High-affinity von Willebrand factor binding does not affect the anatomical or hepatocellular distribution of factor VIII in rats.

UNLABELLED: Essentials Von Willebrand factor (VWF) stabilizes factor VIII (FVIII) and prevents its premature clearance. Rat anatomical and hepatocellular distribution studies assessed the VWF effect on FVIII clearance. Hepatocytes and liver sinusoidal endothelial cells play a key role in FVIII clearance. Anatomical and hepatocellular distribution of FVIII is independent of high-affinity VWF binding. ABSTRACT: Background Von Willebrand factor (VWF) stabilizes factor VIII in the circulation and prevents its premature clearance. Objective To study the effects of VWF on FVIII clearance in rats with endogenous VWF. Methods Anatomical and hepatocellular distribution studies were performed in rats following intravenous administration of glycoiodinated recombinant FVIII (rFVIII) and a FVIII variant, FVIII-Y1680F, lacking high-affinity VWF binding. Radioactivity was quantified in organs, and in distinct liver cell populations. The role of VWF binding was also studied by immunohistochemical staining of rat livers perfused ex vivo with rFVIII alone or with a FVIII-binding VWF fragment. Results The liver was the predominant organ of rFVIII distribution, and a radioactivity peak was also observed in the intestines, suggesting FVIII secretion to the bile by hepatocytes. In the liver, ~60% of recovered radioactivity was associated with hepatocytes, 32% with liver sinusoidal endothelial cells (LSECs), and 9% with Kupffer cells (KCs). When calculated per cell, 1.5-fold to 3-fold more radioactivity was associated with LSECs than with hepatocytes. The importance of hepatocytes and LSECs was confirmed by immunohistochemical staining; strong staining was seen in LSECs, and less intense, punctate staining in hepatocytes. Minor staining in KCs was observed. Comparable anatomical and hepatocellular distributions were observed with rFVIII and FVIII-Y1680F, and the presence of the VWF fragment, D'D3A1, did not change the FVIII staining pattern in intact livers. Conclusions The present data support FVIII clearance via the liver, with hepatocytes and LSECs playing a key role. High-affinity VWF binding did not alter the anatomical or hepatocellular distribution of FVIII.

In vivo clearance and metabolism of recombinant activated factor VII (rFVIIa) and its complexes with plasma protease inhibitors in the liver.

INTRODUCTION: Recombinant activated factor VII (rFVIIa, NovoSeven®) is injected intravenously for the treatment of haemophilia patients with inhibitory antibodies. In plasma, rFVIIa forms complexes with protease inhibitors, primarily antithrombin III (ATIII). The liver is believed to be involved in clearance of rFVIIa, however, it is not known whether the liver is also involved for the clearance of the rFVIIa-ATIII complex. In this study, we explored the fate of intravenously injected rFVIIa from plasma to the hepatic lysosomes. MATERIALS AND METHODS: A novel method using magnetic chromatography was used to isolate catabolic organelle (CO) fractions from mouse liver following injection of superparamagnetic dextran (SPD)-coated iron oxide particles and rFVIIa. The effect of co-circulating SPD particles on rFVIIa pharmacokinetic (PK) parameters was evaluated by ELISA. Cryo-immuno transmission electron microscopy (TEM) was used to study hepatic distribution of SPD particles and rFVIIa. The isolated hepatic CO fractions were characterized using Western Blotting (WB). RESULTS: Cryo-immuno TEM of the liver confirmed hepatic co-localisation of SPD particles and rFVIIa in identical endosomes and lysosomes of both hepatocytes and Kupffer cells. SPD particles did not affect the PK parameters of rFVIIa. WB analysis of plasma and CO fractions detected rFVIIa as the full-length protein and also in high molecular weight (HMW) complexes with ATIII and α-2 macroglobulin (α-2M). CONCLUSIONS: Following injection, both hepatocytes and Kupffer cells appeared to be involved in the hepatic clearance and metabolism of both full-length rFVIIa and rFVIIa in complex with at least two plasma protease inhibitors; ATIII and α-2M.

Role of the nonspecific DNA-binding region and alpha helices within the core domain of retroviral integrase in selecting target DNA sites for integration.

Retroviral integrase plays an important role in choosing host chromosomal sites for integration of the cDNA copy of the viral genome. The domain responsible for target site selection has been previously mapped to the central core of the protein (amino acid residues 49-238). Chimeric integrases between human immunodeficiency virus type 1 (HIV-1) and feline immunodeficiency virus (FIV) were prepared to examine the involvement of a nonspecific DNA-binding region (residues 213-266) and certain alpha helices within the core domain in target site selection. Determination of the distribution and frequency of integration events of the chimeric integrases narrowed the target site-specifying motif to within residues 49-187 and showed that alpha 3 and alpha 4 helices (residues 123-166) were not involved in target site selection. Furthermore, the chimera with the alpha 2 helix (residues 118-121) of FIV identity displayed characteristic integration events from both HIV-1 and FIV integrases. The results indicate that the alpha 2 helix plays a role in target site preference as either part of a larger or multiple target site-specifying motif.

Molecular genetics and target site specificity of retroviral integration.

Integration is an essential step in the life cycle of retroviruses, resulting in the stable joining of the viral cDNA to the host cell chromosomes. While this critical process makes retroviruses an attractive vector for gene delivery, it also presents a potential hazard. The sites where integration occurs are nonspecific. Therefore,it is possible that integration of retroviral DNA will affect host gene expression and disrupt normal cellular functions. The mechanism by which integration sites are chosen is not well understood, and is influenced by several factors, including DNA sequence and structure, DNA-binding proteins, DNA methylation, and transcription. Integrase, the viral enzyme responsible for catalyzing integration, also plays a key role in controlling the choice of target sites. The integrase domain responsible for target site selection has been mapped to the central core region. A better understanding of the interaction between the target-specifying motif of integrase and the target DNA may allow a means to manipulate integration into particular chromosomal sites. Another approach to directing integration is to fuse integrase with a sequence-specific DNA-binding protein, which results in a bias of integration in vitro into the recognition site of the fusion partner. Successful incorporation of the fusion protein into infectious virions and the identification of optimal proteins that can be fused to integrase will advance the development of site-specific vectors. Retroviruses are promising for the delivery of genes in experimental and therapeutic protocols. A better understanding of integration will aid in the design of safer and more effective gene transfer vectors.

Postnatal testicular secretions partially restore the disturbances in reproductive activity caused by prenatal hormonal manipulation.

Male rats that prenatally had been exposed to an antiestrogen, nitromifene citrate (CI 628), showed evidence of impaired defeminization and masculinization in adulthood, suggesting a role of endogenous estrogen for the sexual differentiation of the male. The present study was undertaken to investigate a possible role of postnatal testicular secretions for the above behavioral effects. Male rats were exposed prenatally to CI 628 (1 mg/rat) or saline, and castrated on Day 0, Day 10, or Day 90 after birth. After treatment with gonadal hormones in adulthood, the males were tested for feminine and masculine sexual behavior and for sexual orientation, both when sexually naive and after they had acquired sexual experience. The following conclusions were drawn: 1. Permanent deficits of lordotic behavior were observed in all experimental groups, suggesting the importance of prenatal estrogen for the defeminization process. 2. Hop/darting and ear wiggling behaviors were enhanced in Day-0 and Day-10 castrates, and blocked in the Day-90 castrates. The restitution of these behaviors to normal levels in Day-90 castrates suggests that, in addition to prenatal estrogen, postnatal testicular secretions also are involved in the behavioral defeminization process. 3. Prenatal estrogen contributes to masculinization as evidenced by the impaired ejaculatory behavior observed in all experimental groups. 4. Male-typical sexual orientation toward the female seems to be facilitated by prenatal estrogen. Both the masculinization and the defeminization of male-typical sexual orientation toward a female were impaired by castration at birth and at Day 10 in the experimental animals, but a full restoration of the sexual orientation toward females was seen in Day-90 castrates, suggesting the restorative role of postnatal testicular secretions for these processes.

Characterization of feline immunodeficiency virus integrase and analysis of functional domains.

The wild-type and mutant derivatives of the integrase protein of feline immunodeficiency virus (FIV) were cloned and expressed in Escherichia coli. The purified proteins were examined using various model DNA substrates for their catalytic activities: 3'-end processing, 3'-end joining, and disintegration. The reactions required the presence of either Mn2+ or Mg2+ as a divalent cation. The N-terminal and C-subterminal domains (residues 1-52 and 189-235, respectively) were necessary for 3'-end processing and joining reactions but not for disintegration. Substitution of asparagine for the highly conserved aspartic acid at position 118 resulted in a complete loss of all three activities, confirming that the catalytic domain resides in the central core region (residues 53-188) of the protein. Deletion of the C-terminus (residues 236-281) resulted in a FIV integrase mutant that had efficient 3'-end processing and disintegration activities but weak 3'-end joining activity, a finding that has not been reported previously with other retroviral integrases. The result suggests that the C-terminus is the primary binding site for target DNA. Attachment of a histidine-tag at the N-terminus of the wild-type and deletion derivatives increased the binding affinity to the DNA substrate, resulting in altered levels of catalytic activities and selection of integration sites. Similar to other retroviral integrases, certain pairs of mutant derivatives of FIV integrase could complement each other to restitute 3'-end processing and joining activities, suggesting that formation of functional multimers is a general feature of proteins in the integrase family.