Effect Of Serine Protease Inhibitor Research Articles

Karl landsteiner and his major contributions to haematology

Karl landsteiner and his major contributions to haematology

A Mouse Serine Protease TESP5 Is Selectively Included into Lipid Rafts of Sperm Membrane Presumably as a Glycosylphosphatidylinositol-anchored Protein

We have previously indicated that at least in mouse, sperm serine protease(s) other than acrosin probably act on the limited proteolysis of egg zona pellucida to create a penetration pathway for motile sperm, although the participation of acrosin cannot be ruled out completely. A 42-kDa gelatin-hydrolyzing serine protease present in mouse sperm is a candidate enzyme involved in the sperm penetration of the zona pellucida. In this study, we have PCR-amplified an EST clone encoding a testicular serine protease, termed TESP5, and then screened a mouse genomic DNA library using the DNA fragment as a probe. The DNA sequence of the isolated genomic clones indicated that the TESP5 gene is identical to the genes coding for testicular testisin and eosinophilic esp-1. Immunochemical analysis using affinity-purified anti-TESP5 antibody revealed that 42- and 41-kDa forms of TESP5 with the isoelectric points of 5.0 to 5.5 are localized in the head, cytoplasmic droplet, and midpiece of cauda epididymal sperm probably as a membranous protein. Moreover, these two forms of TESP5 were selectively included into Triton X-100-insoluble microdomains, lipid rafts, of the sperm membranes. These results show the identity between TESP5/testisin/esp-1 and the 42-kDa sperm serine protease. When HEK293 cells were transformed by an expression plasmid carrying the entire protein-coding region of TESP5, the recombinant protein produced was released from the cell membrane by treatment with Bacillus cereus phosphatidylinositol-specific phospholipase C, indicating that TESP5 is glycosylphosphatidylinositol-anchored on the cell surface. Enzymatic properties of recombinant TESP5 was similar to but distinguished from those of rat acrosin and pancreatic trypsin by the substrate specificity and inhibitory effects of serine protease inhibitors.

Inhibitory effect of serine protease inhibitors on neutrophil-mediated endothelial cell injury

To investigate the inhibitory effect of serine protease inhibitors (SPI) on neutrophil-mediated endothelial cell (EC) injury, we analyzed the in vitro cytotoxicity of radiolabeled human umbilical vein EC (HUVEC) mediated by neutrophils in the presence of SPI. The EC injury was inhibited dose-dependently by urinary trypsin inhibitor (ulinastatin, UTI) and ONO-5046, which have the ability to inactivate neutrophil elastase, but not by gabexate mesilate, nafamostat mesilate, aprotinin, and argatroban, which have no ability to inactivate neutrophil elastase. In addition, when UTI and ONO-5046 were added to the tumor necrosis factor alpha-primed neutrophils alone, they showed a dose-dependent inhibition of the intracellular elastase activity, but the other SPI did not, for either flow cytometry or confocal microscopy. Therefore, UTI and ONO-5046 may protect EC against the neutrophil-mediated injury not only by inactivating the extracellular elastase secreted by neutrophils, but also by acting directly on neutrophils and suppressing the production and secretion of activated elastase from them.

Effect of Serine Protease Inhibitors on Posttraumatic Brain Injury and Neuronal Apoptosis

N-Tosyl-l-phenylalanyl chloromethyl ketone (TPCK), an inhibitor of chymotrypsin-like serine protease (CSP), prevents DNA fragmentation and apoptotic cell death in certain blood cell lines and was reported to reduce hippocampal neuronal damage caused by cerebral ischemia. We examined the role of CSP on recovery after lateral fluid percussion-induced traumatic brain injury (TBI) in rats, as well as on cell survival in various in vitro models of neuronal cell death. TBI caused significant time-dependent upregulation of CSP activity, but not trypsin-like serine protease activity in injured cortex. Intracerebroventricular administration of TPCK to rats after TBI did not significantly affect deficits of spatial learning but exacerbated motor dysfunction after injury. Moreover, TPCK did not prevent apoptotic neuronal cell death caused by serum/K+ deprivation or by application of staurosporine or etoposide in cultured rat cerebellar granule cells, rat cortical neurons, or in the human neuroblastoma SH-SY5Y cell line. Instead, at doses from 10 to 100 μM, TPCK was cytotoxic in all cultures tested. Similar results were obtained in cultures treated with another CSP inhibitor, 3,4-dichloroisocoumarin. Cell death caused by CSP inhibitors was neither caspase-dependent nor associated with oligonucleosomal DNA fragmentation. Taken together, these data do not support a neuroprotective role for CSP inhibitors. Rather, they suggest that CSPs may serve an endogenous neuroprotective role, possibly by modulating necrotic cell death.

Inhibition of Melanosome Transfer Results in Skin Lightening1

The chemical basis of melanogenesis is well documented, but the mechanism of melanosome transfer and the regulation of pigmentation by keratinocyte-melanocyte interactions are not well understood. Therefore we examined the effects of serine protease inhibitors on skin pigmentation and found that the protease-activated receptor 2, expressed on keratinocytes, may regulate pigmentation via keratinocyte-melanocyte interactions. Here we show that modulation of protease-activated receptor 2 activation affects melanosome transfer into keratinocytes, resulting in changes in pigment production and deposition. SLIGRL, the protease-activated receptor 2 activating peptide, enhanced melanosome ingestion by keratinocytes, thus increasing pigment deposition. RWJ-50353, a serine protease inhibitor, led to reduced pigment deposition in melanocytes and depigmentation. Electron microscopy studies illustrated an accumulation of immature melanosomes inside melanocytes and abnormal dendrite dynamics in RWJ-50353-treated epidermal equivalents. RWJ-50353 induced a visible and dose-dependent skin lightening effect in the dark-skinned Yucatan swine. Examinations by electron microscopy indicated that the in vivo transfer of melanosomes from melanocytes to keratinocytes was affected. Our data suggest that modulation of keratinocyte-melanocyte interactions via the protease-activated receptor 2 pathway affects melanosome transfer. The use of RWJ-50353 to modulate protease-activated receptor 2 activation could lead to a new class of depigmenting agents.

4-(2-Aminoethyl)benzenesulfonyl fluoride attenuates tumor-necrosis-factor-α-induced blood–brain barrier opening

The effect of serine protease inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF) was investigated on the prevention of tumor-necrosis-factor-α (TNF-α)-induced blood–brain barrier opening. TNF-α (10,000 IU) was injected intracarotidly to newborn pigs pretreated with 0, 2.4, 4.8, 9.6 and 19.2 mg/kg AEBSF ( n=6 in each group). AEBSF dose-dependently inhibited the TNF-α-induced increase in the blood–brain barrier permeability for sodium fluorescein (MW=376) in all of the five brain regions examined, while only 19.2 mg/kg AEBSF could significantly ( P<0.05) decrease the change in Evan's blue-albumin (MW=67,000) transport in two regions. In conclusion, AEBSF attenuates vasogenic brain edema formation.

Effect of serine protease inhibitors on the expression of cell surface and soluble forms of two types of tumor necrosis factor receptors

Effect of serine protease inhibitors on the expression of cell surface and soluble forms of two types of tumor necrosis factor receptors

Effect of serine protease inhibitors on the expression of cell surface and soluble forms of two types of tumor necrosis factor receptors

Effect of serine protease inhibitors on the expression of cell surface and soluble forms of two types of tumor necrosis factor receptors

Mathematical Model of Serine Protease Inhibition in the Tissue Factor Pathway to Thrombin

A mathematical model has been developed to simulate the generation of thrombin by the tissue factor pathway. The model gives reasonable predictions of published experimental results without the adjustment of any parameter values. The model also accounts explicitly for the effects of serine protease inhibitors on thrombin generation. Simulations to define the optimum affinity profile of an inhibitor in this system indicate that for an inhibitor simultaneously potent against VIIa, IXa, and Xa, inhibition of thrombin generation decreases dramatically as the affinity for thrombin increases. Additional simulations show that the reason for this behavior is the sequestration of the inhibitor by small amounts of thrombin generated early in the reaction. This model is also useful for predicting the potency of compounds that inhibit thrombosis in rats. We believe that this is the first mathematical model of blood coagulation that considers the effects of exogenous inhibitors. Such a model, or extensions thereof, should be useful for evaluating targets for therapeutic intervention in the processes of blood coagulation.

Effects of serine protease inhibitors on accumulation of polymorphonuclear leukocytes in the lung induced by acute pancreatitis in rats

The administration of a high-dose of a serine protease inhibitor is recommended in patients complicated by multiple organ failure (MOF), including adult respiratory distress syndrome (ARDS), induced by acute pancreatitis. The accumulation of polymorphonuclear leukocytes (PMN) in affected organs is considered to be one of the causative factors of MOF. Adhesion to endothelial cells (EC), via adhesion molecules, and the transendothelial migration of PMN is closely associated with the accumulation of PMN. We examined the effects of two serine protease inhibitors, ulinastatin (UT) and gabexate mesilate (GM), on EC-PMN adhesion and transendothelial migration in human umbilical vein EC and 51Cr-labeled PMN in vitro. EC-PMN adhesion, and the expression of intercellular adhesion molecule-1 (ICAM-1) and endothelial cell adhesion molecule-1 (ELAM-1) on EC induced by IL-1 beta and TNF alpha, were reduced by the pretreatment of EC with these inhibitors. The transendothelial migration of PMN stimulated by IL-8 was also inhibited by pretreating PMN with UT or GM. We also examined whether these inhibitors reduced PMN accumulation in the lung in rats with acute pancreatitis induced by a closed duodenal loop. The myeloperoxidase activity in and histological findings of the lung suggested that UT and GM reduced PMN accumulation. In conclusion, serine protease inhibitors may inhibit PMN accumulation in ARDS due to acute pancreatitis.

Effect of Serine Protease Inhibitor, N-α-Tosyl-L-lysyl-Chloromethyl Ketone (TLCK), on Cell-Mediated and Cell-Free HIV-1 Spread

The effect of a serine protease inhibitor, N-α-tosyl-L-lysyl-chloromethyl ketone (TLCK), on cell adhesion and resulting cell-to-cell HIV-1 transmission was examined. Cell-mediated vital spread was blocked by 10-4M TLCK—the nontoxic dose at which the maximum inhibition of cell adhesion was achieved. TLCK (10-4-10-8M range) exhibited a similar dose-dependent effect when tested against cell-free virus infection. These findings suggest that TLCK acts by preventing both cell-cell adhesion and viral binding to the target cell. The effect of TLCK could be attributed to an irreversible modification of surface and/or intracellular proteases required for functional activation of HIV-1 envelope glycoproteins.

Effects of serine protease inhibitor, tame, on IL-1? in LPS-stimulated human monocytes: Relationship between synthesis and release of a 33-kDa precursor and the 17-kDa biologically active species

LPS stimulation of human monocytes in vitro induced release of the 17-kDa mature IL-1 beta (mIL-1 beta) but did not result in release of precursor IL-1 beta (pIL-1 beta). In contrast, the presence of a serine protease inhibitor, N alpha-(p-toluene sulfonyl)-L-arginine methyl ester (TAME; 10 mM) for 6 or 18 h was associated with the LPS-stimulated release of the 33-kDa pIL-1 beta as well. These effects were initially discerned from observations that the fraction of the total IL-1 beta produced (as detected by ELISA) that was released from monocytes increased in the presence of TAME, and immunoblot assays confirmed that this fraction was predominantly 33-kDa IL-1 beta. A global decrease in monocyte protein synthesis was also observed after prolonged (18-h) exposure to TAME and was associated with a decrease in IL-1 beta synthesis, predominantly affecting 31-kDa pIL-1 beta, and a dose-dependent inhibition of TNF-alpha production. Parallel examination of lactate dehydrogenase (LDH) release indicated that pIL-1 beta release was unrelated to cell lysis. These results demonstrate that TAME-inhibitable serine proteases are probably involved in the production and eventual proteolysis of the 33-kDa pIL-1 beta in situ but are probably not mechanistically related to either maturation of the IL-1 beta molecule or signaling of IL-1 beta release. IL-1 beta release appears to be dependent on the amount of total IL-1 beta synthesized. Serine proteolysis may constitute a degradative pathway for excess precursor, which, if interfered with, could result in release of the higher-molecular-weight forms of IL-1 beta.

Functional and structural domains of the sixth component (C6) of human complement.

The effects of serine protease inhibitors, diisopropyl fluorophosphate (DFP) and phenylmethanesulfonyl fluoride (PMSF), on hemolytic activity of C6 were reinvestigated. C6 was inactivated in a range of 1-10 mM by both of the inhibitors as previously reported. Limited proteolytic digestion was also studied to elucidate the functional and structural domains of C6. The major fragments produced by trypsin, plasmin, or lysyl endopeptidase could not be separated unless disulfide bonds were disrupted, but Staphylococcus aureus V8 protease yielded several fragments, each of which was not linked by disulfide bond. When C6 labeled with [3H]DFP was subjected to limited digestion with V8 protease, a fragment with a molecular weight of 38 kilodaltons (kDa) was mainly labeled and other fragments of 53 kDa and 26.4 kDa were also faintly labeled, while fragment 35 kDa wasn't labeled, indicating specific domains reactive with DFP. On the other hand, when C6 with or without DFP treatment was digested with V8 protease and those fragments were incubated with C5 and subjected to sucrose density ultracentrifugation, fragments 53, 38, 35 and 27.5 kDa interacted with C5 in both cases. These results suggest that C6 modified by DFP can interact with C5, and the amino-terminal sequences of fragment 38 and 35 kDa suggest the binding domain of C6 with C5 takes place within the two short consensus repeats.

Effects of serine protease inhibitors on oxyradical burst from phagocytizing neutrophils--analysis by chemiluminescence counting and its microscopic imaging.

Reaction difference of oxyradical generation and luminol-dependent photoemission of zymosan- and phorbol ester-treated neutrophils were investigated using a conventional photomultiplier and ultrasensitive photonic imaging technique. Zymosan-treated cells released a concentrated photonic burst corresponding to the cellular distribution. In contrast, phorbol ester-treated cells produced a negligible level of photoemission, and the additional application of Ca2+ ionophore enhanced the photonic burst, which was gradually spread out into extracellular space. Serine protease inhibitors did not attenuate PMA-induced chemiluminescence but did attenuate zymosan-induced chemiluminescence. This suggests the involvement of serine protease in the respiratory burst of phagocytizing neutrophils.

Studies on the effect of serine protease inhibitors on activated contact factors. Application in amidolytic assays for factor XIIa, plasma kallikrein and factor XIa.

Amidolytic assays have been developed to determine factor XIIa, factor XIa and plasma kallikrein in mixtures containing variable amounts of each enzyme. The commercially available chromogenic p-nitroanilide substrates Pro-Phe-Arg-NH-Np (S2302 or chromozym PK), Glp-Pro-Arg-NH-Np (S2366), Ile-Glu-(piperidyl)-Gly-Arg-NH-Np (S2337), and Ile-Glu-Gly-Arg-NH-Np (S2222) were tested for their suitability as substrates in these assays. The kinetic parameters for the conversion of S2302, S2222, S2337 and S2366 by beta factor XIIa, factor XIa and plasma kallikrein indicate that each active enzyme exhibits considerable activity towards a number of these substrates. This precludes direct quantification of the individual enzymes when large amounts of other activated contact factors are present. Several serine protease inhibitors have been tested for their ability to inhibit those contact factors selectively that may interfere with the factor tested for. Soybean trypsin inhibitor very efficiently inhibited kallikrein, inhibited factor XIa at moderate concentrations, but did not affect the amidolytic activity of factor XIIa. Therefore, this inhibitor can be used to abolish a kallikrein and factor XIa contribution in a factor XIIa assay. We also report the rate constants of inhibition of contact activation factors by three different chloromethyl ketones. D-Phe-Pro-Arg-CH2Cl was moderately active against contact factors (k = 2.2 X 10(3) M-1 s-1 at pH 8.3) but showed no differences in specifity. D-Phe-Phe-Arg-CH2Cl was a very efficient inhibitor of plasma kallikrein (k = 1.2 X 10(5) M-1 s-1 at pH 8.3) whereas it slowly inhibited factor XIIa (k = 1.4 X 10(3) M-1 s-1) and factor XIa (k = 0.11 X 10(3) M-1 s-1). Also Dns-Glu-Gly-Arg-CH2Cl was more reactive towards kallikrein (k = 1.6 X 10(4) M-1 s-1) than towards factor XIIa (k = 4.6 X 10(2) M-1 s-1) and factor XIa (k = 0.6 X 10(2) M-1 s-1). Since Phe-Phe-Arg-CH2Cl is highly specific for plasma kallikrein it can be used in a factor XIa assay selectively to inhibit kallikrein. Based on the catalytic efficiencies of chromogenic substrate conversion and the inhibition characteristics of serine protease inhibitors and chloromethyl ketones we were able to develop quantitative assays for factor XIIa, factor XIa and kallikrein in mixtures of contact activation factors.

Ｓｅｒｉｎｅ ｐｒｏｔｅａｓｅ ｉｎｈｉｂｉｔｏｒの血小板凝集抑制機序に関する検討

We reported the effects of serine protease inhibitors on the interaction of platelet cytoskeletal proteins. Serine protease inhibitors such as PMSF, FOY-007 or FUT-175 and aspirin inhibited the incorporation of myosin heavy chain into the 1% Triton X-100 insoluble residue during the aggregation induced by ADP. FUT-175 also suppressed the incorporation of actin binding protein and fibrinogen into the cytoskeletons. This may result in the inhibition of not only the second aggregation but also the primary wave of aggregation. Anti-hydrolytic activity of FUT-175 against trypsin was recovered in cytosol fraction at least 2×10-6M, when platelets were incubated with this agent, 10-4M. This seems to indicate that FUT-175 has unique site (s) of action in cytosol which is related to the inhibition of integration of cytoskeletons during the process of platelet aggregation besides it's action on arachidonate metabolism. In fact, zymogram of platelet cytosol fraction with Tosyl-L-lysine α-naphthyl ester as substrate, demonstrated 5 active bands (pl. 9.2, 8.1, 6.25, 5.9, 5.3, 4.4). Some of these cytosomal serine proteases might act as an important role in the formation of cytoskeletons during the process of platelet reaction.

The effects of serine protease inhibitors on morphological differentiation of murine neuroblastoma cells (NB 15)

Morphological differentiation of neuroblastoma cells (NB15) was induced by cAMP effectors in the presence and absence of serine protease inhibitors. In all conditions tested, the percent differentiation was inhibited by protease inhibitors antipain, diisopropylfluorophosphate (DFP), leupeptin, and soybean trypsin inhibitor (SBTI). The level of morphological differentiation obtained in medium containing fetal calf serum was significantly less than the percent differentiation obtained with serum-free medium alone, so serum-free medium was the principal method of induction and comparisons were made to control uninduced cultures or cultures induced with the phosphodiesterase inhibitor R020-1724. Secreted or cell surface caseinolytic protease activity was higher in differentiating cells than in control cultures and was inhibited by the serine protease inhibitors. The effects of the protease inhibitors on growth and differentiation are discussed.