Cloning of human anti-factor XIII monoclonal antibody dissects mechanisms of polyclonal antibodies in a single patient.

BACKGROUND: Coagulation factor XIII (FXIII) consists of 2 A (FXIII-A) and 2 B (FXIII-B) subunits that cross-link and strengthen the hemostatic fibrin thrombus; thus, abnormal bleeding occurs when FXIII is significantly reduced. Autoimmune-acquired FXIII deficiency (AiF13D) is characterized by lethal bleeding secondary to the development of autoantibodies against FXIII. However, since anti-FXIII autoantibodies are polyclonal, the mechanism underlying FXIII dysfunction is unclear. OBJECTIVES: The objective of this study was to dissect the inhibitory mechanisms of polyclonal anti-FXIII autoantibodies. METHODS: In this study, we prepared the human monoclonal antibodies (hmAbs) from the peripheral blood of an 86-year-old man with AiF13D by using a new complementary DNA cloning method and analyzed the properties of each autoantibody. RESULTS: Seventeen clones obtained from hmAbs were divided into the following 3 groups: dissociation inhibitors of FXIII-A2B2 (6 clones), assembly inhibitors of FXIII-A2B2 (3 clones), and nonneutralizing/inhibitory hmAbs (8 clones). Dissociation inhibitors strongly inhibited fibrin cross-linking and amine incorporation. Assembly inhibitors extracted FXIII-A from FXIII-A2B2, strongly inhibited binding of FXIII-A to FXIII-B, and activation peptide cleavage. However, the patient's plasma presented a strong inhibition of A2B2 heterodimer assembly but only a slight inhibition of thrombin-Ca2+-dependent dissociation, suggesting that the assembly inhibitors concealed the effect of dissociation inhibitors in plasma. By contrast, nonneutralizing antibodies had little effect on the function of FXIII, suggesting that nonneutralizing hmAbs (and/or dissociation inhibitors and/or assembly inhibitors) promoted the clearance of FXIII-A from the blood. CONCLUSION: Cloning of anti-FXIII autoantibodies enabled us to not only elucidate the mechanism and pathophysiology of AiF13D but also develop a completely new type of anticoagulant.

The Plasma Level of Interleukin-1ß Can Be a Biomarker of Angiopathy in Systemic Chronic Active Epstein-Barr Virus Infection.

Systemic chronic active Epstein-Barr virus infection (sCAEBV) is an EBV-positive T- or NK-cell neoplasm revealing persistent systemic inflammation. Twenty-five percent of sCAEBV patients accompany angiopathy. It is crucial to clarify the mechanisms of angiopathy development in sCAEBV because angiopathy is one of the main causes of death. Interleukin-1ß (IL-1ß) is reported to be involved in angiopathy onset. We investigated if IL-1ß plays a role as the inducer of angiopathy of sCAEBV. We detected elevated IL-1ß levels in four out of 17 sCAEBV patient's plasma. Interestingly, three out of the four had clinically associated angiopathy. None of the other patients with undetectable level of IL-1ß had angiopathy. In all patients with high plasma levels of IL-1ß and vascular lesions, EBV-infected cells were CD4-positive T cells. In one patient with high plasma IL-1ß, the level of IL-1ß mRNA of the monocytes was 17.2 times higher than the level of the same patient's EBV-infected cells in peripheral blood. In Ea.hy926 cells, which are the models of vascular endothelial cells, IL-1ß inhibited the proliferation and induced the surface coagulation activity. IL-1ß is a potent biomarker and a potent therapeutic target to treat sCAEBV accompanying angiopathy.

Excessive Prolongation of Coagulation Time During Treatment With Direct Oral Anticoagulants in Patients With Nonvalvular Atrial Fibrillation.

BACKGROUND: Conventional coagulation assays have poor sensitivity and specificity for assessing the anticoagulant effect of direct oral anticoagulants (DOACs). This study aimed to evaluate the causes and consequences of the excessive prolongation of coagulation time in patients with nonvalvular atrial fibrillation who receive DOACs. METHODS: We retrospectively analysed 1521 patients (age, 66 ± 12 years). The prothrombin time (PT) and activated partial thromboplastin time (APTT) were averaged if they were measured more than twice depending on the respective DOAC and dosage across individuals. Excessive coagulation time prolongation was defined as PT or APTT of >2 standard deviations over the median for each DOAC. RESULTS: In all, 1913 DOAC cases were found. Excessive prolongation (EP), which was noted in 88 patients (5.8%), was found to be significantly associated with inappropriately high DOAC dosage and body weight (≤ 60 kg). During follow-up (median, 8.9 months), thromboembolisms developed in 10 patients (0.66%) and bleeding events in 85 (5.6%). Bleeding events were significantly higher in patients with excessive prolongation (EP group) than in those without (P = 0.013). Of the 53 patients in the EP group, 15 (28%) were positive for antiphospholipid antibodies, 6 (11%) had inappropriately high prescription dosages, 4 (8%) had coagulation factor deficiencies, and 3 (6%) had severe liver dysfunction. CONCLUSIONS: Bleeding event rates were remarkably higher in patients receiving DOACs that caused EP of PT or APTT. Thus, following the current guidelines and administering the recommended dose of DOACs are fundamentally important. Patients with the body weight of <60 kg should be considered for dosage reduction or DOAC withdrawal.

Author Correction: Differential Assessment of Factor Xa Activity and Global Blood Coagulability Utilizing Novel Dielectric Coagulometry.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Differential Assessment of Factor Xa Activity and Global Blood Coagulability Utilizing Novel Dielectric Coagulometry.

An easy-to-use assessment for activated factor X (FXa) is lacking despite its pivotal role in the coagulation. Dielectric blood coagulometry (DBCM) was recently invented as a novel assessment tool for determining the whole blood coagulability by measuring the temporal change in the permittivity of blood. We previously reported that it could evaluate the global blood coagulability. This study aimed to apply the DBCM for assessing FXa activity and its inhibition by anticoagulants. We performed the DBCM analysis along with measurement of the FXa activity by a fluorometric assay in samples from healthy subjects, and identified a new index named maximum acceleration time (MAT) that had a correlation to the FXa activity. Next the DBCM analysis was performed using blood samples mixed with anticoagulants (unfractionated heparin, dalteparin, and edoxaban). Blood samples with three anticoagulants had different profiles of the temporal change in the permittivity, reflecting their different selectivity for FXa. We compared the MAT with the anti-FXa activity assay, and found that the prolongation of MAT was similarly correlated with the anti-FXa activity regardless of the type of anticoagulants. In conclusion, the DBCM has the possibility for evaluating the innate FXa activity and effect of anticoagulants focusing on their FXa inhibition.

STAT3 is constitutively activated in chronic active Epstein-Barr virus infection and can be a therapeutic target.

Chronic active Epstein-Barr virus infection (CAEBV) is a lymphoproliferative disorder characterized by the clonal proliferation of EBV-infected T or NK cells and is related to severe systemic inflammation. This study aims to investigate STAT3 to elucidate the mechanism underlying the CAEBV development. We determined that STAT3 was constitutively activated in EBV-positive T- or NK-cell lines. We also determined that STAT3 was activated in the peripheral blood mononuclear cells (PBMCs) containing EBV-infected clonally proliferating T or NK cells in six of seven patients with CAEBV. We conducted direct sequencing of the STAT3 Src homology 2 (SH2) domain, which has previously been reported to be mutated in T- or NK-cell neoplasms. No mutation was detected in the STAT3 SH2 domain in patients with CAEBV. Next, we investigated the effects of ruxolitinib, an inhibitor of both JAK1 and JAK2, which phosphorylates and activates STAT3. Ruxolitinib suppressed the phosphorylation of STAT3 in EBV-positive T- or NK-cell lines. Ruxolitinib also decreased the viable cell number of EBV-positive T- or NK-cell lines and PBMCs from patients with CAEBV. Furthermore, ruxolitinib suppressed the production of inflammatory cytokines in the cell lines and CAEBV patient-derived cells. In conclusion, constitutively activated STAT3, which promotes survival and cytokine production, could be a therapeutic target for CAEBV.

Correction: EBV induces persistent NF-κB activation and contributes to survival of EBV-positive neoplastic T- or NK-cells.

[This corrects the article DOI: 10.1371/journal.pone.0174136.].

Cross-reacting Material-positive Hemophilia A Diagnosed in a Patient with a Spontaneous Thigh Hemorrhage.

A 53-year-old man, who had been diagnosed with mild hemophilia A (HA) at 35 years of age, was hospitalized with a thigh hematoma. His bleeding continued despite the administration of recombinant factor VIII (FVIII). The results of an FVIII/von Willebrand factor binding assay were normal. The patient's FVIII coagulant activity (FVIII:C) was low, but his FVIII antigen levels were within the normal limits, suggesting FVIII protein dysfunction. The FVIII:C measurements obtained by one-stage clotting and chromogenic assays were different. An FVIII gene analysis revealed a missense mutation p.Ser308Leu, which is rare in Japan. This case highlights that gene analyses and chromogenic assays are necessary to interpret the discrepancies between FVIII:C and the bleeding phenotype of patients with mild HA.

Virus-specific cytotoxic T cells in chronic active Epstein-Barr virus infection.

Chronic active Epstein-Barr virus infection (CAEBV) is a disease characterized by clonally proliferating and activated EBV-infected T or NK cells accompanied by chronic inflammation and T- or NK-cell neoplasms. However, the mechanism for developing CAEBV has not been clarified to date. Because the decreased number or inactivation of EBV-specific cytotoxic T lymphocytes (CTLs) resulted in the development of EBV-positive B-cell neoplasms, we investigated the number of CTLs in CAEBV patients using the tetrameric complexes of HLA-restricted EBV-specific peptides. Among the seven patients examined, EBV-specific CTLs were detected in the peripheral blood mononuclear cells (PBMCs) of four cases but were not detected in three cases. The ratio of EBV-specific CTLs in PBMCs tended to be higher in the patients with active disease than in those with inactive disease. In two patients in whom EBV-specific CTLs had not been detected, CTLs appeared after the eradication of EBV-infected T cells by allogeneic bone marrow transplantation. These results suggested that the failure of CTLs had a role in developing CAEBV, although the induction number and function of EBV-specific CTLs might vary in each patient.

Inflammatory cytokine production in chronic active Epstein-Barr virus infection.

In order to clarify the mechanisms underlying the development of inflammation in chronic active Epstein-Barr virus infection (CABEV), we examined cytokine production using patient samples. Eleven patients were analyzed. The serum concentrations of IFN-γ, TNF-α, and IL-6 were significantly higher in patients than in healthy donors. The mRNAs of these cytokines in peripheral blood mononuclear cells were elevated in patients as compared with healthy donors. The mRNA of IFN-γ was significantly higher in patients than in healthy donors. We examined which fraction produced the cytokines in the CD4-, CD8-, and CD56-positive fractions of PBMCs. The mRNAs of IFN-γ, TNF-α, and IL-6 were highly expressed in EBV-infected cells, whereas expression was also observed in non-infected cells. We performed in vitro infection of EBV on a T-cell line, MOLT4. EBV infection enhanced the mRNA expressions of IFN-γ and TNF-α. These results suggest that the inflammatory cytokines in CAEBV are produced not only by EBV-infected but also non-infected cells. EBV itself may have roles in the cytokine production observed in infected cells.

Cell-based evaluation of changes in coagulation activity induced by antineoplastic drugs for the treatment of acute myeloid leukemia.

Idarubicin (IDR), cytarabine (AraC), and tamibarotene (Am80) are effective for treatment of acute myeloid leukemia (AML). In acute leukemia, the incidence of venous thromboembolism or disseminated intravascular coagulation is associated with induction chemotherapy. Procoagulant effects of IDR, AraC, and Am80 were investigated in a vascular endothelial cell line EAhy926 and AML cell lines HL60 (AML M2), NB4 (AML M3, APL), and U937 (AML M5), focusing on tissue factor (TF), phosphatidylserine (PS), and thrombomodulin (TM). IDR induced procoagulant activity on the surface of vascular endothelial and AML cell lines. Expression of TF antigen, TM antigen, and PS were induced by IDR on the surface of each cell line, whereas expression of TF and TM mRNAs were unchanged. Conversely, Am80 decreased TF exposure and procoagulant activity, and increased TM exposure on NB4 cells. In NB4 cells, we observed downregulation of TF mRNA and upregulation of TM mRNA. These data suggest IDR may induce procoagulant activity in vessels by apoptosis through PS exposure and/or TF expression on vascular endothelial and AML cell lines. Am80 may suppress blood coagulation through downregulation of TF expression and induction of TM expression. Our methods could be useful to investigate changes in procoagulant activity induced by antineoplastic drugs.

EBV induces persistent NF-κB activation and contributes to survival of EBV-positive neoplastic T- or NK-cells.

Epstein-Barr virus (EBV) has been detected in several T- and NK-cell neoplasms such as extranodal NK/T-cell lymphoma nasal type, aggressive NK-cell leukemia, EBV-positive peripheral T-cell lymphoma, systemic EBV-positive T-cell lymphoma of childhood, and chronic active EBV infection (CAEBV). However, how this virus contributes to lymphomagenesis in T or NK cells remains largely unknown. Here, we examined NF-κB activation in EBV-positive T or NK cell lines, SNT8, SNT15, SNT16, SNK6, and primary EBV-positive and clonally proliferating T/NK cells obtained from the peripheral blood of patients with CAEBV. Western blotting, electrophoretic mobility shift assays, and immunofluorescent staining revealed persistent NF-κB activation in EBV-infected cell lines and primary cells from patients. Furthermore, we investigated the role of EBV in infected T cells. We performed an in vitro infection assay using MOLT4 cells infected with EBV. The infection directly induced NF-κB activation, promoted survival, and inhibited etoposide-induced apoptosis in MOLT4 cells. The luciferase assay suggested that LMP1 mediated NF-κB activation in MOLT4 cells. IMD-0354, a specific inhibitor of NF-κB that suppresses NF-κB activation in cell lines, inhibited cell survival and induced apoptosis. These results indicate that EBV induces NF-κB-mediated survival signals in T and NK cells, and therefore, may contribute to the lymphomagenesis of these cells.

The expression and localization of RNase and RNase inhibitor in blood cells and vascular endothelial cells in homeostasis of the vascular system.

RNA may be released from vascular cells including endothelial cells in the event of injury and in vascular disease. Extracellular RNAs have been recognized as novel procoagulant and permeability-increasing factors. Extracellular RNA may function as inflammatory host alarm signals that serve to amplify the defense mechanism, but it may provide important links to thrombus formation. Extracellular RNA is degraded by RNase. We propose that RNase and its inhibitor RNase inhibitor (RI) act as modulators of homeostasis in the vasculature to control the functions of extracellular RNA. We aimed to investigate the expression and localization of RNase 1 and RI in cells that contact blood, such as platelets, mononuclear cells, polymorphonuclear cells, and red blood cells. RNase 1 and RI expression and localization in blood cells were compared with those in the human umbilical vein endothelial cell line, EAhy926. Additionally, we further investigated the effect of thrombin on the expression of RNase 1 and RI in platelets. We used an RNase activity assay, reverse transcription-polymerase chain reaction, western blot, immunocytochemistry, transmission electron microscopy, and immunoelectron microscopy (pre- and post-embedding methods). RNase activity in the supernatant from EAhy926 cells was 50 times than in blood cells (after 60 min). RNase 1 mRNA and protein expression in EAhy926 cells was highest among the cells examined. However, RI mRNA and protein expression was similar in most cell types examined. Furthermore, we observed that RNase 1 and von Willebrand factor were partially colocalized in EAhy926 cells and platelets. In conclusion, we propose that high RNase activity is ordinarily released from endothelial cells to support anticoagulation in the vasculature. On the other hand, platelets and leukocytes within thrombi at sites of vascular injury show very low RNase activity, which may support hemostatic thrombus formation. However, activated platelets and leukocytes may accelerate pathologic thrombus formation.

Cell-based laboratory evaluation of coagulation activation by antineoplastic drugs for the treatment of lymphoid tumors.

OBJECTIVES: Combining vorinostat, L-asparaginase, and doxorubicin (Dox) led to improved response rates in the treatment of lymphoid tumors. However, deep-vein thrombosis has been noted as one of the most serious side effects with these drugs, and how these regimens cause deep-vein thrombosis is unclear. METHODS: We investigated the procoagulant effects of vorinostat, L-asparaginase, and doxorubicin in lymphoid tumors, focusing on tissue factor, phosphatidylserine, and antithrombin. The human vascular endothelial cell line EAhy926 as well as the lymphoid neoplastic cell lines HUT78 (cutaneous T-cell lymphoma), Molt4 (acute T-lymphoblastic leukemia), and Ramos (Burkitt lymphoma) were employed to investigate these procoagulant effects. RESULTS: Vorinostat, L-asparaginase, and doxorubicin induced exposure of phosphatidylserine and procoagulant activity on the surface of lymphoid tumor cells. Vorinostat and doxorubicin also induced phosphatidylserine exposure and increased procoagulant activity on EAhy926 cells. Expression of tissue factor antigen was induced by doxorubicin on the surface of each type of cells, whereas expression of tissue factor mRNA was unchanged. Secretion of antithrombin from HepG2 cells was reduced only by L-asparaginase. CONCLUSION: These data suggest that vorinostat and doxorubicin may induce procoagulant activity in vessels through apoptosis of tumor cells and through phosphatidylserine exposure and/or tissue factor expression on vascular endothelial cells. L-asparaginase may induce a thrombophilic state by reducing the secretion of anticoagulant proteins such as antithrombin. The laboratory methods described here could be useful to evaluate the procoagulant effects of antineoplastic drugs.

Novel Dielectric Coagulometer Identifies Hypercoagulability in Patients with a High CHADS2 Score without Atrial Fibrillation.

BACKGROUND: Recent reports showed that the CHADS2 score predicted the risk of strokes in patients without atrial fibrillation (AF). Although the hypercoagulability may contribute to the thrombogenesis, it has not been fully investigated due to a lack of a sensitive evaluation modality. Recently a novel dielectric blood coagulometry (DBCM) was invented for evaluating the coagulability by measuring the temporal change in whole blood dielectric permittivity. OBJECTIVE: We evaluated the utility of the DBCM for identifying the coagulability. PATIENTS/METHODS: For fundamental experiments, 133 citrated blood samples were drawn from subjects with or without heparin administration. A DBCM analysis was performed to find the adequate coagulation index, and to delineate its measurement range by adding recombinant human tissue factor (TF) or heparin. Then the coagulability was assessed by DBCM and conventional coagulation assays in 84 subjects without AF, who were divided into 3 groups by their CHADS2 score. Another 17 patients who received warfarin were also assessed by DBCM to evaluate the effect of anticoagulants. RESULTS AND CONCLUSIONS: We calculated the derivative of the dielectric permittivity change after recalcification, and extracted the end of acceleration time (EAT) as a novel index. The EAT showed a dose-dependent shortening with the addition of serial dilutions of TF (×10-2 to ×10-4), and a dose-dependent prolongation with the addition of heparin (0.05 to 0.15 U/ml). The EAT was significantly shorter in the higher CHADS2 score group (19.8 ± 4.8, 18.6 ± 3.1, and 16.3 ± 2.7 min in the CHADS2 = 0, 1, and ≥2 groups, respectively, p = 0.0065 by ANOVA). Patients receiving warfarin had a significantly more prolonged EAT than those without warfarin (18.6±4.2 vs. 25.8±7.3 min, p <0.001). DBCM detected the whole blood coagulability with a high sensitivity. Subjects with higher CHADS2 scores exhibited hypercoagulability without AF.

Autoimmune Hemorrhaphilia Resulting from Autoantibody against the A Subunit of Factor XIII.

A 65-year-old woman was admitted with acute intramuscular hemorrhage of the left gluteus medius and piriformis muscles and associated anemia. Blood tests showed low plasma factor XIII (FXIII) antigen and activity. A cross-mixing test revealed a concave "inhibitor" pattern and anti-FXIII-A subunit antibody was detected. The patient was diagnosed with autoimmune hemorrhaphilia resulting from anti-FXIII antibody. The bleeding has not recurred since the initiation of treatment with oral immunosuppressive agents. Although hemorrhagic acquired FXIII deficiency is a rare disorder, prompt recognition of the underlying mechanism can save lives.

P-glycoprotein is expressed and causes resistance to chemotherapy in EBV-positive T-cell lymphoproliferative diseases.

Epstein-Barr virus-positive T-cell lymphoproliferative diseases (EBV-T-LPDs) are rare lymphomas with poor prognosis. Although chemotherapeutic strategies such as CHOP have been often selected, they have exhibited only limited efficacy. To clarify the mechanism of chemoresistance, we examined P-glycoprotein (P-gp) expression. P-gp acts as an energy-dependent efflux pump that excretes drugs from the cytoplasm, resulting in low-intracellular drug concentrations and poor sensitivity to chemotherapy. We examined P-gp expression in EBV-positive cells by immunohistochemistry staining in three patients of EBV-T-LPDs and the expression was detected in all patients. We also examined mdr1 mRNA expression by reverse-transcriptase polymerase-chain reaction (RT-PCR) in EBV-positive tumor cells from these patients and additional three patients. The expression was detected in all examined patients. In five EBV-T-LPDs patients, P-gp function was detected by Rhodamine-123 efflux assay in these cells. The efflux was inhibited by treatment with a P-gp inhibitor, cyclosporine A (CsA). We also examined and detected P-gp expression in EBV-positive T-cell lines SNT8 and SNT16 established from EBV-T-LPDs patients, by RT-PCR and western blotting. The function was also detected by Rhodamine-123 efflux in these cell lines. Inhibition and knock down of P-gp by CsA and siRNA, respectively, enhanced etoposide- and doxorubicin-induced cell death in the EBV-positive T-cell lines. Finally, we infected the T-cell line MOLT4 with EBV, and found that mdr1 mRNA expression and Rhodamine 123 efflux were upregulated after infection. These results indicated that enhanced P-gp expression contributed to the chemoresistance of EBV-T-LPDs.

CD137 expression is induced by Epstein-Barr virus infection through LMP1 in T or NK cells and mediates survival promoting signals.

To clarify the mechanism for development of Epstein-Barr virus (EBV)-positive T- or NK-cell neoplasms, we focused on the costimulatory receptor CD137. We detected high expression of CD137 gene and its protein on EBV-positive T- or NK-cell lines as compared with EBV-negative cell lines. EBV-positive cells from EBV-positive T- or NK-cell lymphoproliferative disorders (EBV-T/NK-LPDs) patients also had significantly higher CD137 gene expression than control cells from healthy donors. In the presence of IL-2, whose concentration in the serum of EBV-T/NK-LPDs was higher than that of healthy donors, CD137 protein expression was upregulated in the patients' cells whereas not in control cells from healthy donors. In vitro EBV infection of MOLT4 cells resulted in induction of endogenous CD137 expression. Transient expression of LMP1, which was enhanced by IL-2 in EBV-T/NK-LPDs cells, induced endogenous CD137 gene expression in T and NK-cell lines. In order to examine in vivo CD137 expression, we used EBV-T/NK-LPDs xenograft models generated by intravenous injection of patients' cells. We identified EBV-positive and CD8-positive T cells, as well as CD137 ligand-positive cells, in their tissue lesions. In addition, we detected CD137 expression on the EBV infected cells from the lesions of the models by immune-fluorescent staining. Finally, CD137 stimulation suppressed etoposide-induced cell death not only in the EBV-positive T- or NK-cell lines, but also in the patients' cells. These results indicate that upregulation of CD137 expression through LMP1 by EBV promotes cell survival in T or NK cells leading to development of EBV-positive T/NK-cell neoplasms.

Acquired factor X deficiency associated with atypical AL-amyloidosis.

We herein describe the case of a 77-year-old woman with acquired factor X deficiency that was likely caused by atypical amyloidosis. The patient developed severe gastrointestinal bleeding as a result of a significant decrease of factor X activity. Neither proteinuria nor diarrhea was observed as an initial manifestation. Although a bone marrow examination revealed direct fast scarlet-positive extracellular deposits, they did not exhibit red-to-green dichroism under polarized light. Immunofluorescence microscopy showed that the fibrillar proteins were positive for CD138 but negative for ß2-microglobulin or amyloid A antibodies. These atypical pathological features of immunoglobulin light chain-amyloidosis in this patient might be related to its unique clinical presentation.

Physiological fluctuations of human plasma total salusin-ß, an endogenous parasympathomimetic/proatherosclerotic peptide.

Salusin-ß is an endogenous bioactive peptide that systemically exerts acute parasympathomimetic hemodynamic actions and locally induces atherogenesis. Due to its unique physicochemical characteristics to immediately adhere to all types of plastic and glassware, its plasma concentrations have only been successfully determined very recently. Using a total of 50 healthy adults (median age 28 years, range 24-57 years), we evaluated whether circulating salusin-ß levels are affected by the autonomic nervous functions. Plasma total salusin-ß levels obtained during daytime ambulatory monitoring of heart rate variability showed strong negative correlations with variables reflecting parasympathetic nervous activity, high frequency amplitude (HF; r=-0.27, p=0.0018) and the square root of the mean squared differences of successive normal-to-normal intervals (RMSSD; r=-0.19, p=0.0292), but did not with low frequency amplitude (LF) or LF/HF, variables influenced by sympathetic nervous activity. Because early morning nadir in the diurnal variation of plasma total salusin-ß levels appeared to follow the nighttime parasympathetic nervous activity peak as quantified by HF and RMSSD, we determined whether parasympathetic stimulation reduces plasma salusin-ß levels. Both Valsalva maneuver (p<0.05) and urination (p<0.05) significantly reduced plasma total salusin-ß levels. Despite the fact that salusin-ß is the sole endogenous parasympathomimetic peptide identified to date, the current results argue against the contention that physiological parasympathetic augmentation is the consequences of upregulated circulating salusin-ß. Rather, circulating salusin-ß levels are suppressed following physiological parasympathetic stimulation and appear to constitute a negative feedback relationship with the parasympathetic nervous system.