CATHEPSIN A - LIKE ACTIVITY IN THROMBIN - ACTIVATED HUMAN PLATELETS SUBSTRATE SPECIFICITY, pH DEPENDENCE, AND INHIBITORY PROFILE

TbmmbosisResearch,Vol.S6,No.5, pp.393-404,1997 Copyright01997 Ek.evierScienceLtd Printedin tie USA. Allrightsxwexved 0049-3848/97$17.00+ .00

Pergamon

PII S0049-3848(97)00084-4

CATHEPSIN A - LIKE ACTIVITY IN THIU)MBIN - ACTIVATED HUMAN PLATELETS SUBSTRATESPECIFICITY,pH DEPENDENCE,AND INHIBITORYPROFILE HalinaOstrowska Departmentof InstrumentalAnalysis, MedicalAcademy,Bialystok,Poland (Received2Januay 1997by EditorC. Cierniewski:revisadlaccepted14 April 1997)

Abstract CathepsinA-like enzymereleasedfrom human plateletsby thrombinhydrolyzedat the highest rate Cbz-Phe-Ala, Cbz-Phe-Met and Cbz-Phe-Leu, did not require activatorsand was inhibitedby DFP, DCI and mercurialcompounds(mersalylacid, PCMS, PCMB and HgC12).The optimumactivity of secreted enzyme was at pH 5.0-6.0. Cbz-Glu-Tyrwas also hydrolyzedat lower pH with optimum at pH 3.5. These enzymaticpropertiesare the same as those of cathepsinA solubilizedfrom whole plateletsand purifiedfrom other mammaliancells and tissues. High specific activity of secreted cathepsin A, and a broad pH range of activity may have a significancein extracellularproteolysisin local sites of ischemia.Large portion of cathepsinA-like activitywas not secretableby high concentrationof thrombin and was sedimented with platelet aggregates. No activity of lysosomal carboxypeptidasesB and prolylcarboxypeptidasewas detectablein supematantsand pelletsof thrombin-stimulatedplatelets. @1997 Elsevier Science Ud ----------------------------------

Key words: cathepsinA, thrombin-stimulatedhumanplatelets,enzymaticcharacteristics Abbreviations:Cbz, carbobenzoxy;Cbz-Glu-Tyr,N-Cbz-L-glutamyl-L-tyrosine,Cbz-Glu-Phe, N-Cbz-L-glutamyl-L-phenylalanine;Cbz-Phe-Ala, N-Cbz-L-phenylalanyl-alanine;Cbz-PheMetj N-Cbz-L-phenylakmyl-L-metionine; Cbz-Phe-Leu,N-Cbz-L-phenylalanyl-L-leucine;CbzPhe-Gly,N-Cbz-L-phenylalanyl-L-glycine;Cbz-Pro-Phe,N-Cbz-L-prolyl-L-phenylalanine;Bz, berrzoyl;Bz-Gly-Arg, N-Bz-L-glycin-L-arginineFA, N-(3-(2-furyl)-acryloyl;FA-Phe-Phe, NFA-L-phenylalanyl-L-phenylalanine; DFP, diisopropyl fluorophosphate; DCI, 3,4dichloroisocoumarin; EDTA, etylenediaminetetraaceticacid disodium salt; PCMS, pchloromercuriphenylsulphonicacid; PCMB, p-chloromercuribenzoicacid; TCA, trichloroacetic acid. Corresponding author: Dr Halina Ostrowska, Department of Instrumental Analysis, Medical Academy,Mickiewicza2, 15-230Bialystok,Poland,FAX (085)4249 07,teL(085)422021 393

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Mammalian cathepsin A (E.C.3.4.16.1)is described as lysosomalserine carboxypeptidasethat specifically cleaves C-terminal amino acid from Cbz-Glu-Tyrat pH 5.0, it does not require activatorsand is inhibitedby serineproteasesinhibitor(DFP) and inhibitorsreacting with a free SH group essential for its activity (PCMS, PCMB, HgC12)(l). The enzyme preferentially cleaves C-terminalamino acid from N-blockedhydrophobicdipeptideshaving phenylakmineat P2 position such as Z-Phe-Ala, Z-Phe-Leu, Z-Phe-Met, Z-Phe-Phe (l-5). Though at present the physiological and pathological significanceof cathepsin A remains unclear, in vitro the enzyme is able to hydrolyze natural bioactive peptides such as angiotensin I, bradykinin, endothelin, substance P, both at acidic pH (carboxypeptidaseactivity) and at neutrality (amidaseactivity) (1, 3, 6, 7). CathepsinA/lysosomalprotectiveprotein also plays a structural functionby forming a high molecular-weightcomplexeswith &galactosidase(EC 3.2.1.23)and neuraminidase(EC 3.2.1.18) essential for the stabilizationand activation of these enzymes in lysosomes of various cells (5, 8-9). Since lysosomal enzymes are secreted from the cells in different pathological circumstances, cathepsin A may also be involved in extracellular proteolysis.Blood plateletsare activatedin variousvasculardisorderssuch as atherosclerosisand thrombosis and they release many of the granule contents including acid hydrolyses (10-13). While most of these enzymeshave been extensivelystudied,little is known about cathepsin A released from activated platelets. Recently it was found that cathepsin A-like enzyme was the main acid carboxypeptidasefrom detergent solublized whole platelets (14-15). Platelets in responseto strongphysiologicalstimuliare knownto releaseonly their granulecontentswhereas membrane and cytosolic substancesdo not participatein release reaction (11-13). Therefore in the present study, acid carboxypeptidasereleased from the human platelets stimulatedby high concentration of thrombin has been characterized as a fhnction of its substrate specificities, optimum pH and inhibitory profile. These enzymaticpropertiesof lysosomalcathepsin A-like enzymewere comparedwith those of the enzymesolubilizedfrom whole plateletsand cathepsin A purifiedfrom other mammaliancells and tissues. MATERIALSAND METHODS Cbz-Glu-Tyr, Cbz-Glu-Phe, Cbz-Phe-Ala, Cbz-Phe-Leu, Cbz-Phe-Met, Bz-Gly-Phe, Bz-G1yArg, Cbz-Pro-Phe, FA-Phe-Phe; DFP, DCI, mersalyl acid, PCMS, PCMB, EDTA, pepstatin, leupeptin, calcium chloride, cystein, ninhydrin reagent solution, calcium ionophore A23187, ADP were from Sigma ChemicalCo (St Louis, MO, USA); dithiotreitoland Triton X-1OOwere from Serva Feinbiochemica(Heidelberg);bovinethrombinwas from BiomedLublin,Poland. Preparationof human platelets Blood obtainedfrom healthyhuman volunteerswas collectedin polystyrenetubes containing 1/6 volume of acid citrate dextrose (85 mM trisodium citrate, 64 mM citric acid and 111 mM glucose) and centrifuged at 100 x g for 15 min at room temperatureto obtain platelet rich. plasma (PRP). Plateletsprepared by centrifugingof PRP (1500 x g, 20 rein), were suspendedin phosphatebuffer consistingof 21mMNa2HP04, 117mM NaCl, 15mM citrate, 5mM dextrose, 5mM EDTA, pH 7.4 (platelet buffer), and they were washed three times with this buffer by repeating the centrifugationstep (250xg, 5min, 200C). The platelet suspension was free of erythrocytes,and the contaminationof leukocyteswas lessthen 1 cell per 104platelets.

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Thrombin-inducedreleasereaction Human platelets, finally resuspendedin the plateletbuffer (withoutEDTA) to concentrationof 12 x 108ceIIs/mL were used for activation, as a source of secreted cathepsin A for its biochemicalinvestigation. Thrombin-inducedrelease reaction was performed accordingto the method of Holmsen et al (16) with slight modification.To choose the concentration of the thrombin that induced maximum secretion of cathepsin A-like enzyme, the concentrated plateletswere incubatedat 370C in the absenceand presenceof thrombin(0.05, 0.5, 5.0 and 10 units/mL)for 5 and 15 min with continousstirring.The plateletrelease reactionwas stoppedby the additionof O.lmol/LEDTA (pH 7.4), the mixturescooledin ice, and centrifugedat 12000x g for 15 min at 4°C. The supernatantswere immediatelyused for enzymaticinvestigation.The remainedpellets were resuspendedto the startingvolumein the platelet buffer containing0.5’?/o Triton X-1OO,then left for 15 min at 4oC, and centrifugedat 12000 x g for 15min to obtain supematant@atelet lysate).The whole plateletsIysedwith 0.5’Yo Triton X-1OO(15 min at 4oC) were used to enabledeterminationof the extentof the enzymerelease. To test cathepsinA secretionunder more physiologicalconditions,the plateletsat concentration of 1.5 -2.0 x 108 cells/mLwere incubatedfor 5 minutesat 370C with thrombin (0.05, 0,2, 0,5 units/mL),ADP (1, 20vM) or calciumionophoreA23187(1-1OLM)as desribedabove. Assay of cathepsinA CathepsinA activity was assayed at pH 5.5 using its specificsubstrates:Cbz-Phe-Alaand CbzGlu-Tyr accordingto the method of Kawamuraet al (2) with slight modification.The reaction mixtures had a final volume of 500 IJLand consistedof 50pL of the supematantof thrombinstimulated platelets (total protein 20 or 40 pg), 5mmol/L substrate and 50 mmol/L sodium acetatebuffer containing100rmnoULNaCl, 500 mmol/Lsucroseand lmmol/L EDTA at pH 5.5 (“assaybuffer”)at pH 5.5. Buffer and substratewere warmedto 37°C for 1 min prior to addition of the enzyme extracts and incubationwas allowed to proceed at 370C for 30- 120 min. The reaction was stoppedby the additionof 500 pL of 10°/0TCA. Samplesprecipitatedat zero time as well as incubated without substrates were used as a control. After centrifigation of the precipitated proteins, 100 pL of aliquots diluted 1:5 (v/v) with 0.1 mol/L acetate buffer were used to measure the amount of amino acid (in triplicate)by the calorimetricninhydrin method (17). Enzyme activity was expressed as nmoles of C-terminal amino acids released from substrateper minute (nmoles/min).The hydrolysisrate of other N-blockeddipeptidesat pH 5.5 were examinedin the sameway. Cathepsin A activity was also measured spectrophotometrically(A330) with FA-Phe-Phe as a substrate,which was used ealier for determinationof carboxypeptidaseactivityof human platelet deamidase(7) and cathepsinA/protectiveprotein(18). The reactionmixtureshad a final volume of lmL and consistedof 0.1 mmol/L FA-Phe-Phe,10-20VLof supematants(20 or 40 pg total protein) and “assay buffer” ( pH 5.5). Enzyme activity was expressed as nmoles of substrate converted by enzyme per min. CathepsinA activity in supematantsof the platelets lysed with Triton X-100 was assayedin the sameway. Undertheseexperimentalconditionsthe liberationof C-terminal amino acid from substratewas proportionalto the time of incubation and platelet proteinconcentration. The effect of pH on the activity of thrombin-secretedcathepsin A was determined in a 0.06 mmol/L buffer consisting of citric acid, potassiumphosphateand boric acid (19 ) in the pH range from 3.0 to 8.0 for Cbz-Glu-Tyrand from pH 5.0 to 8.0 for Cbz-Phe-Ala hydrolysis.This substratewas not dissolvedat pH below 5.0.

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The influence of different activators (3mmol/L cysteine, 10 mrnol/L dithiotreitol and 1-10 mmol/L calcium chloride) and inhibitors(listed in table III) on carboxypeptidaseactivity was examined in the supernatantof thrombin-activatedplatelets (total protein 40~g) preincubated with suitablecompoundfor 15 min at 250C, and reactionswere startedby the additionof CbzPhe-Ala. Assay of other lysosomalcarboxypeptidases Lysosomal carboxypeptidaseB (EC 3.4.18.1)was assayed at pH 5.0 using Bz-Gly-Arg in the presence of activating mixture consistingof IOmmol/Ldithiotreitoland IOmmol/L EDTA (l). Prolylcarboxypeptidase(EC 3.4.16.2) was determined with Cbz-Pro-Phe in supernatant of stimulatedplateletspreviouslyheatedto 650Cfor 15min in orderto inactivatecathepsinA (19). Protein concentrationin platelet supernatantwas measured by the method of Lowry et al (20) with bovine serumalbuminas a standard. All experimentswere repeated at least three times with each point run in triplicate.The figures and tables show mean valuesof these experiments.

RESULTS CathepsinA activity in supernatantsand pelletsof the human plateletsstimulatedwith different concentrationof thrombinfor 15 minutesis shown in Fig.1. When platelets(12 x 108 cells/mL) were incubated with 0.05 units/mL of thrombin, low level of cathepsin A-like activity was presentin the supernatants,and almost all activityof this enzymewas observedin the platelet

r-l

L 0,05

0,5

5

10

Thrombin concentration, units/mL

FIG.1. CathepsinA-like activity in supernatantsand pellets of human plateletsstimulatedbv thrombin. Platelets(12 x 108cells~mL)were incubated~ith thrombinat ~ifferentconcentratio&(0.05, 0.5 5.0 and 10 units/mL) for 15 minutesat 37 oC as describedin MATERIALSAND METHODS. Enzyme activity was assayed using Cbz-Phe-Ala(5mM) at pH 5.5 in supernatants(-B-) and pellets (-0-) after 30 min incubationat 37 oC and was expressedas nmoles/min/108platelets. Resultsare mean valuesof experimentsdoneat leastin triplicate.

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pellets. Treatmentof plateletswith 0.5 units/mLof thrombinincreasedthe extent of the enzyme secretion. The maximum release was obtained using 5.0 units/mL of thrombin since higher concentrationof this inducer(10 units/mLand more) did not significantlyaffect the extent of the secretionand almosthalf of cathepsinA-likeactivitystillremainedin plateletpellets. Comparisonof cathepsinA-like activityin the supematantsof native platelets,plateletstreated with Triton X-100 (total activity) or activated by high concentration of thmmbin (secreted activity) is shown in Table I. No activity of cathepsin A in the supernatant solution before activation of the platelets was detectableusing both Cbz-Phe-Alaand FA-Phe-Phe suggesting that spontaneousactivationof the plateletsdid not occur during their preparation.Thrombin at concentration of 5.0 units/mL released about 50°/0of the total cathepsin A-like activity solubilizedfrom whole platelets.Howeverits specificactivity(amountof activityper mg of total protein) was about 6-times higher in supematant of thrombin-stimulatedplatelets than in supematant of detergent solubilized cells. The ninhydrin method for determination of the hydrolysis of various Cbz-blockeddipeptideswas suitable only for concentratedplatelets, but was too insensitivefor the plateletsat their physiologicalconcentration.Thereforethe combined supematantsof the plateletsat concentrationof 12x 108cells/mLstimulatedwith 5.0 units/mL TABLEI Comparisonof cathepsinA-like activity in supematantsof the native plateletsand the platelets stimulatedwith thrombinor treatedwith TritonX-lOO.* Enzyme activityagainst: Platelets : FA-Phe-Pheb Cbz-Phe-Ala’ units/108platelets units/mgprotein units/108platelets units/mgprotein

o

0

0

0

Triton X-1OO (0.5%)

4.3

8.7

1.31

3.57

Thrombin (5.0 u/mL)

2.0

48

0.69

19.2

Native Treatedwith:

*Platelets (12 x 108cells/mL)were incubated in the absence or presence of thrombin (5.0 units/mL) or they were treated with Triton X-1OO(0.50A)as described in MATERIALS AND METHODS. Cathepsin A activity was assayed in supematantsat pH 5.5 by ninhydrinmethod using Cbz-Phe-Ala (5mM) or spectrophotometricallyusing FA-Phe-Phe (O.lmM). Incubation was 30 min at 370C. One unit is definedas the enzymeactivitythat releases 1 nmole of alan[ne per min (a) or that convert 1 nmole of substrateper min (b). The data are mean values of three separateexperimentsperformedin triplicate.

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thrombin, that caused maximal secretion were used for examination of the biochemical propertiesof cathepsinA-like enzymein thesework. The hydrolysis of various N-blocked dipeptides at pH 5.5 in supernatant of the platelets stimulated with thrombin is shown in Table II. Rates of hydrolysiswere compared at pH 5.5 using Cbz-Glu-Tyr as the standard substrate of platelet cathepsinA (21). Only N- blocked hydrophobic dipeptides, specific for cathepsin A were hydrolyzed. Substrates having phenylakmine(Phe) at penultimateposition(P2) (i.e. Cbz-Phe-Ala,Cbz-Phe-Met,Cbz-Phe-Leu, Cbz-Phe-Gly)were hydrolyzedat higher rate than those with a hydrophilicamino acids in this position(i.e.Cbz-Glu-Tyr,Cbz-Glu-Phe,Cbz-Gly-Phe).Cbz-Phe-Alawas hydrolyzednearly 10fold faster than Cbz-Glu-Tyr. No hydrolysisof Bz-Gly-Arg,a specific substrate of lysosomal carboxypeptidaseB in the presence or absence of dithiotreitol (lOmM) and Cbz-Pro-Phe, a specific substrate of prolylcarboxypeptidasewere found suggestingthat these enzymes did not occur in human plateletlysosomal granules. The hydrolysisof Cbz-Phe-Alaand Cbz-Glu-Tyrby thrombin-secretedcathepsinA-like enzyme at differentpH values is presentedin Fig.2. Two peaks of the carboxypeptidaseactivity assayed with Cbz-Glu-Tyr were noted, one major at pH 5.0-5.5 and one at pH 3.5. Cbz-Phe-Ala washydrolyzedat the highest rate at pH 5.5-6.0.The same pH optimumwas found using FAPhe-Phe(not shown). TABLE II. Hydrolysisof variousN-blockeddipeptidesin the supernatantof thrombin-stimulatedhuman - plateletsat pH 515*

Substrate

Cbz-Glu-Tyr Cbz-Glu-Phe Cbz-Gly-Phe Cbz-Phe-Ala Cbz-Phe-Met Cbz-Phe-Leu Cbz-Phe-Gly Cbz-Pro-Phe Bz-Gly-Arg

Hydrolysisrate nmoles/min

Relativeactivity

0.16 0.1 0.0 1.76 1.26 0.5 0.27 o o

100 62 0 1135 813 316 172 0 0

‘?/0

*The reaction mixture (0.5 ml) containing substrate (5mM) and 0.05 ml of supernatant of thrombin-stimulatedplatelets ( total protein 40 ~g) in assay buffer at pH 5.5 was incubated for 60 min at 370 C. The data are mean valuesof three separateexperimentsperformedin triplicate. The value of 100% corresponds to hydrolysis rate of Cbz-Glu-Tyr as standard substrate of cathepsinA.

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,., (1 +

3

--—+—

,

4

5

I

6

-m

-–+-–---+--—

7

.:.

8

pH

FIG,2. Effect of pH on hydrolysisrate of Cbz-Phe-Alaand Cbz-Glu-Tyrby cathepsinA in supernatant of thrombin-stimulatedhuman platelets.Supernatant( total protein 20 pg ) was incubatedwith 5mM Cbz-Glu-Tyr(-=-) for 90 min and with 5 mM Cbz-Phe-Ala(-0-) for 30 min at 370C in a 0.06 mmol/L buffer consistingof citric acid, potassiumphosphateand boric acid. Results are mean value of experimentsdone three times. The hydrolysisof Cbz-Phe-Alaat lower pH values could not be shown sinceit was not dissolvedat pH below 5.0. As shown in table III, the hydrolysisrate of Cbz-Phe-Ala was decreasedin the presenceof DFP (70%) and DCI (51%) which indicatesthat the enzyme is serine carboxypeptidase.The strong inhibition of this activity was also observed in the presence of mercurial compounds such as mersalyl acid (92’%o),PCMS (720A), PCMB (620A)and HgC12 (74°ZJ)suggestingthat platelet carboxypeptidasehas a free SH group essential for its activity. Other inhibitors: pepstatin (aspartylproteases),leupeptin(cysteineproteases)and EDTA (metallo-proteases)did not affect the rate of hydrolysis of Cbz-Phe-Ala. The same inhibitory effects were also observed in hydrolysisof FA-Phe-Pheat pH 5.5 and Cbz-Glu-Tyr both at pH 3.5 and 5.0 (not shown).The following synthetic inhibitors were tested but did not affect the Cbz-Phe-Ala-hydrolyzing enzyme at acidic pH: TPCK (N-ctp-tosyI-L-phenyIakmine chloromethylketone),TLCK (N-atosyl-L-lysine chloromethylketone) and E-64 (trans-epoxysuccinyl-L-leucylamido-(4guanidino)butane)(data not shown).The hydrolysisof Cbz-Phe-Alaand Cbz-Glu-Tyrdid not increasein the presenceof activatorsof cysteineproteases:3 mmol/Lof cysteineand 10 mmol/L of dithiotreitol/lOmmol/LEDTA, and in the presence of calcium chloride (1-10 mM), an activatorof plateletcalpains (22) (not shown).

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TABLEIII. Effect of inhibitorson activityof cathepsinA-likeenzymein supernatantof thrombin-stimulated platelets*

Inhibitor

Without DFP DCI Mersalylacid PCMB PCMS Leupeptin EDTA Pepstatin HgC12

Final concentration (mol/L)

Hydrolysisrate nmoles/min

Residualactivity

o 10-3 10-5 10-4 10-3 10-3 10-4 10-3 10-3 10-5

1.73 0.52 0.85 0.13 0.66 0.49 1.77 1.73 1.77 0.44

100 30 49 7.6 38 28 102 100 102 26

%0

*Inhibitorswere preincubatedwith supernatantof thrombin-stimulatedplatelets (tOtalprotein +g) for 15 rein, and reactionswere startedby the additionof Cbz-Phe-Ala(final concentration 5mM),pH 5.5. Incubationwas 30 min at 370C.Resultsare mean values of experimentsdone at leastthree times.

The test cathepsinA secretionfrom the physiologicalconcentrationof the plateletsby thrombin, ADP or calcium ionophoreis shown in Table IV. CathepsinA was assayed using Cbz-Phe-Ala since this substratewas hydrolyzedby plateletenzymeat the highestrate. As seen in this table, cathepsin A-like enzyme was released from the platelets only by strong agonists such as thrombin and A23187. Incubation of the platelets with 0.2 units/mL of thrombin or 5pM of A23187 released about 35% of the total cathepsin A activity. Higher concentrationsof these inducers did not significantlyincrease secretionof this enzyme.The weak agonist ADP, which induces dens granule, and et-granule secretion had no effect. It is also noteworthy that the secretion of cathepsin A from the platelets was slow and required about 5 minutes of the activation(not shown).Further studieson the secretionof cathepsinA from the human platelets by differentinducersand in the presenceof the inhibitorsof the plateletrelease reaction are in progress. DISCUSSION In the present study it was shown that acid carboxypeptidasereleased from human platelet granules hydrolyzed only N-blocked hydrophobic dipeptides with strong preferences for substrateshaving phenylalanineat P2 positionsuch as Cbz-Phe-Ala,Cbz-Phe-Leu,Cbz-Phe-Met and FA-Phe-Phe.The enzymedid not requireactivators(dithiotreitol,calciumchloride)and was

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TABLE IV Releaseof cathepsinA-1ikeenzymefrom the physiologicalconcentrationof human platelets*

Inducer

Concentration

None

PercentcathepsinA activity 0.0

TritonX-1OO

0.5%

100

Thrombin

0.05 U/mL 0.2 U/mL 0.5 U/mL

10 31 35

Calciumionophore

lpM 5pM IOpM

13 37 41

ADP

5pM 20pM

5.3 7.0

*Washedhuman platelets(1.5 - 2.OX108cells/mL)were incubatedwith the inducerat 370C for 5 minuteswith continousstirring,as describedin MATERIALSAND METHODS. CathepsinA activitywas assayedin the supernatants(50pL)using Cbz-Phe-Ala, pH 5.5 after 120minutes of incubation at 370C. ‘A release of the enzyme activity was calculated in relation to the total enzymeactivityin washedplateletslysedwith TritonX-1OO.The resultsare mean values fkomat least three experiments. inhibited only by serine protease inhibitors (DFP, DCI) and inhibitors reacting with SH group(mersalylacid, PCMS, PCMB, HgC12).These findingsindicatethat acid carboxypeptidase releasedfrom the plateletgranuleshas many propertiesin commonwith cathepsinA-like activity solubilized from whole cells (14-15), cathepsin A purified fi-omother mammalian cells and tissues (2-5), carboxypeptidaseactivity of human platelet deamidase(7) and human lysosomal protective protein (5, 8). Since no activities of both lysosomal carboxypeptidase B and prolylcarboxypeptidasewere found, it seems likely that only cathepsinA-like enzyme may be responsiblefor hydrolysisof N-blockedhydrophobicdipeptidesin human plateletsat acidic PH. Besides, the activator and inhibitory experiments indicate that these substrates were not hydrolyzedby other plateletproteases(22-24).It is in agreementwith previousreports in which cathepsinA had been shownto be the only acidiccarboxypeptidaseable to cleave Z-Phe-Ala,ZPhe-Leu, Z-Phe-Phe and Cbz-Glu-Tyrin crude extractsof human placentaand fibroblastssince these substrateswere not hydrolyzedin galactosialidosiscells in which cathepsinA is genetically deficient,but all the otherproteasesare present.(5, 18). Human platelets stimulated with calcium ionophore release a carboxypeptidasenamed desLeulO-angiotensinI-generating activity, which is susceptibleto inhibition by mersalyl acid, leupeptinand iodoacetamide(25). AlthoughcathepsinA-likeenzymereleasedfrom the activated platelets as well as the porcine kidney cathepsinA that convertsangiotensinI to angiotensinII

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(6) were completely inhibited by mersalyl acid, they were not inhibited by leupeptin and

iodoacetamide. Therefore ftier work should be undertaken to find out if cathepsin A is responsiblefor des-Leu10-angiotensinI-generatingactivityin humanplatelets. As mammalian cathepsin A, the thrombin-secretedcarboxypeptidasehydrolyzed its specific substratesat optimumpH 5.0-6.0. Cbz-Glu-Tyrwas also hydrolyzedat about 60°A at pH 3.5. The same pH optima for this substratehad been reported previouslyfor cathepsin A from pig platelet (14), pig kidney (4), and rat liver (3). The acid pH optimum suggests that the platelet cathepsin A has a lysosomal localization. In addition, cathepsin A secretion required high concentrationof thrombin, it was slow and incomplete(about 50°/0)- findings which are also consistentwith the platelet lysosomesas a sourceof secretedenzymes(11-13).The observation that large portion of cathepsinA-like activitywas unsecretableby thrombinand sedimentedwith platelet pellets suggests that differentpools of this activity may be present in human platelets. Blood platelets activated in response to strong physiologicalstimuli do not release membrane and cytosol components(11-12).It is well-knownthat cathepsinA exists in a free form as dimer (100 kDa) and in a membrane-bound high-molecular-mass(>660kDa) complexes with ~galactosidaseand neuraminidasein lysosomesof variouscells (5, 8, 9). Sincehuman plateletsdo contain ~-galactosidase(16), it is likely that such complexis also formed in lysosomesof these cells. On the other hand, the resultspresentedhere indicatethat plateletcathepsinA-like activity closely resembles that of chymotrypsin-likeactivity of the proteasome, a cytosolic high molecular-weightmulticatalyticproteinase complex (26-27) including preferential cleavage of the peptide bond after a hydrophobicamino acids in substratesboth at neutral and acid pH, and strong inhibition by 3,4-dichloroisocumarin(DCI) and mercurial compounds. Recently it has been found that the lactacystin,the most specific proteasomeinhibitor (28) strongly inhibited human platelet cathepsin A-like activity against Cbz-Phe-Ala and FA-Phe-Phe (29). These findings,and the fact that the lysosomalenzymesand the plateletgranulecontentscan leak into the cytosol during activation of the lysosomes(30) or during platelet storage (13) suggest that cathepsinA may be also involvedin the chymotrypsin-likeactivityof the proteasomein human platelets. On the basis of these results it appearsthat cathepsinA-like enzyme is the major (probablythe only) acid carboxypeptidase released from the human platelets by thrombin. The strong preferencesof the platelet enzyme for substrateshaving aromaticand large hydrophobicamino acids at carboxyterminalends may have significancein hydrolysisof variousbiologicallyactive peptidesas it was reportedfor mammaliancathepsinA (1, 6) and human platelet deamidase(7). The high specificactivityof cathepsinA-likeenzymeafter its secretionfrom the platelets,and its activity in a broad pH range indicatethat this enzymemay also act extracellularyunder various pathological conditions, such as the local sites of inflammation,thrombosis, atherosclerosis, ischemiaor necrosis,where pH may be lowereddue to lactateaccumulation.It is noteworthythat the plasma inhibitorsdid not affect pig platelet cathepsinA-like activity against low molecular weight substrates (14). The hydrolysisrate of the Cbz-Phe-Alain human platelet rich plasma (PRP) previously treated with Triton X-1OOamounted to 5.Onmoles/min/108cells,and was similar to that in detergent-solubilizedplatelets (Ostrowska H., unpublished data). The observationthat high level of the cathepsinA-like activity was unsecretableby thrombin may have a significancein clearing of plateletthrombiin vivo. Howeverto clarifj a possible role of both secretableand unsecretableforms of cathepsinA-like activityin physiologyand pathology of plateletsfirther work will be undertaken.

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