Crystal structure of a cold-active protease (Pro21717) from the psychrophilic bacterium, Pseudoalteromonas arctica PAMC 21717, at 1.4 Å resolution: Structural adaptations to cold and functional analysis of a laundry detergent enzyme.

Enzymes isolated from organisms found in cold habitats generally exhibit higher catalytic activity at low temperatures than their mesophilic homologs and are therefore known as cold-active enzymes. Cold-active proteases are very useful in a variety of biotechnological applications, particularly as active ingredients in laundry and dishwashing detergents, where they provide strong protein-degrading activity in cold water. We identified a cold-active protease (Pro21717) from a psychrophilic bacterium, Pseudoalteromonas arctica PAMC 21717, and determined the crystal structure of its catalytic domain (CD) at a resolution of 1.4 Å. The Pro21717-CD structure shows a conserved subtilisin-like fold with a typical catalytic triad (Asp185, His244, and Ser425) and contains four calcium ions and three disulfide bonds. Interestingly, we observed an unexpected electron density at the substrate-binding site from a co-purified peptide. Although the sequence of this peptide is unknown, analysis of the peptide-complexed structure nonetheless provides some indication of the substrate recognition and binding mode of Pro21717. Moreover, various parameters, including a wide substrate pocket size, an abundant active-site loop content, and a flexible structure provide potential explanations for the cold-adapted properties of Pro21717. In conclusion, this is first structural characterization of a cold-adapted subtilisin-like protease, and these findings provide a structural and functional basis for industrial applications of Pro21717 as a cold-active laundry or dishwashing detergent enzyme.

Distinct roles of transforming growth factor-ß signaling and transforming growth factor-ß receptor inhibitor SB431542 in the regulation of p21 expression.

Transforming growth factor-ß (TGF-ß) has both tumor suppressive and oncogenic activities. Autocrine TGF-ß signaling supports tumor survival and growth in certain types of cancer, and the TGF-ß signaling pathway is a potential therapeutic target for these types of cancer. TGF-ß induces p21 expression, and p21 is considered as an oncogene as well as a tumor suppressor, due to its anti-apoptotic activity. Thus, we hypothesized that autocrine TGF-ß signaling maintains the expression of p21 at levels that can support cell growth. To verify this hypothesis, we sought to examine p21 expression and cell growth in various cancer cells following the inhibition of autocrine TGF-ß signaling using siRNAs targeting TGF-ß signaling components and SB431542, a TGF-ß receptor inhibitor. Results from the present study show that p21 expression and cell growth were reduced by knockdown of TGF-ß signaling components using siRNA in MDA-MB231 and A549 cells. Cell growth was also reduced in p21 siRNA-transfected cells. Downregulation of p21 expression induced cellular senescence in MDA-MB231 cells but did not induce apoptosis in both cells. These data suggest that autocrine TGF-ß signaling is required to sustain p21 levels for positive regulation of cell cycle. On the other hand, treatment with SB431542 up-regulated p21 expression while inhibiting cell growth. The TGF-ß signaling pathway was not associated with the SB431542-mediated induction of p21 expression. Specificity protein 1 (Sp1) was downregulated by treatment with SB431542, and p21 expression was increased by Sp1 knockdown. These findings suggest that downregulation of Sp1 expression is responsible for SB43154-induced p21 expression.

The ADAM15 ectodomain is shed from secretory exosomes.

We demonstrated previously that a disintegrin and metalloproteinase 15 (ADAM15) is released into the extracellular space as an exosomal component, and that ADAM15-rich exosomes have tumor suppressive functions. However, the suppressive mechanism of ADAM15-rich exosomes remains unclear. In this study, we show that the ADAM15 ectodomain is cleaved from released exosomes. This shedding process of the ADAM15 ectodomain was dramatically enhanced in conditioned ovarian cancer cell medium. Proteolytic cleavage was completely blocked by phenylmethylsulfonyl fluoride, indicating that a serine protease is responsible for exosomal ADAM15 shedding. Experimental evidence indicates that the ADAM15 ectodomain itself has comparable functions with those of ADAM15-rich exosomes, which effectively inhibit vitronectininduced cancer cell migration and activation of the MEK/extracellular regulated kinase signaling pathway. We present a tumor suppressive mechanism for ADAM15 exosomes and provide insight into the functional significance of exosomes that generate tumor-inhibitory factors.

Plasminogen activator inhibitor-1 regulates infiltration of macrophages into melanoma via phosphorylation of FAK-Tyr9²5.

Tumor-infiltrating macrophages are potential candidates for cancer immunotherapy. However, the detailed molecular mechanism underlying macrophage infiltration into tumors is poorly understood. Based on our previous finding that plasminogen activator inhibitor-1 (PAI-1) enhances vitronectin-dependent migration of macrophages, we investigated the potential role of PAI-1 in macrophage invasion into melanoma. Experimental evidence obtained from spheroid confrontation assay clearly showed that PAI-1 overexpression significantly enhanced the invasion of RAW 264.7 cells into B16F10 melanoma. We further demonstrated that PAI-1 induces phosphorylation of focal adhesion kinase (FAK) at Tyr(925), which, in turn, mediated the invasion of macrophages into the melanoma. This work further illustrates that low-density lipoprotein receptor related-protein 1 (LRP1) is essential for PAI-1-mediated FAK phosphorylation and macrophage invasion into melanoma. In conclusion, our study demonstrates a novel role of PAI-1 in macrophage invasion into melanoma and provides insights into the underlying molecular mechanism.

Dimerization of matrix metalloproteinase-2 (MMP-2): functional implication in MMP-2 activation.

Matrix metalloproteinase-2 (MMP-2) functions in diverse biological processes through the degradation of extracellular and non-extracellular matrix molecules. Because of its potential for tissue damage, there are several ways to regulate MMP-2 activity, including gene expression, compartmentalization, zymogen activation, and enzyme inactivation by extracellular inhibitors. Enzyme regulation through zymogen activation is important for the regulation of MMP-2 activity. In our previous studies, we showed that thrombin directly cleaved the propeptide of MMP-2 at specific sites for enzyme activation. We also demonstrated that heparan sulfate was required for thrombin-mediated activation of pro-MMP-2 by binding to thrombin, presumably through conformational changes at the active site of the enzyme. This suggests a regulatory mechanism for thrombin-mediated activation of pro-MMP-2. In this study, we found that MMP-2 formed a reduction-sensitive homodimer in a controlled manner and that Ca(2+) ion was essential for homodimerization of MMP-2. Homodimerization was not associated with protein kinase C-mediated phosphorylation of MMP-2. MMP-2 formed a homodimer through an intermolecular disulfide bond between Cys(102) and the neighboring Cys(102). Homodimerization of MMP-2 enhanced thrombin-mediated activation of pro-MMP-2. Moreover, the MMP-2 homodimer could cleave a small peptide substrate without removal of the propeptide. Taken together, our experimental data suggest a novel regulatory mechanism for pro-MMP-2 activation that is modulated through homodimerization of MMP-2.

Exosome release of ADAM15 and the functional implications of human macrophage-derived ADAM15 exosomes.

A disintegrin and metalloproteinase 15 (ADAM15), the only ADAM protein containing an Arg-Gly-Asp (RGD) motif in its disintegrin-like domain, is a widely expressed membrane protein that is involved in tumor progression and suppression. However, the underlying mechanism of ADAM15-mediated tumor suppression is not clearly understood. This study demonstrates that ADAM15 is released as an exosomal component, and ADAM15 exosomes exert tumor suppressive activities. We found that exosomal ADAM15 release is stimulated by phorbol 12-myristate 13-acetate, a typical protein kinase C activator, in various tumor cell types, and this results in a corresponding decrease in plasma membrane-associated ADAM15. Exosomes rich in ADAM15 display enhanced binding affinity for integrin αvß3 in an RGD-dependent manner and suppress vitronectin- and fibronectin-induced cell adhesion, growth, and migration, as well as in vivo tumor growth. Exosomal ADAM15 is released from human macrophages, and macrophage-derived ADAM15 exosomes have tumor inhibitory effects. This work suggests a primary role of ADAM15 for exosome-mediated tumor suppression, as well as functional significance of exosomal ADAM protein in antitumor immunity.

Thrombin-dependent MMP-2 activity is regulated by heparan sulfate.

Like most metalloproteases, matrix metalloprotease 2 (MMP-2) is synthesized as a zymogen. MMP-2 propeptide plays a role in inhibition of catalytic activity through a cysteine-zinc ion pairing, disruption of which results in full enzyme activation. A variety of proteases have been shown to be involved in the activation of pro-MMP-2, including metalloproteases and serine proteases. In the previous study we showed that MMP-2 activation occurred via specific cleavages of the propeptide by thrombin followed by intermolecular autoproteolytic processing for full enzymatic activity. Thrombin also degraded MMP-2, but this degradation was reduced greatly under cell-associated conditions with a concomitant increase in activation, prompting us to elucidate the molecular mechanisms underlying thrombin-mediated MMP-2 activation. In the present study we demonstrate that heparan sulfate is essential for thrombin-mediated activation of pro-MMP-2. Binding of heparan sulfate to thrombin is primarily responsible for this activation process, presumably through conformational changes at the active site. Furthermore, interaction of MMP-2 with exosites 1 and 2 of thrombin is crucial for thrombin-mediated MMP-2 degradation, and inhibition of this interaction by heparan sulfate or hirudin fragment results in a decrease in MMP-2 degradation. Finally, we demonstrated interaction between exosite 1 and hemopexin-like domain of MMP-2, suggesting a regulatory role of hemopexin-like domain in MMP-2 degradation. Taken together, our experimental data suggest a novel regulatory mechanism of thrombin-dependent MMP-2 enzymatic activity by heparan sulfate proteoglycans.

ADAMTS9 is a cell-autonomously acting, anti-angiogenic metalloprotease expressed by microvascular endothelial cells.

The metalloprotease ADAMTS9 participates in melanoblast development and is a tumor suppressor in esophageal and nasopharyngeal cancer. ADAMTS9 null mice die before gastrulation, but, ADAMTS9+/- mice were initially thought to be normal. However, when congenic with the C57Bl/6 strain, 80% of ADAMTS9+/- mice developed spontaneous corneal neovascularization. beta-Galactosidase staining enabled by a lacZ cassette targeted to the ADAMTS9 locus showed that capillary endothelial cells (ECs) in embryonic and adult tissues and in capillaries growing into heterotopic tumors expressed ADAMTS9. Heterotopic B.16-F10 melanomas elicited greater vascular induction in ADAMTS9+/- mice than in wild-type littermates, suggesting a potential inhibitory role in tumor angiogenesis. Treatment of cultured human microvascular ECs with ADAMTS9 small-interfering RNA resulted in enhanced filopodial extension, decreased cell adhesion, increased cell migration, and enhanced formation of tube-like structures on Matrigel. Conversely, overexpression of catalytically active, but not inactive, ADAMTS9 in ECs led to fewer tube-like structures, demonstrating that the proteolytic activity of ADAMTS9 was essential. However, unlike the related metalloprotease ADAMTS1, which exerts anti-angiogenic effects by cleavage of thrombospondins and sequestration of vascular endothelial growth factor165, ADAMTS9 neither cleaved thrombospondins 1 and 2, nor bound vascular endothelial growth factor165. Taken together, these data identify ADAMTS9 as a novel, constitutive, endogenous angiogenesis inhibitor that operates cell-autonomously in ECs via molecular mechanisms that are distinct from those used by ADAMTS1.

Cell-surface processing of the metalloprotease pro-ADAMTS9 is influenced by the chaperone GRP94/gp96.

A disintegrin-like and metalloprotease domain with thrombospondin type 1 motifs 9 (ADAMTS9) is a highly conserved metalloprotease that has been identified as a tumor suppressor gene and is required for normal mouse development. The secreted ADAMTS9 zymogen undergoes proteolytic excision of its N-terminal propeptide by the proprotein convertase furin. However, in contrast to other metalloproteases, propeptide excision occurs at the cell surface and leads to decreased activity of the zymogen. Here, we investigated the potential cellular mechanisms regulating ADAMTS9 biosynthesis and cell-surface processing by analysis of molecular complexes formed by a construct containing the propeptide and catalytic domain of pro-ADAMTS9 (Pro-Cat) in HEK293F cells. Cross-linking of cellular proteins bound to Pro-Cat followed by mass spectrometric analysis identified UDP-glucose:glycoprotein glucosyltransferase I, heat shock protein gp96 (GRP94), BiP (GRP78), and ERdj3 (Hsp40 homolog) as associated proteins. gp96 and BiP were present at the cell surface in an immunoprecipitable complex with pro-ADAMTS9 and furin. Treatment with geldanamycin, an inhibitor of the HSP90alpha family (including gp96), led to decreased furin processing of pro-ADAMTS9 and accumulation of the unprocessed pro-ADAMTS9 at the cell surface. gp96 siRNA down-regulated the levels of cell-surface pro-ADAMTS9 and furin, whereas the levels of cell-surface pro-ADAMTS9, but not of cell-surface furin, were decreased upon treatment with BiP siRNA. These data identify for the first time the cellular chaperones associated with secretion of an ADAMTS protease and suggest a role for gp96 in modulating pro-ADAMTS9 processing.

Membrane type-1 matrix metalloprotease-independent activation of pro-matrix metalloprotease-2 by proprotein convertases.

Matrix metalloprotease-2 is implicated in many biological processes and degrades extracellular and non-extracellular matrix molecules. Matrix metalloprotease-2 maintains a latent state through a cysteine-zinc ion pairing which, when disrupted, results in full enzyme activation. This pairing can be disrupted by a conformational change or cleavage within the propeptide. The best known activation mechanism for pro-matrix metalloprotease-2 occurs via cleavage of the propeptide by membrane type-1 matrix metalloprotease. However, significant residual activation of pro-matrix metalloprotease-2 is seen in membrane type-1 matrix metalloprotease knockout mice and in fibroblasts treated with metalloprotease inhibitors. These findings indicate the presence of a membrane type-1 matrix metalloprotease-independent activation mechanism for pro-matrix metalloprotease-2 in vivo, which prompted us to explore an alternative activation mechanism for pro-matrix metalloprotese-2. In this study, we demonstrate membrane type-1 matrix metalloprotease-independent propeptide processing of matrix metalloprotease-2 in HEK293F and various tumor cell lines, and show that proprotein convertases can mediate the processing intracellularly as well as extracellularly. Furthermore, processed matrix metalloprotease-2 exhibits enzymatic activity that is enhanced by intermolecular autolytic cleavage. Thus, our experimental data, taken together with the broad expression of proprotein convertases, suggest that the proprotein convertase-mediated processing may be a general activation mechanism for pro-matrix metalloprotease-2 in vivo.

Regulatory mechanism of matrix metalloprotease-2 enzymatic activity by factor Xa and thrombin.

Matrix metalloprotease (MMP)-2 plays a key role in many biological and pathological processes related to cell migration, invasion, and mitogenesis. MMP-2 is synthesized as a zymogen that is activated through either a conformational change or proteolysis of the propeptide. Several activating enzymes for pro-MMP-2 have been proposed, including metalloproteases and serine proteases. The mechanism of pro-MMP-2 activation by metalloproteases is well established, and the most studied activation mechanism involves cleavage of the propeptide by membrane type 1-MMP (MT1-MMP). In contrast, serine protease activation has not been thoroughly studied, although studies suggest that MT1-MMP may be involved in activation by thrombin and plasmin. Here, we demonstrate that factor Xa mediates MT1-MMP-independent processing of pro-MMP-2 in vascular smooth muscle cells and endothelial cells. Factor Xa and thrombin directly cleaved the propeptide on the carboxyl terminal sides of the Arg(98) and Arg(101) residues, whereas plasmin only cleaved the propeptide downstream of Arg(101). Moreover, processed MMP-2 showed enzymatic activity that was enhanced by intermolecular autoproteolytic processing at the Asn(109)-Tyr peptide bond. In addition to its role in activation, factor Xa rapidly degraded MMP-2, thereby restricting excessive MMP-2 activity. Thrombin also degraded MMP-2, but the degradation was reduced greatly under cell-associated conditions, resulting in an increase in processed MMP-2. Overall, factor Xa and thrombin regulate MMP-2 enzymatic activity through its activation and degradation. Thus, the net enzymatic activity results from a balance between MMP-2 activation and degradation.

Characterization of proADAMTS5 processing by proprotein convertases.

ADAMTS5 (aggrecanase-2), a key metalloprotease mediating cartilage destruction in arthritis, is synthesized as a zymogen, proADAMTS5. We report a detailed characterization of the propeptide excision mechanism and demonstrate that it is a major regulatory step with unusual characteristics. Using furin-deficient cells and a furin inhibitor, we found that proADAMTS5 was processed by proprotein convertases, specifically furin and PC7, but not PC6B. Mutagenesis of three sites containing basic residues within the ADAMTS5 propeptide (RRR(46), RRR(69) and RRRRR(261)) suggested that proADAMTS5 processing occurs after Arg(261). That furin processing was essential for ADAMTS5 activity was illustrated using the known ADAMTS5 substrate aggrecan, as well as a new substrate, versican, an important regulatory proteoglycan during mammalian development. When compared to other ADAMTS proteases, proADAMTS5 processing has several distinct features. In contrast to ADAMTS1, whose furin processing products were clearly present intracellularly, cleaved ADAMTS5 propeptide and mature ADAMTS5 were found exclusively in the conditioned medium. Despite attempts to enhance detection of intracellular proADAMTS5 processing, such as by immunoprecipitation of total ADAMTS5, overexpression of furin, and secretion blockade by monensin, neither processed ADAMTS5 propeptide nor the mature enzyme were found intracellularly, which was strongly suggestive of extracellular processing. Extracellular ADAMTS5 processing was further supported by activation of proADAMTS5 added exogenously to HEK293 cells stably expressing furin. Unlike proADAMTS9, which is processed by furin at the cell-surface, to which it is bound, ADAMTS5 does not bind the cell-surface. Thus, the propeptide processing mechanism of ADAMTS5 has several points of distinction from those of other ADAMTS proteases, which may have considerable significance in the context of osteoarthritis.

ADAMTSL3/punctin-2, a gene frequently mutated in colorectal tumors, is widely expressed in normal and malignant epithelial cells, vascular endothelial cells and other cell types, and its mRNA is reduced in colon cancer.

ADAMTSL3/punctin-2 is a secreted glycoprotein that resembles the ADAMTS proteases. Recently, identification of frequent ADAMTSL3 mutations in colorectal cancer suggested it might have a regulatory role in cellular homeostasis in colorectal epithelium or in pathways to colorectal malignancy. Here, we used in situ hybridization to validate ADAMTSL3 antibodies for IHC of a variety of normal and malignant tissues, including colon cancer. Quantitative real-time PCR (RTQ-PCR) was used to compare mRNA expression levels in colon carcinoma (n = 10) and adjacent normal colon. ADAMTSL3 is expressed in epithelial cells of the colon, fallopian tube, skin, breast, prostate, epididymis, liver, pancreatic islets and bile ducts, as well as by vascular endothelial cells, smooth muscle cells, fibroblasts, cortical and ganglionic neurons and cardiac myocytes. Malignant epithelial cells in colon cancer, as well as breast, prostate, renal and skin tumors expressed ADAMTSL3. Normal colon showed stronger immunostaining of surface than basal crypt epithelium and staining of a variety of cells within the lamina propria and submucosa. Colon carcinomas demonstrated weaker staining in tumor cells than normal colon epithelium and weak stromal staining. RTQ-PCR comparison of ADAMTSL3 mRNA in colon carcinoma and adjacent normal colon demonstrated a statistically significant reduction in the tumors, possibly reflecting their decreased stromal content and lack of complete differentiation of tumor samples. The major findings of these studies are that ADAMTSL3 is expressed in numerous tissues, suggesting a broader regulatory role than in colorectal epithelium alone, and that colorectal cancer has both structural mutations as well as decreased expression of ADAMTSL3.

ADAMTS-like 2 (ADAMTSL2) is a secreted glycoprotein that is widely expressed during mouse embryogenesis and is regulated during skeletal myogenesis.

ADAMTS-like 2 (ADAMTSL2), is a secreted protein resembling the ancillary domains of the ADAMTS proteases, but with distinct structural features. It has 7 thrombospondin type-1 repeats (TSRs), but an unusually long spacer module, which in both humans and mice, contains a novel insertion bearing six N-glycosylation sites. The ADAMTSL2 protein expressed in HEK293F and COS-1 cells, is a cell-surface and extracellular matrix binding glycoprotein, with N-linked carbohydrate constituting approximately 20% by mass. The 4.0 kb Adamtsl2 mRNA is found most abundantly in adult mouse liver, lung and spleen by northern blotting. During mouse embryogenesis, Adamtsl2 was expressed most strongly in the third week of gestation. Adamtsl2 mRNA was detected by in situ hybridization in developing skeletal muscle, liver, bronchial and arterial smooth muscle, skin, intervertebral disc, perichondrium, pancreas and spinal cord. Immunohistochemical localization of ADAMTSL2 protein was similar to mRNA expression. Detection of Adamtsl2 mRNA and protein in developing skeletal myotubes, but not undifferentiated myogenic precursors led us to investigate its regulation during in vitro myogenic differentiation. In C2C12 and 23A2 myogenic cells, but not in 23A2 cells rendered non-myogenic by expression of G12V:H-Ras (9A2 cells), differentiation induced by serum starvation triggered expression of Adamtsl2 mRNA, coordinately with Myog, a marker of muscle differentiation. Furthermore, activation of the key myogenic determinant MyoD in 10T1/2 fibroblasts also triggered expression of Adamtsl2 mRNA. Collectively, the data suggest that induction of Adamtsl2 mRNA is an integral feature of myogenesis.

Regulation of ADAMTS9 secretion and enzymatic activity by its propeptide.

ADAMTS9 is a secreted, cell-surface-binding metalloprotease that cleaves the proteoglycans versican and aggrecan. Unlike most precursor proteins, the ADAMTS9 zymogen (pro-ADAMTS9) is resistant to intracellular processing. Instead, pro-ADAMTS9 is processed by furin at the cell surface. Here, we investigated the role of the ADAMTS9 propeptide in regulating its secretion and proteolytic activity. Removal of the propeptide abrogated secretion of the ADAMTS9 catalytic domain, and secretion was inefficiently restored by expression of the propeptide in trans. Substitution of Ala for Asn residues within each of three consensus N-linked glycosylation sites in the propeptide abrogated ADAMTS9 secretion. Thus, the propeptide is an intramolecular chaperone whose glycosylation is critical for secretion of the mature enzyme. In addition to two previously identified furin-processing sites (Arg74 downward arrow and Arg287 downward arrow) the ADAMTS9 propeptide was also furin-processed at Arg209. Substitution of Ala for Arg74, Arg209, and Arg287 resulted in secretion of an unprocessed zymogen. Unexpectedly, versican incubated with cells expressing this pro-ADAMTS9 was processed to a greater extent than when incubated with cells expressing wild-type, furin-processable ADAMTS9. Moreover, cells and medium treated with the proprotein convertase inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone had greater versican-cleaving activity than untreated cells. Following furin processing of pro-ADAMTS9, propeptide fragments maintained a non-covalent association with the catalytic domain. Collectively, these observations suggest that, unlike other metalloproteases, furin processing of the ADAMTS9 propeptide reduces its catalytic activity. Thus, the propeptide is a key functional domain of ADAMTS9, mediating an unusual regulatory mechanism that may have evolved to ensure maximal activity of this protease at the cell surface.

Cell-surface processing of pro-ADAMTS9 by furin.

Processing of polypeptide precursors by proprotein convertases (PCs) such as furin typically occurs within the trans-Golgi network. Here, we show in a variety of cell types that the propeptide of ADAMTS9 is not excised intracellularly. Pulse-chase analysis in HEK293F cells indicated that the intact zymogen was secreted to the cell surface and was subsequently processed there before release into the medium. The processing occurred via a furin-dependent mechanism as shown using PC inhibitors, lack of processing in furin-deficient cells, and rescue by furin in these cells. Moreover, down-regulation of furin by small interference RNA reduced ADAMTS9 processing in HEK293F cells. PC5A could also process pro-ADAMTS9, but similarly to furin, processed forms were absent intracellularly. Cell-surface, furin-dependent processing of pro-ADAMTS9 creates a precedent for extracellular maturation of endogenously produced secreted proproteins. It also indicates the existence of a variety of mechanisms for processing of ADAMTS proteases.

Factor Xa induces mitogenesis of vascular smooth muscle cells via autocrine production of epiregulin.

Factor Xa has been reported to elicit smooth muscle cell proliferation via autocrine release of platelet-derived growth factor. However, this study has shown that factor Xa-induced mitogenesis of rat aortic smooth muscle cell is independent of platelet-derived growth factor. We also could not observe any platelet-derived growth factor isoforms in the cultured medium of factor Xa-stimulated cells. Our finding that the cultured medium of factor Xa-stimulated cells strongly induces rat aortic smooth muscle cell mitogenesis in the absence of factor Xa activity led us to explore the existence of a novel autocrine pathway. The autocrine growth factor was purified from the cultured medium and was identified to be epiregulin. Recombinant epiregulin was also able to induce the mitogenesis. The secretion of epiregulin from factor Xa-stimulated rat aortic smooth muscle cell required mRNA expression and protein synthesis of the growth factor. The mitogenic effect of factor Xa on rat aortic smooth muscle cell was significantly reduced by anti-epiregulin antibody or by antisense oligodeoxynucleotide to epiregulin. Several lines of experimental evidence clearly indicate that the autocrine production of epiregulin, an epidermal growth factor-related ligand, is induced in the factor Xa-stimulated mitogenic process of rat aortic smooth muscle cell.

Deficiency of von Willebrand factor-cleaving protease activity in the plasma of malignant patients.

von Willebrand factor (vWF) multimeric pattern and von Willebrand factor-cleaving protease activity (vWF-cp) were studied using plasmas from patients with advanced stage- and limited stage-malignant tumors. Deficiency of highly polymeric forms of vWF was observed in plasmas from 7 of 11 patients tested. vWF-cp activity was deficient in plasma samples of six patients with advanced stage-malignant tumors (ranging from 6% to 30% activity of normal plasma), whereas an essentially normal vWF-cp activity was observed in samples taken from patients with limited stage-malignant tumors. Inhibitor of vWF-cp was not detected in any plasma samples tested. To further analyze the relevance of this enzymatic activity in metastatic diagnosis, a study of vWF-cp activity was conducted in 17 patients with colon cancer, and it was shown that deficiency of vWF-cp was associated with the progression of the disease.

Factor Xa induces mitogenesis of coronary artery smooth muscle cell via activation of PAR-2.

Factor Xa-induced stimulation of coronary artery smooth muscle cells (CASMC) was investigated by analyzing [(3)H]thymidine incorporation, cell proliferation, and ERK-1/2 activation. Exposure of the cells to factor Xa evoked a time-dependent activation of ERK-1/2 with increased [(3)H]thymidine incorporation and cell proliferation. The factor Xa-induced ERK-1/2 activation was not desensitized by preincubation of the cells with thrombin. However, ERK-1/2 activation was markedly attenuated by prior exposure of the cells to protease-activated receptor-2 (PAR-2) activating peptide, SLIGKV. The mitogenic effect of factor Xa was significantly reduced in the presence of anti-PAR-2 monoclonal antibody. Several lines of experimental evidence indicate that factor Xa-induced mitogenesis of CASMC is a cellular process mediated by PAR-2 activation.

Characterization and cDNA cloning of halyxin, a heterogeneous three-chain anticoagulant protein from the venom of Agkistrodon halys brevicaudus.

We report upon the isolation, characterization, and cDNA cloning of an anticoagulant protein, halyxin from Agkistrodon halys brevicaudus venom. The protein exists as a 29kDa protein, and is separated into three chains on SDS-PAGE under reducing conditions. However, we cloned only two cDNAs encoding halyxin from the cDNA library of the snake venom gland, on the basis of the determined amino acid sequences. The complete amino acid sequences were deduced from their nucleotide sequences and named halyxin A (129 amino acid residues) and B chain (123 amino acid residues). The deduced amino acid sequence of halyxin A chain corresponds to the two smaller chains. Thus, it is considered that halyxin A chain could be synthesized as a single-chain protein that is subsequently cleaved to yield the mature two-chain protein. The amino acid sequence of halyxin is similar to that of other snake venom proteins of the C-type lectin superfamily, and prolongs plasma-clotting time. In the presence of Ca(2+) ions, halyxin binds to coagulation factors IX, X, IXa, and Xa, but not to other vitamin K-dependent coagulation factors. It also inhibits factor Xa in a non-competitive manner but does not affect other activated coagulation factors.