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Introduction Emerging lines of evidence have suggested that certain dysfibrinogens present a significant risk of thrombosis. Patient/methods The thrombophilic nature of a new-type of dysfibrinogen Kagoshima identified in a 36-year-old female with deep vein thrombosis during the postpartum period was studied. Results/discussion Based on the analyses of the patient fibrinogen and the fibrinogen genes, fibrinogen Kagoshima was shown to have the amino acid substitution of γThr-314 to Ile that resulted in impaired function and hypofibrinogenemia. Polymerization of fibrin monomers derived from patient fibrinogen was severely impaired with a partial correction in the presence of calcium ions, causing very low clottability and delayed cross-linking of patient fibrin catalyzed by activated factor XIII. Because of the low clottability, a large amount of soluble fibrin was formed upon thrombin treatment, resulting in an increase of thrombin in the soluble fraction. Additionally, tPA-mediated plasmin generation on fibrin was impaired and calcium-ion-dependent integrity of the γ-chain D domain of Kagoshima fibrinogen was perturbed. The presence of many tapered-fiber ends inside the tangled fibrin networks, observed by scanning electron microscopy, suggested early termination of fibrin polymerization and the structural alteration. Conclusion These data suggest that fibrinogen Kagoshima is dysfunctional, giving rise to formation of fibrinolysis-resistant soluble fibrin polymers and entrance of soluble fibrin associating with thrombin to the circulation, partly accounting for the thrombophilic nature of the affected fibrinogen and fibrin molecules.

Adeno-associated virus (AAV) vectors carrying the B domain-deleted canine FVIII (BDD cFVIII) gene utilizing the beta-actin minimum promoter (167b) pseudotyped with serotype 1 (AAV1-beta-actin-cFVIII) and serotype 8 (AAV8-beta-actin-cFVIII) were developed to express cFVIII in hemophilia A mice. FVIII clotting activities measured by the APTT method increased in hemophilia A mice with intramuscular injection of AAV1-beta-actin-cFVIII in a dose-dependent manner. Therapeutic FVIII levels (2.9+/-1.0%) in hemophilia A mice with the AAV1-beta-actin-cFVIII dose of 1 x 10(12) gc/body were achieved, suggesting partial correction of the phenotype with AAV1-beta-actin-cFVIII vectors. FVIII clotting activity levels in hemophilia A mice with intravenous injection of AAV8-beta-actin-cFVIII also were increased dose-dependently, achieving therapeutic FVIII levels (5-90%) in hemophilia A mice with the AAV8-beta-actin-cFVIII doses of 1-3 x 10(11) gc/body and supernormal FVIII levels (180-670%) in hemophilia A mice with the AAV8-beta-actin-cFVIII dose of 1 x 10(12) gc/body. Transduction of the liver with AAV8-beta-actin-cFVIII is superior to transduction of skeletal muscles with AAV1cFVIII regarding the FVIII production and antibody formation. These data suggested that both AAV1 and AAV8 vectors carrying the FVIII gene utilizing a minimum promoter have a potential for hemophilia A gene therapy.

We analyzed the intracellular action of sphingosine 1-phosphate (Sph-1-P), formed from sphingosine (Sph) by sphingosine kinase (SPHK), in platelets. When sphingosine kinase activity was inhibited by N,N-dimethylsphingosine (DMS), Ca2+ mobilization induced by convulxin, an agonist of the collagen receptor glycoprotein VI (GPVI), was moderately but specifically abolished; that induced via G protein-coupled receptors was not affected. Under the same conditions, however, tyrosine phosphorylation of Syk and phospholipase Cgamma2, which is essential for the GPVI-mediated signaling, was not inhibited. Sphingosine kinase activity of the platelet membrane fraction increased specifically upon stimulation with convulxin or collagen. Our results suggest that intracellular sphingosine 1-phosphate is related to Ca2+ mobilization in GPVI-mediated signaling pathways.

Sphingosine 1-phosphate (Sph-1-P) is a bioactive lipid released from activated platelets, which may be involved in angiogenesis. We, hence, investigated Sph-1-P effects on human umbilical vein endothelial cells (HUVECs) from a viewpoint of angiogenesis. Sph-1-P facilitated HUVEC spreading on the basement membrane component Matrigel, at concentrations ranging from 10 to 250 nM. This stimulatory response induced by Sph-1-P was blocked by pertussis toxin and C3 transferase (from Clostridium botulinum), which inactivate Gi-type heterotrimeric G protein and Rho, respectively. Furthermore, Sph-1-P, in the modified Boyden's chamber assay, stimulated HUVEC migration in a concentration-dependent manner, up to 250 nM. Checkerboard analysis revealed that Sph-1-P markedly induces directional migration (chemotaxis), but a random motility (chemokinesis) was also enhanced. The stimulatory effect of Sph-1-P on HUVEC migration was much stronger than that of other bioactive lipids, and again inhibited by pertussis toxin and by C3 transferase. Our present results that Sph-1-P induces endothelial spreading and migration through Gi-coupled cell surface receptor(s) and Rho are consistent with a recent report on the role of this platelet-derived sphingolipid as a novel regulator of angiogenesis.

Fibrin degradation products (FDP) are an important marker of coagulopathy. We assessed the reactivity of the monoclonal antibodies used in clinical laboratory testing (6 D-dimer reagents, D-dimer-1-6; 4 plasma FDP reagents, plasma FDP-1-4) to quantify FDP using in vitro-generated FDP as well as FDP in clinical samples. The monoclonal antibodies used in D-dimer-1, -2, -5, and -6 reacted poorly to the low molecular weight forms of in vitro-generated FDP. The monoclonal antibodies used in D-dimer-3 and -4 had better reactivity to the low molecular weight forms of in vitro-generated FDP. The monoclonal antibodies used in plasma FDP-2, -3, and -4 reacted well to the high and low molecular weight FDP forms, while the monoclonal antibody in plasma FDP-1 reacted poorly to the low molecular weight FDP forms. Analysis of clinical samples revealed deviations in FDP molecular weight forms in DIC samples. The reactivity of the monoclonal antibodies of laboratory FDP testing to FDP variants in clinical samples was similar to that of in vitro-generated FDP. In conclusion, the monoclonal antibodies used in clinical laboratories to detect FDP have distinct reactivity to the molecular variants of FDP generated in vitro as well as those present in clinical samples. Our findings support the consensus for the standardization of D-dimer and plasma FDP testing.

Introduction Factor VIII (FVIII) treatment for hemophilia A has difficulties in correcting bleeding diathesis in the presence of inhibitors. Materials and Methods An adeno-associated virus type 8 (AAV8) vector containing the factor VII (FVII) gene or the activated factor VII (FVIIa) gene was used to investigate the therapeutic effect of FVII or FVIIa overexpression in FVIII-deficient mice with inhibitors. Results Following repeated human FVIII injection, FVIII-deficient mice developed anti-human FVIII antibodies that cross-reacted with mouse FVIII. High transgene expression of murine FVII or murine FVIIa was achieved using the AAV8 vector and resulted in increased blood FVII activity greater than 800% of normal murine FVII levels in vector-injected FVIII-deficient mice. Thromboelastography analysis showed significant improvements in clotting time, clot formation time, α angle, and mean clot firmness in AAV8 vector-injected FVIII-deficient mice with inhibitors. Overexpression of FVIIa ameliorated the bleeding phenotype of FVIII-deficient mice with inhibitors and significantly increased the survival rate after tail clipping. In addition, overexpression of FVII increased the survival rate of FVIII-deficient mice with inhibitors after tail clipping though it was not as efficient as FVIIa overexpression. Conclusions These data suggest that FVII overexpression is an alternative strategy for the treatment of hemophilia A with inhibitors.

High residual platelet aggregability during thienopyridine treatment occurs because of low levels of the active drug metabolite, and is associated with an increased rate of major adverse cardiovascular events. Recent findings suggest that paraoxonase-1 (PON1) is a major determinant for clopidogrel efficacy. The aim of this study was to assess the impact of serum PON1 activity on platelet aggregability in thienopyridine-treated patients. In 72 patients receiving treatment with aspirin and ticlopidine after acute coronary syndrome, various laboratory data including the formation of platelet aggregations induced by agonists were compared with serum PON1 activities, measured as paraoxonase and homocysteine thiolactone hydrolase (HTLase). Serum paraoxonase activity was significantly associated with HTLase activity (R=0.4487, P0.0001). These PON1 activities were not correlated with any parameters for platelet aggregation, hypertension, sleep apnea, and diabetes mellitus. In contrast, serum PON1 activities seemed to be involved in cardiac function, with brain natriuretic peptide and ejection fraction being significantly correlated with serum HTLase activity (R=-0.2767, P=0.0214) and paraoxonase activity (R=0.2558, P=0.0339), respectively. Paraoxonase activity also demonstrated a significant association with increased levels of ankle-brachial index (R=0.267, P=0.0255). Serum PON1 activities did not influence platelet aggregability during treatment with thienopyridine. However, they might modulate cardiac function after acute coronary syndrome and progression of atherosclerosis.

Pulmonary embolism development may be prevented if asymptomatic venous thromboembolism (VTE) can be predicted and treated preoperatively or soon after total knee arthroplasty (TKA). The purpose of this study was to evaluate whether asymptomatic VTE can be predicted by blood coagulation markers preoperatively or early after TKA. This prospective single-centre study enrolled 68 patients (6 men, 62 women; mean age: 71 years) who underwent TKA between September 2004 and August 2009. Sixteen-row multidetector computed tomography was performed 4 days before and after surgery for diagnosis of asymptomatic VTE. Blood samples were taken to measure the plasma levels of soluble fibrin monomer complex (SFMC), D-dimer and cross-linked fibrin degradation products by leukocyte elastase (e-XDP) at 4 days preoperatively, and at 1 hour, 1 day and 4 days postoperatively. The preoperative SFMC, D-dimer and e-XDP levels did not differ significantly between the thrombus (n=36) and no-thrombus (n=32) groups. D-dimer and e-XDP levels showed the most significant increases at days 4 and 1, respectively, after surgery in the thrombus group. With cut-off points of 7.5 μg/ml for D-dimer and 8.2 U/ml for e-XDP, the sensitivities were 75% and 75%, and the specificities were 63% and 59%, respectively. By multiple logistic regression analysis, D-dimer at day 4 and e-XDP at day 1 postoperatively were independent markers for early diagnosis of VTE (odds ratio=1.61 and 1.19, P=0.01 and 0.04, respectively). The postoperative occurrence of new asymptomatic VTE may be predicted by D-dimer at day 4 and e-XDP at day 1 after TKA.

Neutrophil elastase released from activated neutrophils contributes in combating bacterial infection. While chronic inflammation results in anemia and decreased bone marrow activities, little is known about the effect of neutrophil elastase on hematological cell growth in severe inflammatory states. Here, we demonstrated that α1-antitrypsin, a physiological inhibitor of neutrophil elastase, functions as a regulator for cell growth by neutralizing neutrophil elastase activity in lipopolysaccharide-primed hematological cells. HL-60 cells were resistant to neutrophil elastase, as they also expressed α1-antitrypsin. The growth of HL-60 cells transduced with a LentiLox-short hairpin α1-antitrypsin vector was significantly suppressed by neutrophil elastase or lipopolysaccharide. When CD34(+) progenitor cells were differentiated towards a granulocytic lineage, they concomitantly expressed neutrophil elastase and α1-antitrypsin and prevented neutrophil elastase-induced growth inhibition. These results suggest that granulocytes might protect themselves from neutrophil elastase-induced cellular damage by efficiently neutralizing its activity through the simultaneous secretion of endogenous α1-antitrypsin.

An alternative pathway for fibrinolysis that comprises leukocyte elastase and its interaction with the plasminogen activator-plasmin system has been suggested. Plasma levels of cross-linked fibrin degradation product by leukocyte elastase (e-XDP) were significantly increased in patients with sepsis induced disseminated intravascular coagulation (DIC) compared with healthy subjects (18.6±19.9 vs 0.58±0.47U/mL, p0.001). Twenty seven unique spots were identified from e-XDP dominant patients by immune-purification and two-dimensional difference gel electrophoresis, and they contained fibrinogen Bβ-chain derived fragments Bβ Asp-164, Ser-200, Gln-301, Ala-354, Ile-484 and γ-chain derivatives γ Val-274 at their amino-termini by acquired and processed tandem mass spectrometer. The Sequential Organ Failure Assessment Scores in patients with e-XDPs levels 3-10U/mL were significantly lower than those with e-XDPs levels -3U/mL, 10-30U/mL, and 30- U/mL. The adjusted odds for 28-day mortality rate in patients with e-XDP levels less than 3U/mL (hazard ratio, 4.432; 95% CI, 1.557-12.615 [p=0.005]) were significantly higher than those in patients with e-XDP levels of 3-10U/mL. These data suggest that leukocyte elastase might contribute to the degradation of cross-linked fibrin in sepsis-induced DIC.

Introduction Gene therapy is expected to be the next generation therapy for hemophilia, and a good animal model is required for hemophilia gene therapy preclinical studies. Methods Taking advantage of the human factor IX (FIX) specificity of monoclonal antibody 3A6, the epitope of which resides in the amino acid polypeptide segment including Ala 262 of human FIX, mutant macaque FIX with an amino acid substitution of Thr 262 to Ala (macaque FIX T262A) was generated and its reactivity to monoclonal antibody 3A6, biological activity and expression in vivo were studied. Results Enzyme-linked immunosorbent assays (ELISAs) and Western blot analyses showed that monoclonal antibody 3A6 bound to human FIX and macaque FIX T262A but not to wild-type macaque FIX. Recombinant macaque FIX T262A exhibited a comparable coagulation activity to wild-type macaque FIX and human FIX. High expression of macaque FIX T262A was achieved in mice by injection of AAV8 vectors carrying the macaque FIX T262A gene and reached levels of up to 31.5 µg/mL (1050% of the normal human FIX concentration). Macaque FIX T262A expressed in the liver of mice was as biologically active as that expressed in vitro . In addition, the macaque FIX T262A concentrations determined by a 3A6-based ELISA were not influenced by the presence of normal macaque plasma. Conclusions The results of the present study suggest that macaque FIX T262A may be processed appropriately in vivo and that the macaque FIX T262A concentration in the macaque circulation can be quantified precisely by a monoclonal antibody 3A6-based ELISA.

Introduction Secondary ADAMTS13 deficiency may occur in septic patients. The expression of ADAMTS13 in mouse endotoxinemia was studied. Methods The blood and mRNA expression levels of ADAMTS13 and von Willebrand factor were measured in lipopolysaccharide-injected mice. Results The plasma ADAMTS13 activity in wild-type mice was significantly decreased at 2 h after lipopolysaccharide injection, and this decrease in ADAMTS13 activity preceded the decrease in ADAMTS13 mRNA expression in the liver and continued for 24 h. However, no decreases in the plasma ADAMTS13 activity after lipopolysaccharide injection were observed in mice pretreated with a neutrophil elastase inhibitor or in plasminogen-deficient mice, suggesting that the decrease in ADAMTS13 activity was processed efficiently by the coordinated actions of plasmin and neutrophil elastase. von Willebrand factor mRNA was abundantly expressed in the lung and moderately in the kidney, but showed relatively low expression in the liver without lipopolysaccharide injection. However, von Willebrand factor mRNA expression in the liver was significantly increased after lipopolysaccharide injection and this high expression level continued for 24 h after the injection. The von Willebrand factor and ADAMTS13 mRNA expression levels in these organs changed in the opposite manners following lipopolysaccharide administration. Furthermore, the blood von Willebrand factor level increased after lipopolysaccharide administration, in contrast to the decrease in the blood ADMTS13 level after lipopolysaccharide administration. Conclusion These data suggest that imbalance between the blood von Willebrand factor and ADAMTS13 levels may occur in endotoxinemia, and that this may partly contribute to the thrombotic state associated with endotoxinemia.

Vascular intimal carcinomatosis refers to a characteristic tumor proliferation on vascular intima that replaces normal endothelium. This pathological event of unknown cause is quite different from tumor thrombotic microangiopathy due to the absence of thrombi on the tumor cell surfaces. We analyzed renal transitional cell carcinoma cases with metastasis to the main pulmonary arteries and marked hyperfibrino(geno)lysis. The fibrinogen-derived products from patients' plasma were identified as D1A/γ, D1/γ, and D1/β by immunoblotting with the NH2-terminus of the fragment D specific antibody JIF-23. In all cases, the neoplastic cells with vascular intimal carcinomatosis were stained positive for anti-human annexin 2, which is a unique cell surface co-receptor for plasminogen and tissue-type plasminogen activator. In contrast, normal renal pelvic mucosa or renal transitional cell carcinoma without vascular intimal carcinomatosis did not express any annexin 2. The isolated transitional cell carcinoma cells contained annexin 2 mRNA and expressed its protein. Anti-annexin 2 antibody and transfection of annexin 2 small interfering RNA into these carcinoma cells significantly inhibited tissue-type plasminogen activator dependent plasmin generation. These findings suggest that annexin 2 mediated fibrinolysis on the transitional cell carcinoma cells may play a role in inducing hemorrhagic disorder in vascular intimal carcinomatosis.

Sphingosine 1-phosphate (Sph-1-P) is now regarded as a lysophospholipid mediator which is present in plasma and other body fluids, and exerts potent and pleiotropic biological effects [1,2]. Such extracellular mediator activities of Sph-1-P are mainly mediated by subfamilies of G proteincoupled receptors, of which the most completely characterized are those encoded by the endothelial differentiation genes (EDGs) [2,3]. Previously, we showed that in platelets, Sph-1-P is rapidly formed from sphingosine (Sph) by the action of Sph kinase, abundantly stored intracellularly, and released into the extracellular environment upon stimulation [1,4]. Furthermore, vascular endothelial cells express the EDG family Sph-1-P receptors, i.e., EDG-1 and EDG-3 [5]. Consistent with the expression of the high-affinity Sph-1-P receptors in these cells, nanomolar Sph-1-P reportedly induces a variety of vascular endothelial cell responses [5–9]. Sph-1-P stimulates endothelial cell survival or proliferation through a Gi-coupled receptor, probably EDG-1 [5,7]. Furthermore, Sph-1-P induces adherens junction assembly, migration, capillary tube formation, and promotion of angiogenesis [5,8,9]. In this case, the signals mediated via EDG-1 (coupled with Gi) and EDG-3 (coupled with G13 and Gq) are both necessary, and the small GTPase Rhoand Rac-mediated signalings are also involved [5,8]. Very recently, Sph-1-P has been shown to activate endothelial nitric oxide synthase through a novel mechanism involving Akt activation and produce nitric oxide, which plays an important role in endothelial survival and maintenance of the endothelial function [10]. Accordingly, Sph-1-P should be added to the list of platelet-derived bioactive mediators and may play an important role in platelet–endothelial cell interactions, which constitute a central part of vascular biology. To obtain insight into the physiological and pathophysiological role(s) of Sph-1-P in the vascular system, information about the regulation of endothelial cell expression of EDG family Sph-1-P receptors, i.e., EDG-1 and EDG-3, is very important. In fact, the EDG-1 cDNA was originally isolated as a phorbol ester-inducible immediate early transcript from vascular endothelial cells; morphogenetic differentiation of vascular endothelial cells into capillary-like tubules was induced under the conditions [11]. Furthermore, recent data have shown that the mechanical force associated with blood flow, i.e., the fluid shear stress, modulates vascular structure and function, and plays an important role in the pathogenesis of vascular diseases, including atherosclerosis, hypertension, and restenosis [12]. Shear stress has been reported to affect endothelial cell gene expression, and EDG-1 has been cloned as one of two cDNAs encoding a G protein-coupled receptor from a cDNA library of human umbilical vein endothelial cells (HUVECs) exposed to fluid shear stress; EDG-1 mRNA levels increase markedly in response to fluid flow [13]. Accordingly, the modulation of Sph-1-P/EDG signaling may be involved in important vascular responses related to angiogenesis and blood flow conditions. In this study, we examined the possibility that EDG expression, and hence, responsiveness to Sph-1-P in vascular endothelial cells, may be modulated by tumor necrosis factor-a (TNF-a), which is a pleiotropic cytokine that