Publikationsserver der RWTH Aachen University
Cloning and targeted deletion of the mouse histidine rich glycoprotein gene and functional characterization of HRG in mice
Abstract
dc:descriptionHuman histidine-rich glycoprotein (HRG) is a single-chain glycoprotein with a molecular weight of 67,000 composed of 507 amino acid residues. By it’s structural similarity, it is classified to belong to the cystatin superfamily like fetuin-A, fetuin-B and kininogen. Mature HRG circulates in blood at relatively high concentrations of up to 150 mg/l (~2 µM). HRG is exclusively synthesized in the liver, but it is stored in platelets and megakaryocytes where it can be released after thrombin stimulation. HRG may play a regulatory role in hemostasis and innate immunity. However, this role is uncertain for lack of rigorous testing in an animal model. To elucidate the in vivo function(s) of HRG, we cloned the mouse Hrg gene and generated HRG–deficient mice. As a first step, overlapping phage clones including mouse Hrg gene were isolated and subcloned. A complete nucleotide sequence of 13,591 bases was determined (Tsuchida, Master’s thesis, Himeji Institute of Technology, 2000). To analyze the 5-prime upstream region of the mouse Hrg gene, pPAC4 clone was isolated, subcloned and as a result of this a 1,889 nucleotides sequence was determined. The sequencing of the murine Hrg gene revealed that it consists of seven exons and six introns like its human counterpart. Like in other members of the cystatin superfamily, the cystatin-like domains D1 and D2 of HRG are encoded by three exons, exons I to III and IV to VI, respectively. The C-terminal HRG-specific portion of the molecule is encoded by one large exon VII. Next, the gene targeting vector was constructed by replacing a 1.8-kb XbaI-BamHI fragment containing exon I with a 1.8-kb neomycin cassette for positive selection of resistant clones. Three correctly targeted embryonic stem cell clones were identified among a total of 144 neomycin resistant clones. We generated mice lacking the translation start point of exon I of the Hrg gene, effectively resulting in a null mutation (Hrg-/-). Homozygous HRG-deficient mice were viable and fertile, but had no HRG in their blood. The histological analysis of organs revealed no gross differences between wild-type and HRG-deficient mice. Since it is known that HRG binds to fibrinogen, plasminogen and heparin, it is thought that HRG regulates blood coagulation and fibrinolysis. In addition, three cases of hereditary histidine-rich glycoprotein deficiency with familial thrombophilia have been reported. Therefore, we analyzed mainly effects of HRG on blood coagulation and fibrinolysis. Antithrombin activity in plasma of Hrg-/- mice was higher than in plasma of heterozygous Hrg+/- or wild-type Hrg+/+ mice in the presence of a fixed amount of heparin. The prothrombin time was shorter in Hrg-/- mice than in Hrg+/- and Hrg+/+ mice. Bleeding time after tail tip amputation in Hrg-/- mice was shorter than in Hrg+/+ mice. The spontaneous fibrinolytic activity in clotted blood of Hrg-/- mice was higher than in Hrg+/+ mice. The platelet aggregation activity of platelet rich plasma in the presence of thrombin, ADP and collagen in Hrg-/- mice was significantly higher than in Hrg+/+ mice. These findings suggest that HRG plays a role both as an anticoagulant and antifibrinolytic modifier. In addition it may function as an anti-platelet factor in vivo.
Degree
thesis:*- Grantor dc:publisher
- Publikationsserver der RWTH Aachen University
- Year dc:date
- 2005
Author and committee
dc:creator, dc:contributor.*- Author dc:creator
-
- Tsuchida-Straeten, Nobuko
- Contributors dc:contributor
-
- Jahnen-Dechent, Wilhelm
Subjects
dc:subject × 10Rights
dc:rights- Statement dc:rights
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- info:eu-repo/semantics/openAccess
- Language dc:language
- eng