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.

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.

High-resolution genomic copy number profiling of primary intraocular lymphoma by single nucleotide polymorphism microarrays.

Primary intraocular lymphoma (PIOL) is a rare lymphoma. Because of difficulties in obtaining tissue samples, little is known about the disease's genetic features. In order to clarify these features, we carried out single nucleotide polymorphism array karyotyping of IOL using genomic DNA extracted from vitreous fluid. We analyzed 33 samples of IOLs consisting of 16 PIOLs, 12 IOLs with a central nervous system (CNS) lesion at diagnosis (IOCNSL), and five secondary IOLs following systemic lymphoma. All were B-cell type. We identified recurrent copy number (CN) gain regions in PIOLs, most frequently on chromosome 1q followed by 18q and 19q. Chromosome 6q was the most frequent loss region. Although these CN gain regions of PIOL were in common with those of IOCNSL, loss of 6q22.33 containing PTPRK and 9p21.3 containing CDKN2A were more frequently deleted in IOCNSL. Large CN loss in 6q was detected in three of four PIOL patients who had early CNS development and short survival periods, whereas long-term survivors did not have such deletions. There was a correlation between gain of the IL-10 gene located on 1q and intravitreal interleukin-10 concentration, which was higher in IOL than in benign uveitis. The results suggest that IOCNSL is a highly malignant form of PIOL that infiltrates into the CNS at an early stage. They also indicate that genetic differences between PIOL and primary CNS lymphoma need to be clarified.

Severe postoperative hemorrhage caused by antibody-mediated coagulation factor deficiencies: report of two cases.

Antibody-mediated coagulation factor deficiencies constitute a rare disorder that may develop in elderly patients without any history of a bleeding diathesis. Patients may present with severe and sometimes catastrophic bleeding. We report two cases of postoperative hemorrhage caused by a coagulation factor deficiency. In Case 1, massive intraabdominal bleeding occurred on day 3 after pancreaticoduodenectomy for bile duct cancer, and was caused by an acquired inhibitor of coagulation factor VIII. Hemostasis was achieved and the factor VIII inhibitor titer decreased to zero with activated prothrombin complex concentrates, prednisolone, and cyclophosphamide. In Case 2, intraabdominal bleeding occurred on day 7 after hepatectomy for hepatocellular carcinoma, and was caused by an acquired inhibitor against factors II (prothrombin) and V. This patient was treated with hemostatic agents containing bovine thrombin during surgery and also with prednisolone. We report these cases to highlight that antibody-mediated coagulation factor deficiencies should be considered when an elderly patient suffers sudden postoperative hemorrhage and to stress the importance of prompt diagnosis because of the risk of potentially life-threatening hemorrhage.

Antagonism of sphingosine 1-phosphate receptor 2 causes a selective reduction of portal vein pressure in bile duct-ligated rodents.

UNLABELLED: Sinusoidal vasoconstriction, in which hepatic stellate cells operate as contractile machinery, has been suggested to play a pivotal role in the pathophysiology of portal hypertension. We investigated whether sphingosine 1-phosphate (S1P) stimulates contractility of those cells and enhances portal vein pressure in isolated perfused rat livers with Rho activation by way of S1P receptor 2 (S1P(2) ). Rho and its effector, Rho kinase, reportedly contribute to the pathophysiology of portal hypertension. Thus, a potential effect of S1P(2) antagonism on portal hypertension was examined. Intravenous infusion of the S1P(2) antagonist, JTE-013, at 1 mg/kg body weight reduced portal vein pressure by 24% without affecting mean arterial pressure in cirrhotic rats induced by bile duct ligation at 4 weeks after the operation, whereas the same amount of S1P(2) antagonist did not alter portal vein pressure and mean arterial pressure in control sham-operated rats. Rho kinase activity in the livers was enhanced in bile duct-ligated rats compared to sham-operated rats, and this enhanced Rho kinase activity in bile duct-ligated livers was reduced after infusion of the S1P(2) antagonist. S1P(2) messenger RNA (mRNA) expression, but not S1P(1) or S1P(3) , was increased in bile duct-ligated livers of rats and mice and also in culture-activated rat hepatic stellate cells. S1P(2) expression, determined in S1P 2LacZ/+ mice, was highly increased in hepatic stellate cells of bile duct-ligated livers. Furthermore, the increase of Rho kinase activity in bile duct-ligated livers was observed as early as 7 days after the operation in wildtype mice, but was less in S1P 2-/- mice. CONCLUSION: S1P may play an important role in the pathophysiology of portal hypertension with Rho kinase activation by way of S1P(2) . The S1P(2) antagonist merits consideration as a novel therapeutic agent for portal hypertension.

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.

A key role for Toll-like receptor-3 in disrupting the hemostasis balance on endothelial cells.

Various virus infections cause dysfunctional hemostasis and in some instances lead to the development of viral hemorrhagic fever syndrome. How do diverse viruses induce the expression of tissue factor on vascular cells? We hypothesize that a direct stimulation of pattern recognition receptors (PRR) by viral nucleic acids may be the key. Double-stranded RNA (dsRNA) is produced by many viruses and is recognized by various PRR, including Toll-like receptor-3 (TLR3). We have investigated whether poly I:C, a model for viral dsRNA, can influence cellular hemostasis. Poly I:C could up-regulate tissue factor and down-regulate thrombomodulin expression on endothelial cells but not on monocytes. The response to poly I:C was diminished upon small interfering RNA (siRNA)-mediated inhibition of TLR3, but not other PRR. In vivo, application of poly I:C induced similar changes in the aortic endothelium of mice as determined by enface microscopy. D-dimer, a circulating marker for enhanced coagulation and fibrinolysis, and tissue fibrin deposition was elevated. All the hemostasis-related responses to poly I:C, but not cytokine secretion, were blunted in TLR3(-/-) mice. Hence, the activation of TLR3 can induce the procoagulant state in the endothelium, and this could be relevant for understanding the mechanisms of viral stimulation of hemostasis.

[Physicochemical characteristics of salusin-beta and establishment of the radioimmunoassay].

Salusin-beta is a 20 amino acid bioactive peptide originally identified using bioinformatic analyses of human full-length enriched cDNA library. Salusin-beta has been shown to exert potent hypotensive, bradycardic, and pro-atherosclerotic effects. The form in which it exists in biological fluids remained undetermined due to technical difficulties originating from its unexpected physicochemical properties. Salusin-beta peptide adheres to polypropylene, glass and polystyrene, so that the aliquoted peptide dissolved in distilled water may rapidly disappear from the dissolved solution. Strategies to circumvent such problems in biological experiments include use of low doses of NP-40 or Tween-20 which alleviates its adhesiveness. Addition of 0.1% of NP-40 to the radioimmunoassay buffer markedly reduced non-specific binding of both labeled and unlabeled salusin-beta to the assay tubes without interfering the binding of salusin-beta to its antibody. Successful establishment of a specific radioimmunoassay suitable for detection of immunoreactive human salusin-beta allowed to characterize the molecular form of salusin-beta released from a human-derived cultured cell line.

[Analyses on activation and regulation of blood coagulation].

We have been investigating the mechanism of blood coagulation and anticoagulation related to pathogenesis and the regulation of thrombosis and bleeding disorders. Since our focus is basically internal medicine and hematology, the contribution to this field is clinically based and aimed to help patients and maintain public health. In this review some of our achievements associated with the activation and regulation of blood coagulation are introduced, mainly based on our research with post-graduate medical technologists. The main topics are the structural and functional relationship of thrombomodulin (TM), the regulation of tissue factor (TF) and TM expression, TM gene therapy, activation of the extrinsic coagulation system due to increased TF-bearing leukocytes during and after cardiac surgery, endoplasmic reticulum-associated degradation of protein C and plasmin inhibitor mutants leading to protein deficiency, activation of blood coagulation with exposure of cellular or viral RNA, and the detection of novel bioactive peptides salusin-alpha and -beta.

[Proteasome degradation of protein C and plasmin inhibitor mutants: molecular mechanism of congenital protein deficiency].

In many inherited disorders, protein deficiency is one of the major aetiologies, but the molecular and cellular mechanisms remain unclear. We investigated the intracellular degradation of mutant proteins, using naturally occurring PC and PI mutants that lead to congenital deficiencies. We have shown that proteasomes are very important for the degradation of PC and PI mutants, irrespective of the presence or absence of N-glycosylation moieties. Furthermore, mannose trimming after glucose removal is very important for initiation of the degradation. Inhibition of glucose trimming of the mutant proteins accelerated degradation by the proteasomes, and initiation of the degradation occurs after mannose trimming of the middle chain of N-linked glycosylation by mannosidase I. The binding of molecular chaperons influenced by the presence of N-glycosylation moieties may affect the efficient degradation of the mutant proteins. Cotransfection of endoplasmic reticulum (ER) degradation enhancing alpha-mannosidase like protein (EDEM) accelerated the degradation of N-glycosylated PC. The mutant PC or PI molecules were ubiquitin-independently degraded by proteasomes. Autophagy does not appear to contribute to the degradation of PC and PI mutants. These findings might help to elucidate the molecular mechanisms and potential treatments of congenital deficiencies of proteins in a system of coagulation and fibrinolysis.

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.

Proteasome degradation of protein C and plasmin inhibitor mutants.

Protein C (PC) deficiency and plasmin inhibitor (PI) deficiency are inherited thrombotic and haemorrhagic disorders. We investigated the intracellular degradation of mutant proteins, using naturally occurring PC and PI mutants that lead to congenital deficiencies. To examine the necessity of N-linked glycosylation for the proteasomal degradation of PC and PI, PC178 and PC331 mutants treated with tunicamycin and N-glycosylation-lacking mutants, PC92Stop and PI-America were pulse chased. The analysis revealed that the speed of degradation of the tunicamycin-treated PC mutants, PC92Stop and PI-America lacking glycosylation, was slower than that of N-glycosylated mutants. Immunoprecipitation and immunoblot analysis showed that PC178 and PC331 mutants were associated with molecular chaperones, Bip, GRP94, and calreticulin. PI-America was associated with only Bip. Although degradation of mutants was mediated by proteasomes, no association with ubiquitin was detected. Cotransfection of endoplasmic reticulum (ER) degradation enhancing alpha-mannosidase-like protein (EDEM) accelerated the degradation of N-glycosylated PC. In the absence of autophagy using Atg5-deficient cell lines, the degradation of the PC331 mutant was mildly accelerated but that of PC178, PI-America and PI-Okinawa mutants was not influenced. While the degradation of the PC and PI mutants was facilitated by N-glycosylation moieties, they were ubiquitin-independently degraded by proteasomes, irrespective of the presence or absence of N-glycosylation. Molecular chaperone binding was influenced by the presence of N-glycosylation moieties. When the misfolded or truncated mutant proteins are functionally active, proteasome inhibitors such as bortezomib may have therapeutic potential for treatment of protein deficiencies.

Serum salusin-alpha levels are decreased and correlated negatively with carotid atherosclerosis in essential hypertensive patients.

Salusin-alpha is a new bioactive peptide with mild hypotensive and bradycardic effects. Our recent study showed that salusin-alpha suppresses foam cell formation in human monocyte-derived macrophages by down-regulating acyl-CoA:cholesterol acyltransferase-1, contributing to its anti-atherosclerotic effect. To clarify the clinical implications of salusin-alpha in hypertension and its complications, we examined the relationship between serum salusin-alpha levels and carotid atherosclerosis in hypertensive patients. The intima-media thickness (IMT) and plaque score in the carotid artery, blood pressure, serum levels of salusin-alpha, and atherosclerotic parameters were determined in 70 patients with essential hypertension and in 20 normotensive controls. There were no significant differences in age, gender, body mass index, fasting plasma glucose level, or serum levels of high-sensitive C-reactive protein, high- or low-density lipoprotein (LDL) cholesterol, small dense LDL, triglycerides, lipoprotein(a), or insulin between the two groups. Serum salusin-alpha levels were significantly lower in hypertensive patients than in normotensive controls. The plasma urotensin-II level, maximal IMT, plaque score, systolic and diastolic blood pressure, and homeostasis model assessment for insulin resistance (HOMA-IR) were significantly greater in hypertensive patients than in normotensive controls. In all subjects, maximal IMT was significantly correlated with age, systolic blood pressure, LDL cholesterol, urotensin-II, salusin-alpha, and HOMA-IR. Forward stepwise multiple linear regression analysis revealed that salusin-alpha levels had a significantly independent and negative association with maximal IMT. Serum salusin-alpha levels were significantly lower in accordance with the severity of plaque score. Our results suggest that the decrease in serum salusin-alpha, an anti-atherogenic peptide, may be associated with carotid atherosclerosis in hypertensive patients.

Biosynthesis and secretion of salusin-alpha from human cells.

Salusins originally identified using bioinformatics analyses have been shown to act on the cardiovascular and endocrine systems. Although the hypotensive activity of salusin-alpha is limited, it exerts a significant anti-atherosclerotic effect via suppression of foam cell formation in human monocyte-derived macrophages by down-regulating acyl-CoA:cholesterol acyltransferase-1. Furthermore, serum salusin-alpha levels show a close negative correlation with the severity of atherosclerotic diseases. However, biosynthesis and secretion of salusin-alpha peptide from cultured mammalian cells have not been demonstrated to date. We examined the expression, synthesis and release of salusin-alpha in human-derived cell lines. Preprosalusin mRNA and protein were detected ubiquitously in all cells tested, whereas the processing of preprosalusin into salusin-alpha peptide is dependent upon each cell type. Immunohistochemical study revealed the most abundant salusin-alpha-like immunoreactivity to be present in HeLa cells which released salusin-alpha-like immunoreactivity into the culture supernatant. Analysis of extracted conditioned media from HeLa cells by reverse-phase high performance liquid chromatography coupled with radioimmunoassay detection revealed a single immunoreactive component that co-eluted with authentic salusin-alpha. These results present the first evidence that salusin-alpha is biosynthesized and released from human-derived cells.

A variant-type MLL/SEPT9 fusion transcript in adult de novo acute monocytic leukemia (M5b) with t(11;17)(q23;q25).

As a result of recurrent chromosomal translocations in acute leukemias, the mixed-lineage-leukemia (MLL) gene fuses with a variety of partner genes, which include several members of the septin gene family. SEPT9 is a very rare but recurrent fusion partner of MLL, and has recently been implicated in the oncogenesis of various malignancies. Herein, we report a case of de novo acute monocytic leukemia (M5b) with t(11;17)(q23;q25). MLL involvement was revealed by fluorescent in situ hybridization (FISH) analysis, and an MLL/SEP9 fusion transcript was detected by RT-PCR. Sequencing analysis further showed that, in contrast to originally reported cases, MLL exon 8 was fused not with SEPT9 exon 3 but with exon 2, which codes for the unique N-terminal region of the SEPT9_v1 isoform, the region implicated in the regulation of gene expression and cell proliferation. We did not detect any mutation of FLT3, which was expressed at a relatively low level in the leukemic cells. Relapsing after a very short complete remission, the leukemia progressed rapidly and became fatal in spite of intensive therapies including hematopoietic stem cell transplantation. It is thus suggested that, in common with the original MLL/SEPT9 cases, monocytic differentiation and a poor prognosis may also be associated with acute myeloid leukemia with the variant MLL/SEPT9 fusion transcript.

[The effect of air massage on serum constituents].

This study investigated the effect of air massage of the legs on serum constituents. Five volunteers (aged 48.8+/-12.98 years, n=5) served as subjects. The study consisted of three periods as follows: control period for one month(no-mas), massage period receiving the massage for 15 minutes per day for one month(mas), and another massage period receiving the massage for 15 minutes per day along with 40% oxygen uptake for one month (O2-mas). Venous blood samples were drawn every week during the study periods. Serum constituents were examined using an auto analyzer(Hitachi Ltd., JEOL Ltd.). Albumin concentration significantly increased under the O2-mas condition, whereas free-cholesterol concentration was significantly decreased under the same condition. In addition, significant increases in concentrations of creatinine and calcium were seen in men under the O2-mas condition. Furthermore, there was a significant decrease in the activity of alkaline phosphatase in women under both the mas and O2-mas conditions. These findings suggested that massage has positive effects on nutritional recovery and metabolism of lipids, bone and muscle. Thus, it may be argued that massage could potentially be effective as a form of exercise.

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.