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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.

Although vinculin is used frequently as a marker for integrin-mediated focal adhesion complexes, how it regulates the activation of integrin is mostly unknown. In this study, we examined whether vinculin would activate integrin in Chinese hamster ovary (CHO) cells expressing human integrin αIIbβ3. Silencing of vinculin by lentiviral transduction with a short hairpin RNA sequence affected the binding of PAC-1 (an antibody recognizing activated human αIIbβ3) to a constitutively active form of αIIbβ3 (α6Bβ3) expressed on CHO cells, while its inhibitory effects were much weaker than those of talin-1. Overexpression of an active form of vinculin without intramolecular interactions, but not the full length one, induced PAC-1 binding to native αIIbβ3 expressed on CHO cells in a manner dependent on talin-1. On the other hand, silencing of talin-1, but not vinculin, failed to induce cell spreading of α6Bβ3-CHO cells on fibrinogen, even in the presence of PT 25-2, a monoclonal antibody that activates αIIbβ3. Thus, an active form of vinculin could induce αIIbβ3 inside-out signaling through the actions of talin-1, while vinculin was dispensable for outside-in signaling.

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.

We studied restoration of the coagulation and fibrinolysis system in pediatric patients following liver transplantation and biomarkers of blood coagulation and fibrinolysis for suspecting the occurrence of acute cellular rejection. Coagulation activity recovered rapidly within two days following transplantation, but it took approximately 21-28 days for full recovery of the coagulation and fibrinolysis factors synthesized in the liver. PAI-1 levels were significantly higher in patients at the time of acute cellular rejection compared with levels after control of AR, and levels on days 14 and 28 in patients without AR. Plasma protein C and plasminogen levels at the time of rejection were significantly lower than those on day 14 in patients without AR. Statistical analysis suggested that an increase in plasma PAI-1 at a single time point in the post-operative period is a reliable marker among the coagulation and fibrinolysis factors for suspecting the occurrence of acute cellular rejection. These data suggested that appropriate anticoagulation may be required for 14 days after liver transplantation in order to avoid vascular complications and measurement of plasma PAI-1 levels may be useful for suspecting the occurrence of acute cellular rejection in pediatric patients following liver transplantation.

Background Gene therapy for hemophilia A with adeno-associated virus (AAV) vectors involves difficulties in the efficient expression of factor VIII (FVIII) and in antibody formation against transgene-derived FVIII. Methods AAV8 vectors carrying the canine B domain deleted FVIII (cFVIII) gene under the control of the ubiquitous β-actin promoter, the liver-specific human α1 anti-trypsin promoter (HAAT) and the liver-specific hepatic control region (HCR) enhancer/human α1 anti-trypsin promoter complex (HCRHAAT) were used for the expression of cFVIII in FVIII deficient (fviii−/−) mice. Results Addition of the hepatic control region enhancer element to the HAAT promoter successfully augmented HAAT promoter activity without loss of liver-specificity in vivo. Using this enhancer/promoter complex, a high cFVIII transgene expression was achieved, resulting in increased blood cFVIII activities to more than 100% of the normal canine FVIII levels in fviii−/− mice at a 1 : 10 lower dose of the AAV8 vector carrying the cFVIII gene driven by the HAAT promoter. Under short-term immunosuppression, neutralizing antibodies against cFVIII developed in only one out of six mice when the HAAT promoter was used for cFVIII expression, whereas all the mice developed neutralizing antibodies against cFVIII when the β-actin promoter was used for cFVIII expression. No neutralizing antibodies against cFVIII developed in fviii−/− mice that received the AAV8 vector carrying the cFVIII gene driven by the HCRHAAT enhancer/promoter complex without immunosuppression. Conclusions These data suggest that AAV8 vector-mediated liver-restricted cFVIII gene expression is sufficient for immune hypo-responsiveness to transgene-derived cFVIII in fviii−/− mice. Copyright © 2009 John Wiley & Sons, Ltd.

Summary. Background: Hemophilia A is a congenital bleeding disorder caused by a deficiency of coagulation factor VIII. Approximately 30% of hemophilia A patients develop inhibitors against FVIII following replacement therapy. We have reported that neonatal exposure of FVIII antigen can induce antigen-specific immune tolerance by interferon-γ (IFN-γ)-dependent T-cell anergy in hemophilia A mice. Objective: The thymus plays crucial roles in self-tolerance, with negative selection of self-reactive effector T cells and positive selection of self-reactive regulatory T cells. We investigated the possibility of the induction of antigen-specific immune tolerance by intrathymic injection of FVIII in hemophilia A mice. Methods: Hemophilia A mice were injected with recombinant FVIII into the thymus under real-time high-resolution image guidance. Results: Anti-FVIII inhibitory antibody titers in mice challenged with intravenous administration of FVIII were significantly lower in mice (n = 22) that had received thymic FVIII injection than in mice (n = 18) without thymic injection (9.4 ± 2.3 vs. 122.5 ± 27.6 BU mL−1, respectively, P = 0.00078). The CD4+ T cells from thymic-injected mice could not proliferate or produce interleukin (IL)-2, IL-12 and IFN-γ in response to FVIII. The CD4+CD25+ T cells generated from thymic-treated mice but not from naive mice efficiently suppressed the in vitro proliferative response of CD4+ T cells and blocked the in vivo development of anti-FVIII antibodies in the adoptive transfer. Conclusion: These data suggest that intrathymic administration of FVIII could result in immune tolerance by induction of FVIII-specific regulatory T cells.

Background and Purpose— We have previously shown that the sphingosine 1-phosphate (S1P)/S1P receptor-1 (S1P 1 R) axis contributes to the migration of transplanted neural progenitor cells (NPCs) toward areas of spinal cord injury. In the current study, we examined a strategy to increase endogenous NPC migration toward the injured central nervous system to modify S1PR. Methods— S1P concentration in the ischemic brain was measured in a mouse thrombosis model of the middle cerebral artery. NPC migration in vitro was assessed by a Boyden chamber assay. Endogenous NPC migration toward the insult was evaluated after ventricular administration of the S1P 2 R antagonist JTE-013. Results— The concentration of S1P in the brain was increased after ischemia and was maximal 14 days after the insult. The increase in S1P in the infarcted brain was primarily caused by accumulation of microglia at the insult. Mouse NPCs mainly expressed S1P 1 R and S1P 2 R as S1PRs, and S1P significantly induced the migration of NPCs in vitro through activation of S1P 1 R. However, an S1P 1 R agonist failed to have any synergistic effect on S1P-mediated NPC migration, whereas pharmacologic or genetic inhibition of S1P 2 R by JTE-013 or short hairpin RNA expression enhanced S1P-mediated NPC migration but did not affect proliferation and differentiation. Interestingly, administration of JTE-013 into a brain ventricle significantly enhanced endogenous NPC migration toward the area of ischemia. Conclusions— Our findings suggest that S1P is a chemoattractant for NPCs released from an infarcted area and regulation of S1P 2 R function further enhances the migration of NPCs toward a brain infarction.

Platelets are receiving much attention as novel target cells to secrete a coagulation factor for hemophilia gene therapy. In order to extend the application of platelet-directed gene therapy, we examined whether ectopic expression of activated factor VII (FVIIa) in platelets would result in an efficient bypass therapy to induce sufficient thrombin generation on platelet surfaces in mice with hemophilia A. Transduction of bone marrow cells with a simian immunodeficiency virus (SIV)-based lentiviral vector harboring the platelet-specific GPIb alpha promoter resulted in efficient transgene expression in platelets. FVIIa antigen was expressed in platelets by this SIV system; FVII transgene products were found to localize in the cytoplasm and translocate toward the sub-membrane zone and cell surface after activation. Although FVII antigen levels in platelets did not reach the therapeutic levels seen with FVIIa infusion therapy, whole-blood coagulation, as assessed by thromboelastography, was significantly improved in mice with hemophilia A. Further, we observed correction of the bleeding phenotype in mice with hemophilia A after transplantation, even in the presence of FVIII-neutralizing antibodies. Our results demonstrate that FVIIa-expressing platelets can strengthen hemostatic function and may be useful in treating hemophilia and other inherited bleeding disorders. These findings are comparable to the proven therapeutic effects of FVIIa infusion.

Since the discovery of anti-HIV activity in oligo(tyrosine sulfate)s in our laboratory, we have been interested in their potential as heparin pentasaccharide mimics. In this study, we investigated their interactions with synthetic heparin-binding peptides, derived from human antithrombin III (hAT III) and heparin-interacting protein (HIP), using surface noncovalent affinity mass spectrometry. We compared binding affinities to those heparin-binding peptides between oligo(tyrosine sulfate)s and several known sulfated compounds and found that oligo(tyrosine sulfate)s bind to hAT III (123–139) more strongly than a heparin-derived hexasaccharide dp6. Moreover, we found longer oligo(tyrosine sulfate) has higher binding affinity to hAT III (123–139).

Platelet-derived mediators may play an important role in the development of renal diseases through interaction with glomerular mesangial cells (MCs), and we, in this study, examined the effect of sphingosine 1-phosphate (Sph-1-P), a bioactive lipid released from activated platelets, on the contraction of MCs. Sph-1-P was found to induce MC contraction through mediation of Rho kinase both in cell shape change and collagen gel contraction assays. The specific antagonist of the Sph-1-P receptor S1P 2 inhibited the response. Similar results were obtained when the supernatant from activated platelet suspensions were used instead of Sph-1-P. Our findings suggest that platelet-derived Sph-1-P may be involved in MC contraction via S1P 2 and that regulation of this receptor might be useful therapeutically.

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.

Summary. Objective: The main objective was to study the relationships of the molecular defects in 38 dysfibrinogens with their fibrin networks. Methods and results: Scanning electron microscopic analyses revealed that all the fibrins formed under the same conditions had networks composed of either normal thickness fibers or thin fibers, accompanied by a variety of alterations in the network structure and characteristics. We classified these fibrin networks into five classes, designated normal, less-ordered, porous A, porous B and lace-like networks. The dysfibrinogens with defects in fibrinopeptide A release or the E:D binding sites formed normal or less-ordered networks, while those with defects in the D:D association formed porous A networks composed of many tapered terminating fibers, despite having fibers of normal width, and containing many pores or spaces. The porous B and lace-like networks were composed of highly branched thin fibers because of defects in the lateral association among protofibrils, and the major difference between them was the porosity of the porous B networks. All the porous B networks were easily damaged by mechanical stress, whereas the lace-like networks retained high resistance to such stress, indicating that the network strength was not dependent on the fiber width, but on the porosity that led to fragility of the network. Conclusion: Impairment of the D:D association is the major disturbing factor that leads to the formation of porous fibrin networks. The porosity may be introduced by severe impairment of the D:D association, as well as the lateral association, as has often been observed by extra glycosylation or defects in Ca2+ binding.

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.

Deficiency of ADAMTS13 is found in patients with thrombotic thrombocytopenic purpura (TTP), and the genetic defects in the ADAMTS13 gene or the autoantibody against ADAMTS13 is thought to be responsible for the development of TTP. The clinical correlation and mechanisms of secondary ADAMTS13 deficiency in other disease states were investigated. In addition to TTP, ADAMTS13 levels were severely decreased in patients with sepsis-induced disseminated intravascular coagulation (DIC). The incidence of acute renal failure and serum creatinine levels in patients with ADAMTS13 activity levels lower than 20% (incidence, 41.2%; creatinine, 160 ± 150 μM [1.81 ± 1.70 mg/dL]) (P < .05) were significantly higher than they were in patients with ADAMTS13 activity levels higher than 20% (incidence, 15.4%; creatinine, 84 ± 67 μM [0.95 ± 0.76 mg/dL]) (P < .01). Additionally, unusually large von Willebrand factor multimers were detected in 26 (51.0%) of 51 patients with ADAMTS13 activity levels lower than 20%. Lower molecular weight forms of ADAMTS13 were found in the plasma of patients with sepsis-induced DIC, suggesting that the deficiency of ADAMTS13 was partially caused by its cleavage by proteases in addition to decreased synthesis in the liver. These data suggested that severe secondary ADAMTS13 deficiency can be associated with sepsis-induced DIC and may contribute to the development of renal failure.

Sphingosine 1-phosphate (Sph-1-P), a bioactive lipid derived from activated platelets, may play an important role in coronary artery spasm and hence the pathogenesis of ischemic heart diseases, since we reported that a decrease in coronary blood flow was induced by this lysophospholipid in an in vivo canine heart model [Cardiovasc. Res. 46 (2000) 119]. In this study, metabolism related to and cellular responses elicited by Sph-1-P were examined in human coronary artery smooth muscle cells (CASMCs).[3H]Sphingosine (Sph), incorporated into CASMCs, was converted to [3H]Sph-1-P intracellularly, but its stimulation-dependent formation and extracellular release were not observed. Furthermore, the cell surface Sph-1-P receptors of S1P family (previously called EDG) were found to be expressed in CASMCs. Accordingly, Sph-1-P seems to act as an extracellular mediator in CASMCs. Consistent with Sph-1-P-elicited coronary vasoconstriction in vivo, Sph-1-P strongly induced CASMC contraction, which was inhibited by JTE-013, a newly-developed specific antagonist of S1P(2) (EDG-5). Furthermore, C3 exoenzyme or Y-27632 inhibited the CASMC contraction induced by Sph-1-P, indicating Rho involvement. Finally, exogenously-added [3H]Sph-1-P underwent a rapid degradation. Since lipid phosphate phosphatases, ectoenzymes capable of dephosphorylating Sph-1-P, were expressed in CASMCs, Sph-1-P may be dephosphorylated by the ectophosphatases.Sph-1-P, derived from platelets and dephosphorylated on the cell surface, may induce the contraction of coronary artery smooth muscle cells through the S1P(2)/Rho signaling.

Platelets play important roles in thrombosis and hemostasis, and their abnormalities lead to hemostatic disorders or thrombotic diseases. Due to genetic technique development, the study of genetic factors related to the hereditary platelet dysfunction, and the evaluation of gene polymorphisms have been reported recently. In this article, we will focus mainly on hereditary platelet disorders and genetic variations of platelet glycoproteins involved in the pathogenesis of thrombotic diseases.

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

Sphingosine 1-phosphate (Sph-1-P) is a bioactive sphingolipid, acting both as an intracellular second messenger and extracellular mediator, in mammalian cells. In cell types where Sph-1-P acts as an intracellular messenger, stimulation-dependent synthesis of Sph-1-P, possibly resulting from sphingosine (Sph) kinase activation, is essential. Since this important kinase has recently been cloned, precise regulation of intracellular Sph-1-P synthesis will be clarified in the near future. As an intercellular mediator, elucidation of sources for extracellular Sph-1-P is important, in addition to identification of the cell surface receptors for this phospholipid. Blood platelets are very unique in that they store Sph-1-P abundantly (possibly due to the existence of highly active Sph kinase and a lack of Sph-1-P lyase) and release this bioactive lipid extracellularly upon stimulation. It is likely that platelets are an important source for extracellular Sph-1-P, especially for plasma and serum Sph-1-P. Platelet-derived Sph-1-P seems to play an important role in vascular biology.

The serum-borne lysophospholipid mediators sphingosine 1-phosphate (Sph-1-P) and lysophosphatidic acid (LPA) have been shown to be released from activated platelets and to act on endothelial cells. In this study, we employed the repeated lipid extraction (under alkaline and acidic conditions), capable of detecting Sph-1-P, LPA, and possibly structurally similar lysophospholipids, whereby a marked formation of [32P]Sph-1-P, but not [32P]LPA, was observed in [32P]orthophosphate-labeled platelets. Platelet Sph-1-P release, possibly mediated by protein kinase C, was greatly enhanced in the presence of albumin, which formed a complex with Sph-1-P. This finding suggests that platelet Sph-1-P may become accessible to depletion by albumin when its transbilayer movement (flipping) across the plasma membrane is enhanced by protein kinase C. Although human umbilical vein endothelial cells expressed receptors for both Sph-1-P and LPA, Sph-1-P acted much more potently than LPA on the cells in terms of intracellular Ca++ mobilization, cytoskeletal reorganization, and migration. The results suggest that Sph-1-P, rather than LPA, is a major bioactive lysophospholipid that is released from platelets and interacts with endothelial cells, under the conditions in which critical platelet-endothelial interactions (including thrombosis, angiogenesis, and atherosclerosis) occur. Furthermore, albumin-bound Sph-1-P may account for at least some of the serum biological activities on endothelial cells, which have been ascribed to the effects of albumin-bound LPA, based on the similarities between LPA and serum effects.

Elevated Expression of Mitochondrial NADH Dehydrogenase Subunit 4/4L Genes in Vascular Endothelial Cells Undergoing Sphingosine-induced Apoptosis

Hemophilia A is a life-threatening bleeding disorder caused by mutations in the factor VIII (FVIII) gene that lead to deficiency of FVIII. Gene therapy is respected to provide an alternative to current FVIII supplemental therapy. Among a variety of vectors, AAV vectors are thought to be ideal for transfer of therapeutic genes since they are derived from non-pathogenic virus and have been demonstrated to provide sustained transgene expression in non-dividing cells with little toxicity, though, delivery of the FVIII gene using AAV vectors are limited by its small packaging capacity. To overcome the packaging capacity limit, we developed AAV1 vectors and AAV8 vectors carrying the B domain deleted canine FVIII gene (BDD cFVIII) utilizing a minimum promoter (150b) and expressed canine FVIII in hemophilia A mice. Previous reports suggested AAV1 serotype is suitable for transduction of skeletal muscles and AAV8 serotype is superior to other AAV serotypes for transduction of the liver, thus, AAV1 vectors carrying the BDD cFVIII gene (AAV1 cFVIII) were injected to the skeletal muscles of hemophilia A mice. Since the liver could be transduced with intravenously injected AAV8 vectors carrying the Lac Z gene as efficiently as intraportally injected vectors, AAV8 vectors carrying the BDD cFVIII gene (AAV8 cFVIII) were injected to hemophilia A mice intravenously. FVIII clotting activities measured by the APTT method increased in mice injected with AAV1cFVIII in a dose dependent manner. The FVIII activity level in peripheral blood increased to 2.9±1.0% in hemophilia A mice with the AAV1cFVIII dose at 1x1012 gc/body, suggesting partial correction of phenotype with AAV1cFVIII vectors. The FVIII clotting activity levels in hemophilia A mice injected with AAV8cFVIII increased also in a dose dependent manner, achieving supernormal FVIII levels (436±219 %) in hemophilia A mice with the AAV8 cFVIII dose at 1x1012 gc/body. It is apparent that transduction of the liver with AAV8cFVIII is superior to transduction of skeletal muscles with AAV1cFVIII regarding FVIII production, however, AAV1 vectors have advantages of removing the transgenes in case of inconvenience and of less systemic distribution of vectors. 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.

The level of plasma dopamine-beta-hydroxylase (DBH) activity in subjects at rest was found to be significantly lower in 12 patients on long-term hemodialysis than in a healthy 8-member control group: 28.3 +/- 7.2 and 13.6 +/- 7.6 IU/1, respectively (p less than 0.01). Following immersion of one hand of each subject into cold water (4 degrees C) for 1 min, a significant rise was observed in both groups, 6.1 +/- 4.8 IU/1 for the control and 1.6 +/- 1.4 IU/1 for the patient group (p less than 0.01). Upon tilting up the head of all subjects, activity in both groups increased significantly, but a markedly smaller rise was found in the patient group: 5.8 +/- 4.8 and 1.1 +/- 1.6 IU/1 for the two groups, respectively (p less than 0.01). The data suggest an autonomic nervous dysfunction in patients on long-term hemodialysis.

Introduction Current criteria for early diagnosis of coagulopathy in sepsis are limited. We postulated that coagulopathy is already complicated with sepsis in the initial phase, and severe coagulopathy or disseminated intravascular coagulation (DIC) becomes overt after progressive consumption of platelet and coagulation factors. To determine early diagnostic markers for severe coagulopathy, we evaluated plasma biomarkers for association with subsequent development of overt DIC in patients with sepsis. Methods A single-center, prospective observational study was conducted in an adult ICU at a university hospital. Plasma samples were obtained from patients with sepsis at ICU admission. Fourteen biomarkers including global markers (platelet count, prothrombin time, activated partial thromboplastin time, fibrinogen and fibrin degradation product (FDP)); markers of thrombin generation (thrombin-antithrombin complex (TAT) and soluble fibrin); markers of anticoagulants (protein C (PC) and antithrombin); markers of fibrinolysis (plasminogen, α2-plasmin inhibitor (PI), plasmin-α2-PI complex, and plasminogen activator inhibitor (PAI)-1); and a marker of endothelial activation (soluble E-selectin) were assayed. Patients who had overt DIC at baseline were excluded, and the remaining patients were followed for development of overt DIC in 5 days, and for mortality in 28 days. Results A total of 77 patients were enrolled, and 37 developed overt DIC within the following 5 days. Most patients demonstrated hemostatic abnormalities at baseline with 98.7% TAT, 97.4% FDP and 88.3% PC. Most hemostatic biomarkers at baseline were significantly associated with subsequent development of overt DIC. Notably, TAT, PAI-1 and PC discriminated well between patients with and without developing overt DIC (area under the receiver operating characteristic curve (AUROC), 0.77 (95% confidence interval, 0.64 to 0.86); 0.87 (0.78 to 0.92); 0.85 (0.76 to 0.91), respectively), and using the three together, significantly improved the AUROC up to 0.95 (vs. TAT, PAI-1, and PC). Among the significant diagnostic markers for overt DIC, TAT and PAI-1 were also good predictors of 28-day mortality (AUROC, 0.77 and 0.81, respectively). Conclusions Severe coagulation and fibrinolytic abnormalities on ICU admission were associated with subsequent development of overt DIC. A single measurement of TAT, PAI-1, and PC activity could identify patients with ongoing severe coagulopathy, early in the course of sepsis.