Recurrent Cerebral Hemorrhaging with Platelet Dysfunction Accompanied by Anti-glycoprotein VI Autoantibodies in a Patient with TAFRO Syndrome.

Thrombocytopenia, anasarca, fever, renal dysfunction, and organomegaly (TAFRO) syndrome is an inflammatory disorder with an unclear pathogenesis. We herein report a case of TAFRO syndrome in remission in a patient who experienced recurrent intracranial bleeding despite a normal platelet count and coagulation system. A further investigation suggested the presence of anti-glycoprotein VI (GPVI) autoantibodies in the plasma, which induced platelet dysfunction and bleeding tendency. No new bleeding or relapse of TAFRO syndrome occurred after immunosuppressive therapy was initiated. These findings may help elucidate the autoimmune pathogenesis of TAFRO syndrome.

C-type lectin-like receptor-2 in platelets mediates ferric chloride-induced platelet activation and attenuates ferroptosis of endothelial cells.

BACKGROUND: An iron overload status induces ferroptosis, an iron-dependent nonapoptotic cell death, in various pathological conditions. We previously reported that hemin (heme), protoporphyrin-IX with ferric iron, activates platelets via C-type lectin-like receptor-2 (CLEC-2) and glycoprotein VI/FcRγ, but protoporphyrin-IX alone blocks CLEC-2-dependent platelet activation. Therefore, we hypothesized that free iron has the ability to activate platelets. OBJECTIVES: This study aimed to elucidate platelet activation mechanisms of iron (ferric chloride), including the identification of signaling pathways and receptors, and to examine whether platelets regulate ferroptosis. METHODS: Platelet aggregometry, platelet activation marker expression, and protein phosphorylation were examined in ferric chloride-stimulated human and murine platelets. Inhibitors of platelet activation signaling pathways and receptor-deleted platelets were utilized to identify the responsible signaling pathway and receptor. The effect of platelets on ferroptosis of endothelial cells was investigated in vitro. RESULTS: Ferric chloride induced platelet activation dependent on Src family kinase pathways in humans and mice. Ferric chloride-induced platelet aggregation was almost lost in CLEC-2-depleted murine platelets and wild-type platelets preincubated with recombinant CLEC-2 proteins. Furthermore, coculture of wild-type platelets, but not CLEC-2-deficient platelets, attenuated ferroptosis of endothelial cells in vitro. CONCLUSION: Ferric chloride activates platelets via CLEC-2 and Src family kinase pathways, and platelets have a protective role in the ferroptosis of endothelial cells dependent on CLEC-2.

Abnormalities in C-type lectin-like receptor 2 in a patient with Gorham-Stout disease: the first case report.

Background: Gorham-Stout disease (GSD) is a form of lymphangiomatosis of unknown etiology, characterized by abnormal distribution of lymphatic vessels. Platelets and lymphangiogenesis are closely related via C-type lectin-like receptor 2 (CLEC-2)/podoplanin. Key Clinical Question: Despite similarities between abnormal lymphatic vessels in CLEC-2-deficient mice and patients with GSD, whether CLEC-2 on platelets is involved in GSD pathogenesis is unknown. Clinical Approach: We examined CLEC-2 expression in platelets of a patient with lethal GSD. Most of the patient's platelets expressed aberrant CLEC-2 that was not detectable by certain monoclonal antibodies for human CLEC-2. Further, this population was not activated by a CLEC-2-activating snake venom, rhodocytin. Possible causes of abnormal CLEC-2 including anti-CLEC-2 autoantibodies, podoplanin binding to CLEC-2, and pathogenic CLEC1B gene alteration were excluded. Conclusions: We believe that this is the first report of a patient with structurally and functionally abnormal CLEC-2. CLEC-2 abnormality may be associated with dysregulated lymphangiogenesis in GSD.

C-type lectin-like receptor-2 (CLEC-2) is a key regulator of kappa-carrageenan-induced tail thrombosis model in mice.

Kappa-carrageenan (KCG), which is used to induce thrombosis in laboratory animals for antithrombotic drug screening, can trigger platelet aggregation. However, the cell-surface receptor and related signaling pathways remain unclear. In this study, we investigated the molecular basis of KCG-induced platelet activation using light-transmittance aggregometry, flow cytometry, western blotting, and surface plasmon resonance assays using platelets from platelet receptor-deficient mice and recombinant proteins. KCG-induced tail thrombosis was also evaluated in mice lacking the platelet receptor. We found that KCG induces platelet aggregation with α-granule secretion, activated integrin αIIbß3, and phosphatidylserine exposure. As this aggregation was significantly inhibited by the Src family kinase inhibitor and spleen tyrosine kinase (Syk) inhibitor, a tyrosine kinase-dependent pathway is required. Platelets exposed to KCG exhibited intracellular tyrosine phosphorylation of Syk, linker activated T cells, and phospholipase C gamma 2. KCG-induced platelet aggregation was abolished in platelets from C-type lectin-like receptor-2 (CLEC-2)-deficient mice, but not in platelets pre-treated with glycoprotein VI-blocking antibody, JAQ1. Surface plasmon resonance assays showed a direct association between murine/human recombinant CLEC-2 and KCG. KCG-induced thrombosis and thrombocytopenia were significantly inhibited in CLEC-2-deficient mice. Our findings show that KCG induces platelet activation via CLEC-2.

Thrombosis is a serious medical condition that occurs when blood clots form in the blood vessels and can lead to heart attacks or strokes. Animal models are important for evaluating the effectiveness of drugs in thrombosis treatment. Kappa-carrageenan (KCG) is a food thickener and a substance used to induce clot formation in laboratory animals. In this study, we investigated the molecular basis of KCG-induced platelet activation, which is an important step in thrombosis development. We found that KCG activates platelets via a receptor called C-type lectin-like receptor 2 (CLEC-2), leading to a prothrombotic state in mice. We also showed that KCG-induced tail thrombosis (CTT) is significantly inhibited in CLEC-2 deficient mice. Our findings suggest that CLEC-2-mediated platelet activation plays a key role in the pathogenesis of thrombosis and CLEC-2 May participate in innate immunity as a receptor for sulfate-polysaccharide.Abbreviation; CLEC-2: C-type lectin-like receptor 2; CRP: collagen-related peptide; CTT: KCGN-induced tail thrombosis; DIC: disseminated intravascular coagulation; EDTA: ethylenediaminetetraacetic acid; GPVI: glycoprotein VI; HRP: horseradish peroxidase; KCG: Κ-Carrageenan; LAT: linker activated T cells; LDS: lithium dodecyl sulfate; LTA: light-transmittance aggregometry; MFI: mean fluorescence intensity; PFA: paraformaldehyde; PLCγ2: phospholipase C gamma 2; PS: phosphatidylserine; Syk: spleen tyrosine kinase; Co-HP: Cobalt-hematoporphyrin.

Cancer-associated fibroblasts promote venous thrombosis through podoplanin/CLEC-2 interaction in podoplanin-negative lung cancer mouse model.

BACKGROUND: Cancer-associated thrombosis (CAT) is the leading cause of morbidity and mortality. Cancer-associated fibroblasts (CAFs) are a prominent component of the tumor microenvironment that contributes to cancer progression through direct cell-cell interactions and the release of extracellular vesicles (EVs). However, the role of CAFs in CAT remains unclear. OBJECTIVE: This study aims to investigate whether CAFs aggravate CAT and the underlying molecular mechanism using a preclinical mouse lung cancer model. METHODS: We designed a Lewis lung carcinoma (LLC) tumor-bearing mouse model. CAFs were characterized using fluorescence immunohistostaining. The presence of podoplanin, a platelet-activating membrane protein through C-type lectin-like receptor 2 (CLEC-2), in EVs isolated from primary CAFs or LLC tumor tissues was assessed by immunoblotting. The platelet activation and aggregation abilities of the EVs were quantified using flow cytometry. Podoplanin plasma levels were measured by enzyme-linked immunosorbent assay. Venous thrombosis was induced in the femoral vein using 2.5% ferric chloride. The anti-CLEC-2 monoclonal antibody 2A2B10 was used to deplete CLEC-2 on the surface of the platelets. RESULTS: CAFs expressing CD90, PDGFRß, HSP47, CD34, and vimentin, co-expressed podoplanin and induced platelet activation and aggregation in a CLEC-2-dependent manner. Tumor-bearing mice showed elevated podoplanin plasma levels. CAF-EV injection and tumor-bearing mice showed shorter occlusion time in the venous thrombosis model. Although tumor growth was not altered, antibody-induced CLEC-2 depletion suppressed venous thrombosis in the tumor-bearing state but not in the healthy condition. CONCLUSION: CAFs and CAF-derived EVs induce CLEC-2-dependent platelet aggregation and aggravate venous thrombosis.

Heme activates platelets and exacerbates rhabdomyolysis-induced acute kidney injury via CLEC-2 and GPVI/FcRγ.

There is increasing evidence that platelets participate in multiple pathophysiological processes other than thrombosis and hemostasis, such as immunity, inflammation, embryonic development, and cancer progression. A recent study revealed that heme (hemin)-activated platelets induce macrophage extracellular traps (METs) and exacerbate rhabdomyolysis-induced acute kidney injury (RAKI); however, how hemin activates platelets remains unclear. Here, we report that both C-type lectin-like receptor-2 (CLEC-2) and glycoprotein VI (GPVI) are platelet hemin receptors and are involved in the exacerbation of RAKI. We investigated hemin-induced platelet aggregation in humans and mice, binding of hemin to CLEC-2 and GPVI, the RAKI-associated phenotype in a mouse model, and in vitro MET formation. Using western blotting and surface plasmon resonance, we showed that hemin activates human platelets by stimulating the phosphorylation of SYK and PLCγ2 and directly binding to both CLEC-2 and GPVI. Furthermore, hemin-induced murine platelet aggregation was partially reduced in CLEC-2-depleted and FcRγ-deficient (equivalent to GPVI-deficient) platelets and almost completely inhibited in CLEC-2-depleted FcRγ-deficient (double-knockout) platelets. In addition, hemin-induced murine platelet aggregation was inhibited by the CLEC-2 inhibitor cobalt hematoporphyrin or GPVI antibody (JAQ-1). Renal dysfunction, tubular injury, and MET formation were attenuated in double-knockout RAKI mice. Furthermore, in vitro MET formation assay showed that the downstream signaling pathway of CLEC-2 and GPVI is involved in MET formation. We propose that both CLEC-2 and GPVI in platelets play an important role in RAKI development.

CLEC-2 stimulates IGF-1 secretion from podoplanin-positive stromal cells and positively regulates erythropoiesis in mice.

BACKGROUND: Erythropoiesis is a complex multistep process by which erythrocytes are produced. C-type lectin-like receptor 2 (CLEC-2) is a podoplanin (PDPN) receptor almost exclusively expressed on the surface of platelets and megakaryocytes. Deletion of megakaryocyte/platelet CLEC-2 was reported to cause anemia along with thrombocytopenia in mice. PDPN-expressing stromal cells in the bone marrow (BM) were also reported to facilitate megakaryocyte expansion and maturation depending on the CLEC-2/PDPN interaction. OBJECTIVES: We investigated how specific deletion of CLEC-2 in megakaryocytes/platelets leads to anemia. METHODS: We used flow cytometry to analyze maturation of erythroblasts, apoptotic cell death, and cell cycle distribution. CLEC-2 stimulated PDPN-expressing stromal cell-conditioned medium was analyzed by cytokine array and ELISA, and co-cultured with immature erythroblasts. Cytokine levels in serum and BM extracellular fluid were quantified by ELISA. RESULTS: We observed increased apoptosis of BM erythroblasts in megakaryocyte/platelet-specific CLEC-2 conditional knockout (Clec1bΔPLT ) mice. Moreover, PDPN-expressing stromal cells in the BM secreted insulin-like growth factor 1 (IGF-1) depending on the CLEC-2/PDPN interaction. Pretreatment with IGF-1 receptor inhibitor increased apoptosis rate and decreased the proliferation of erythroblasts in vitro. Furthermore, in Clec1bΔPLT mice, IGF-1 concentrations in serum and BM extracellular fluid were decreased, and IGF-1 replacement in Clec1bΔPLT mice attenuated anemia. CONCLUSIONS: Our findings suggest that IGF-1 secretion from PDPN-expressing stromal cells by CLEC-2 stimulation positively regulates erythroblasts. This novel mechanism of erythropoiesis regulation indicates that a microenvironment consisting of megakaryocytes and PDPN-expressing stromal cells supports erythropoiesis.

Soluble CLEC-2 is generated independently of ADAM10 and is increased in plasma in acute coronary syndrome: comparison with soluble GPVI.

Soluble forms of platelet membrane proteins are released upon platelet activation. We previously reported that soluble C-type lectin-like receptor 2 (sCLEC-2) is released as a shed fragment (Shed CLEC-2) or as a whole molecule associated with platelet microparticles (MP-CLEC-2). In contrast, soluble glycoprotein VI (sGPVI) is released as a shed fragment (Shed GPVI), but not as a microparticle-associated form (MP-GPVI). However, mechanism of sCLEC-2 generation or plasma sCLEC-2 has not been fully elucidated. Experiments using metalloproteinase inhibitors/stimulators revealed that ADAM10/17 induce GPVI shedding, but not CLEC-2 shedding, and that shed CLEC-2 was partially generated by MMP-2. Although MP-GPVI was not generated, it was generated in the presence of the ADAM10 inhibitor. Moreover, antibodies against the cytoplasmic or extracellular domain of GPVI revealed the presence of the GPVI cytoplasmic domain, but not the extracellular domain, in the microparticles. These findings suggest that most of the GPVI on microparticles are induced to shed by ADAM10; MP-GPVI is thus undetected. Plasma sCLEC-2 level was 1/32 of plasma sGPVI level in normal subjects, but both soluble proteins significantly increased in plasma of patients with acute coronary syndrome. Thus, sCLEC-2 and sGPVI are released by different mechanisms and released in vivo upon platelet activation.

Combined Coagulopathy Can Induce Both Hemorrhagic and Thrombotic Complications in Multiple Myeloma.

Coagulation abnormalities are a rare but critical complication associated with plasma cell diseases. We herein present a case of multiple myeloma (MM) with complicated coagulopathy. Initially, the patient showed severe bleeding tendency due to concomitant acquired hemophilia A and acquired von Willebrand syndrome. Interestingly, the patient also exhibited hyperactivation of factor IX. During treatment for MM, the bleeding complications were ameliorated; however, the patient had central retinal vein occlusion. All of the coagulation abnormalities were completely resolved after the complete remission of MM. This case suggests that MM patients may have concomitant risks for both bleeding and thromboembolic complications.

Mutation in a highly conserved glycine residue in strand 5B of plasminogen activator inhibitor 1 causes polymerisation.

Serpinopathy is characterised as abnormal accumulation of serine protease inhibitors (SERPINs) in cells and results in clinical symptoms owing to lack of SERPIN function or excessive accumulation of abnormal SERPIN. We recently identified a patient with functional deficiency of plasminogen activator inhibitor-1 (PAI-1), a member of the SERPIN superfamily. The patient exhibited life-threatening bleeding tendencies, which have also been observed in patients with a complete deficiency in PAI-1. Sequence analysis revealed a homozygous single-nucleotide substitution from guanine to cytosine at exon 9, which changed amino acid residue 397 from glycine to arginine (c.1189G>C; p.Gly397Arg). This glycine was located in strand 5B and was well conserved in other serpins. The mutant PAI-1 was polymerised in the cells, interfering with PAI-1 secretion. The corresponding mutations in SERPINC1 (anti-thrombin III) at position 456 (Gly456Arg) and SERPINI1 (neuroserpin) at position 392 (Gly392Glu) caused an anti-thrombin deficiency and severe dementia due to intracellular retention of the polymers. Glycine is the smallest amino acid, and these mutated amino acids were larger and charged. To determine which factors were important, further mutagenesis of PAI-1 was performed. Although the G397A, C, I, L, S, T, and V were secreted, the G397D, E, F, H, K, M, N, P, Q, W, and Y were not secreted. The results revealed that the size was likely triggered by the polymerisation of SEPRINs at this position. Structural analyses of this mutated PAI-1 would be useful to develop a novel PAI-1 inhibitor, which may be applicable in the context of several pathological states.

Aspirin Half Maximal Inhibitory Concentration Value on Platelet Cyclooxygenase1 in Severe Type-2 Diabetes Mellitus is not Significantly Different from that of Healthy Individuals.

It is implicated that diabetic patients are more resistant to aspirin therapy than patients with other diseases or healthy individuals. We evaluated the inhibitory effects of aspirin on aggregation and the cyclooxygenase activity of platelets of 10 patients with severe type-2 diabetes mellitis (DM) and compared the results with those of healthy individuals. Although platelet aggregation had a tendency to be more resistant to aspirin with the DM group, there was no significant difference in half maximal inhibitory concentration 50 values of aspirin on the cyclooxygenase activity between the patients with DM and healthy individuals. Thus, the residual platelet aggregability uninhibited by aspirin appears to be independent of the cyclooxygenase activity. Since adenosine diphosphate (ADP) receptor blocking almost completely inhibited the residual platelet aggregability, it is suggested that hyperreactivity to ADP is more prevalent in patients with DM.

VacA, the vacuolating cytotoxin of Helicobacter pylori, binds to multimerin 1 on human platelets.

Platelets were activated under the infection with H. pylori in human and mice. We investigated the role of VacA, an exotoxin released by H. pylori in this context. Acid-activated VacA, but not heated VacA, induced platelet CD62P expression. However, VacA reacted with none of the alleged VacA receptors present on platelet membranes. We therefore analyzed VacA associated proteins obtained through VacA affinity chromatography, using MALDI-TOF-MS. Multimerin1 was detected in two consecutive experiments, as the binding protein for VacA. Plasmon resonance confirmed their binding, and dot blot analysis revealed that the peptide sequence AA 321-340 of multimerin 1 is the binding site for VacA. In conclusion, we propose a new interaction between multimerin1 and VacA , which may give another insight into H. pylori-induced platelet activations under H. pylori infection.

Platelet activation receptor CLEC-2 regulates blood/lymphatic vessel separation by inhibiting proliferation, migration, and tube formation of lymphatic endothelial cells.

The platelet activation receptor CLEC-2 plays crucial roles in thrombosis/hemostasis, tumor metastasis, and lymphangiogenesis, although its role in thrombosis/hemostasis remains controversial. An endogenous ligand for CLEC-2, podoplanin, is expressed in lymphatic endothelial cells (LECs). We and others have reported that CLEC-2-deficiency is lethal at mouse embryonic/neonatal stages associated with blood-filled lymphatics, indicating that CLEC-2 is essential for blood/lymphatic vessel separation. However, its mechanism, and whether CLEC-2 in platelets is necessary for this separation, remains unknown. We found that specific deletion of CLEC-2 from platelets leads to the misconnection of blood/lymphatic vessels. CLEC-2(+/+) platelets, but not by CLEC-2(-/-) platelets, inhibited LEC migration, proliferation, and tube formation but had no effect on human umbilical vein endothelial cells. Additionally, supernatants from activated platelets significantly inhibited these three functions in LECs, suggesting that released granule contents regulate blood/lymphatic vessel separation. Bone morphologic protein-9 (BMP-9), which we found to be present in platelets and released upon activation, appears to play a key role in regulating LEC functions. Only BMP-9 inhibited tube formation, although other releasates including transforming growth factor-ß and platelet factor 4 inhibited proliferation and/or migration. We propose that platelets regulate blood/lymphatic vessel separation by inhibiting the proliferation, migration, and tube formation of LECs, mainly because of the release of BMP-9 upon activation by CLEC-2/podoplanin interaction.

Liver-restricted expression of the canine factor VIII gene facilitates prevention of inhibitor formation in factor VIII-deficient mice.

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.

[Detection of the preservative chlorphenesin in cosmetics by high-performance liquid chromatography].

A simple determination method for preservative chlorphenesin in cosmetics was developed. Cosmetic samples were dissolved in methanol. The sample solution was analyzed by high-performance liquid chromatography (HPLC) with ODS column, using water-methanol (55:45) or water-acetonitrile (3:1) adjusted to pH 2.5 with phosphoric acid as the mobile phase. Chlorphenesin was detected with ultraviolet light detection at 280 nm. A linear relation was obtained between the peak areas and the concentrations of chlorphenesin in the range of 1-500 microg/ml. The determination limit of chlorphenesin was 1-2 microg/ml. Recoveries of chlorphenesin spiked in lotion and milky lotion at the levels of 0.03% and 0.3% were 98.8-100.0%. This method was applied for cosmetics including 0.03% and 0.3% of chlorphenesin and their content corresponded with the determined values.

Double-labelled in situ hybridization reveals the lack of co-localization of mRNAs for the circadian neuropeptide PDF and FMRFamide in brains of the flies Musca domestica and Drosophila melanogaster.

Many lines of evidence have suggested that neuropeptides other than pigment-dispersing factor (PDF) are involved in regulating insect circadian rhythms, and FMRFamide-related peptides are additional candidates acting as such neuromodulators. Double-immunolabelling in insect brains with anti-crustacean beta-PDH and anti-FMRFamide antibodies had previously suggested that insect PDF and FMRFamide-like peptides may coexist in the same cells. However, it is critical for this kind of comparative investigations to use antibodies of proven specificity, to eliminate the possibility of both reciprocal cross-reactivity and the detection of unknown peptides. In the present study, we achieved the cDNA cloning of an fmrf mRNA from the housefly Musca domestica, for which co-localization of FMRFamide and PDF peptides was previously suggested. In order to examine the possible co-expression of this gene with the pdf gene, we carried out double-labelled in situ hybridization for simultaneous detection of both pdf and fmrf mRNAs in housefly, Musca brains. The results clearly indicated that they occur in distinctly different cells. This was also proven for the fruit fly Drosophila melanogaster by similar double-labelled in situ hybridization. The results thus revealed no reason to evoke the physiological release of FMRFamide and PDF peptides from the same neurons.

Efficient expression of a transgene in platelets using simian immunodeficiency virus-based vector harboring glycoprotein Ibalpha promoter: in vivo model for platelet-targeting gene therapy.

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.

Severe secondary deficiency of von Willebrand factor-cleaving protease (ADAMTS13) in patients with sepsis-induced disseminated intravascular coagulation: its correlation with development of renal failure.

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.

Phenotype correction of hemophilia A mice with adeno-associated virus vectors carrying the B domain-deleted canine factor VIII gene.

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.

Sustained transgene expression by human cord blood derived CD34+ cells transduced with simian immunodeficiency virus agmTYO1-based vectors carrying the human coagulation factor VIII gene in NOD/SCID mice.

BACKGROUND: Gene therapy is being studied as the next generation therapy for hemophilia and several clinical trials have been carried out, albeit with limited success. To explore the possibility of utilizing autologous bone marrow transplantation of genetically modified hematopoietic stem cells for hemophilia gene therapy, we investigated the efficacy of genetically engineered CD34+ cell transplantation to NOD/SCID mice for expression of human factor VIII (hFVIII). METHODS: CD34+ cells were transduced with a simian immunodeficiency virus agmTYO1 (SIV)-based lentiviral vector carrying the enhanced green fluorescent protein (eGFP) gene (SIVeGFP) or the hFVIII gene (SIVhFVIII). CD34+ cells transduced with SIV vectors were transplanted to NOD/SCID mice. Engraftment of transduced CD34+ cells and expression of transgenes were studied. RESULTS: We could efficiently transduce CD34+ cells using the SIVeGFP vector in a dose-dependent manner, reaching a maximum (99.6 +/- 0.1%) at MOI of 5 x 10(3) vector genome/cell. After transducing CD34+ cells with SIVhFVIII, hFVIII was produced (274.3 +/- 20.1 ng) from 10(6) CD34+ cells during 24 h in vitro incubation. Transplantation of SIVhFVIII-transduced CD34+ cells (5-10 x 10(5)) at a multiplicity of infection (MOI) of 50 vector genome/cell into NOD/SCID mice resulted in successful engraftment of CD34+ cells and production of hFVIII (minimum 1.2 +/- 0.9 ng/mL, maximum 3.6 +/- 0.8 ng/mL) for at least 60 days in vivo. Transcripts of the hFVIII gene and the hFVIII antigen were also detected in the murine bone marrow cells. CONCLUSIONS: Transplantation of ex vivo transduced hematopoietic stem cells by non-pathogenic SIVhFVIII without exposure of subjects to viral vectors is safe and potentially applicable for gene therapy of hemophilia A patients.