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P Savi

Publications and source records attributed to P Savi.

At least 37 records · Page 2Linked to original sources
PubMed1998

Role of P2Y1 purinoceptor in ADP-induced platelet activation.

ADP acts as an agonist of platelet aggregation via specific receptors which are still to be characterised. Amplification by PCR of a human platelet cDNA library confirmed the presence of mRNA of the P2Y1 receptor in platelets. In order to determine if these P2Y1 receptors were involved in ADP-induced platelet activation, we determined the effects of A3P5PS, an antagonist of the P2Y1 receptor, on the binding of [33P]2-MeS-ADP, a potent analogue of ADP. We found that A3P5PS displaced about 27% of [33P]2-MeS-ADP binding, a receptor population which has been shown to be resistant to treatment with clopidogrel, a selective anti-ADP agent. A3P5PS specifically inhibited 2-MeS-ADP-induced shape change and calcium increase but did not affect adenylyl cyclase down-regulation. 2-MeS-ADP-induced platelet aggregation was also inhibited by A3P5PS but was restored when platelets were further activated by serotonin, a non-aggregating compound, therefore suggesting that P2Y1-mediated stimulation is an absolute prerequisite for ADP to induce platelet aggregation and a key event for platelet activation and aggregation to occur. These results therefore show that ADP-induced aggregation cannot be attributed to activation of P2Y1 alone, but must be attributed to the simultaneous activation of the high affinity receptor (P2Y1) and a low affinity receptor of ADP (still to be discovered), each of them essential, but neither able to trigger aggregation alone.

Adenosine Diphosphate↗
PubMed1998

Clopidogrel: a review of its mechanism of action.

The search for active antiplatelet drugs within the original chemical class of the thienopyridines, led to the discovery of clopidogrel, a novel ADP-selective agent whose antiaggregating properties are several times higher than those of ticlopidine. The antiaggregating properties of this compound are well known and, very recently, new results have clarified its mechanism of action. Clopidogrel is active only after intravenous or oral administration, and no circulating activity has been found in the plasma of treated animals or human volunteers. Experiments in rats have demonstrated that the antiaggregating activity was caused by a shortlasting metabolite generated in the liver by a cytochrome P450-dependent pathway. The antiaggregating property of clopidogrel is caused by an inhibition of the binding of ADP to its platelet receptors, and more specifically to the low affinity receptors, the high affinity binding sites being unaffected by clopidogrel. Several events in the ADP activation process, including adenylyl cyclase down-regulation, protein tyrosine phosphorylation, activation of the GPIIb-IIIa complex, fibrinogen binding, aggregation and release, were inhibited by clopidogrel and indicate their close relationship with the activation of a low affinity receptor by ADP. In contrast, binding of ADP to its high affinity binding sites (clopidogrel-resistant receptors) induced shape change, cytosolic calcium increase and phosphorylations of several other proteins, some events which were clopidogrel-sensitive. Thus, clopidogrel not only constitutes a potent antithrombotic drug in humans but also a good tool to study the effect of ADP on platelets.

Journal Article↗
PubMed1998

Effects of a new platelet glycoprotein IIb/IIIa antagonist, SR121566, on platelet activation, platelet-leukocyte interaction and thrombin generation.

The effects of SR121566, a new inhibitor of the glycoprotein (GP) IIb/IIIa complex on platelet activation and platelet-leukocyte interactions, as well as on thrombin generation were investigated. SR121566 dose-dependently inhibited adenosine diphosphate (ADP)-induced platelet fibrinogen binding determined either by flow cytometry analysis (IC50=50 nmol/l) or by measuring the binding of 125I-fibrinogen to activated human gel-filtered platelets (IC50=20 nmol/l). Consistent with its inhibitory effects on platelet fibrinogen binding, SR121566 demonstrated a dose-dependent inhibition of collagen-, ADP- or thrombin-induced platelet aggregation with IC50 values ranging between 20 and 60 nmol/l. SR121566, even tested at high concentrations, did not significantly affect ADP-induced platelet-leukocyte aggregate formation. The GPIIb/IIIa antagonist strongly inhibited thrombin generation in both native clotting blood and recalcified whole blood, suggesting that SR121566, by interfering with the platelet-activation events involved in facilitating thrombin generation, may also function as an anticoagulant, an effect which may contribute to its antithrombotic properties in humans.

Adenosine Diphosphate↗
PubMed1998

Effect of SR121566A, a potent GP IIb-IIIa antagonist on platelet-mediated thrombin generation in vitro and in vivo.

The effect of SR121566A, a new non-peptide GP IIb-IIIa antagonist was studied in vitro with regard to thrombin generation in platelet rich plasma and in vivo on stasis-induced venous thrombosis in the rabbit. SR121566A inhibited ADP-, arachidonic acid- and collagen-induced human platelet aggregation with IC50 values of 46 +/- 7.5, 56 +/- 6 and 42 +/- 3 nM, respectively. In the same experimental conditions, SR121566A strongly inhibited thrombin generation triggered by low concentrations of tissue factor. SR121566A reduced in a dose-dependent manner both the area under the curve and the thrombin peak concentration but did not affect the lag phase (defined as the time until 10 nM thrombin was generated). Aspirin (100 microg/ml) did not affect thrombin generation. One hour after intravenous administration to rabbits, SR121566A exhibited a potent ex vivo inhibitory effect against ADP-, arachidonic acid- and collagen-induced platelet aggregation. The ID50 were 0.6 +/- 0.25, 0.7 +/- 0.08 and 0.13 +/- 0.08 mg/kg, respectively. The ability of aspirin and SR121566A to affect venous stasis was determined in a stasis-induced venous thrombosis model in rabbits under high and low thrombogenic challenges. While aspirin was ineffective in both conditions, SR121566A significantly inhibited thrombus formation under low thrombogenic challenge demonstrating for the first time that a potent non-peptide platelet GP IIb-IIIa antagonist inhibits thrombin generation in vivo and exhibits a strong antithrombotic effect with regard to stasis-induced venous thrombosis. These results therefore confirm the existence of a close relationship between platelet activation and thrombin generation leading to blood coagulation but also emphasise the key role of platelets in the development of venous thrombosis, most likely through activation of the GP IIb-IIIa complex.

Animals↗
PubMed1998

Prevention of thrombosis and enhancement of thrombolysis in rabbits by SR 121787, a glycoprotein II/III antagonist.

SR 121566, a novel nonpeptide antiplatelet agent with high affinity and specificity for the GP IIb/IIIa complex, exhibits potent in vitro antiaggregating activity in rabbit platelets. This paper reports results from a study in rabbits about the efficacy and tolerability of SR 121787, the prodrug of SR 121566. After p.o. pretreatment with SR 121787, ADP-, arachidonic acid- and collagen-induced rabbit platelet aggregation was inhibited ex vivo in a dose-dependent manner (ED50 between 2.3 and 6.1 mg/kg). Collagen-induced thrombocytopenia was totally abolished by SR 121787 at 20 mg/kg p.o. In a carotid artery lesion model of arterial thrombosis, p.o. administration of SR 121787 resulted in a dose-dependent inhibition of thrombosis with a maximum effect of 68% (ED50 = 16.0 +/- 0.3 mg/kg). Recombinant tissue plasminogen activator-induced thrombolysis of a preformed thrombus in the jugular vein was potentiated by SR 121787 at doses between 1 and 6 mg/kg i.v. In an ear incision bleeding model, SR 121787 at doses up to 15 mg/kg p.o. did not cause an increase in blood loss. These results demonstrate that SR 121787 exerts oral antiplatelet, antithrombotic and thrombolysis-enhancing efficacy in rabbits. SR 121787 appears to be a promising compound for evaluation under clinical conditions in the therapy of acute coronary syndromes.

Animals↗
PubMed1998

Effect of SR121566A, a potent GP IIb-IIIa antagonist, on the HIT serum/heparin-induced platelet mediated activation of human endothelial cells.

Heparin-induced thrombocytopenia (HIT) is a common adverse effect of heparin therapy that carries a risk of serious thrombotic events. This condition is caused by platelet aggregation, which is mediated by anti-heparin/platelet factor 4 antibodies. Sera from patients with HIT in the presence of platelets, induced the expression of E-selectin, VCAM, ICAM-1 and tissue factor and the release of IL1beta, IL6, TNFalpha and PAI-1 by human umbilical vein endothelial cells (HUVECs) in vitro and initiated platelet adhesion to activated HUVECs. These effects which occurred in a time-dependent manner were significant in the first 1-2 h of incubation and reached a maximum after 6 to 9 h. The GP IIb-1IIa receptor antagonist SR121566A which has been shown to block platelet aggregation induced by a wide variety of agonists including HIT serum/heparin, reduced in a dose-dependent manner the HIT serum/heparin-induced, platelet mediated expression and release of the above mentioned proteins. The IC50 for inhibition of HIT serum/ heparin-induced platelet dependent HUVEC activation by SR121566A was approximately 10-20 nM. ADP, but not serotonin release, also appeared to be involved as apyrase and ATPgammaS blocked platelet-dependent, HIT serum/heparin-induced cell surface protein expression and cytokine release by HUVECs. Increased platelet adherence to HIT serum/heparin-activated HUVECs was inhibited by SR121566A and, to a lesser extent, by apyrase and ATPgammaS, showing that platelet activation and release was at the origin of the HIT serum/heparin-induced expression of these proteins by HUVECs. Thus, sera from patients with HIT induced the expression of adhesive and coagulation proteins and the release of cytokines by HUVECs through the activation of platelets which occurred in a GP IIb-IIIa-dependent manner, a process that could be selectively blocked by SR121566A.

Adenosine Diphosphate↗
PubMed1998

SR 121787, a new orally active fibrinogen receptor antagonist.

The aim of this study was to describe the pharmacological properties of SR 121787, a new antiaggregating drug which is metabolized in vivo into SR 121566, a potent non-peptide antagonist of Gp IIb/IIIa. In vitro, SR 121566 antagonized the binding of [125I]-fibrinogen (IC50 = 19.8+/-6.3 nM) and of [125I]-L-692,884, an RGD-containing peptide (IC50 = 291+/-96 nM) to activated human platelets. SR 121566 inhibited the aggregation of human platelets induced by ADP, collagen, thrombin, arachidonic acid and PAF at concentrations lower than 0.1 microM. Adhesion of human platelets to adhesive proteins was inhibited by SR 121566 (IC50 = 40.3+/-2.5 nM) only when Gp IIb/IIIa and fibrinogen were involved. No effect was found with regard to other adhesive proteins and/or other integrins. SR 121787 demonstrated a potent and sustained antiaggregating effect when administered intravenously to baboons at a dose 50 microg/kg, and eight hours after the administration of 100 microg/kg, ADP-induced aggregation was still strongly inhibited (more than 80%). A single oral administration of 2 mg/kg of SR 121787 produced a nearly complete inhibition of platelet aggregation for up to 8 h (ED50 at 8 h = 193+/-20 microg/kg), a significant residual antiaggregating activity being still observed 24h after the administration. When administered orally to rabbits, SR 121787 exhibited a potent antiaggregating (ED50 = 2.3+/-0.3 mg/kg) and antithrombotic activity in an arterio-venous shunt thrombosis model (ED50 = 10.4+/-0.8 mg/kg). After oral and IV administration, SR 121787 was well tolerated suggesting that SR 121787, the most potent and long lasting orally active Gp IIb/IIIa antagonist described to date, is a promising antithrombotic compound.

Administration, Oral↗
PubMed1998

The antiaggregating and antithrombotic activity of clopidogrel is potentiated by aspirin in several experimental models in the rabbit.

It is unknown whether the addition of aspirin might increase both the efficacy and the potency of clopidogrel, a potent and selective ADP blocker. For that purpose, the efficacy of clopidogrel (1-20 mg/kg, p.o.) administered orally to rabbits alone or in combination with aspirin (0.1-10 mg/kg, p.o.) was determined in several experimental models. A potent synergistic effect of the clopidogrel/aspirin association was demonstrated with regard to collagen-induced platelet aggregation measured ex vivo. Similarly, aspirin potentiated the antithrombotic activity of clopidogrel measured with regard to experimental thrombosis induced by a silk thread or on stents placed in an arteriovenous shunt, thrombus formation following electrical stimulation of the rabbit carotid artery and with regard to 111In-labeled platelet deposition on a stent implanted in an arteriovenous shunt or on the subendothelium following air drying injury of the rabbit carotid artery. A similar potentiating effect of aspirin was obtained with regard to myointimal proliferation (restenosis) in the femoral arteries of atherosclerotic rabbits which occurred as a consequence of stent placement. The clopidogrel/aspirin combination showed only additive-type effects on bleeding time prolongation induced by ear transection in the rabbit, therefore showing that combined inhibition of cyclooxygenase and ADP's effects provide a marked enhanced antithrombotic efficacy. Such a combination may provide substantial protection against platelet aggregation leading to thrombotic occlusion at sites of endothelial injuries and coronary artery stenosis in humans.

Animals↗
PubMed1997

New orally active non-peptide fibrinogen receptor (GpIIb-IIIa) antagonists: identification of ethyl 3-[N-[4-[4-[amino[(ethoxycarbonyl) imino]methyl]phenyl]-1,3-thiazol-2-yl]-N-[1-[(ethoxycarbonyl)methyl]pip erid -4-yl]amino]propionate (SR 121787) as a potent and long-acting antithrombotic agent.

The platelet fibrinogen receptor GpIIb-IIIa is currently considered a target of choice for drugs used in the prevention and treatment of thrombosis. Ethyl 3-[N-[4-[4-[amino[(ethoxycarbonyl)-imino] methyl]phenyl]-1,3-thiazol-2-yl]-N-[1-[(ethoxycarbonyl)methyl] piperid-4-yl] amino]propionate (6, SR 121787) is a new antiaggregating agent which generates in vivo the corresponding diacid 19d (SR 121566), non-peptide GpIIb-IIIa antagonist. In vitro, 19d inhibited ADP-induced aggregation of human and baboon platelets (IC50 = 46 +/- 11 and 54 +/- 6 nM, respectively), and on human platelets, 19d antagonized the binding of 125I-labeled fibrinogen (IC50 = 19.2 +/- 6.2 nM). Ex vivo, 8 h after an i.v. administration of 19d (100 micrograms/kg, i.v.) to baboons, ADP-induced aggregation was strongly inhibited (more than 90%). At 8 h, the ED50 value was 24 +/- 3.3 micrograms/kg), and even 24 h after the administration of a single dose of 100 micrograms/kg of 19d, platelet aggregation was still significantly inhibited (50 +/- 6% inhibition, P < 0.05). In the same species, the oral administration of 500 micrograms/kg of 6 produced a nearly complete inhibition of aggregation for up to 8 h (ED50 at 8 h was 193 +/- 20 micrograms/kg). After an oral dose of 2 mg/kg of 6, an antiaggregating effect was still observed at 24 h (44 +/- 12% inhibition, P < 0.05). 6 was well tolerated in animals, showing that, on the basis of these studies, it is a suitable candidate for development as an orally active antithrombotic agent.

Adenosine Diphosphate↗
PubMed1997

Heparin inhibits the binding of basic fibroblast growth factor to cultured human aortic smooth-muscle cells.

Basic fibroblast growth factor (bFGF) and its specific receptors have diverse roles on a variety of cell types, such as the induction of vascular smooth-muscle cell proliferation which contributes to restenosis after coronary balloon angioplasty. bFGF is also known to interact with heparan sulphate proteoglycans present on the cell surface or in the extracellular matrix. In this study, the binding of 125I-bFGF to human aortic smooth-muscle cells was investigated. 125I-bFGF binding to these cells was reversible and saturable. Scatchard analysis revealed the presence of two distinct binding sites: a high-affinity receptor (Kd=38+/-7 pM; 1480+/-220 sites/cell) and a low-affinity non-saturable binding site (Kd=8. 0+/-2.0 nM). Pretreatment of the cells with heparinase resulted in a large reduction of 125I-bFGF binding to its low-affinity receptors, suggesting that they are heparin-like molecules. The specificity of the low- and high-affinity binding sites for bFGF was determined with acidic FGF, platelet-derived growth factor-BB and epidermal growth factor, which did not compete for 125I-bFGF binding. Expression of FGF receptor isoforms analysed by reverse transcriptase-PCR revealed the presence of only the type-1 receptor. Binding to low-affinity binding sites was antagonized by heparin, suramin, protamine sulphate and platelet factor 4. Unexpectedly, these molecules also reduced the binding of 125I-bFGF to its high-affinity sites. Consistent with these results, heparin, suramin, protamine sulphate and platelet factor 4 inhibited bFGF-induced proliferation of human aortic smooth-muscle cells. Heparin abrogated bFGF-induced release of tissue-type plasminogen activator by these cells. These observations suggest that the interaction of bFGF with human aortic smooth-muscle cells is different from that described for other cells such as endothelial cells, in which heparin acts as a potentiating factor of the mitogenic activity of bFGF.

Aorta↗
PubMed1997

Intimal hyperplasia following vascular injury is not inhibited by an antisense thrombin receptor oligodeoxynucleotide.

Thrombin is a multifunctional serine protease with central functions in hemostasis, but demonstration of its role in the initiation and maintenance of cell proliferation which occurs following vascular injury is still lacking. To determine the role played by thrombin and its receptor in neointimal accumulation of smooth muscle cells in a rabbit carotid artery model, we have used an 18 mer antisense phosphorothioate oligonucleotide (ODN) directed against the translation initiation region of the human thrombin receptor gene. The antisense ODN inhibited in a dose-dependent manner thrombin- or thrombin receptor activating peptide-induced human aortic smooth muscle cell proliferation. The growth-inhibitory effect of thrombin receptor antisense ODN was preventable by an excess of sense oligomer and specific for thrombin. The suppression of growth was accompanied by a marked decrease of the level of thrombin receptor expression as evidenced by [125I]-thrombin binding to smooth muscle cells. Under the same experimental conditions, the corresponding sense ODN was inactive. The effect of the antisense ODN on intimal smooth muscle hyperplasia in rabbit carotid arteries subjected to endothelial injury was then investigated. The topical application of the antisense (500 microg/artery) but not the sense ODN dissolved in F127 pluronic gel around the injured artery resulted, 2 weeks after the application, in a dramatic reduction of the expression of the thrombin receptor mRNA and protein levels as determined by in situ hybridization and immunohistochemistry. However, intimal smooth muscle cell accumulation as estimated by an intimal to medial cross-sectional area ratio was reduced only by 2.7% (vs. 10.3% for the sense ODN), whereas r-hirudin (200 microg/kg/day, s.c.), a potent direct thrombin inhibitor significantly reduced the formation of neointima in denuded carotid arteries (35.4% inhibition, P = 0.03).

Amino Acid Sequence↗
PubMed1997

Characterization of P2x1 purinoreceptors on rat platelets: effect of clopidogrel.

This study aimed to determine the binding characteristics of [3H]alpha,beta-Me-ATP, a specific ligand of the P2x1 receptors to rat platelets, and to investigate the effect of clopidogrel, a thienopyridine compound which has been found to selectively inhibit ADP-induced platelet aggregation and adenylyl cyclase ex vivo. Binding of [3H]alpha,beta-Me-ATP to rat platelets was time-dependent and saturable. Scatchard analysis of the saturation binding data indicated that [3H]alpha,beta-Me-ATP bound to one population of specific binding sites with high affinity (KD = 23.6 +/- 1.6 nM; Bmax = 690 +/- 24 fmole/10[8]cells) (n=3). Unlabelled alpha,beta-Me-ATP as well as 2-MeS-ADP and ADP competitively inhibited the specific binding of [3H]alpha,beta-Me-ATP with IC50 values of 19.0 +/- 6.6, 103 +/- 20 and 1120 +/- 80 nM respectively (n=3). Other nucleotide analogues such as ATP, ATP-gammaS, UTP and GTP also antagonized [3H]alpha,beta-Me-ATP binding. When administered orally (10mg/kg, p.o.), clopidogrel inhibited ADP- or 2-MeS-ADP-induced platelet aggregation but did not affect the binding of [3H]alpha,beta-Me-ATP to rat platelets ex vivo. In vitro, alpha,beta-Me-ATP did not induce the aggregation or shape change of rat platelets and did not interfere with ADP-induced platelet aggregation.

Adenosine Triphosphate↗
PubMed1997

Effect of clopidogrel treatment on ADP-induced phosphorylations in rat platelets.

Phosphorylations induced by 2-MeS-ADP, a potent agonist of platelet ADP receptors, have been studied in rat platelets, and the effect of clopidogrel, a compound which inhibits platelet aggregation by selectively reducing the binding of ADP to its low affinity receptors on platelets, has been determined. 2-MeS-ADP induced platelet activation (shape change and aggregation) simultaneously with the phosphorylation of myosin light chain (P20) and plekstrin (P47). Phosphorylation of P20 and P47 was transient, a maximum being observed 10 s after addition of the agonist when shape change reached its maximum. P20 and P47 phosphorylations were not strongly affected by clopidogrel treatment. Following stimulation of platelets with 2-MeS-ADP, several proteins were phosphorylated at tyrosine residues. Clopidogrel treatment inhibited the increase in phosphorylation of P140, P100, P80/85, P66 and P55 concomitantly with the inhibition of platelet aggregation. However, clopidogrel did not interfere with the early phosphorylation of the P80/85 kD doublet which occurs at the time of the shape change. P80/85, identified by immunodetection as cortactin, could be involved in the reorganization of the cytoskeleton necessary for morphological changes. Thus, by using clopidogrel-treated rat platelets, we were able to determine some of the phosphorylations coupled either to clopidogrel-resistant high-affinity ADP receptors leading to shape change or to clopidogrel sensitive low-affinity ADP receptors coupled to the aggregation process.

Adenosine Diphosphate↗
PubMed1997

Antiplatelet and antithrombotic efficacy of SR 121787, a nonpeptide orally active GP IIb/IIIa antagonist, in rabbits: comparison with clopidogrel and aspirin.

Previous reports indicate that rabbits are not an appropriate species for testing glycoprotein (GP) IIb/IIIa antagonists because tested compounds caused only a weak inhibitory action on aggregation of rabbit platelets. SR 121566 is a novel low-molecular-weight antiplatelet agent with high affinity and specificity for the GP IIb/IIIa complex. In this study, SR 121566 exhibited a potent dose-dependent inhibition of adenosine diphosphate (ADP)-induced aggregation of rabbit platelets in vitro [median inhibitory concentration (IC50), 0.8 +/- 0.04 microM], whereas the GP IIb/IIIa antagonists SC 52012A and GR 144053F were devoid of antiplatelet activity. After oral administration of SR 121787, the prodrug of SR 121566, rabbit platelet aggregation ex vivo was inhibited in a dose-dependent manner [median effective dose (ED50) for ADP- arachidonic acid (AA)- and collagen-induced aggregation 2 h after gavage, 2.3 +/- 0.3, 6.1 +/- 0.9, and 2.5 +/- 0.4 mg/kg], a 58% inhibition of ADP-induced platelet aggregation being still observed 6 h after single oral treatment with 20 mg/kg. In an arteriovenous-shunt model, oral administration of SR 121787 resulted in a dose-dependent inhibition of thrombus growth (ED50, 10.4 +/- 0.8 mg/kg). Clopidogrel revealed a maximal inhibitory efficacy of 40% at 20 mg/kg p.o., and aspirin, at 100 mg/kg p.o., was without effect in this model. SR 121787 at oral doses < or = 10 mg/kg, did not cause an increase in blood loss after incision of the rabbit ear. In conclusion, SR 121787 is the first GP IIb/IIIa antagonist with oral antiplatelet and antithrombotic activity in rabbits.

Animals↗
PubMed1996

The mitogenic effect of H2O2 for vascular smooth muscle cells is mediated by an increase of the affinity of basic fibroblast growth factor for its receptor.

Increased generation of active oxygen species such as hydrogen peroxide (H202) may be important in vascular smooth muscle cell growth associated with atherosclerosis and restenosis. In this work, we showed that H202 was a potent mitogen for growth-arrested cultured human aortic smooth muscle cells (SMC), stimulating an increase in cell number at 10 nM to 100 microM concentration. This effect was inhibited in a dose-dependent manner by catalase, deferoxamine, dimethylthiourea or probucol showing that it was dependent on the oxidative activity of H202. H202-induced SMC proliferation was strongly and specifically inhibited by a neutralizing monoclonal antibody directed against basic fibroblast growth factor (bFGF) but was not due to increased expression of bFGF or the bFGF receptor-1 (FGFR-1) by SMC. H202 strongly increased the affinity of bFGF for its receptor-1 at the surface of the SMC, therefore showing that the mitogenic effect of H202 might occur through a direct effect on the bFGF receptor.

Antioxidants↗
PubMed1996

Purification and characterization of a novel protease from culture filtrates of a Streptomyces sp.

A fibrinolytic protease has been isolated from Streptomyces sp. culture filtrate by successive chromatography on Mono S and Sephadex G50. The purified protease had a molecular mass of 33 kDa and had an isoelectric point of 6.7. It showed a sharp pH optimum at 7.8 with maximal protease activity between 35 degrees C and 50 degrees C. Its amino acid composition and amino-terminal sequence (17 residues) were determined. The protein exhibited marked hydrolytic activity toward the substrates N-Succ-(Ala)2-Pro-Phe-pNA (K(m) = 0.77 mM, Vmax = 24.2 mumol mg-1 min-1) and N-Succ-(Ala)2-Pro-Leu-pNA (K(m) = 0.92 mM, Vmax = 7.7 mumol mg-1 min-1). It was totally inhibited by alpha 1-antitrypsin, D-Phe-Pro-Arg-chloromethylketone and sodium dodecyl sulfate but was insensitive to EDTA, dithiothreitol, phenylmethylsulfonyl fluoride, soybean trypsin inhibitor, pepstatin or elastatinal. In this respect, this protease differed in its physico-chemical and biochemical properties from other extracellular proteases previously found in bacteria and fungi. The results suggest that it has properties of chymotrypsin-like serine-type proteases.

Amino Acid Sequence↗