Unusual proteolytic activation of pro-hepatocyte growth factor by plasma kallikrein and coagulation factor XIa

Hepatocyte growth factor (HGF), the ligand for the receptor tyrosine kinase c-Met, is composed of an alpha-chain containing four Kringle domains (K1-K4) and a serine protease domain-like beta-chain. Receptor activation by HGF is contingent upon prior proteolytic conversion of the secreted inactive single chain form (pro-HGF) into the biologically active two chain form by a single cleavage at the Arg(494)-Val(495) bond. By screening a panel of serine proteases we identified two new HGF activators, plasma kallikrein and coagulation factor XIa (FXIa). The concentrations of kallikrein and FXIa to cleave 50% (EC(50)) of (125)I-labeled pro-HGF during a 4-h period were 10 and 17 nm. Unlike other known activators, both FXIa and kallikrein processed pro-HGF by cleavage at two sites. Using N-terminal sequencing they were identified as the normal cleavage site Arg(494)-Val(495) and the novel site Arg(424)-His(425) located in the K4 domain of the alpha-chain. The identity of this unusual second cleavage site was firmly established by use of the double mutant HGF(R424A/R494E), which was completely resistant to cleavage by kallikrein and FXIa. Experiments with another mutant form, HGF(Arg(494) --> Glu), indicated that cleavage at the K4 site was independent of a prior cleavage at the primary, kinetically preferred Arg(494)-Val(495) site. The cleavage at the K4 site had no obvious consequences on HGF function, because it was fully capable of phosphorylating the c-Met receptor of A549 cells. This may be explained by the disulfide bond network in K4, which holds the cleaved alpha-chain together. In conclusion, the ability of plasma kallikrein and FXIa to activate pro-HGF in vitro raises the possibility that mediators of inflammation and blood coagulation may also regulate processes that involve the HGF/c-Met pathway, such as tissue repair and angiogenesis.     

Anionic regulation of biological systems: the special role of chloride in the coagulation cascade

The discovery that previously unidentified allosteric properties of several proteins, such as fibrinogen and myoglobin, can be triggered by anions binding, has suggested the possibility to design a new "active" role of chloride in the modulation of a broad range of biological systems. The molecular bases of the anions binding to proteins depends by their charge density in turn regulating the ability to bind water molecules and interact with basic groups on proteins. This review reports the role of the physiologically relevant chloride, and of other anions, in the regulation of several proteins, with special attention to the coagulation cascade. Moreover, possible mechanisms of modification of plasma, intra- or extracellular chloride concentration are listed.     

The matriptase-prostasin proteolytic cascade in epithelial development and pathology

The type II transmembrane serine protease matriptase has an essential role in the integrity and function of multiple epithelial tissues. In the epidermis, matriptase activates the glycosylphosphatidylinositol (GPI) anchored membrane serine protease prostasin to initiate a proteolytic cascade that is required for the development of the stratum corneum barrier function. Accordingly, mice deficient for matriptase phenocopy mice deficient for epidermal prostasin and present with impaired corneocyte differentiation, imparied lipid matrix formation, loss of profilaggrin processing and loss of tight junction formation and function. Together, these defects lead to a compromised epidermal barrier and result in fatal dehydration during the neonatal period. Proteolytic activity of the matriptase-prostasin cascade is regulated in the epidermis via inhibition by the Kunitz-type serine protease inhibitor hepatocyte growth factor activator inhibitor-1 (HAI-1). Importantly, targeted post-natal ablation of matriptase in mice perturbs the function of multiple adult tissues, indicating an ongoing requirement for matriptase proteolysis in the maintenance of diverse types of epithelia. Impaired matriptase proteolytic activity has been linked to human Autosomal Recessive Icthyosis with Hypotrichosis (ARIH), whereas aberrant matriptase activity has been implicated in Netherton’s Syndrome. This review will summarize information pertaining to the role of matriptase in epithelial biology and will discuss recent advancements in our understanding of how matriptase activity is regulated and the down-stream effectors of matriptase proteolysis.     

Half-of-the-sites and all-of-the-sites reactivity in human plasma blood coagulation factor XIIIa

The reactivities of human plasma factor XIIIa toward iodoacetic acid and toward alpha-bromo-4-hydroxy-3-nitroacetophenone have been studied under conditions where this dimeric enzyme reacts with the reagents in half-of-the-sites fashion and under conditions where it reacts with the reagents in all-of-the-sites fashion. Direct measurements of alkylation of active site -- SH groups in the apparently identical subunits of the enzyme as functions of remaining catalytic activity are in agreement with the observed reactivities. In addition to extending earlier evidence for half-of-the-sites reactions in factor XIIIa (Chung, S. I., Lewis, M. S., and Folk, J. E. (1974) J. Biol. Chem. 249, 940-950), the present findings suggest that the all-of-the-sites reactivity results from a positively cooperative interaction between enzyme subunits.     

Tissue kallikrein proteolytic cascade pathways in normal physiology and cancer

Human tissue kallikreins (KLKs or kallikrein-related peptidases) are a subgroup of extracellular serine proteases that act on a wide variety of physiological substrates, while they display aberrant expression patterns in certain types of cancer. Differential expression patterns lead to the exploitation of these proteins as new cancer biomarkers for hormone-dependent malignancies, in particular. The prostate-specific antigen or kallikrein-related peptidase 3 (PSA/KLK3) is an established tumor marker for the diagnosis and monitoring of prostate cancer. It is well documented that specific KLK genes are co-expressed in tissues and in various pathologies suggesting their participation in complex proteolytic cascades. Here, we review the currently established knowledge on the involvement of KLK proteolytic cascades in the regulation of physiological and pathological processes in prostate tissue and in skin. It is well established that the activity of KLKs is often regulated by auto-activation and subsequent autolytic internal cleavage leading to enzymatic inactivation, as well as by inhibitory serpins or by allosteric inhibition by zinc ions. Redistribution of zinc ions and alterations in their concentration due to physiological or pathological reasons activates specific KLKs initiating the kallikrein cascade(s). Recent studies on kallikrein substrate specificity allowed for the construction of a kallikrein interaction network involved in semen liquefaction and prostate cancer, as well as in skin pathologies, such as skin desquamation, psoriasis and cancer. Furthermore, we discuss the crosstalks between known proteolytic pathways and the kallikrein cascades, with emphasis on the activation of plasmin and its implications in prostate cancer. These findings may have clinical implications for the underlying molecular mechanism and management of cancer and other disorders in which KLK activity is elevated.     

b-chains prevent the proteolytic inactivation of the a-chains of plasma factor XIII

While the transglutaminase activity is associated exclusively with the thrombin-cleaved a chains of plasma Factor XIII, there is little information regarding the role of the b-chains. The present investigations were undertaken to clarify the role of the b-chains during proteolytic activation of plasma factor XIII a-chains. The a-chains of platelet Factor XIII (a2) were extremely sensitive to alpha-thrombin proteolysis, especially in the presence of 5 mM EDTA, resulting in two major fragments with molecular masses 51 +/- 3 kDa and 19 +/- 4 kDa. Furthermore, fibrin enhanced the alpha-thrombin proteolysis of thrombin-cleaved platelet Factor XIII a-chains in presence of CaCl2 or EDTA, resulting in several peptide fragments with molecular masses from 51 +/- 3 kDa to 14 +/- 4 kDa. By contrast, thrombin-cleaved a-chains of plasma Factor XIII (a2b2) were not further degraded by alpha-thrombin in presence of 5 mM EDTA. Even in the combined presence of 5 mM EDTA and 0.1 mg/ml fibrin, alpha-thrombin proteolysis of plasma Factor XIIIa was limited to the formation of a 76 kDa fragment (= Factor XIIIa), a 51 +/- 3 kDa fragment and trace amounts of a 14 +/- 4 kDa species. Platelet Factor XIII proteolyzed by 500 nM alpha-thrombin in presence of 5 mM EDTA expressed less than 20% of enzymatic activity obtained when platelet Factor XIII was activated in presence of 5 mM CaCl2. In contrast, plasma Factor XIII activated by 500 nM apha-thrombin in presence of 5 mM EDTA expressed nearly 65% of original transglutaminase activity. Likewise, when plasma Factor XIII was proteolyzed by 100-1000 nM gamma-thrombin in presence of 5 mM CaCl2 or 5 mM EDTA, maximal transglutaminase activity was observed. However, when platelet Factor XIII was similarly treated with gamma-thrombin in presence of 5 mM EDTA, only one-half the original transglutaminase activity was obtained. The b-chains thus appear to mimic the function of Ca2+ in preserving transglutaminase activity of thrombin-cleaved a-chains. The b-chains of plasma Factor XIII were not degraded by either alpha- or gamma-thrombin treatment, in presence of 5 mM EDTA or 5 mM CaCl2. Both platelet and plasma Factor XIII a-chains were degraded by trypsin to fragments with molecular masses of 51 +/- 3 kDa and 19 +/- 4 kDa in presence of 5 mM CaCl2 and to fragments with molecular masses of 19 +/- 4 kDa and lower, in presence of 5 mM EDTA.(ABSTRACT TRUNCATED AT 400 WORDS)     

Introduction to the blood coagulation cascade and cloning of blood coagulation factors

The coagulation cascade that occurs in mammalian plasma involves a large number of plasma proteins that participate in a stepwise manner and eventually give rise to the formation of thrombin. This enzyme then converts fibrinogen to an insoluble fibrin clot. This series of reactions involves a number of glycoproteins that particupate as enzymes as well as cofactors. These proteins that circulate in the blood in a precursor or zymogen form are multifunctional proteins that share many common segments or domains. One group includes the vitamin K-dependent glycoproteins (prothrombin, factor IX, factor X, and protein C) that show considerable homology in both their amino acid sequences and their gene structures. The proteins that participate in the contact or early phase of the blood coagulation cascade include plasma prekallikrein, factor XII, and factor IX. The amino-terminal regions of both factor XI and plasma prekallikrein contain four tandem repeats of about 90 amino acids, and these tandem repeats show considerable amino acid sequence homology. Factor XII contains four different domains in the amino-terminai region of the protein, including a kringle structure, two growth factor domains, and type I and type II finger domains. The finger domains were first identified in fibronectin. The carboxyl-terminal portion of plasma prekallikrein, factor XII, and factor XI contains the serine or protease portion of the molecule. These various plasma proteins that share common domains appear to have evolved by gene shuffling that may have, in some cases, involved introns.     

Proteomic analysis of human plasma in chronic rheumatic mitral stenosis reveals proteins involved in the complement and coagulation cascade

Background

Rheumatic fever in childhood is the most common cause of Mitral Stenosis in developing countries. The disease is characterized by damaged and deformed mitral valves predisposing them to scarring and narrowing (stenosis) that results in left atrial hypertrophy followed by heart failure. Presently, echocardiography is the main imaging technique used to diagnose Mitral Stenosis. Despite the high prevalence and increased morbidity, no biochemical indicators are available for prediction, diagnosis and management of the disease. Adopting a proteomic approach to study Rheumatic Mitral Stenosis may therefore throw some light in this direction. In our study, we undertook plasma proteomics of human subjects suffering from Rheumatic Mitral Stenosis (n = 6) and Control subjects (n = 6). Six plasma samples, three each from the control and patient groups were pooled and subjected to low abundance protein enrichment. Pooled plasma samples (crude and equalized) were then subjected to in-solution trypsin digestion separately. Digests were analyzed using nano LC-MS^E. Data was acquired with the Protein Lynx Global Server v2.5.2 software and searches made against reviewed Homo sapiens database (UniProtKB) for protein identification. Label-free protein quantification was performed in crude plasma only.

Results

A total of 130 proteins spanning 9–192 kDa were identified. Of these 83 proteins were common to both groups and 34 were differentially regulated. Functional annotation of overlapping and differential proteins revealed that more than 50% proteins are involved in inflammation and immune response. This was corroborated by findings from pathway analysis and histopathological studies on excised tissue sections of stenotic mitral valves. Verification of selected protein candidates by immunotechniques in crude plasma corroborated our findings from label-free protein quantification.

Conclusions

We propose that this protein profile of blood plasma, or any of the individual proteins, could serve as a focal point for future mechanistic studies on Mitral Stenosis. In addition, some of the proteins associated with this disorder may be candidate biomarkers for disease diagnosis and prognosis. Our findings might help to enrich existing knowledge on the molecular mechanisms involved in Mitral Stenosis and improve the current diagnostic tools in the long run.

Electronic supplementary material

The online version of this article (doi:10.1186/1559-0275-11-35) contains supplementary material, which is available to authorized users.     

Evidence for a matriptase-prostasin proteolytic cascade regulating terminal epidermal differentiation

Recent gene ablation studies in mice have shown that matriptase, a type II transmembrane serine protease, and prostasin, a glycosylphosphatidylinositol-anchored membrane serine protease, are both required for processing of the epidermis-specific polyprotein, profilaggrin, stratum corneum formation, and acquisition of epidermal barrier function. Here we present evidence that matriptase acts upstream of prostasin in a zymogen activation cascade that regulates terminal epidermal differentiation and is required for prostasin zymogen activation. Enzymatic gene trapping of matriptase combined with prostasin immunohistochemistry revealed that matriptase was co-localized with prostasin in transitional layer cells of the epidermis and that the developmental onset of expression of the two membrane proteases was coordinated and correlated with acquisition of epidermal barrier function. Purified soluble matriptase efficiently converted soluble prostasin zymogen to an active two-chain form that formed SDS-stable complexes with the serpin protease nexin-1. Whereas two forms of prostasin with molecular weights corresponding to the prostasin zymogen and active prostasin were present in wild type epidermis, prostasin was exclusively found in the zymogen form in matriptase-deficient epidermis. These data suggest that matriptase, an autoactivating protease, acts upstream from prostasin to initiate a zymogen cascade that is essential for epidermal differentiation.     

Haloarchaeal proteases and proteolytic systems

Proteases play key roles in many biological processes and have numerous applications in biotechnology and industry. Recent advances in the genetics, genomics and biochemistry of the halophilic Archaea provide a tremendous opportunity for understanding proteases and their function in the context of an archaeal cell. This review summarizes our current knowledge of haloarchaeal proteases and provides a reference for future research.     

Interconnections between autophagy and the coagulation cascade in hepatocellular carcinoma

Autophagy has an important role in tumor biology of hepatocellular carcinoma (HCC). Recent studies demonstrated that tissue factor (TF) combined with coagulation factor VII (FVII) has a pathological role by activating a G-protein-coupled receptor called protease-activated receptor 2 (PAR2) for tumor growth. The present study aimed to investigate the interactions of autophagy and the coagulation cascade in HCC. Seventy HCC patients who underwent curative liver resection were recruited. Immunohistochemical staining and western blotting were performed to determine TF, FVII, PAR2 and light chain 3 (LC3A/B) expressions in tumors and their contiguous normal regions. We found that the levels of autophagic marker LC3A/B-II and coagulation proteins (TF, FVII and PAR2) were inversely correlated in human HCC tissues. Treatments with TF, FVII or PAR2 agonist downregulated LC3A/B-II with an increased level of mTOR in Hep3B cells; in contrast, knockdown of TF, FVII or PAR2 increased LC3A/B. Furthermore, mTOR silencing restored the impaired expression of LC3A/B-II in TF-, FVII- or PAR2-treated Hep3B cells and activated autophagy. Last, as an in vivo correlate, we administered TF, FVII or PAR2 agonist in a NOD/severe combined immunodeficiency xenograft model and showed decreased LC3A/B protein levels in HepG2 tumors with treatments. Overall, our present study demonstrated that TF, FVII and PAR2 regulated autophagy mainly via mTOR signaling. The interaction of coagulation and autophagic pathways may provide potential targets for further therapeutic application in HCC.     

A highly sensitive fluorometric assay for determination of human coagulation factor XIII in plasma

Based on the iso-peptidase activity of human plasma FXIII, a novel fluorometric assay that determines FXIII concentrations in human plasma below 0.05 IU/ml is introduced. We considered a peptide sequence derived from alpha(2)-antiplasmin (n =12) to yield high sensitivity. Peptide Abz-NE(Cad-Dnp)EQVSPLTLLK exhibits a K(m) value of 19.8+/-2.8 microM and is used in a concentration of 50 microM. The assay design is suitable for measurements in cuvettes (1 ml volume) as well as for the microtiter plate (MTP) format (0.2 ml volume). It provides linear dose-response curves over a wide range of FXIII concentrations (0.05-8.8 IU/ml). The assay was validated with respect to precision, detection and quantitation limits, accuracy/specificity, linearity, and range. A comparison of the fluorometric assay with the photometric assay for FXIII determinations in plasma pools as well as single donor plasma revealed suitability of the fluorometric assay for FXIII determination in plasma of healthy individuals. FXIII concentrations in plasma samples of patients with severe FXIII deficiency are discussed in the context of FXIII antigen levels. These assays correlate well in the critical range below 0.1 IU/ml, whereas the photometric assay may overestimate residual FXIII activity in severe FXIII-deficient patients.