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1.
J Thromb Haemost ; 17(2): 350-360, 2019 02.
Artículo en Inglés | MEDLINE | ID: mdl-30525289

RESUMEN

Essentials Factor (F)VIII with an intermediate-length B-domain showed higher levels in murine gene therapy. FVIII with different B-domain lengths were analysed. FVIII variants with B-domains between 186 and 240 amino acids (aa) have extended half-life in mice. Reduced cell binding of FVIII with a 237aa B-domain may explain the extended half-life. SUMMARY: Background Factor VIII consists of the A1-domain, A2-domain, B-domain, A3-domain, C1-domain, and C2-domain. FVIII with an intermediate-length B-domain of 226 amino acids (aa) has previously been evaluated in murine gene therapy studies. Objective To characterize FVIII with intermediate-length B-domains in vitro and in vivo in F8-knockout (KO) mice. Methods and results FVIII molecules with B-domains of 186-240aa had longer half-lives in F8-KO mice than FVIII molecules with shorter or longer B-domains. FVIII with a B-domain containing the 225 N-terminal aa fused to the 12 C-terminal aa of the wild-type B-domain (FVIII-237) had a 1.6-fold extended half-life in F8-KO mice as compared with FVIII with a 21aa B-domain (FVIII-21). The in vitro and in vivo activity of FVIII-237 were comparable to those of FVIII-21, as was binding to von Willebrand factor. Cell binding to LDL receptor-related protein 1 (LRP-1)-expressing cells was markedly reduced for FVIII-237 as compared with FVIII-21, whereas the affinity for LRP-1 was not reduced in surface plasmon resonance (SPR) studies. FVIII-21 cell binding and internalization could be inhibited by a fragment consisting of the 226 N-terminal aa of the FVIII B-domain, and SPR analysis suggested that this B-domain fragment might bind with weak affinity to FVIII-21. Conclusion Reduced cell binding of FVIII-237 might explain the observed extended half-life in F8-KO mice. This may contribute to the increased FVIII levels measured in murine gene therapy studies using FVIII constructs with similar B-domain lengths.


Asunto(s)
Coagulantes/farmacocinética , Factor VIII/farmacocinética , Hemofilia A/tratamiento farmacológico , Animales , Línea Celular , Coagulantes/sangre , Modelos Animales de Enfermedad , Factor VIII/genética , Técnicas de Inactivación de Genes , Semivida , Hemofilia A/sangre , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Ratones Noqueados , Unión Proteica , Dominios Proteicos , Proteínas Recombinantes/farmacocinética
2.
Biochim Biophys Acta ; 1375(1-2): 61-72, 1998 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-9767112

RESUMEN

P1,P4-Diadenosine 5'-tetraphosphate (Ap4A) acts as an extracellular modulator through its interaction with purinoceptors. Our laboratory has demonstrated the presence of an Ap4A receptor in cardiac tissue [1,2]. Due to the rapid hydrolysis of ATP by cardiac membranes the relationship of ATP and Ap4A binding to purinoceptors on cardiac membranes has not been characterized. In this communication we used two approaches to determine the relationship of ATP to the Ap4A receptor. Radioligand binding carried out with [alpha-32P]Ap4A and adenosine 5'-O-¿3-thiotriphosphate¿ ([gamma-35S]ATPgammaS) demonstrates the presence of a single high affinity binding site for Ap4A and the presence of two binding sites for ATPgammaS. The second approach utilized immunoaffinity purified Ap4A receptor that was shown to be free of ATPase and Ap4Aase activities. Non-radiolabeled Ap4A and ATPgammaS effectively inhibited photocrosslinking of [alpha-32P]8-N3Ap4A to the receptor polypeptide while ATP was a much less effective inhibitor. Furthermore, on plasma membranes [alpha-32P]8-N3Ap4A photocrosslinked to only a 50 kDa polypeptide. These data are consistent with Ap4A interacting with a homogeneous population of receptors on cardiac plasma membranes but with ATP having a low affinity for the receptor.


Asunto(s)
Adenosina Trifosfato/agonistas , Membrana Celular/metabolismo , Fosfatos de Dinucleósidos/farmacología , Miocardio/metabolismo , Agonistas del Receptor Purinérgico P2 , Receptores Purinérgicos P2/metabolismo , Animales , Fosfatos de Dinucleósidos/metabolismo , Hidrólisis , Ratones , Ensayo de Unión Radioligante , Transducción de Señal/efectos de los fármacos
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