• Factor XII can contribute to thrombus formation in human and nonhuman primate blood.• An antibody that blocks factor XII activation (15H8) produces an antithrombotic effect in a primate thrombosis model.The plasma zymogens factor XII (fXII) and factor XI (fXI) contribute to thrombosis in a variety of mouse models. These proteins serve a limited role in hemostasis, suggesting that antithrombotic therapies targeting them may be associated with low bleeding risks.Although there is substantial epidemiologic evidence supporting a role for fXI in human thrombosis, the situation is not as clear for fXII. We generated monoclonal antibodies (9A2 and 15H8) against the human fXII heavy chain that interfere with fXII conversion to the protease factor XIIa (fXIIa). The anti-fXII antibodies were tested in models in which anti-fXI antibodies are known to have antithrombotic effects. Both anti-fXII antibodies reduced fibrin formation in human blood perfused through collagen-coated tubes. fXII-deficient mice are resistant to ferric chloride-induced arterial thrombosis, and this resistance can be reversed by infusion of human fXII. 9A2 partially blocks, and 15H8 completely blocks, the prothrombotic effect of fXII in this model. 15H8 prolonged the activated partial thromboplastin time of baboon and human plasmas. 15H8 reduced fibrin formation in collagen-coated vascular grafts inserted into arteriovenous shunts in baboons, and reduced fibrin and platelet accumulation downstream of the graft. These findings support a role for fXII in
Hepatocyte growth factor activator (HGFA) is responsible for proteolytic activation of the precursor form of hepatocyte growth factor in injured tissues. To date, two specific inhibitors of HGFA have been identified, namely HGFA inhibitor type 1 (HAI-1) and type 2 (HAI-2)/placental bikunin (PB). Both inhibitors are first synthesized as integral membrane proteins having two Kunitz domains and a transmembrane domain, and are subsequently released from cell surface by shedding. Here we show that an active form of HGFA is specifically complexed with membrane-form HAI-1, but not with HAI-2/PB, on the surface of epithelial cells expressing both inhibitors. This binding required the enzyme activity of HGFA. The selective binding of HGFA to the cell surface HAI-1 was further confirmed in an engineered system using Chinese hamster ovary cells, in which only the cells expressing HAI-1 retained exogenous HGFA. The binding of HGFA to HAI-1 was reversible, and no irreversible modifications affecting the enzyme activity occurred during the binding. Importantly, HAI-1 and the HGFA⅐HAI-1 complex were quickly released from the cell surface by treatment with phorbol 12-myristate 13-acetate or interleukin 1 accompanying the generation of 58-kDa fragments of HAI-1, which are less potent against HGFA, as well as significant recovery of HGFA activity in the culture supernatant. This regulated shedding was completely inhibited by BB3103, a synthetic zinc-metalloproteinase inhibitor. We conclude that HAI-1 is not only an inhibitor but also a specific acceptor of active HGFA, acting as a reservoir of this enzyme on the cell surface. The latter property appears to ensure the concentrated pericellular HGFA activity in certain cellular conditions, such as tissue injury and inflammation, via the up-regulated shedding of HGFA⅐HAI-1 complex. These findings shed light on a novel function of the integral membrane Kunitz-type inhibitor in the regulation of pericellular proteinase activity.
SUMMARYWe used a specific monoclonal antibody to human hepatocyte growth factor activator inhibitor type 1 (HAI-1) in immunohistochemical procedures to determine the distribution and localization of HAI-1 in human tissues. In normal adult tissues, HAI-1 was predominantly expressed in the simple columnar epithelium of the ducts, tubules, and mucosal surface of various organs. In all cases, HAI-1 was localized predominantly on the cellular lateral (or basolateral) surface. By contrast, hepatocytes, acinar cells, endocrine cells, stromal mesenchymal cells, and inflammatory cells were hardly stainable with the antibody, and stratified squamous epithelium showed only faint immunoreactivity on the surface of cells of the basal layer. In the gastrointestinal tract, the surface epithelium was strongly stained. RNA blot analysis confirmed the presence of specific mRNA transcript in the gastrointestinal mucosa, and in situ hybridization revealed that HAI-1 mRNA showed a similar cellular distribution pattern. Although HAI-1 was not expressed in normal hepatocytes, strong immunoreactivity was observed on the epithelium of pseudo-bile ducts and on the surface of scattered hepatocytes in fulminant hepatitis. The enhanced expression was also noted in regenerating tubule epithelial cells of the kidney after infarction. We conclude that HAI-1 is preferentially expressed in the simple columnar epithelium of the mucosal surface and duct, that the predominant localization of HAI-1 is the cell surface, and that the expression of HAI-1 can be modulated by tissue injury and regeneration.
The growth, survival, and metabolic activities of multicellular organisms at the cellular level are regulated by intracellular signaling, systemic homeostasis and the pericellular microenvironment. Pericellular proteolysis has a crucial role in processing bioactive molecules in the microenvironment and thereby has profound effects on cellular functions. Hepatocyte growth factor activator inhibitor type 1 (HAI-1) and HAI-2 are type I transmembrane serine protease inhibitors expressed by most epithelial cells. They regulate the pericellular activities of circulating hepatocyte growth factor activator and cellular type II transmembrane serine proteases (TTSPs), proteases required for the activation of hepatocyte growth factor (HGF)/scatter factor (SF). Activated HGF/SF transduces pleiotropic signals through its receptor tyrosine kinase, MET (coded by the proto-oncogene MET), which are necessary for cellular migration, survival, growth and triggering stem cells for accelerated healing. HAI-1 and HAI-2 are also required for normal epithelial functions through regulation of TTSP-mediated activation of other proteases and protease-activated receptor 2, and also through suppressing excess degradation of epithelial junctional proteins. This review summarizes current knowledge regarding the mechanism of pericellular HGF/SF activation and highlights emerging roles of HAIs in epithelial development and integrity, as well as tumorigenesis and progression of transformed epithelial cells.
Hepatocyte growth factor/scatter factor (HGF/SF) plays critical roles in cancer progression through its specific receptor, MET. HGF/SF is usually synthesized and secreted as an inactive proform (pro-HGF/SF) by stromal cells, such as fibroblasts. Several serine proteases are reported to convert pro-HGF/SF to mature HGF/SF and among these, HGF activator (HGFA) and matriptase are the most potent activators. Increased activities of both proteases have been observed in various cancers. HGFA is synthesized mainly by the liver and secreted as an inactive pro-form. In cancer tissues, pro-HGFA is likely activated by thrombin and/or human kallikrein 1-related peptidase (KLK)-4 and KLK-5. Matriptase is a type II transmembrane serine protease that is expressed by most epithelial cells and is also synthesized as an inactive zymogen. Matriptase activation is likely to be mediated by autoactivation or by other trypsin-like proteases. Recent studies revealed that matriptase autoactivation is promoted by an acidic environment. Given the mildly acidic extracellular environment of solid tumors, matriptase activation may, thus, be accelerated in the tumor microenvironment. HGFA and matriptase activities are regulated by HGFA inhibitor (HAI)-1 (HAI-1) and/or HAI-2 in the pericellular microenvironment. HAIs may have an important role in cancer cell biology by regulating HGF/SF-activating proteases.
Hepatocyte growth factor activator inhibitor-1 (HAI-1), encoded by the serine protease inhibitor Kunitz type 1 (SPINT1) gene, is a membrane-associated proteinase inhibitor that potently inhibits a variety of serine proteinases, including those that are membrane bound. Although HAI-1/SPINT1 is widely expressed by epithelial cells and cancer cells, its functional role is still unclear, particularly in cancer. Here, we show that stable knockdown of HAI-1/SPINT1 in the human pancreatic cancer cell line SUIT-2 induces an elongated spindle-like morphology associated with accelerated invasion, thereby mimicking an epithelial to mesenchymal transition (EMT). We found that HAI-1/SPINT1 knockdown significantly reduced the expression of E-cadherin and was accompanied by up-regulation of Smad-interacting protein 1 (SIP1), an E-cadherin transcriptional repressor. In addition, matrix metalloproteinase-9 (MMP-9) was up-regulated. Similar results were obtained in the HLC-1 lung carcinoma cell line. Moreover, a metastatic variant of SUIT-2 (S2-CP8) that showed loss of E-cadherin expression also showed a significantly reduced level of HAI-1/SPINT1. Engineered overexpression of HAI-1/SPINT1 in S2-CP8 resulted in reversion of E-cadherin expression and SIP1 down-regulation, which accompanied reestablishment of epithelial morphology in culture. The EMT caused by HAI-1/SPINT1 knockdown seemed to be mediated, at least partly, by membrane-bound serine proteinases, matriptase/ST14 and TMPRSS4, as knockdown of matriptase/ST14 or TMPRSS4 in HAI-1/SPINT1 knockdown SUIT-2 cells and HLC-1 cells resulted in reversion of SIP1 and/or MMP-9 expression levels. We suggest that interactions between HAI-1/SPINT1 and membrane-bound serine proteinases contribute to transcriptional and functional changes involved in EMT in certain carcinoma cells.
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