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Histidine-rich glycoprotein (HRG), also known as histidine-proline-rich glycoprotein, is a relatively abundant plasma protein that belongs to the cystatin superfamily. A clear picture of the physiological role(s) of HRG is still emerging. In vitro studies of the molecular interactions of HRG and its effects on cells have suggested a function in host defense, as a modulator of the coagulation and fibrinolytic systems, as well as of innate immunity. ‘Classic’ HRG ligands include heparin, plasminogen, fibrinogen, and thrombospondin. New activities for this protein are continuously unraveled. HRG was recently found to exert antiangiogenic properties in vitro [1] and antitumor effects in vivo [2]. It acts as an opsonin by bridging FcγRI receptors on macrophages to DNA on apoptotic cells, stimulating phagocytosis [3], and modulates the binding of IgG and immune complexes to FcγRI [4].

The hallmark feature of HRG is a central domain rich in histidine and proline, which is composed of tandem pentapeptide repeats with the consensus sequence ‘GHHPH’. This conserved motif has no homology to other protein sequences and may exhibit a characteristic new structural fold. Owing to an abundance of histidine residues, the histidine domain binds transitional metal cations, such as zinc(II) or copper(II), which promotes HRG interactions with negatively charged ligands, particularly sulfated glycosaminoglycans. The histidine-proline-rich domain acquires a large positive charge upon protonation of histidine residues [5]. Consequently, a drop in pH values below the physiological range facilitates HRG binding to heparin, heparan sulfate, or dermatan sulfate [6]. This exquisite sensitivity to pH changes and the presence of transitional metal ions was postulated to regulate the localization and activity of HRG [6].

The modular domain structure of HRG allows simultaneous interactions with multiple ligands, suggesting that HRG may function as an adapter molecule. For instance, HRG interaction with plasminogen is mediated by lysine residues in its amino and/or carboxyl-terminal domains, and can occur concomitantly with binding to thrombospondin [7] or glycosaminoglycans [8]. Under conditions of low pH or elevated Zn2+, HRG associates with cell-surface heparan sulfates [9] and acts as a high affinity receptor for plasminogen [10]. Because generation of plasmin on cell surfaces has been implicated in cell migration associated with wound healing, angiogenesis, and tumor metastasis, it is of interest that HRG strongly stimulates plasminogen activation when bound to a surface [8,11]. In contrast, HRG in solution weakly inhibits plasminogen activation. This may provide a regulatory mechanism for HRG activity, analogous to surface-dependent activation of many components of the coagulation and fibrinolytic systems.

However intriguing or appealing, all biological functions of HRG inferred from in vitro studies must ultimately be validated by in vivo findings. To date, the lack of an HRG-knockout animal model has hampered a direct experimental approach. Nevertheless, several families with congenital HRG deficiency have been described, and studies of their clinical phenotype may provide clues about the physiological role of HRG. In some patients, reduced levels of HRG are associated with a thrombophilic phenotype; however, a causal connection has not been proved. Although such observations suggest that HRG may modulate the hemostatic balance, the evidence remains circumstantial. Furthermore, as patients still have ∼20%–50% of the normal levels of HRG, which may be sufficient to provide adequate function, the phenotype in the complete absence of HRG remains unknown.

In this issue of the journal, Tsuchida-Straten et al. [12] report the long-awaited generation of a transgenic mouse with a targeted deletion of HRG, and provide an initial characterization of its phenotype. The HRG-knockout mice were viable and fertile with no apparent abnormalities. Changes in hemostatic parameters pointed to a mild anticoagulant activity of HRG in vivo. Plasma from HRG-deficient mice had higher antithrombin activity and significantly shorter prothrombin time, suggesting that HRG deficiency is associated with faster blood clotting. This observation is rationalized in terms of HRG neutralizing heparin-like cofactors that accelerate the formation of a complex between antithrombin and thrombin or factor X. Bleeding time after tail tip amputation, measuring combined plasma and platelet-mediated blood coagulation, was shorter. HRG is known to bind to platelets and may inhibit their aggregation by inactivating thrombospondin. On the other hand, spontaneous clot lysis was faster in HRG-deficient mice, implying that HRG has a mild antifibrinolytic activity in vivo, attributable to its binding to plasminogen in plasma. The overall findings are consistent with other in vitro studies and clinical findings in HRG-deficient patients, and provide independent support for the hypothesis that HRG has a modulator role as an anticoagulant and antifibrinolytic in vivo.

The initial characterization of the phenotype in HRG-knockout mice has focused largely on changes in hemostatic parameters [12]. It is expected that future characterization of these mice will reveal additional functions of HRG in vivo. One should recall that many biological activities of HRG are predicted to be manifest only under rather specific circumstances. Thus, further refinements of this model may include challenging the HRG-deficient mice under conditions that mimic various physiological and pathological events. Studies in mice deficient for plasminogen, plasminogen activators, and their inhibitors can provide a paradigm for future studies of HRG functions. These studies have demonstrated that the role of the plasminogen activation system is not limited to fibrinolysis, but is also implicated as a mediator of inflammation and infection, vascular remodeling, wound healing, excitotoxin-induced neurodegeneration, axonal degeneration and demyelination, cerebral ischemic infarction, cancer growth and metastasis, glomerulonephritis, and pulmonary fibrosis [13]. Given the prominent place of plasminogen among HRG ligands, and the ability of immobilized HRG to accelerate plasmin generation, one can hypothesize that HRG modulates some of the non-fibrinolytic activities of the plasminogen–plasmin system. Generation of an HRG-knockout mice line provides an important tool for future studies about the biological functions of HRG, including its presumed roles in innate immunity, angiogenesis, and other physiological and pathological processes.

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    Olsson AK, Larsson H, Dixelius J, Johansson I, Lee C, Oellig C, Bjork I, Claesson-Welsh L. A fragment of histidine-rich glycoprotein is a potent inhibitor of tumor vascularization. Cancer Res 2004; 64: 599605.
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