- Email: [email protected]
Cryobiology of platelets
(~llI'OBI OLOGY
Vol. 6, No. 3, 196,9
CRYOBIOIA)GY I. A n ~ i , m p e l ) t i ( l a s e
OF
I LA I I_,I,E'I S
A c t i v i t y its a 3 I e a s u r e of Platelets in Vitro*
of Intactness
1 )()N :'il]I.) (; !~I:,IFV, D ( ) I l l S .B [-I(K)I,,I:.I,, ' " - " ' t ANI) SIIII~LEY 3IA(,KI,,5. t d)cporttn('nl oJ" ])alhulogll , :llar(l~C/tc School of .'lfedicine, Inc., .ll ilwoul:c,~:, lliae'~,,n.sit~ 53:?33
4-nmti,t~xy-2-1mpl~t, byl't~,~icte i l('l !w tlu? n m i n o pel)tidases ,K platelcts. We ]lave inv,~..qignte(l the effect,s of fhe following \'ari.~l)lo.~: 1) 1)II, q) motnrity of }mfl'er, 3) t y l w ,,f tml'fer, t) l y p e of ~ubsi,rale, 5) slll)s~.t'at,: e~)twel~Ir:~ti~)~i, 6) stop regictio~ witl~ lriehlor:l,'elie acid (T('.\), 7) interfereiwe l~y red lfloo~t ...ell.-, A) interfl,rel~('e by wliite blood e,,lis, 9) l~l:tieh:t (e~lzynw) c(.m. eentratim~.
()t~' st,~l~ties of the effect.- of freezillg, storage. ;it low i e ~ p e r a t u r e s , :,~(t ~,,.,~ .... ;",,' ,,,._ t~;" .. subliination in rm'im on the integrity of is,~l.tte(t blood platelets were impe(le~] gre'itly I,y t,lw al~seuee of :t ilua~t.itntive lest, for ~,wa'~uring in iff6"o ihe ~umhers of intavt p!atelels. ('lvt ret,r'lei,i~,n 'is a fir.~t :tl)proxilnafion of i)~tegrity was used i~ (r,n' e~rly inves(,igations. The sul>jecLive gr:t(ling vf the extent, of elof retractions, even trader stt, ndard t.est con(liticms, 4 alut th,, relatively l.~rge range i~ t.lm mtmbers ,d lflatelet,s for each
[~'[ATEI'~[A],S AND
),[12TII~)I}S
A frc-~]l pl,'ilelet eolc'r,-ed in 500 Inl of 0.25 .st 1)h,,.,q*ll:ih; I,uffer (t,]l 7.0): ('eJ~t,rifuge~t at, 8000 rlml fiw 2,1} rain, tlw Slll~('l'lt:it{llll. I'C,11IOVL:'(], ltll(I tlw i)hdeleI~ disl-,er.~e~l J~ sufth'ie1~l, phosl)hate tmffc, r or olber dillu,uis to g.ive tile fin:t] eolment r'di(),t (t(:-qle
w "l "~ i
,.qh.rp re,:elf on. i E t h y l :~('etat e 7 E.rtraclcd ¢'o~qffed split pro&~cl
The absortm,my of tlw ext.raeLed coupled spli~ product was rea(t by mea~s of a sp.eeLrophofomelm'. ]~ ],:s.'ULT8
Rc¢~.'tion. ]"cr our initial studies, 2 ml of washed platelets, 2 )< 1,0~ per rnm ~, were added to 2 ml of a. ]'eaeI.iol~ mixl, urc col~sisting of 0.8, mg of 1-ieucyl-fl-n:,i,ht,},ylamide HCI c.," 1-1eucyl 4194
CItYOBI()IX)(IY OF P L A T E I , E T S .
r,:el.hoxy-2-natflit, hylamide H('I in I nil of 0.25 .~r potassium phosl)hate buffer, pH' 6.5, 1)his l I;fl of pl~.ysi: exlraetod with 10 nil (if ethyl acetate. Tile :i,1)s(n'bam.y of the colored l)rofiucl in ei.hyl lt¢'Ct,:ttt' w : t s deler!lliliOll fit, 540 m#< The b l a l l k I'~U" zernill~ t i l e SlWetrol)hotoniete.r c~n~siste(l of:
S UBS'r R,,'Vl ~7 I -tC'~cvt
0-~--
nl>orli:tlicv t,t' r!)e n:ei,hoxy deriv:itive ~A;is iillOal" wiili rO~l'~(X'l, to tinie; LIuIL of 1-]euexq-
ffl
llllf] 3)
"File
LJ-iiut)}liitylailii~lo I I('l was lied (Fi~. 1). Becnuse (if" i{rt?:ti'ily :!till / r e a r e r lwodlict.ion of slJlii, !)r(ul:l(-{, [ i l r l i i e r slu, lies w e r e earrie~l out. with 1-h,u<..vl..l-lnoi ll~tx)-2-11ntJll( hylgtn-ih]o Il( :1 as sut)c[ 1':I t O.
7sll. t ' s i i l X ilie t.<,ln] sysl,-~ri ns
libovo :l!il{ :i lW,:!elj(ll/ lii'l(, <>I' 1,~ lllill, the. el'feels of ().25 M ]t~q.il.'-,'.itil~t t)[l<>q)ll:i.le ].~tlfl'or lti t)ll 6.(), 6.25,
-2 -,~ap:',hy h:,';1-de. ' t~C !
]. -l¢,i~:}| - ~i-,h.4iilbyl,l,-,a0.,:
--0
IIC}
E 0 0.30 ,-',r >-
2) T C . \ ,
~ rl-:,~e:i.,.~>~
0.40
ethyl
]) lilt' r('lJ(°.li,)li !alixluro, fl(:('~{ 11t (• .
195
1
1
0
0.20
n-" O cO
0.10
o2
1
0.00/-" 0
~ix(.i~
_
~
, I0
5
TIME
, .... 15
OF I N C U B A T I O N
l 20
(MINUTES)
FIe:. 1. Tim lime course :if ehange:: in M)sorlt.
ILS(), 7.{1, 7.25, ,)r 7.50 were (h,lerniilie~t. Tile
an('v usilsg [ - l e u c y l - . l - m o l , h o x y - 2 - i l l t l ) h l . h y t a l i i i d e , , ]ICi (Jl" l-leticyl-fl-naphih3"l,+unide, l l C l a8 s u t >
~l't':t!0.'-{ :ttli, ltlliI +ll" ,',pill l)i'u,Jucl l~II <,f t.;.5()
s(raie. The, complele re,act.ion rliixtilrt: co/itaine.d w:t+slled plalele.ts SUSl)en~led ilt 2 nil f If lfllysioh@cal sa.lirm, 2 Y, l() ~ per II1111;1; 0,~ Ill!f,(if silbsirill.o ill l
w a s f(~ttml :tt ~t
.l/ol,.,.ri/jt qf h,(/i?.r. 'l'he et]'eets (Jl' l'Oai31iOlt I':I(('S ,,l" ().t25, ().2h(J, ().'{7.~, ().5()(1, 0.d25, o," ().750 iii(,b:r c(tiivoiVr:ttiOli.~ o1' lwJl.nssitiiit t)jlOSlJll:ile t)llt~l't'-I", l~ll +i.5, w e r e ~h?ttq'iliilletl..\ i-!l(ll:lr ('o111,ott{ t'~.lI it JJl ill '< l)._o '~- ~:tVC Ill("
~qJtiilillili
mined al 5-t0 niv.
ro:lct,ioil
,Sub,~Lrate corwerdralion.
1'~i I p.
tI'm',' t< ~,gl/,. [:silig tile fnst t)iue t i-.',t)lit, l)ro/hi0L c,~:~ifl('.'~ fl-,.~li: till, tesl sy>.4eiil al)ove r(>it{:i(,(t :it. l~If ii.,5, il: 0.25 -~i ])otnssitnn pht)slfl~vt(;" luiffer, tilt:, :l]).-,~;rl)li~Jli slX'('{l'tllll betweel~ 450 ::nd 600 Inp wa < (]eli,rlttitio(t. 'l'lw r('a(]illg [
itll Of 0.D M p~das.Sillna l)ho.~pll,'tti; b u f f e r , p l [ 6.5; I m] (if p h y s i o l o g i e a l ' s n l i i i a , T i m ' re;te.tion ,n;ixttlre was n : M n l a i u e d at, 3 7 ° C . A l i k o r l m u e y wns dote, r-
:,vii.-: ol)t,:till(,(t
af, :l w n v o
[eragth of
5-I() m#.
:l'ypc of b~(t]".:r. In ,..})is tesl. ~ff t,}W effects of ,lifferel~t. 1)tlff(,rs, {he nunabors of 1)]afe!ets us(,+t was re(iur'exl to 1()~ per l l l l l l r'~. T I l e rea,ction i.Jn-ie was 30 thin. "J']l,' i)lfl]'0rs user] u o r e : 0.25 M pot:l,,,. Sitlhi t)tanSlflutte, 0.25 ~r Tris-nut]ee,,to> 0.25 5I
eitrale, and 0.25 M l~host-,h:it(.,-eigrate. The pI-[s ~tsod we,re 6.25, ti.50, and tj.75. "['tie :l.t-l)Olllil~ Of split 1)roduct~ pro(luted were r~mximum ;it, pll, 6.50 for potns.-iun~ pllosplmte and 1)hosptuttec..itr
'l'he depen~lence of tim rate of re:~etim: on the emmentral, ion of substrato, t.teucyl-4-1netlioxy-2-tmphthylanfidv, wus studied (Fig. 2), Subst, rat,e COlWelltrilLion wits vn,ricd fl't)iti ().5 X 1.0-a 5l kl').|6 ntg per ml) io -
2.0 X. t0 -'~ ~r
[
i
0.):65
li:g per ml). ;l'heso dnta ilMi.,
care t.hat, at. :lit initial eoneolit.raiioll O[ fl.D X 1()-~ M (0.10 nN per .~nl) t,he reaot-~orl became linfit, ing with respect to SII}.)S(;Fltl(. ~. (Ifl[.10r ,+{0 Ji'iill, There was evi{lenee nlso thiit, higher (-Oll,(~orii.l-l-i, fions of ,~ubstl~d;e interfered wilh tile l.esi,'sygten'a. A eoncerlLrlttioll of silL)strut0, ill' 0,0 ;K 10 "~ N (0.19 lng t w r nil) appe,tle]' ' " c ]~O.S.'t}'or o l l r piiYposo,% TCA and fast t)i,m 13. The, adding of ,'FCA to stop tim reaction prior t0 llae additkm of fiisl, blue ti resulted in lower v~lluos for opth,al dcn,,dt.y when comp/u'ocl wii.h (hose ob{,ailied witch 'J'(:A was added :filer the. t'asl; hlue B, I t was forrod also l:hl:tB a eorieetlfc, rr~l, ioti
produet.
] 96
GI{ E II:F, ]:11100KEI{, AN 1) MACK]:;Y
0.80 ...D
0,70 ,#
0.60 /
1.0 >.-
/
0.40
o co CD
0.30
/
/
•
/
/
/
///
CD rl-
/
.,~
/
.--,
/
°//
0.50
z<
Ill ,." I~J
.
E o,q,-
/ /
.."
,
."
,"
I
//¢5-;> SUBS1 RAI [ 60NCL N [ RAI I0.'I
0------0
020 ........Ill
0.10
0 .0 0
} I
0
I
I0
[
20
'
II
0.5~10 "3 ~0.15-;~1 ;roll 0.6×I0-3M
{) ~'~"~u",';'
0 75xIO'3M
'I} 24mq'ml)
E)- ..... --42]
1.0~10-3M
X-----X
"1.5~IO'3tJ (0.4St;,C, 'rn~l
+'---+
2 9~10"3M (O.b5"n~/I~t)
]
30
1
40
~,0 32'nq,mI'~
-
,
[
50
TIME OF INCUBATION (MINUTES)
Fie. 2. The effeH:s of coxicentratiolis of l-le.ucyl-4-met, h(~xy-2-1mlflttlLvlanlide.]l('l ,,I: the t i m e course of c h a n g e s in a,b s o r b a u c v . T h e c( replete, r e a c t i u n m i x / u r e c,~n(.ui~w(I wasl:e(t
t)latelets ml-~pended in I ml of physiological saline, 10 Gper ram:"; wu-iu,ls o,,!,ccl~tt'~ti.l~,-, ,,f substrale ifl t nfi of 0.5 ~i pobassilma phosphate buffer, 1)II (;.5. The react, toil wa,<~maimailmd ,'it. ',:lT°C anti absorlm~ey ream tit 540 ln~. The c,mcentrat iuv.s of subst rate are bhowll eli t.hc graph.
[nlerfcre~cc by red blood cells and while blood cells. Coneel~tn~tions of red blood cells from 2 X 1()a per m m a to tO 4 per nmP (lid not produce suiEcient split, product of ,~ubstrate to be detected by the test above. Small 'tmounts of split i}roduct were defected when white blood {:ells in colmentrations of 4 X 103 per nmP to 104 per m m ~ were tet~ted. The minhnum number of white blood cells necessary ~o produce a reaction exceeded by a, factor oI".J00 the lmmber in our platelet preparatiol~ and was not, therefore, considered to be ;m iuterferiug entity of significance. Plalelet (e~zymc) conce~b'aEon. A test system using the p,n'ameters above was used to determine the effects of different concentrations of platelets (enzyme.) on the production and visualizatioa of sp]i~ producL Washed platelets suspended in sufticienl, amounts of 0.25 -~ phosphate buffer, plI 6.5, to make a. final volume of 1 mI were added to I ml of a reaction mixture of 0.38 mg of t-leueyl-4-methoxy-2-naph~hylamide ]-ICI
in 0.25 ~[ phoslJmtc buffer, ]~II 6.5; t.lm fin'al concentration of .~ubsh'ate :::~, therefore,, 0.19 mg per m]. (N.t{. In lifts s t u d y ;vhell the conccitt,rat, ion oF platelets was 3.1 X 10 '~ p(:r mm :~, the alitollnt; of substrate was iI~cre<<~sedby 100 :,<,.) The re'te(,ion mixture wits placed in a 37°C' water bad~ for 30 mil~. The system was ' @ t a r e d during incubation. At the end of 30 rain, 3 mg of fl;tsfo b h m B , dissolved in 1 ml of distilled water, were added to the above, and the combination of dye with the splil product, methoxy-2-naphthylamine, was allowed to proceed rot i mi~u One milliliter of trMfloracetie acid was added to stop the re'lotion of enzymes with substr'~te. "Fen milliliters of ethyl a c e t a t e were added to the above, and following vigorous agitation for several mill the mixture was centrifuged at 2000 rpm for 5 rain; t,he fast blue B-split product complex was present in the ethyl acetate layer. The extracted complex was read at 540 mlx. A linear relationship was found between ab-
(.,l,'i OBIOI~CGS. OF . ~_,..,~, ,,.~_,,,.. s~n'l)mwy and llul,tt)ers of 1)latelet.s.; a doubling of llatelc(,~' ~ ' ~m~nbers resulted in n doubling of nbs.orbal,:y (Fig. 3). Clot retraction t,s. aminopeptidasc activity. '['he l}roeeclures used for clot, retraction were those dew'lolwd bV ]['trtmcmu "m{l ( o n l e y . 4 Tlie degrees of r('lra('ti(m were })ased on 1,'igm:e [ ()f their putflie:ltion" neg'd, ive, the tube on the righl,; 1-+-, i.he tul,e see(m(l frorn l,lie rig;tit,; 2-1-, the t,ul)e thir(l fro~ll the rigtit; 3-t-, lhe tube Lhir(l front the left; -t q-, tim first or second t,ul)es frol]t tlle M't. The retatioilsttips botweell degrees of clot ret.raetiol~, ml1,.~t)ers of phttelets per cubic millililc.ter, atlJ(t l('vt'lq of :u~iiln~pel)tidase activities (absor}):tnvy at. 5t(1 m#) "tre. shown in Tal)te 1. Wlmreas t lic nil'iil!)ors (it" 1)]ttt.elels for ettch deg,ree of (',l,~t ri't,r'lctjoti vn fled froni 210,000 for I. + , 260,000 for off_,_ 1,000,000 for 3 + 'rod ,0,9100,000 t'()r 4 + retraction, :tntinol)el)~,id:t.~e act, ivit, ies shower [ :t raiigt, of a l)sorl)alic.y values eorresl)ondii~<,',, to l,he liilli-ibt~l'.,~ of tflatelets ln'esent. .t minop~'pt[d<.~.,~'e actit:'t:/ as a me(t,surc, of pla, leh>,t dama, qc or drstr~u.'tio~. To dete)'inille if the test (ies('rit.)ed pi'evi~usly coithl be used (:o (;vahiate ill(; dl:greo of lfl'lt.ehq, de.%ruct, ion, t)rol)arat, ioris nf plateh:l~ were disrupted by 1) osmotic shook and 2) soiiific..-&iol~. In the first, inst, P~lico, 1.0~ l)latelet, s per n ] l l l 3 were suspelided in 1 inl of di.-:tilled water or in t Inl of differenTo molaril.ies of N a ( ' l for 30 lnin; in the seool~d t-ml 1)rep aratiolis of phitelets simihu' to l.he above were exposed to ult, t;asonie os(tillatioi~s for retrying 1)eriods of time. I)uring sonifical, ion d~.. tubes co~ttaini~g t l , j SUSlmnsio~ts of platelets were submerged in a bath of melting ice. Following exposure to the colidit, ions above the suspensions were eelltrifuged at, 8000 rpm for 30 mh]. In bot, h instances the supernattu~ts were carefully removed and saved. T h e platelet, b u t t o n s of the os,notie stress experiments were resuspended in sufficient, amourlts of 0.25 a~ phosphate buffe,', pH 6.5, to obtain the original volume and tlm activities of aminopep.~idascs measured. T h e s u p e r n a t a n t s were diluted with 1 ml of 0.5 5, phosphate buffer, p H 6.5, prior to the determination of a e t M t i e s ; the r a b i e s obtained were corrected for dilution. A q u a n t i t a t i v e reciprocal relationship ,between the activities of the p l a t d e t s and the a c t M t i e s of the s u p e n m t a n t s for each hypotonie contend tration o f Na:CI and for distilled water was oh•
x.
1~
1.20
l
t( "
-
/
1.00 E
o 0.80 u') >-. (..) Z <:I:
0.60
or"
0
O0 £13 <1:
0.40 0.20 _
QO0
l
..
l
~
I
I0
20
l
I
....i
30
PLATELETS x 105/ram Fro. 3. The effect of platelet coltcem, rat.ion on absorbancy. The complei.e rcactAon mixhn'e cont,aiaed washed ph~telets suspended in 1 ml of 0.25 M potassium phosphate buffer, pH 6.5; 0.38 mg of 1 - leucyl -4 - met.hoxy - 2- n a p h t h y h u n i d e : I IrCl. The react.ion was m a i n t a i n e d al, 37°C for 30 min aim reltd at 5-I0 nm. The concenlr~tions of platelets are noted on t:he graph. A(, l)latelet, concentrations of 3.1 X 10 ~ per mm a, the amount of s u b s t r a t e was increased t,o 0,76 rag. served (Fig. 4). Platelets suspended in distilled water ( m a x i m u m osmot, ic shock) lost all aminopeptidase tmt, ivit,y and the total a c t i v i t y was found in the s u p e r n a t a n t . Platelets suspended in physiological saline (minilnum osmotic shock) lost little activiW and no measurable aminopeptidase activiW could be found in t,h(~ s u p e r n a t a n t . T h e nminopeptid:~se acfdvii;ies for the h y p o t o n i e sohd, ions of N a C [ were" pai'titioned between 1)latelets a n d sut)e:'natan t."in a qua n ti ta give ma nnet. 1
TABLE CORRELATION
~2/UDIllS
OF
. . . . ~'" '
Pr, aTELm'S, I ) E G a E E Or," Ot, oT I{ETRACT I E S , XND 2kMINOPEPTIDASE A C T I V I T y Clot Retraction*
0
1+ 2+ 3+ 4+
I
No. of Platelets per Cubic Millimeter (Range)
Aminopeptidase Activi ty, Absorbancy at 540 m,, (Range).
8.6 .o. 2 ,t.6 8.0 2
0.025-0.058 0.061-0,140 0, ! 4 5 - 0 . 3 0 0 0.325-:0: 580 0._620-1.40
X X X X X
104-2.0 I 0 -s4 . 3 " 10~-7.2 105-1.8 106-4.1
× X X X X
l05 l0 s 10 ~ l0 G l0 G
* Adt~pted from the, s t u d y of_ I~l~rtma, n, and Conley."
1 98
(.; II E 1FF, B P,O()KE R, AN 1) 5Ie~,CI,:],~Y
050
• 4:I
:~ 0 4 0
\
E >_ o
LETS
\o
/<
""
,,
....
050
z
<
cn
/,,.
020
0
o ,,
/
0 I0
O, O0
potential and ~,.s used successfully for the eolorb met,tic estiTnnt,ion of :m~iJlopel)tidases il~ tissue homogenates, seruin, and urilie, a .Ill the adopt.toll of t,his chromogenic substrate for lfistochen~ic:d meth-dology it, was fotm(l tlmt althoitgh ('nzylnic. sites ('o,ihl be visualized ill t,issu(, set,tit,as, the slow r:tte of ('ot,'plhL~ (ff the ~ty(', let.mz()/,ize(t (tiorthoanisi(lille (fasL 1)lue l~), ,vit,]l file split 1,ro~luet, ~-Ilat)ht.hyh~mine, result~_,d ill (lifhlsioli artifacts. To milffnfize these : t r t i [ a e t s , :t new sulostrate ~ itll a fasl er Cal)iure reaction, l-leuc:yl... -l-methoxy-2-nal)hthyhtn~i(le 11(1, x~:ls synitiesize(l, s ~l'lle sl>lit, l)l'o(llwt ~)f this SllbSlm[!,, 4metl l(2xy-,,-lmj)tllllylnlnin(,, ') was fomld t~, ('otlt)le with tlte letr:tzotized (linrt, h()atlisi(line ,14) t,il~es ' faster than th(; uil.,qtbst.it~lte(] tllllill,:. Ollr sl.tt(iies ill(tie:ire thai, tliis new :,ubstrat(,' ('all be used ,,tlso for the (;ol()rinlet.ri<', (leterlu hint hJl~ of the a('{ ivities of the anii)~ol)ei)tid't.'-e~ ii) -~()iutim,. "I'll('. lfi,,zlmr levels (,f aclivil.y we l'(nitl(l witl~ l-tmie.yl--lnleiil{~xy,2-1~ll)hl,ltyl,ai~lide ll('l (lo lint illdi,~'ate lm('('ssqril 3" lhat, it is a .-~lp('ri()r sul~sir'~te fc)r tim ailiiii+)l)ept, idases t Jill r:tlllor lhnt, the :q)tit 1)roduet,
UPERNATANT
'
o,o
I
..
02
i
I
o.4
0.6
..,'-,7-r "° os
I
% NoCI (W/v) ] ' q c . . L T h e eft'eel, of ()shier, i t sh()ck on the n,nlii~ol)Olil, itlase a c t i v i t i e s of l)ropltrill.ions ~lf ilit,n,et; p l M e l e l s n,it(t SltlJerllttt:lnts, The filial (,olieolil.rll{.iOli ~)f tj]:il.(,lOls was .5 X ] 0 ' per innl'L T h e re't(.lioit I n i x l u r o arid t.hc e(,ll{titioils of rolicLiolt were the same ,'ts tlmso of F'iguro '3.
]:({,stilts simil'ir i,o llie ~bove were-foull~l ,.vhe~ l)]aLelet,~ wove de,%royed or dam'l/ed l)y expos~ure LO StlliersolliO oscillations.
'l'he values of ;lii-lillo-
l)epti(hi~e activities in lihitelet,s rum ill StlpOrll,'.itatitS were a ftit~etioll of tinlo of ('X[IOStll'O {-O Stll)OrSOIl it; VvILVOS. J)lscVssIoN
Several reviews of the :~minotml~t,i(htses serve l:o emphasiz(: lhe eornplexil ies of enzylnes belonging 1.o this g r o u p . : ' , n-n J-ii ge~el'al, these enzymes •wt. only on subst.rates wl~ich possess m~e or more free lenni~ml polar R r o u l ) s , s u c h :is c e - a m h i o , I n addii,ion stone of t.hese enzymes require metal ions for act.iwtt, ion or for nmximum aetivit,y. 1t~ amy be noted, however, t h a i , eerUdn of the a~ninopept.idases were much less stable wl~en fully. activat.e(l l)y cations aim tha~ the eoneentral, ions of ions in the reacting system were erit, ical. I t has been reported also tha.l, the degree of act.teat.ion incre,'ises with 1,he purity of t,he enzyme preparat.ion. ]Tecause we were measuring t h e aetivit, ies of antinopeptidases in relat, ively crude prepa,rat~ions, wil,h varying amount, s of enzyme present, in iM~,i
,
}la; a sill>crier /;ltl)l I11"O ("l I llt} )J]il J{'~ l'(-ir l lie Jil(iJ<'a l~or
,t3-e, The ('xeetlell{ Col'rel<
ot)t'line.d in otlr st,u(lies of f.he, iovels ,~f a,.'t,ivifies Of nn-iii~o|)epl.Jclases iri platelets ~llid i1/ Sll[)Orlh~t~llitS of pl.telet t)repn ratiolis ,~posed to OSItlot,]0 sliock and t{~ ill[rasonio vil)ratioils, it. would nI)i)eiu' that. the amhlopeptidase ac'tivit, ie~ of suspeiisions of l~latelets Call ])e lised to nieasure the ;lttlrnt)or8 o{" intaot, ph~teiets romahthlg in li suspension of pl'itelets exposed to abnormal colMiLions Of erivh'ol~n~en{8,
The corltJnuous changes in values for amino1)eptidase act.Jetties in rel,'~tion to. numbers of pt,ttelets i~) w-~shed suspm~sions in contrast to t:}le diseoutinugus, s:ubjeot, ive ela~sifieat, ion based on degrees of c:lot~ ret.rael~ion, emphasize fi.,,rt.her t,he usefulness of the test described for studies of platelets exposed 1.o potentially hosi,ile enviromuent,8. SUMMARY
In order to achieve t,he discriminat.ion neceseesary to evah.mte t.he effects of freezing, stornge
CIlYOI31()I,O(;Y ()l,' PI,ATI,;IA~;TS. ab l()w l.e))~peragures, n)l(l drying by sublimttl, i(m i', vac,o <)~ the i~ltegrily of isolate¢[ bh>od platelels, an e~z3'nfi<; [es(, systen~ was developed. The met.i)od was b.Jse(l o1~ tim hydroly.sis of l-leueyl-4nmt.hoxy-2-n:~l)h(Jtyla~)tide ][C1 I)y l,lm ,n~i~o1)cptidases. <)t' l)lateh,ts. Such l a)a)ncters)' " a,'s 1) gyl)e of l)~fffe,', 2) n,)larit.y (>I" buffer, 3) u,)ve le)~gth for dcI.(:rn,ini~)g the a m o m ) t of split l ) r o d u,;t, (,l-nm(,hoxy-2-)ml)t~l hyhunille)-dye (fust blue 11) co)ul)lex, ,1) .-,ubst,rate co))centration, a))d 5) l>l:ttele(, (enzyme) c()ncentratio~ ~ e r e inve~ti:'/ ' ~ . .lhe lest, s~'s((,)~ . ...at(.,l as (]ev(;loped. meastlred with good l>recisio~) tlm a)~tout~b of d-mmge to ]Wel>ar'tei-its (~t" isohtted )>latclets exl)osed to osm,)tic sh,,<-k or-t<) mechanical disrul)Lion. l i e ['"Jc,"I,EN ' ' " CES " "' 1. ]~alogh, I(. C vt~whcn~cal de~m)nstra(,io~ of and~,,i~epI, idas;. :u,livity iu I>I<,M ptatelels. Naiuro, 19c~: 1 [91i, 1.)b,~. '2. ' "" " ' .\I., m , t Yh~ut>i~, B. Chimie ~h's phtlo)-imet,'io det, er)ninaii(qt of le~tcine ami~)-pel)tidase activity with ]-teut'y t-/.'-n:)l>hthyla~uide ]Lvdr()chlr)ride. Arch. t{ioehen). BioI>hys., o+..J;5S-47.t, 1'355. l, }IHI'IIII&tl, ][ . (.: ,, a~)d Contey, C.' L. Clot ' retr:-tct.i()n a,.5: :l lltC'tl~tll'O ()f platelet fmwtit)~:. 1. l..;ITecls iff cerl aiit exlJerin~enl al eondit.ions
I
19!)
on l)latelets in vilro. ,}ohns ]Iopkins [l,)sp. Btdl., 8,3: ;;55-369, 1953, 5. ,Johnston, 51. J., aml Berger, J. The et~zvmatie. ('l ln'ol.)cri, ies ~,f pe.plhhtse. Adv. Lnzymol., 2. ¢~q_n9 19.t.> 6..K.ocholaty, W. PoptMuse,,,, in mammalian plalelets. ]. The hydrolysis of glycytglyeine. aud gl3,cyt-l-lmteilm by human !:qntele.t,s, U , S, Army Medical llese~reh Laboratory Report. 377, March 195:). 7. l ( o c h o l a t y , W. Pepi, idases in mammaJian plat.elets. I.I. The hydrolysis of 1-leueyi. glycine, al~d olher di- and tripep~ides by h~lmaJi platelets. U. S, Army Medical l~ese.u'ch L a b o r a t o r y t~el)o)'t 451) November, ] 9(30.
S N:tchtas, M. M., 3'Ionis, B:,g liosenblatl~, ])., and bcligmau, A . M. hnprovemel~t, i)~ Lhe histoclmmieM localization of leuci~m ; ~ m i m , ~ . peptidase with a new substr~t.e, ldeu<,yl-,t= methoxy-2-nat)hthylamide. J. ]~;iophys. B i o chem. Cytol 7: 2bl---fN, lg(i0. 9. Smith, E. I,. Tim specificity of certain peptidases. Adv. l:mzynml.," 12: J,u-...,)¢,'°" 9~" i95t. 10. Smith, E. L. _Pept,ide bond c'lt;avag('. (siIrvey). In "Fb.e enzymes, rot. 4, 1.>. l). l~l()yer, l-[,, L.n'dv. n/)d K. MyrbSck, ed,'.... ~ op.. 1-,36. A c a demic Press, New York, 1 9 0 0 . I1. SmiLh, E. I~., and ltill, 1[. L. Leueine. amino., peptidase, irt The enzymes, vol..t, P, ].). 13oyer, 1t. Lardy, and K. MyrbSck, eds., pp. 37-62. Academic Press, New York, 1 9 t ; 0 . 12. Zucker, M. B., and Bm'relli, J. A survey of some platelet enzymes and funetiolis: The pla',elets as the source ~)f normal ,serun~ acid glycerophosph~tase. Ann. N. Y. Acad, ScI., ' '" 75: 90',. o1'-~ 1958.
Vol. 6, No. 3, 196,9
CRYOBIOIA)GY I. A n ~ i , m p e l ) t i ( l a s e
OF
I LA I I_,I,E'I S
A c t i v i t y its a 3 I e a s u r e of Platelets in Vitro*
of Intactness
1 )()N :'il]I.) (; !~I:,IFV, D ( ) I l l S .B [-I(K)I,,I:.I,, ' " - " ' t ANI) SIIII~LEY 3IA(,KI,,5. t d)cporttn('nl oJ" ])alhulogll , :llar(l~C/tc School of .'lfedicine, Inc., .ll ilwoul:c,~:, lliae'~,,n.sit~ 53:?33
4-nmti,t~xy-2-1mpl~t, byl't~,~icte i l('l !w tlu? n m i n o pel)tidases ,K platelcts. We ]lave inv,~..qignte(l the effect,s of fhe following \'ari.~l)lo.~: 1) 1)II, q) motnrity of }mfl'er, 3) t y l w ,,f tml'fer, t) l y p e of ~ubsi,rale, 5) slll)s~.t'at,: e~)twel~Ir:~ti~)~i, 6) stop regictio~ witl~ lriehlor:l,'elie acid (T('.\), 7) interfereiwe l~y red lfloo~t ...ell.-, A) interfl,rel~('e by wliite blood e,,lis, 9) l~l:tieh:t (e~lzynw) c(.m. eentratim~.
()t~' st,~l~ties of the effect.- of freezillg, storage. ;it low i e ~ p e r a t u r e s , :,~(t ~,,.,~ .... ;",,' ,,,._ t~;" .. subliination in rm'im on the integrity of is,~l.tte(t blood platelets were impe(le~] gre'itly I,y t,lw al~seuee of :t ilua~t.itntive lest, for ~,wa'~uring in iff6"o ihe ~umhers of intavt p!atelels. ('lvt ret,r'lei,i~,n 'is a fir.~t :tl)proxilnafion of i)~tegrity was used i~ (r,n' e~rly inves(,igations. The sul>jecLive gr:t(ling vf the extent, of elof retractions, even trader stt, ndard t.est con(liticms, 4 alut th,, relatively l.~rge range i~ t.lm mtmbers ,d lflatelet,s for each
[~'[ATEI'~[A],S AND
),[12TII~)I}S
A frc-~]l pl,'ilelet eol
w "l "~ i
,.qh.rp re,:elf on. i E t h y l :~('etat e 7 E.rtraclcd ¢'o~qffed split pro&~cl
The absortm,my of tlw ext.raeLed coupled spli~ product was rea(t by mea~s of a sp.eeLrophofomelm'. ]~ ],:s.'ULT8
Rc¢~.'tion. ]"cr our initial studies, 2 ml of washed platelets, 2 )< 1,0~ per rnm ~, were added to 2 ml of a. ]'eaeI.iol~ mixl, urc col~sisting of 0.8, mg of 1-ieucyl-fl-n:,i,ht,},ylamide HCI c.," 1-1eucyl 4194
CItYOBI()IX)(IY OF P L A T E I , E T S .
r,:el.hoxy-2-natflit, hylamide H('I in I nil of 0.25 .~r potassium phosl)hate buffer, pH' 6.5, 1)his l I;fl of pl~.ysi
S UBS'r R,,'Vl ~7 I -tC'~cvt
0-~--
nl>orli:tlicv t,t' r!)e n:ei,hoxy deriv:itive ~A;is iillOal" wiili rO~l'~(X'l, to tinie; LIuIL of 1-]euexq-
ffl
llllf] 3)
"File
LJ-iiut)}liitylailii~lo I I('l was lied (Fi~. 1). Becnuse (if" i{rt?:ti'ily :!till / r e a r e r lwodlict.ion of slJlii, !)r(ul:l(-{, [ i l r l i i e r slu, lies w e r e earrie~l out. with 1-h,u<..vl..l-lnoi ll~tx)-2-11ntJll( hylgtn-ih]o Il( :1 as sut)c[ 1':I t O.
7sll. t ' s i i l X ilie t.<,ln] sysl,-~ri ns
libovo :l!il{ :i lW,:!elj(ll/ lii'l(, <>I' 1,~ lllill, the. el'feels of ().25 M ]t~q.il.'-,'.itil~t t)[l<>q)ll:i.le ].~tlfl'or lti t)ll 6.(), 6.25,
-2 -,~ap:',hy h:,';1-de. ' t~C !
]. -l¢,i~:}| - ~i-,h.4iilbyl,l,-,a0.,:
--0
IIC}
E 0 0.30 ,-',r >-
2) T C . \ ,
~ rl-:,~e:i.,.~>~
0.40
ethyl
]) lilt' r('lJ(°.li,)li !alixluro, fl(:('~{ 11t (• .
195
1
1
0
0.20
n-" O cO
0.10
o2
1
0.00/-" 0
~ix(.i~
_
~
, I0
5
TIME
, .... 15
OF I N C U B A T I O N
l 20
(MINUTES)
FIe:. 1. Tim lime course :if ehange:: in M)sorlt.
ILS(), 7.{1, 7.25, ,)r 7.50 were (h,lerniilie~t. Tile
an('v usilsg [ - l e u c y l - . l - m o l , h o x y - 2 - i l l t l ) h l . h y t a l i i i d e , , ]ICi (Jl" l-leticyl-fl-naphih3"l,+unide, l l C l a8 s u t >
~l't':t!0.'-{ :ttli, ltlliI +ll" ,',pill l)i'u,Jucl l~II <,f t.;.5()
s(raie. The, complele re,act.ion rliixtilrt: co/itaine.d w:t+slled plalele.ts SUSl)en~led ilt 2 nil f If lfllysioh@cal sa.lirm, 2 Y, l() ~ per II1111;1; 0,~ Ill!f,(if silbsirill.o ill l
w a s f(~ttml :tt ~t
.l/ol,.,.ri/jt qf h,(/i?.r. 'l'he et]'eets (Jl' l'Oai31iOlt I':I(('S ,,l" ().t25, ().2h(J, ().'{7.~, ().5()(1, 0.d25, o," ().750 iii(,b:r c(tiivoiVr:ttiOli.~ o1' lwJl.nssitiiit t)jlOSlJll:ile t)llt~l't'-I", l~ll +i.5, w e r e ~h?ttq'iliilletl..\ i-!l(ll:lr ('o111,ott{ t'~.lI it JJl ill '< l)._o '~- ~:tVC Ill("
~qJtiilillili
mined al 5-t0 niv.
ro:lct,ioil
,Sub,~Lrate corwerdralion.
1'~i I p.
tI'm',' t< ~,gl/,. [:silig tile fnst t)iue t i-.',t)lit, l)ro/hi0L c,~:~ifl('.'~ fl-,.~li: till, tesl sy>.4eiil al)ove r(>it{:i(,(t :it. l~If ii.,5, il: 0.25 -~i ])otnssitnn pht)slfl~vt(;" luiffer, tilt:, :l]).-,~;rl)li~Jli slX'('{l'tllll betweel~ 450 ::nd 600 Inp wa < (]eli,rlttitio(t. 'l'lw r('a(]illg [
itll Of 0.D M p~das.Sillna l)ho.~pll,'tti; b u f f e r , p l [ 6.5; I m] (if p h y s i o l o g i e a l ' s n l i i i a , T i m ' re;te.tion ,n;ixttlre was n : M n l a i u e d at, 3 7 ° C . A l i k o r l m u e y wns dote, r-
:,vii.-: ol)t,:till(,(t
af, :l w n v o
[eragth of
5-I() m#.
:l'ypc of b~(t]".:r. In ,..})is tesl. ~ff t,}W effects of ,lifferel~t. 1)tlff(,rs, {he nunabors of 1)]afe!ets us(,+t was re(iur'exl to 1()~ per l l l l l l r'~. T I l e rea,ction i.Jn-ie was 30 thin. "J']l,' i)lfl]'0rs user] u o r e : 0.25 M pot:l,,,. Sitlhi t)tanSlflutte, 0.25 ~r Tris-nut]ee,,to> 0.25 5I
eitrale, and 0.25 M l~host-,h:it(.,-eigrate. The pI-[s ~tsod we,re 6.25, ti.50, and tj.75. "['tie :l.t-l)Olllil~ Of split 1)roduct~ pro(luted were r~mximum ;it, pll, 6.50 for potns.-iun~ pllosplmte and 1)hosptuttec..itr
'l'he depen~lence of tim rate of re:~etim: on the emmentral, ion of substrato, t.teucyl-4-1netlioxy-2-tmphthylanfidv, wus studied (Fig. 2), Subst, rat,e COlWelltrilLion wits vn,ricd fl't)iti ().5 X 1.0-a 5l kl').|6 ntg per ml) io -
2.0 X. t0 -'~ ~r
[
i
0.):65
li:g per ml). ;l'heso dnta ilMi.,
care t.hat, at. :lit initial eoneolit.raiioll O[ fl.D X 1()-~ M (0.10 nN per .~nl) t,he reaot-~orl became linfit, ing with respect to SII}.)S(;Fltl(. ~. (Ifl[.10r ,+{0 Ji'iill, There was evi{lenee nlso thiit, higher (-Oll,(~orii.l-l-i, fions of ,~ubstl~d;e interfered wilh tile l.esi,'sygten'a. A eoncerlLrlttioll of silL)strut0, ill' 0,0 ;K 10 "~ N (0.19 lng t w r nil) appe,tle]' ' " c ]~O.S.'t}'or o l l r piiYposo,% TCA and fast t)i,m 13. The, adding of ,'FCA to stop tim reaction prior t0 llae additkm of fiisl, blue ti resulted in lower v~lluos for opth,al dcn,,dt.y when comp/u'ocl wii.h (hose ob{,ailied witch 'J'(:A was added :filer the. t'asl; hlue B, I t was forrod also l:hl:tB a eorieetlfc, rr~l, ioti
produet.
] 96
GI{ E II:F, ]:11100KEI{, AN 1) MACK]:;Y
0.80 ...D
0,70 ,#
0.60 /
1.0 >.-
/
0.40
o co CD
0.30
/
/
•
/
/
/
///
CD rl-
/
.,~
/
.--,
/
°//
0.50
z<
Ill ,." I~J
.
E o,q,-
/ /
.."
,
."
,"
I
//¢5-;> SUBS1 RAI [ 60NCL N [ RAI I0.'I
0------0
020 ........Ill
0.10
0 .0 0
} I
0
I
I0
[
20
'
II
0.5~10 "3 ~0.15-;~1 ;roll 0.6×I0-3M
{) ~'~"~u",';'
0 75xIO'3M
'I} 24mq'ml)
E)- ..... --42]
1.0~10-3M
X-----X
"1.5~IO'3tJ (0.4St;,C, 'rn~l
+'---+
2 9~10"3M (O.b5"n~/I~t)
]
30
1
40
~,0 32'nq,mI'~
-
,
[
50
TIME OF INCUBATION (MINUTES)
Fie. 2. The effeH:s of coxicentratiolis of l-le.ucyl-4-met, h(~xy-2-1mlflttlLvlanlide.]l('l ,,I: the t i m e course of c h a n g e s in a,b s o r b a u c v . T h e c( replete, r e a c t i u n m i x / u r e c,~n(.ui~w(I wasl:e(t
t)latelets ml-~pended in I ml of physiological saline, 10 Gper ram:"; wu-iu,ls o,,!,ccl~tt'~ti.l~,-, ,,f substrale ifl t nfi of 0.5 ~i pobassilma phosphate buffer, 1)II (;.5. The react, toil wa,<~maimailmd ,'it. ',:lT°C anti absorlm~ey ream tit 540 ln~. The c,mcentrat iuv.s of subst rate are bhowll eli t.hc graph.
[nlerfcre~cc by red blood cells and while blood cells. Coneel~tn~tions of red blood cells from 2 X 1()a per m m a to tO 4 per nmP (lid not produce suiEcient split, product of ,~ubstrate to be detected by the test above. Small 'tmounts of split i}roduct were defected when white blood {:ells in colmentrations of 4 X 103 per nmP to 104 per m m ~ were tet~ted. The minhnum number of white blood cells necessary ~o produce a reaction exceeded by a, factor oI".J00 the lmmber in our platelet preparatiol~ and was not, therefore, considered to be ;m iuterferiug entity of significance. Plalelet (e~zymc) conce~b'aEon. A test system using the p,n'ameters above was used to determine the effects of different concentrations of platelets (enzyme.) on the production and visualizatioa of sp]i~ producL Washed platelets suspended in sufticienl, amounts of 0.25 -~ phosphate buffer, plI 6.5, to make a. final volume of 1 mI were added to I ml of a reaction mixture of 0.38 mg of t-leueyl-4-methoxy-2-naph~hylamide ]-ICI
in 0.25 ~[ phoslJmtc buffer, ]~II 6.5; t.lm fin'al concentration of .~ubsh'ate :::~, therefore,, 0.19 mg per m]. (N.t{. In lifts s t u d y ;vhell the conccitt,rat, ion oF platelets was 3.1 X 10 '~ p(:r mm :~, the alitollnt; of substrate was iI~cre<<~sedby 100 :,<,.) The re'te(,ion mixture wits placed in a 37°C' water bad~ for 30 mil~. The system was ' @ t a r e d during incubation. At the end of 30 rain, 3 mg of fl;tsfo b h m B , dissolved in 1 ml of distilled water, were added to the above, and the combination of dye with the splil product, methoxy-2-naphthylamine, was allowed to proceed rot i mi~u One milliliter of trMfloracetie acid was added to stop the re'lotion of enzymes with substr'~te. "Fen milliliters of ethyl a c e t a t e were added to the above, and following vigorous agitation for several mill the mixture was centrifuged at 2000 rpm for 5 rain; t,he fast blue B-split product complex was present in the ethyl acetate layer. The extracted complex was read at 540 mlx. A linear relationship was found between ab-
(.,l,'i OBIOI~CGS. OF . ~_,..,~, ,,.~_,,,.. s~n'l)mwy and llul,tt)ers of 1)latelet.s.; a doubling of llatelc(,~' ~ ' ~m~nbers resulted in n doubling of nbs.orbal,:y (Fig. 3). Clot retraction t,s. aminopeptidasc activity. '['he l}roeeclures used for clot, retraction were those dew'lolwd bV ]['trtmcmu "m{l ( o n l e y . 4 Tlie degrees of r('lra('ti(m were })ased on 1,'igm:e [ ()f their putflie:ltion" neg'd, ive, the tube on the righl,; 1-+-, i.he tul,e see(m(l frorn l,lie rig;tit,; 2-1-, the t,ul)e thir(l fro~ll the rigtit; 3-t-, lhe tube Lhir(l front the left; -t q-, tim first or second t,ul)es frol]t tlle M't. The retatioilsttips botweell degrees of clot ret.raetiol~, ml1,.~t)ers of phttelets per cubic millililc.ter, atlJ(t l('vt'lq of :u~iiln~pel)tidase activities (absor}):tnvy at. 5t(1 m#) "tre. shown in Tal)te 1. Wlmreas t lic nil'iil!)ors (it" 1)]ttt.elels for ettch deg,ree of (',l,~t ri't,r'lctjoti vn fled froni 210,000 for I. + , 260,000 for off_,_ 1,000,000 for 3 + 'rod ,0,9100,000 t'()r 4 + retraction, :tntinol)el)~,id:t.~e act, ivit, ies shower [ :t raiigt, of a l)sorl)alic.y values eorresl)ondii~<,',, to l,he liilli-ibt~l'.,~ of tflatelets ln'esent. .t minop~'pt[d<.~.,~'e actit:'t:/ as a me(t,surc, of pla, leh>,t dama, qc or drstr~u.'tio~. To dete)'inille if the test (ies('rit.)ed pi'evi~usly coithl be used (:o (;vahiate ill(; dl:greo of lfl'lt.ehq, de.%ruct, ion, t)rol)arat, ioris nf plateh:l~ were disrupted by 1) osmotic shook and 2) soiiific..-&iol~. In the first, inst, P~lico, 1.0~ l)latelet, s per n ] l l l 3 were suspelided in 1 inl of di.-:tilled water or in t Inl of differenTo molaril.ies of N a ( ' l for 30 lnin; in the seool~d t-ml 1)rep aratiolis of phitelets simihu' to l.he above were exposed to ult, t;asonie os(tillatioi~s for retrying 1)eriods of time. I)uring sonifical, ion d~.. tubes co~ttaini~g t l , j SUSlmnsio~ts of platelets were submerged in a bath of melting ice. Following exposure to the colidit, ions above the suspensions were eelltrifuged at, 8000 rpm for 30 mh]. In bot, h instances the supernattu~ts were carefully removed and saved. T h e platelet, b u t t o n s of the os,notie stress experiments were resuspended in sufficient, amourlts of 0.25 a~ phosphate buffe,', pH 6.5, to obtain the original volume and tlm activities of aminopep.~idascs measured. T h e s u p e r n a t a n t s were diluted with 1 ml of 0.5 5, phosphate buffer, p H 6.5, prior to the determination of a e t M t i e s ; the r a b i e s obtained were corrected for dilution. A q u a n t i t a t i v e reciprocal relationship ,between the activities of the p l a t d e t s and the a c t M t i e s of the s u p e n m t a n t s for each hypotonie contend tration o f Na:CI and for distilled water was oh•
x.
1~
1.20
l
t( "
-
/
1.00 E
o 0.80 u') >-. (..) Z <:I:
0.60
or"
0
O0 £13 <1:
0.40 0.20 _
QO0
l
..
l
~
I
I0
20
l
I
....i
30
PLATELETS x 105/ram Fro. 3. The effect of platelet coltcem, rat.ion on absorbancy. The complei.e rcactAon mixhn'e cont,aiaed washed ph~telets suspended in 1 ml of 0.25 M potassium phosphate buffer, pH 6.5; 0.38 mg of 1 - leucyl -4 - met.hoxy - 2- n a p h t h y h u n i d e : I IrCl. The react.ion was m a i n t a i n e d al, 37°C for 30 min aim reltd at 5-I0 nm. The concenlr~tions of platelets are noted on t:he graph. A(, l)latelet, concentrations of 3.1 X 10 ~ per mm a, the amount of s u b s t r a t e was increased t,o 0,76 rag. served (Fig. 4). Platelets suspended in distilled water ( m a x i m u m osmot, ic shock) lost all aminopeptidase tmt, ivit,y and the total a c t i v i t y was found in the s u p e r n a t a n t . Platelets suspended in physiological saline (minilnum osmotic shock) lost little activiW and no measurable aminopeptidase activiW could be found in t,h(~ s u p e r n a t a n t . T h e nminopeptid:~se acfdvii;ies for the h y p o t o n i e sohd, ions of N a C [ were" pai'titioned between 1)latelets a n d sut)e:'natan t."in a qua n ti ta give ma nnet. 1
TABLE CORRELATION
~2/UDIllS
OF
. . . . ~'" '
Pr, aTELm'S, I ) E G a E E Or," Ot, oT I{ETRACT I E S , XND 2kMINOPEPTIDASE A C T I V I T y Clot Retraction*
0
1+ 2+ 3+ 4+
I
No. of Platelets per Cubic Millimeter (Range)
Aminopeptidase Activi ty, Absorbancy at 540 m,, (Range).
8.6 .o. 2 ,t.6 8.0 2
0.025-0.058 0.061-0,140 0, ! 4 5 - 0 . 3 0 0 0.325-:0: 580 0._620-1.40
X X X X X
104-2.0 I 0 -s4 . 3 " 10~-7.2 105-1.8 106-4.1
× X X X X
l05 l0 s 10 ~ l0 G l0 G
* Adt~pted from the, s t u d y of_ I~l~rtma, n, and Conley."
1 98
(.; II E 1FF, B P,O()KE R, AN 1) 5Ie~,CI,:],~Y
050
• 4:I
:~ 0 4 0
\
E >_ o
LETS
\o
/<
""
,,
....
050
z
<
cn
/,,.
020
0
o ,,
/
0 I0
O, O0
potential and ~,.s used successfully for the eolorb met,tic estiTnnt,ion of :m~iJlopel)tidases il~ tissue homogenates, seruin, and urilie, a .Ill the adopt.toll of t,his chromogenic substrate for lfistochen~ic:d meth-dology it, was fotm(l tlmt althoitgh ('nzylnic. sites ('o,ihl be visualized ill t,issu(, set,tit,as, the slow r:tte of ('ot,'plhL~ (ff the ~ty(', let.mz()/,ize(t (tiorthoanisi(lille (fasL 1)lue l~), ,vit,]l file split 1,ro~luet, ~-Ilat)ht.hyh~mine, result~_,d ill (lifhlsioli artifacts. To milffnfize these : t r t i [ a e t s , :t new sulostrate ~ itll a fasl er Cal)iure reaction, l-leuc:yl... -l-methoxy-2-nal)hthyhtn~i(le 11(1, x~:ls synitiesize(l, s ~l'lle sl>lit, l)l'o(llwt ~)f this SllbSlm[!,, 4metl l(2xy-,,-lmj)tllllylnlnin(,, ') was fomld t~, ('otlt)le with tlte letr:tzotized (linrt, h()atlisi(line ,14) t,il~es ' faster than th(; uil.,qtbst.it~lte(] tllllill,:. Ollr sl.tt(iies ill(tie:ire thai, tliis new :,ubstrat(,' ('all be used ,,tlso for the (;ol()rinlet.ri<', (leterlu hint hJl~ of the a('{ ivities of the anii)~ol)ei)tid't.'-e~ ii) -~()iutim,. "I'll('. lfi,,zlmr levels (,f aclivil.y we l'(nitl(l witl~ l-tmie.yl--lnleiil{~xy,2-1~ll)hl,ltyl,ai~lide ll('l (lo lint illdi,~'ate lm('('ssqril 3" lhat, it is a .-~lp('ri()r sul~sir'~te fc)r tim ailiiii+)l)ept, idases t Jill r:tlllor lhnt, the :q)tit 1)roduet,
UPERNATANT
'
o,o
I
..
02
i
I
o.4
0.6
..,'-,7-r "° os
I
% NoCI (W/v) ] ' q c . . L T h e eft'eel, of ()shier, i t sh()ck on the n,nlii~ol)Olil, itlase a c t i v i t i e s of l)ropltrill.ions ~lf ilit,n,et; p l M e l e l s n,it(t SltlJerllttt:lnts, The filial (,olieolil.rll{.iOli ~)f tj]:il.(,lOls was .5 X ] 0 ' per innl'L T h e re't(.lioit I n i x l u r o arid t.hc e(,ll{titioils of rolicLiolt were the same ,'ts tlmso of F'iguro '3.
]:({,stilts simil'ir i,o llie ~bove were-foull~l ,.vhe~ l)]aLelet,~ wove de,%royed or dam'l/ed l)y expos~ure LO StlliersolliO oscillations.
'l'he values of ;lii-lillo-
l)epti(hi~e activities in lihitelet,s rum ill StlpOrll,'.itatitS were a ftit~etioll of tinlo of ('X[IOStll'O {-O Stll)OrSOIl it; VvILVOS. J)lscVssIoN
Several reviews of the :~minotml~t,i(htses serve l:o emphasiz(: lhe eornplexil ies of enzylnes belonging 1.o this g r o u p . : ' , n-n J-ii ge~el'al, these enzymes •wt. only on subst.rates wl~ich possess m~e or more free lenni~ml polar R r o u l ) s , s u c h :is c e - a m h i o , I n addii,ion stone of t.hese enzymes require metal ions for act.iwtt, ion or for nmximum aetivit,y. 1t~ amy be noted, however, t h a i , eerUdn of the a~ninopept.idases were much less stable wl~en fully. activat.e(l l)y cations aim tha~ the eoneentral, ions of ions in the reacting system were erit, ical. I t has been reported also tha.l, the degree of act.teat.ion incre,'ises with 1,he purity of t,he enzyme preparat.ion. ]Tecause we were measuring t h e aetivit, ies of antinopeptidases in relat, ively crude prepa,rat~ions, wil,h varying amount, s of enzyme present, in iM~,i
,
}la; a sill>crier /;ltl)l I11"O ("l I llt} )J]il J{'~ l'(-ir l lie Jil(iJ<'a l~or
,t3-e, The ('xeetlell{ Col'rel<
ot)t'line.d in otlr st,u(lies of f.he, iovels ,~f a,.'t,ivifies Of nn-iii~o|)epl.Jclases iri platelets ~llid i1/ Sll[)Orlh~t~llitS of pl.telet t)repn ratiolis ,~posed to OSItlot,]0 sliock and t{~ ill[rasonio vil)ratioils, it. would nI)i)eiu' that. the amhlopeptidase ac'tivit, ie~ of suspeiisions of l~latelets Call ])e lised to nieasure the ;lttlrnt)or8 o{" intaot, ph~teiets romahthlg in li suspension of pl'itelets exposed to abnormal colMiLions Of erivh'ol~n~en{8,
The corltJnuous changes in values for amino1)eptidase act.Jetties in rel,'~tion to. numbers of pt,ttelets i~) w-~shed suspm~sions in contrast to t:}le diseoutinugus, s:ubjeot, ive ela~sifieat, ion based on degrees of c:lot~ ret.rael~ion, emphasize fi.,,rt.her t,he usefulness of the test described for studies of platelets exposed 1.o potentially hosi,ile enviromuent,8. SUMMARY
In order to achieve t,he discriminat.ion neceseesary to evah.mte t.he effects of freezing, stornge
CIlYOI31()I,O(;Y ()l,' PI,ATI,;IA~;TS. ab l()w l.e))~peragures, n)l(l drying by sublimttl, i(m i', vac,o <)~ the i~ltegrily of isolate¢[ bh>od platelels, an e~z3'nfi<; [es(, systen~ was developed. The met.i)od was b.Jse(l o1~ tim hydroly.sis of l-leueyl-4nmt.hoxy-2-n:~l)h(Jtyla~)tide ][C1 I)y l,lm ,n~i~o1)cptidases. <)t' l)lateh,ts. Such l a)a)ncters)' " a,'s 1) gyl)e of l)~fffe,', 2) n,)larit.y (>I" buffer, 3) u,)ve le)~gth for dcI.(:rn,ini~)g the a m o m ) t of split l ) r o d u,;t, (,l-nm(,hoxy-2-)ml)t~l hyhunille)-dye (fust blue 11) co)ul)lex, ,1) .-,ubst,rate co))centration, a))d 5) l>l:ttele(, (enzyme) c()ncentratio~ ~ e r e inve~ti:'/ ' ~ . .lhe lest, s~'s((,)~ . ...at(.,l as (]ev(;loped. meastlred with good l>recisio~) tlm a)~tout~b of d-mmge to ]Wel>ar'tei-its (~t" isohtted )>latclets exl)osed to osm,)tic sh,,<-k or-t<) mechanical disrul)Lion. l i e ['"Jc,"I,EN ' ' " CES " "' 1. ]~alogh, I(. C vt~whcn~cal de~m)nstra(,io~ of and~,,i~epI, idas;. :u,livity iu I>I<,M ptatelels. Naiuro, 19c~: 1 [91i, 1.)b,~. '2. ' "" " ' .\I., m , t Yh~ut>i~, B. Chimie ~h's pht
I
19!)
on l)latelets in vilro. ,}ohns ]Iopkins [l,)sp. Btdl., 8,3: ;;55-369, 1953, 5. ,Johnston, 51. J., aml Berger, J. The et~zvmatie. ('l ln'ol.)cri, ies ~,f pe.plhhtse. Adv. Lnzymol., 2. ¢~q_n9 19.t.> 6..K.ocholaty, W. PoptMuse,,,, in mammalian plalelets. ]. The hydrolysis of glycytglyeine. aud gl3,cyt-l-lmteilm by human !:qntele.t,s, U , S, Army Medical llese~reh Laboratory Report. 377, March 195:). 7. l ( o c h o l a t y , W. Pepi, idases in mammaJian plat.elets. I.I. The hydrolysis of 1-leueyi. glycine, al~d olher di- and tripep~ides by h~lmaJi platelets. U. S, Army Medical l~ese.u'ch L a b o r a t o r y t~el)o)'t 451) November, ] 9(30.
S N:tchtas, M. M., 3'Ionis, B:,g liosenblatl~, ])., and bcligmau, A . M. hnprovemel~t, i)~ Lhe histoclmmieM localization of leuci~m ; ~ m i m , ~ . peptidase with a new substr~t.e, ldeu<,yl-,t= methoxy-2-nat)hthylamide. J. ]~;iophys. B i o chem. Cytol 7: 2bl---fN, lg(i0. 9. Smith, E. I,. Tim specificity of certain peptidases. Adv. l:mzynml.," 12: J,u-...,)¢,'°" 9~" i95t. 10. Smith, E. L. _Pept,ide bond c'lt;avag('. (siIrvey). In "Fb.e enzymes, rot. 4, 1.>. l). l~l()yer, l-[,, L.n'dv. n/)d K. MyrbSck, ed,'.... ~ op.. 1-,36. A c a demic Press, New York, 1 9 0 0 . I1. SmiLh, E. I~., and ltill, 1[. L. Leueine. amino., peptidase, irt The enzymes, vol..t, P, ].). 13oyer, 1t. Lardy, and K. MyrbSck, eds., pp. 37-62. Academic Press, New York, 1 9 t ; 0 . 12. Zucker, M. B., and Bm'relli, J. A survey of some platelet enzymes and funetiolis: The pla',elets as the source ~)f normal ,serun~ acid glycerophosph~tase. Ann. N. Y. Acad, ScI., ' '" 75: 90',. o1'-~ 1958.