大森 司

A mixing test is useful for distinguishing between coagulation factor deficiency and the presence of inhibitor as the cause of coagulopathy. However, we experienced a patient with acquired factor V (FV) inhibitor whose mixing test showed a coagulation factor deficiency pattern. A 65-year-old man with a tendency for bleeding was referred to our center. The laboratory data showed remarkable prolongation of prothrombin time and activated partial thromboplastin time (APTT). FV activity was less than 3%. A mixing test showed a coagulation factor deficiency pattern. However, neither the tendency for bleeding nor the coagulation tests were corrected by transfusion of fresh frozen plasma. A few days later, a positive test for FV inhibitor of 3 Bethesda units was obtained. Therefore, we started prednisolone and plasma exchange, and the coagulation test results normalized after 6 weeks. Although an incubation period is generally not considered necessary in a mixing test for FV inhibitor, we repeated mixing tests with various incubation periods and confirmed an incubation period-dependent prolongation of the APTT. Therefore, a mixing test with an incubation period is recommended for the detection of FV inhibitor, since a mixing test without an incubation period may show a coagulation factor deficiency pattern when the titer of FV inhibitor is low.

BACKGROUND: Tissue type plasminogen activator (tPA) functions as a fibrinolytic factor in the blood and has unique roles in the nervous system. However, the role of tPA in the olfactory epithelium (OE) is still unclear. Generally, surgical ablation of the olfactory bulb (bulbectomy) triggers degeneration followed by regeneration of OE. In this experimental study, we investigated the role of tPA in OE regeneration. METHODS: Wild-type (WT) mice and tPA-knockout (tPA(-/-) ) mice were subjected to bulbectomy. Reverse-transcription polymerase chain reaction (RT-PCR), in situ hybridization, and immunohistochemical examination was done to detect tPA expression in the olfactory bulb and OE. Cellular proliferation and apoptosis was also monitored in the OE. RESULTS: Before bulbectomy, tPA was found to be expressed in the olfactory bulb and OE. OE degenerated to a similar extent in both strains between 0 and 3 days after bulbectomy. However, OE was thicker and contained more cells in tPA(-/-) mice than in WT mice at 7 days after bulbectomy. Moreover, the number of apoptotic bodies was reduced and the number of proliferating cells was increased in the OE of tPA(-/-) mice compared to WT mice, after bulbectomy. Transmission electron microscopy revealed continuous degeneration of the OE for up to 7 days after bulbectomy in WT mice. In contrast, we observed some intact olfactory vesicles and almost normal supporting cells in the OE of tPA(-/-) mice, at 7 days after bulbectomy. CONCLUSION: The current findings show that the tPA-plasmin system plays an inhibitory role in the regulation of regeneration in the OE.

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.

Hemophilia is an X-linked bleeding disorder, and patients with hemophilia are deficient in a biologically active coagulation factor. This study was designed to combine the efficiency of lentiviral vector transduction techniques with murine adipose tissue-derived stem/stromal cells (mADSCs) as a new method to produce secreted human coagulation factor IX (hFIX) and to treat hemophilia B. mADSCs were transduced with simian immunodeficiency virus (SIV)-hFIX lentiviral vector at multiplicities of infection (MOIs) from 1 to 60, and the most effective dose was at an MOI of 10, as determined by hFIX production. hFIX protein secretion persisted over the 28-day experimental period. Cell sheets composed of lentiviral vector-transduced mADSCs were engineered to further enhance the usefulness of these cells for future therapeutic applications in transplantation modalities. These experiments demonstrated that genetically transduced ADSCs may become a valuable cell source for establishing cell-based gene therapies for plasma protein deficiencies, such as hemophilia.

Neutralizing antibodies (NAbs) against adeno-associated viruses (AAVs) are known to interfere with AAV vector-mediated gene transfer by intravascular delivery. Evading the inhibitory effects of antibodies against AAV vectors is necessary for efficient transfer of therapeutic genes clinically. For this purpose, we tested the efficacy of saline flushing in order to avoid contact of vectors with NAbs present in blood. Direct injection of the AAV8 vector carrying the factor IX (FIX) gene into the portal vein of macaques using saline flushing achieved transgene-derived FIX expression (4.7 ± 2.10-10.1 ± 5.45% of normal human FIX concentration) in the presence of NAbs. Expression was as efficient as that (5.43 ± 2.59-12.68 ± 4.83%) in macaques lacking NAbs. We next tested the efficacy of saline flushing using less invasive balloon catheter-guided injection. This approach also resulted in efficient expression of transgene-derived FIX (2.5 ± 1.06-9.0 ± 2.37%) in the presence of NAbs (14-56× dilutions). NAbs at this range of titers reduced the efficiency of transduction in the macaque liver by 100-fold when the same vector was injected into mesenteric veins without balloon catheters. Our results suggest that portal vein-directed vector delivery strategies with flushing to remove blood are efficacious for minimizing the inhibitory effect of anti-AAV antibodies.

Our aim was to examine the pathophysiology of sleep onset of acute coronary syndrome (ACS); in particular, we focused on the association of sleep onset of ACS, sleep-apnea syndrome (SAS), and diurnal variation of hemostasis and adipokine levels. Seventy-four patients (mean 60.0 years; 84% men) with ACS were cross-sectionally examined. They were examined by circulatory levels of hemostasis [plasminogen activator inhibitor-1 (PAI-1), D-dimer, soluble fibrin] and adipokines (adiponectin, visfatin) before and after sleep, and cardiorespiratory function. The severity of SAS was defined as mild to no SAS [apnea-hypopnea index (AHI) <15/h, n = 30], moderate SAS (AHI 15-30/h, n = 26), and severe SAS (AHI >30/h, n = 18). Nineteen patients (26%) were diagnosed with sleep onset of ACS, and these patients had a greater extent of morning increase from the night-time levels of PAI-1 (median PAI-1 increase: +37.1 vs. +27.3 ng/ml; P = 0.01) and visfatin (median visfatin increase: +0.40 vs. +0.00 ng/ml; P = 0.08) than those who had daytime onset of ACS. Among patients who had sleep onset of ACS, 89% were diagnosed with moderate to severe SAS. According to the severity of SAS, the morning increase from the night-time levels of PAI-1 and visfatin became greater (median PAI-1 increase: +23.7 vs. +29.2 vs. +39.3 ng/ml; median visfatin increase: 0.00 vs. 0.00 vs. +0.45 ng/ml; both P < 0.05), and these differences remained unchanged even after adjustment for significant covariates (both P < 0.05). Patients who have sleep onset of ACS are likely to have high prevalence of SAS and abnormal diurnal variations of PAI-1 and visfatin levels.

Haemophilia A is a life long bleeding disorder caused by an inherited deficiency of factor VIII (FVIII). About 30% of haemophilia A patients develop neutralizing antibodies as a consequence of treatment with FVIII concentrates. Immune tolerance protocols for the eradication of inhibitors require daily delivery of intravenous FVIII. We evaluated the immune responses to serial intravenous administration of FVIII in preimmunized haemophilia A mice. We introduced an implantable venous-access device (iVAD) system into haemophilia A mice to facilitate sequential infusion of FVIII. After preimmunization with FVIII, the haemophilia A mice were subjected to serial intravenous administration of FVIII through the iVAD system. In all mice with serial infusion of FVIII, high titers of anti-FVIII inhibitory antibodies developed at 10exposure days (EDs). However, the anti-FVIII IgG titers were decreased after 150EDs of sequential low-dose infusion of FVIII [0.05Ug -1 body weight (BW) five times per week]. Proliferative response to ex vivo FVIII stimulation was significantly suppressed in splenic CD4 + T cells from mice with serial low-dose FVIII infusion compared with those from mice with high-dose FVIII infusion (0.5Ug -1 BW five times per week) or preimmunized mice. Moreover, splenic CD4 + T cells from mice with serial low-dose infusion of FVIII failed to produce interleukin-2 and interferon-γ. These data suggest that serial infusion of FVIII could induce T-cell anergy in haemophilia A mice with inhibitor antibodies. © 2011 Blackwell Publishing Ltd.

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, P<0.0001). 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.

Spinal cord injury (SCI) is an incapacitating injury that can result in limited functional recovery. We have previously shown increases in the lysophospholipid mediator, sphingosine-1-phosphate (S1P), in the spinal cord after contusion injury. To apply S1P receptor modulation to the treatment of SCI, we examined the therapeutic effects of FTY720, an S1P receptor agonist, on locomotor recovery after SCI in mice. Oral administration of FTY720 shortly after contusion SCI significantly improved motor function recovery, as assessed by both Basso Mouse Scale scores and Rotarod Performance test results. FTY720 induced lymphopenia and reduced T-cell infiltration in the spinal cord after SCI but did not affect the early infiltration of neutrophils and the activation of microglia. In addition, plasma levels and mRNA expression of inflammatory cytokines in the spinal cord after SCI were not attenuated by FTY720. Vascular permeability and astrocyte accumulation were both decreased by FTY720 in the injured spinal cord. The therapeutic effects of FTY720 were not solely dependent on immune modulation, as confirmed by the demonstration that FTY720 also ameliorated motor function after SCI in mice with severe combined immunodeficiency. Finally, the S1P(1) receptor agonist, SEW2871, partly mimicked the therapeutic effect of FTY720. Our data highlight the importance of immune-independent functions of FTY720 in decreasing vascular permeability and astrogliosis in the injured spinal cord and promoting locomotor function recovery after SCI.

Hemophilia A is a common X chromosome-linked genetic bleeding disorder caused by abnormalities in the coagulation factor VIII gene (F8). Hemophilia A patients suffer from a bleeding diathesis, such as life-threatening bleeding in the brain and harmful bleeding in joints and muscles. Because it could potentially be cured by gene therapy, subhuman animal models have been sought. Current mouse hemophilia A models generated by gene targeting of the F8 have difficulties to extrapolate human disease due to differences in the coagulation and immune systems between mice and humans. Here, we generated a porcine model of hemophilia A by nuclear transfer cloning from F8-targeted fibroblasts. The hemophilia A pigs showed a severe bleeding tendency upon birth, similar to human severe hemophiliacs, but in contrast to hemophilia A mice which rarely bleed under standard breed conditions. Infusion of human factor VIII was effective in stopping bleeding and reducing the bleeding frequency of a hemophilia A piglet but was blocked by the inhibitor against human factor VIII. These data suggest that the hemophilia A pig is a severe hemophilia A animal model for studying not only hemophilia A gene therapy but also the next generation recombinant coagulation factors, such as recombinant factor VIII variants with a slower clearance rate.

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 mu g/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. (C) 2011 Elsevier Ltd. All rights reserved.

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, p<0.001). 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.

Vinculin is a highly conserved actin-binding protein that is localized in integrin-mediated focal adhesion complexes. Although critical roles have been proposed for integrins in hematopoietic stem cell (HSC) function, little is known about the involvement of intracellular focal adhesion proteins in HSC functions. This study showed that the ability of c-Kit(+)Sca1(+)Lin(-) HSCs to support reconstitution of hematopoiesis after competitive transplantation was severely impaired by lentiviral transduction with short hairpin RNA sequences for vinculin. The potential of these HSCs to differentiate into granulocytic and monocytic lineages, to migrate toward stromal cell-derived factor 1α, and to home to the bone marrow in vivo were not inhibited by the loss of vinculin. However, the capacities to form long term culture-initiating cells and cobblestone-like areas were abolished in vinculin-silenced c-Kit(+)Sca1(+)Lin(-) HSCs. In contrast, adhesion to the extracellular matrix was inhibited by silencing of talin-1, but not of vinculin. Whole body in vivo luminescence analyses to detect transduced HSCs confirmed the role of vinculin in long term HSC reconstitution. Our results suggest that vinculin is an indispensable factor determining HSC repopulation capacity, independent of integrin functions.

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 31.5microg/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 beta-actin promoter, the liver-specific human alpha1 anti-trypsin promoter (HAAT) and the liver-specific hepatic control region (HCR) enhancer/human alpha1 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 beta-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.

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 naïve 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. © 2009 International Society on Thrombosis and Haemostasis.

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.

AIMS: The aim of the study was to assess mechanisms and clinical backgrounds in order to determine residual platelet aggregability in dual antiplatelet therapy and to ascertain whether platelet aggregability is involved in systemic thrombogenicity. METHODS AND RESULTS: A cross-sectional study was conducted in 85 consecutive patients who underwent dual antiplatelet therapy (aspirin and thienopyridine/cilostazol) after percutaneous coronary intervention (PCI). Although serum thromboxane B(2) and dephosphorylation of vasodilator-stimulated phosphoprotein were significantly abolished, the platelet aggregation tests showed inter-individual differences that could be partly explained by plasma glucose levels. Platelet aggregability was not related to other factors involved in thrombogenicity. Thrombin generation assessed by soluble fibrin was independently associated with total cholesterol (beta = 0.349, P < 0.001), brain natriuretic peptide (beta = 0.222, P = 0.018), and ankle-brachial index (beta = -0.330, P = 0.001). Plasminogen activator inhibitor-1 was associated with the apnea-hypopnea index (beta = 0.300, P = 0.006). E-selectin was correlated with diabetes mellitus (beta = 0.279, P = 0.008) and body mass index (beta = 0.323, P = 0.002). CONCLUSION: Although dual antiplatelet therapy effectively inhibited its pharmacological targets, thrombin generation, inhibition of fibrinolytic activity, and endothelial dysfunction were determined by other clinical backgrounds. Our data suggested that some patients remain at risk of thrombotic complications after PCI and that these may benefit from anticoagulant treatment despite adequate dual antiplatelet therapy.

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).

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 gammaThr-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 gamma-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.

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.

OBJECTIVE: Because platelets are anucleate cells having a limited life span, direct gene manipulation cannot in principle be used to investigate the involvement of a specific signal transduction pathway in platelet activation. In this study, we examined whether the expression of a short hairpin RNA (shRNA) sequence in hematopoietic stem cells is maintained during megakaryocyte differentiation, thus resulting in inhibition of targeted protein in platelets. METHODS AND RESULTS: To identify platelets derived from transduced stem cells, we generated a lentiviral vector that simultaneously expresses the shRNA sequence driven by the U6 promoter and GFP under the control of the glycoprotein (GP) Ib alpha promoter. Transplantation of mouse bone marrow cells transduced with the vector facilitated specifically mark platelets derived from the transduced cells. Transplantation of cells transduced with shRNA sequence targeting integrin alphaIIb caused a significant reduction of integrin alphaIIb beta3 (alphaIIb beta3) expression in GFP-positive platelets. It also inhibited alphaIIb beta3 activation assessed by the binding of JON/A, an antibody that recognizes activated alphaIIb beta3. Talin-1 silencing by the same method resulted in normal alphaIIb beta3 expression but deficient inside-out alphaIIb beta3 signaling. CONCLUSIONS: shRNA expression driven by the U6 promoter is preserved during megakaryopoiesis. This method facilitates functional analysis of targeted protein in platelet activation.

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.

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/gamma, D1/gamma, and D1/beta 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.

Neural stem/progenitor cells (NSPCs) migrate toward a damaged area of the central nervous system (CNS) for the purpose of limiting and/or repairing the damage. Although this migratory property of NSPCs could theoretically be exploited for cell-based therapeutics of CNS diseases, little is known of the mechanisms responsible for migratory responses of NSPCs. Here, we found that sphingosine 1-phosphate (Sph-1-P), a physiological lysophospholipid mediator, had a potent chemoattractant activity for NSPCs, in which, of Sph-1-P receptors, S1P(1) was abundantly expressed. Sph-1-P-induced NSPC migration was inhibited by the pretreatment with pertussis toxin, Y-27632 (a Rho kinase inhibitor), and VPC23019 (a competitive inhibitor of S1P(1) and S1P(3)). Sph-1-P does not act as intracellular mediator or in an autocrine manner, because [(3)H]sphingosine, incorporated into NSPCs, was mainly converted to ceramide and sphingomyeline intracellularly, and the stimulation-dependent formation and extracellular release of Sph-1-P were not observed. Further, Sph-1-P concentration in the spinal cord was significantly increased at 7 days after a contusion injury, due to accumulation of microglia and reactive astrocytes in the injured area. This locally increased Sph-1-P concentration contributed to the migration of in vivo transplanted NSPCs through its receptor S1P(1), given that lentiviral transduction of NSPCs with a short hairpin RNA interference for S1P(1) abolished in vivo NSPC migration toward the injured area. This is the first report to identify a physiological role for a lipid mediator in NSPC migration toward a pathological area of the CNS and further indicates that the Sph-1-P/S1P(1) pathway may have therapeutic potential for CNS injuries.

Sepsis-induced disseminated intravascular coagulation (DIC) is a serious condition because it is closely linked to the development of multiple organ dysfunctions. We compared molecular fibrinolysis markers for 117 patients with sepsis-induced DIC and 1627 patients with nonseptic DIC. Levels of fibrinogen and fibrin degradation products and D-dimer were significantly lower in sepsis-induced DIC cases than in nonseptic DIC cases. In septic DIC cases, plasma plasminogen activator inhibitor 1 (PAI-1) levels were significantly higher than in nonseptic DIC cases. D-dimer levels were negatively correlated with plasma PAI-1 levels in septic DIC cases. Multiple Organ Dysfunction Scores were significantly higher in septic DIC patients with PAI-1 levels >90 ng/mL than in the group with PAI-1 levels <30 ng/mL. The Kaplan-Meier survival functions until 28 days after DIC diagnosis were significantly lower in the group with PAI-1 levels >90 ng/mL than in the other groups. In a multivariate analysis, plasma PAI-1 levels at DIC diagnosis were an independent risk factor for mortality in sepsis-induced DIC (hazard ratio, 1.012; P = .008). These data suggest that plasma PAI-1 plays an important role in sustaining DIC in septic DIC cases and contributes to multiple organ failure and decreased survival in such patients.

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.

Platelets release several mediators that modify vascular integrity and hemostasis. In the present study, we developed a technique for efficient transgene expression in platelets in vivo and examined whether this targeted-gene-product delivery system using a platelet release reaction could be exploited for clinical applications. Analysis of luciferase reporter gene constructs driven by platelet-specific promoters (the GPIIb, GPIbalpha, and GPVI) revealed that the GPIbalpha promoter was the most potent in the megakaryoblastic cell line UT-7/TPO and human CD34+-derived megakaryocytes. Transduction of UT-7/TPO; CD34+-derived megakaryocytes; and c-Kit+, ScaI+, and Lineage- (KSL) murine hematopoietic stem cells with a simian immunodeficiency virus (SIV)-based lentiviral vector carrying eGFP resulted in efficient, dose-dependent expression of eGFP, and the GPIbalpha promoter seemed to bestow megakaryocytic-specific expression. Transplantation of KSL cells transduced with SIV vector containing eGFP into mice showed that there was preferable expression of eGFP in platelets driven by the GPIbalpha promoter [7-11% for the cytomeglovirus (CMV) promoter, 16-27% for the GPIbalpha promoter]. Furthermore, transplantation of ex vivo-transduced KSL cells by SIV vector carrying human factorVIII (hFVIII) driven by the GPIbalpha promoter induced the production of detectable transcripts of the hFVIII gene and the hFVIII antigen in bone marrow and spleen for at least 90 days and partially corrected the hemophilia A phenotype. Platelet-targeting gene therapy using SIV vectors appears to be promising for gene therapy approaches toward not only inherited platelet diseases but also other hemorrhagic disorders such as hemophilia A.

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 microM [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 microM [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.

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.

The LIM domain is a protein-protein interaction motif critically involved in a variety of fundamental biological processes, including cytoskeletal organization, cell lineage specification, and organ development. In this study we examined the expression of the LIM proteins paxillin and Hic-5 in adult human tissues by immunohistochemistry and immunoblotting. Paxillin expression was widespread and observed both in non-muscle and muscle tissues. Of the latter, paxillin was mainly expressed in multinuclear striated muscle. In contrast, Hic-5 showed restricted expression and was expressed in muscle tissues, mainly in mononuclear smooth muscle. Taken together with previous findings, it appears likely that the counterbalance between paxillin and Hic-5 may be deeply involved in muscle differentiation.

Objectives: 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) 1191]. In this study, metabolism related to and cellular responses elicited by Sph-1-P were examined in human coronary artery smooth muscle cells (CASMCs). Methods and results: [H-3]Sphingosine (Sph), incorporated into CASMCs, was converted to [H-3]Sph-1-P intracellularly, but its stimulation-dependent formation and extracellular release were not observed. Furthermore, the cell surface Sph-1-P receptors of SIP 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 SIP2 (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. Conclusions: 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. (C) 2003 European Society of Cardiology. Published by Elsevier Science B.V. All rights reserved.

Sphingosine 1-phosphate (Sph-1-P), a bioactive lysophospholipid capable of inducing a wide spectrum of biological responses, acts as an intercellular mediator, through interaction with the endothelial differentiation gene (EDG)/S1P family of G protein-coupled receptors. In this study, the effects of JTE-013, a specific antagonist of the migration-inhibitory receptor EDG-5, on Sph-1-P-elicited responses were examined in human umbilical vein endothelial cells (HUVECs) and vascular smooth muscle cells (SMCs), which expressed EDG-5 protein weakly and abundantly, respectively. This pyrazolopyridine compound reversed the inhibitory effect of Sph-1-P on SMC migration and further enhanced Sph-1-P-stimulated HUVEC migration. In contrast, its effect on Sph-1-P-induced intracellular Ca2+ mobilization was marginal. Our results indicate that specific regulation of Sph-1-P-modulated migration responses in vascular cells can be achieved by EDG-5 antagonists and that manipulation of Sph-1-P biological activities by each EDG antagonist may lead to a therapeutical application to control vascular diseases. (C) 2002 Elsevier Science (USA). All rights reserved.

Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) is a 130K transmembrane glycoprotein that belongs to the immunoglobulin gene superfamily and is expressed on the surface of hematological or vascular cells, including platelets and endothelial cells. Although the importance of this adhesion molecule in various cell-cell interactions is established, its function in platelets remains ill-defined. In the process of clarifying them mechanism by which the lectin wheat germ agglutinin (WGA) activates platelets, we unexpectedly discovered that PECAM-1 is involved in signal transduction pathways elicited by this N-acetyl-D-glucosamine (NAGlu)-reactive lectin. WGA, which is a very potent platelet stimulator, elicited a rapid surge in Syk and phospholipase C (PLC)-gamma2 tyrosine phosphorylation and the resultant intracellular Ca2+ mobilization; collagen, as reported, induced these responses, but in a much slower and weaker manner. WGA strongly induced tyrosine phosphorylation of a 130-140K protein, which was confirmed to, be PECAM-1 by immunoprecipitation and immunodepletion studies. WGA-induced PECAM-1 tyrosine phosphorylation occurred rapidly, strongly and in a manner independent of platelet aggregation or cell-cell contact; these characteristics of PECAM-1 phosphorylation were not mimicked at all by receptor-mediated platelet agonists. In addition, WGA was found to associated with PECAM-1 itself, and anti-PECAM-1 antibody, as well as NAGlu, specifically inhibited WGA-induced platelet aggregation. In PECAM-1 immunoprecipitates, Src family tyrosine kinases existed, and a kinase activity was detected, which increased upon WGA stimulation. Furthermore, the Src family kinase inhibitor PP2 inhibited WGA-induced platelet aggregation, Ca2+ mobilization, and PLC-gamma2 tyrosine phosphorylation. Finally,. WGA induced PECAM-1 tyrosine phosphorylation and cytoskeletal reorganization in vascular endothelial cells. Our results, suggest that (i) PECAM-1 is involved in WGA-induced platelet activation, (ii) PECAM-1 clustering by WGA activates unique and strong platelet signaling pathways, leading to a rapid PLC activation via Src family kinases, and (iii) WGA is a useful tool for elucidating PECAM-1-mediated signaling with wide implications not confined to platelets.

Hic-5 and paxillin, members of the LIM protein family, have been shown to be localized in focal adhesion and to have a role in integrin-mediated signalling. In the present study we examined the involvement of Hic-5 in human platelet activation: platelets express Hic-5 but not paxiliin, whereas human umbilical-vein vascular endothelial cells and MEG-01 cells express mainly paxiliin. When platelets were stimulated with thrombin, collagen or the stable thromboxane A(2) analogue U46619, Hic-5 was markedly tyrosine-phosphorylated, in a manner dependent on integrin alpha IIb beta3-mediated aggregation. In addition, direct activation of protein kinase C with PMA resulted in tyrosine phosphorylation of Hic-5 only when platelets were fully aggregated with the exogenous addition of fibrinogen. Furthermore, PMA-induced Hic-5 tyrosine phosphorylation was also observed when platelets adhered to immobilized fibrinogen. In studies on immunoprecipitation and immunodepletion, Hic-5 seemed to associate with proline-rich tyrosine kinase 2. (Pyk2) but only marginally with focal adhesion kinase. When platelets were stimulated with thrombin, both Hic-5 and Pyk2 translocated to the cytoskeleton from the cytosol and membrane fractions in a manner dependent on alpha II beta3-mediated aggregation. Finally, on stimulation with PMA, Hic-5: as well as Pyk2, translocated to the cell periphery, where a meshwork of actin filaments assembled after adhesion to immobilized fibrinogen. Our results suggest that Hic-5 might be important in platelet aggregation and adhesion, in a manner dependent on alpha IIb beta3-mediated outside-in signalling, through association with Pyk2.

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 intracelullar 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. (C) 2001 Elsevier Science Inc. All rights reserved.

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 [P-32]Sph-1-P, but not [P-32]LPA, was observed In [P-32]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. (C) 2000 by The American Society of Hematology.