to 0.04 on pioglitazone (P = 0.0002), and there were decreases in fasting levels of factor (F) VII:C (102 +/- 17% to 92 +/- 18%, P = 0.001), FVII:Ag (68 +/- 12% to 60 +/- 14%, P = 0.01) and in von Willebrand factor (VWF) (174 +/- 94% to 142 +/- 69%, P = 0.01). Pioglitazone lowered postprandial levels of FVII:Ag, FVII:C, plasminogen activator inhibitor-1, VWF, and triglycerides, and increased high-density lipoproteins (+9%, P = 0.02). CONCLUSIONS: Pioglitazone improves insulin sensitivity and favorably modifies fasting and postprandial lipid, hemostatic and inflammatory markers of the metabolic syndrome in overweight and obese non-diabetic patients with coronary artery disease.

Direct thrombin inhibitors for treatment of heparin induced thrombocytopenia, deep vein thrombosis and atrial fibrillation.

Thrombin is a central enzyme in hemostasis, exerting potent procoagulant effects and activating platelets. Recently, several small molecule direct thrombin inhibitors (DTI's) with important clinical applications have been developed. Both lepirudin and argatroban are effective in treatment of heparin-induced thrombocytopenia resulting in rapid normalization of platelet counts and a reduction in thrombotic events. Because of differences in clearance mechanisms, argatroban is preferable in patients with renal insufficiency and lepirudin if there is hepatic impairment. DTI's have also been evaluated in treatment of venous thromboembolism. Small studies with recombinant hirudin have shown promise. Ximelagatran is a new DTI in late-stage clinical trials with advantages for treatment of venous thromboembolism including oral administration and fixed dosing, making it convenient for long-term treatment. A Phase III trial demonstrated that ximelagatran was superior to placebo for preventing recurrent thrombosis in patients who had undergone six months of standard anticoagulant therapy for venous thromboembolism. Another large trial compared ximelagatran to standard treatment with enoxaparin and warfarin for treatment of symptomatic deep vein thrombosis in a Phase III trial of 2,528 patients. The results showed that ximelagatran administered twice daily was as effective as standard treatment in preventing recurrence with no increase in bleeding complications. Ximelagatran has also been evaluated in two Phase III trials in patients with atrial fibrillation. The primary analysis of both showed that ximelagatran was non-inferior to warfarin for preventing stroke and other embolic events with no increase in bleeding complications. Unexpectedly, elevated serum transaminase levels were observed in 5-10% of patients receiving ximelagatran for over 1 month, and routine monitoring may be necessary. The introduction of DTIs represents an important advance in treatment of heparin-induced thrombocytopenia. The oral direct thrombin inhibitor, ximelagatran, shows promise in providing simplified, effective therapy for venous thromboembolism and atrial fibrillation.

Fibrinogen binding potentiates FGF-2 but not VEGF induced expression of u-PA, u-PAR, and PAI-1 in endothelial cells.

Endothelial cell responses at sites of injury occur in a fibrin matrix and are regulated by growth factors including those of the FGF and VEGF families. The pericellular proteolytic balance is important in these responses, and FGF-2 and VEGF up-regulate endothelial cell u-PA, u-PAR and PAI-1. Because both VEGF and FGF-2 bind to fibrinogen, we have examined the capacity of fibrinogen to modulate the up-regulation of these proteins by FGF-2 and VEGF. Confluent cultures of endothelial cells were exposed to FGF-2, VEGF, and fibrinogen or to combinations of growth factors with fibrinogen. Changes in mRNA levels of u-PA, u-PAR and PAI-1 were measured by Northern blot. FGF-2 increased u-PA, u-PAR, and PAI-1 mRNA, but there was a significantly greater induction when fibrinogen was added to FGF-2 at all concentrations. The potentiation by fibrinogen was particularly evident at an FGF-2 concentration of 0.1 ng mL(-1), which resulted in non-significant change in transcript levels by itself, but significantly increased up to 2.6-fold with fibrinogen. VEGF also increased endothelial cell expression of u-PA, u-PAR and PAI-1, but this effect was not potentiated by fibrinogen. Addition of LM609, a monoclonal antibody to alphaVbeta3, significantly inhibited induction of u-PA mRNA and activity by fibrinogen-bound FGF-2 compared to FGF-2. A monoclonal antibody to FGFR1 also inhibited u-PA mRNA expression induced by fibrinogen-bound FGF-2. We conclude that fibrinogen increases the capacity of FGF-2, but not of VEGF, to up-regulate u-PA, u-PAR, and PAI-1 in endothelial cells and that fibrinogen-bound FGF-2 requires alphaVbeta3 binding to up-regulate endothelial cell u-PA.

Effects of lipids and lipid-lowering therapy on hemostatic factors in patients with myocardial infarction.

BACKGROUND: The risk of cardiovascular disease (CVD) is associated with specific hemostatic markers and lipid profiles, and evidence indicates that there are associations between lipid profiles and the levels of certain hemostatic factors. The disturbances in hemostasis and the risk of CVD can be ameliorated by lipid-lowering therapy. OBJECTIVE: We investigated the associations of lipid profiles with factor (F)VIIa, von Willebrand factor (VWF), D-dimer and plasminogen activator inhibitor-1 (PAI-1), and examined whether lipid-lowering statin therapy would affect the levels of these hemostatic markers. PATIENTS AND METHODS: This cross-sectional study analyzed 1045 postmyocardial infarction patients. RESULTS: In multivariate regression analyses (without adjusting for clinical covariates) HDL-cholesterol (HDL-C) and HDL size were independent and significant predictors of FVIIa; HDL size was a predictor of VWF; HDL size, HDL-C and LDL size were predictors of D-dimer; and triglyceride and HDL size were predictors of PAI-1. After adjusting for clinical covariates, HDL-C, lipoprotein (Lp)(a), apolipoprotein B (apoB) and warfarin were independent and significant predictors of FVIIa; HDL size, age, diabetes mellitus, insulin, race and warfarin were predictors of VWF; HDL-C, HDL size, LDL size, age, warfarin, hypertension and gender were predictors of D-dimer; and triglyceride, HDL size, body mass index, insulin and hypertension were predictors of PAI-1. Patients on statin therapy had significantly lower levels of D-dimer than those who were not on this therapy. CONCLUSION: There are significant associations of lipid profiles with hemostatic factors, the directions of which suggest novel pathways by which dyslipidemia may contribute to coronary heart disease.

Low-intensity ultrasound increases endothelial cell nitric oxide synthase activity and nitric oxide synthesis.

Low-intensity ultrasound (US) increases tissue perfusion in ischemic muscle through a nitric oxide (NO)-dependent mechanism. We have developed a model to expose endothelial cells to well-characterized acoustic fields in vitro and investigate the physical and biological mechanisms involved. Human umbilical vein endothelial cells (HUVEC) or bovine aortic endothelial cells (BAEC) were grown in tissue culture plates suspended in a temperature-controlled water bath and exposed to US. Exposure to 27 kHz continuous wave US at 0.25 W cm(-2) for 10 min increased HUVEC media NO by 102 +/- 19% (P < 0.05) and BAEC by 117 +/- 23% (P < 0.01). Endothelial cell NO synthase activity increased by 27 +/- 24% in HUVEC and by 32 +/- 16% in BAEC (P < 0.05 for each). The cell response was rapid with a significant increase in NO synthesis by 10 s and a maximum increase after exposure for 1 min. By 30 min post-exposure NO synthesis declined to baseline, indicating that the response was transient. Unexpectedly, pulsing at a 10% duty cycle resulted in a 46% increase in NO synthesis over the response seen with continuous wave US, resulting in an increase of 147 +/- 18%. Cells responded to very low intensity US, with a significant increase at 0.075 W cm(-2) (P < 0.01) and a maximum response at 0.125 W cm(-2). US caused minor reversible changes in cell morphology but did not alter proliferative capacity, indicating absence of injury. We conclude that exposure of endothelial cells to low-intensity, low-frequency US increases NO synthase activity and NO production, which could be used to induce vasodilatation experimentally or therapeutically.