Mitsuhiko Sugimoto

ADAMTS13 gene deletion aggravates ischemic brain damage: a possible neuroprotective role of ADAMTS13 by ameliorating postischemic hypoperfusion.

Reperfusion after brain ischemia causes thrombus formation and microcirculatory disturbances, which are dependent on the platelet glycoprotein Ib-von Willebrand factor (VWF) axis. Because ADAMTS13 cleaves VWF and limits platelet-dependent thrombus growth, ADAMTS13 may ameliorate ischemic brain damage in acute stroke. We investigated the effects of ADAMTS13 on ischemia-reperfusion injury using a 30-minute middle cerebral artery occlusion model in Adamts13(-/-) and wild-type mice. After reperfusion for 0.5 hours, the regional cerebral blood flow in the ischemic cortex was decreased markedly in Adamts13(-/-) mice compared with wild-type mice (P < .05), which also resulted in a larger infarct volume after 24 hours for Adamts13(-/-) compared with wild-type mice (P < .01). Thus, Adamts13 gene deletion aggravated ischemic brain damage, suggesting that ADAMTS13 may protect the brain from ischemia by regulating VWF-platelet interactions after reperfusion. These results indicate that ADAMTS13 may be a useful therapeutic agent for stroke.

Identification of a point mutation in type IIB von Willebrand disease illustrating the regulation of von Willebrand factor affinity for the platelet membrane glycoprotein Ib-IX receptor.

von Willebrand factor (vWF) supports platelet adhesion on thrombogenic surfaces by binding to platelet membrane glycoprotein (GP) Ib in the GP Ib-IX receptor complex. This interaction is physiologically regulated so that it does not occur between circulating vWF and platelets but, rather, only at a site of vascular injury. The abnormal vWF found in type IIB von Willebrand disease, however, has a characteristically increased affinity for GP Ib and binds to circulating platelets. We have analyzed the molecular basis of this abnormality by sequence analysis of a type IIB vWF cDNA and have identified a single amino acid change, Trp550 to Cys550, located in the GP Ib-binding domain of the molecule comprising residues 449-728. Bacterial expression of recombinant fragments corresponding to this vWF domain yielded molecules that, whether containing a normal Trp550 or a mutant Cys550 residue, bound directly to GP Ib in the absence of modulators and with similar affinity. In contrast, mammalian cell expression of the same segment of sequence yielded molecules that, when containing the normal Trp550, did not bind to GP Ib directly but, like native vWF, bound in the presence of ristocetin. However, molecules containing the point mutation (Cys550) behaved like type IIB vWF--namely, bound to GP Ib even without ristocetin modulation and, in the presence of ristocetin, had 10-fold higher affinity than molecules with normal sequence. These results identify a region of vWF that, although not thought to be directly involved in binding to GP Ib, may modulate the interaction through conformational changes.

Functional Property of von Willebrand Factor Under Flowing Blood

Abstractvon Willebrand factor (vWF) is produced in megakaryocytes and endothelial cells, is stored in the α-granule of platelets and in the Weibel-Palade body of endothelial cells, and is present in plasma and vascular subendothelium. This huge protein with a unique multimeric structure plays a pivotal role in both hemostasis and pathological intravascular thrombosis, in which vWF contributes to both platelet adhesion/aggregation and blood coagulation through its multiple adhesive functions for the platelet membrane receptors, glycoprotein Ib-IX-V complex, integrin αIIbβ3, heparin, various types of collagen, and coagulation factor VIII. Among various functions, the most characteristic feature of vWF is its determinant role on platelet thrombus formation under high-shear-rate conditions. Indeed, at in vivo rheological situations where platelets are flowing with high speed in the bloodstream, the only reaction that can initiate mural thrombogenesis is the interaction of vWF with platelet glycoprotein Iba. The recent x-ray analysis of the crystal structure of various functional domains and functional studies of this protein under experimental flow conditions have rapidly advanced and revised our knowledge of the structure-function relationships of vWF, a key protein for hemostasis and arterial thrombosis.

Delivery of Full-Length Factor VIII Using a piggyBac Transposon Vector to Correct a Mouse Model of Hemophilia A

Viral vectors have been used for hemophilia A gene therapy. However, due to its large size, full-length Factor VIII (FVIII) cDNA has not been successfully delivered using conventional viral vectors. Moreover, viral vectors may pose safety risks, e.g., adverse immunological reactions or virus-mediated cytotoxicity. Here, we took advantages of the non-viral vector gene delivery system based on piggyBac DNA transposon to transfer the full-length FVIII cDNA, for the purpose of treating hemophilia A. We tested the efficiency of this new vector system in human 293T cells and iPS cells, and confirmed the expression of the full-length FVIII in culture media using activity-sensitive coagulation assays. Hydrodynamic injection of the piggyBac vectors into hemophilia A mice temporally treated with an immunosuppressant resulted in stable production of circulating FVIII for over 300 days without development of anti-FVIII antibodies. Furthermore, tail-clip assay revealed significant improvement of blood coagulation time in the treated mice.piggyBac transposon vectors can facilitate the long-term expression of therapeutic transgenes in vitro and in vivo. This novel gene transfer strategy should provide safe and efficient delivery of FVIII.

Ratio of von Willebrand factor propeptide to ADAMTS13 is associated with severity of sepsis.

ABSTRACT Von Willebrand factor (VWF)–cleaving protease (ADAMTS13) cleaves ultralarge VWF (ULVWF) secreted from endothelium and by which is regulating its physiologic function. An imbalance between ULVWF secretion and ADAMTS13 level occurs in sepsis and may cause multiple organ dysfunction. We evaluated the association between the VWF-propeptide (VWF-pp)/ADAMTS13 ratio and disease severity in patients with severe sepsis or septic shock. In 27 patients with severe sepsis or septic shock and platelet count less than 120 000/&mgr;L, we measured plasma VWF, VWF-pp, and ADAMTS13 levels on hospital days 1, 3, 5, and 7. The VWF-pp/ADAMTS13 ratio was increased greater than 12-fold in patients with severe sepsis or septic shock on day 1 and remained markedly high on days 3, 5, and 7 compared with normal control subjects. The VWF-pp/ADAMTS13 ratio significantly correlated with Acute Physiology and Chronic Health Evaluation II score on days 1 and 5; Sepsis-related Organ Failure Assessment score on days 1, 3, and 5; maximum Sepsis-related Organ Failure Assessment score and tumor necrosis factor &agr; level on days 1, 3, 5, and 7; and creatinine level on days 1, 5, and 7. Patients with greater than stage 1 acute kidney injury had significantly higher VWF-pp/ADAMTS13 ratio than patients without acute kidney injury. In summary, the VWF-pp/ADAMTS13 ratio was associated with disease severity in patients with severe sepsis or septic shock and may help identify patients at risk for multiple organ dysfunction by detecting severe imbalance between ULVWF secretion and ADAMTS13 level.

ADAMTS13 safeguards the myocardium in a mouse model of acute myocardial infarction

Masaaki Doi1,2; Hideto Matsui1; Yukiji Takeda3; Yoshihiko Saito3; Maiko Takeda4; Yasunori Matsunari1,5; Kenji Nishio6; Midori Shima2; Fumiaki Banno7; Masashi Akiyama7; Koichi Kokame7; Toshiyuki Miyata7; Mitsuhiko Sugimoto1 1Department of Regulatory Medicine for Thrombosis, Nara Medical University, Kashihara, Japan; 2Department of Pediatrics, Nara Medical University, Kashihara, Japan; 3Department of Internal Medicine, Nara Medical University, Kashihara, Japan; 4Department of Pathology, Nara Medical University, Kashihara, Japan; 5Department of Anesthesiology, Nara Medical University, Kashihara, Japan; 6Department of General Medicine, Nara Medical University, Kashihara, Japan; 7Department of Molecular Pathogenesis, National Cerebral and Cardiovascular Center, Osaka, Japan

Factor IX Kawachinagano: impaired function of the Gla-domain caused by attached propeptide region due to substitution of arginine by glutamine at position −4

Summary. Factor IX Kawachinagano (KWC) is a mutant factor IX protein initially recognized in a patient with severe haemophilia B, who had 46% of normal factor IX antigen and no detectable clotting activity. Previous studies indicated that factor IX KWC was not activated by factor XIa in the presence of Ca ions. In the present study, we purified and analysed factor IX KWC at a structural level in an attempt to clarify the nature of the impaired reaction with factor XIa. Kinetic studies showed that activation of factor IX KWC by factor X activator from Russells viper venom (RVV‐X) was normal, whereas activation by factor XIa was defective. Amino acid sequence analysis of tryptic peptides and direct analysis of the NH2‐terminal sequence of factor IX KWC demonstrated that this mutant factor IX retained the propeptide region of 18 amino acids due to a substitution of arginine ‐(‐4) by glutamine. These data suggested that the Gla‐domain of factor IX KWC was dysfunctional, although the total γ‐Gla content, measured by alkaline‐hydrolysis, was normal. We assumed that this attached propeptide region of the molecule directly interferes with the adjacent NH2‐terminus and prevents the metal‐induced conformational changes which are essential for biological activity of normal factor IX.

Transient hypoplastic anemia caused by primary human parvovirus B19 infection in a previously untreated patient with hemophilia transfused with a plasma-derived, monoclonal antibody-purified factor VIII concentrate.

BACKGROUND Modern plasma-derived clotting factor concentrates are produced using various virus-inactivation protocols and are assumed to be safer than they were previously with regard to the risk for transmitting viral infections such as human immunodeficiency virus, hepatitis B, and hepatitis C. The risks from viruses that are relatively resistant to the current inactivation procedures remain uncertain. PATIENT A 7-year-old with mild hemophilia A who had not been previously infused with any blood products was treated with a plasma-derived, monoclonal antibody-purified factor VIII concentrate to cover orthopedic surgery after traumatic fracture of his left arm. RESULTS A typical primary human parvovirus (HPV)-B19 infection was observed associated with transient hypoplastic anemia. Retrospective studies including serologic examination and polymerase chain reaction analysis confirmed that the HPV-B19 infection was transmitted by the factor VIII concentrate. CONCLUSIONS Clotting factor concentrates for the treatment of hemophilia retain a risk for HPV-B19 contamination. HPV-B19 viral infection might induce hypoplastic anemia in these patients, particularly during enhanced hemopoiesis after acute blood loss.

A Novel Cell-Sheet Technology That Achieves Durable Factor VIII Delivery in a Mouse Model of Hemophilia A

Gene- or cell-based therapies aimed at creating delivery systems for coagulation factor VIII (FVIII) protein have emerged as promising options for hemophilia A treatment. However, several issues remain to be addressed regarding the efficacies and adverse events of these new classes of therapies. To improve an existing cell-based therapy involving the subcutaneous transplantation of FVIII-transduced blood outgrowth endothelial cells (BOECs), we employed a novel cell-sheet technology that allows individual dispersed cells to form a thin and contiguous monolayer without traditional bioabsorbable scaffold matrices. Compared to the traditional methodology, our cell-sheet approach resulted in longer-term and 3–5-fold higher expression of FVIII (up to 11% of normal) in recipient hemophilia A mice that lacked a FVIII humoral immune response due to transient immunosuppression with cyclophosphamide. Histological studies revealed that the transplanted BOEC sheets were structured as flat clusters, supporting the long-term expression of therapeutic FVIII in plasma from an ectopic subcutaneous space. Our novel tissue-engineering approach using genetically modified BOEC sheets could aid in development of cell-based therapy that will allow safe and effective in vivo delivery of functional FVIII protein in patients with hemophilia A.

Identification of protein Sα gene mutations including four novel mutations in eight unrelated patients with protein S deficiency

Eight distinct and potentially causative mutations were identified in eight unrelated Japanese patients with protein S (PS) deficiency, by direct DNA sequencing of the protein Sα (PSα) gene‐specific polymerase chain reaction products of all 15 exons and exon/intron boundaries. There were five missense mutations, including two novel mutations (Cys80Tyr and Arg314His), and three showed a major impact on the expected gene products: novel mutations of a 5‐bp deletion (delCTCTG887:Cys206Stop) and a nonsense mutation (Glu208Stop), as well as a previously reported splice site (exon 10 +5 A→G) mutation. One of the patients showed compound heterozygosity for delCTCTG887 and 732A→G. Investigation for the cosegregation state of these two mutations with PS deficiency in the patients family suggested that the delCTCTG887 mutation was responsible for the abnormal phenotype and that the 732A→G (Lys155Glu) mutation did not appear to play a key role. However, we also identified the same 732A→G (Lys155Glu) mutation in an unrelated patient with apparent PS deficiency with severe pulmonary embolism, and found that this mutation seemed to cosegregate with a PS‐deficient state in her family members. These data implied that unknown factor(s) other than the 732A→G mutation itself might influence phenotypic expression of PS status in different individuals.