bleeding time aspirin

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1973 41: 369-377

J. Hirsh, D. Street, J. F. Cade and H. AmyAfter AspirinRelation Between Bleeding Time and Platelet Connective Tissue Reaction

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R ela tion B etw een B leed ing T im e and P la te le t C onn ective

Blood, Vo l. 41 , N o . 3 (M a rch ), 1973 36 9

T issu e R eaction A fter A sp ir inBy J. H irsh , D . S tree t, J . F . C ade , and H . Am y

Asp irin p ro longs the b leed ing tim e inno rm a l sub je c ts and inh ib its p la te le tre lease and agg rega tion w ith connec-tiv e tissue and o the r b io log ica l agen ts .W e have inves tiga ted one o f the pos-s ib le m echan ism s by wh ich asp irinp ro longs the b leed ing tim e by com pa r-ing the e ffe c ts o f asp irin and p laceboon the b leed ing tim e and p la te le tagg rega tion w ith connec tiv e tissue inno rm a l vo lun tee rs . Tw o sepa ra tes tud ie s w e re pe rfo rm ed . B o th showedp ro longa tion o f the b leed ing tim e andinh ib itio n o f the p la te le t connec tivetis sue reac tion afte r asp irin , bu t on lythe second s tudy show ed a s ign ifican tco rre la tion betw een these changes.

B o th s tud ie s a re repo rted in de ta il be -cause the d is c repancy be tw een themillu s tra te s som e im po rtan t p rin c ip le stha t requ ire cons ide ra tion w hen re la t-ing the effects of drugs on plateletfunc tion in v itro to the ir e ffe c ts in v ivo .The find ings suggest tha t w hen pa r-ticu la r ca re is ta ken to s tanda rd izethe m easu rem en t o f the p la te le t con -nec tive tis sue reac tion in term s o f thes tim u lus used , sub je c t va r iab ility , andana ly s is o f re su lts , the p ro longedb leed ing tim e a fte r asp irin can beshown to be re la ted to the de fec tp roduced in the p la te le t connec tiv etissue reac tion .

T HE INTERAC TION O F PLA TELETS w ith conn ec tive tis sue , adenosined ip hospha te (AD P ),3 5 and th rom b in67 is thou gh t to b e of fun dam en ta l im -

p ortance in hem os ta tic p lug fo rm atio n . A num ber o f an tiin flamm ato ry d rugs ,inc lud in g asp ir in ,3 h av e been sh ow n to d ecrease p la tele t agg rega tion w ithco nn ec tive tissue an d w ith low concen tratio ns o f th rom bin #{176} and to inh ib it theseco nda ry w av e of agg rega tion w ith A D P 2 4 and w ith ep ineph rin e.# { 1 7 6 } 2 3Th ese e ffec ts are tho ugh t to be d ue to in h ib itio n by asp irin o f re lea se o fAD P from pla tele ts .8 .# {1 76} 2 A sp ir in has been sh ow n to ace ty la te num ero usp la sm a and p late le t p ro tein s,17 and it has been sug gested th at its e ffec t o nh em osta sis m ay be rela ted to th is ace ty la tion reac tion .8 A sp ir in has a lsob een repo rted to inc rea se th e b leed in g tim e in som e no rm al su b jec ts , 2 22as w e ll a s in pa tien ts w ith an unde rly ing hem o static de fec t.212 4 It h as b eensu ggested th at the d e fec t in AD P re lea se p rodu ced by asp ir in m ay berespon sib le fo r the p ro long atio n of b leed ing tim e .1 5 2 H ow eve r, o the rs have

F rom the D epa rtm en t o f L ab ora tor y M ed ic ine , S t. J ose phs H osp ita l, and the D epa rt-m en ts o f P a tho logy and M ed ic ine , M cM as ter U n iver sity , H am il ton , O n tario , C ana da .

S ubm it ted M arch 6 , 1 972; rev ised Ju ly 10 , 1 972; acc ep ted A ug ust 17 , 1 972 .Sup po rted in par t by gran ts from the M ed ic a l R ese arch C o un cil o f C a na da a nd th e

O n tar io H ea rt F ou nd a tio n .J. H irsh , M .D ., F .R .A .C .P . : P ro fessor, D ep artm en ts o f P a tho lo gy a nd M ed ic in e ,

M cM aster U n ive rsi ty , and D irec tor o f H em ato lo gy , M cM aster U niv ers ity M ed ic a l C en tr ean d S t. Jo sep hs H o sp ita l, H am ilto n , O n tario , C an ad a . D . S tree t, B .S c .: Te chn ica l A ss ista n t,H em a to lo gy La bora to ry , S t. Jo sep hs H osp ita l, H am ilto n , O n tario , C an ad a . J. F . C ad e, M .D .,P h.D ., M .R .A .C .P .: Assistan t P ro fessor, D ep ar tm en ts o f M ed ic in e a nd P a th o lo gy , M cM a ste rU n ive rsity , H am ilton , O n tar io , C an ad a . H . Am y , M .Sc .: C hie f M ed ica l Te chn o lo g is t,H em ato log y L abo ra tory , S t . Jo sep hs H osp ita l, H am ilto n , O n tario , C ana da .

1973 b y G run e & Stra t ton , In c .

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3 7 0 H1 R S H E T AL

suggested that aspi r i n may have an addi tional ef f ect on hemostasi s through anef fect on the vessel w al l i tsel f 22.25,26

W e have investigated the mechanism of prolongation of the bleeding timeproduced by aspi r i n, by relati ng the changes in bleeding time to the changesin platelet aggregation w i th connecti ve ti ssue in normal subjects. Tw o separatestudies w ere perf ormed, the f i rst hav ing a number of shortcom ings that w erecorrected in the second. B oth studies are described in detai l because the dis-crepancy in resul ts i l l ustrates some of the di f f i cul ti es in using in v i tro tests toassess platelet f uncti on in v i vo.

MATER IALS AN D METHO DSD esign of Studies

Study I : Forty normal subjects (al l young adul t f emales) w ere studied using a ran-dom ized, double-bl i nd design. Tw enty w ere given placebo and 20 were given aspi rin.Tests w ere performed before treatment i n al l subjects, af ter 2 hr (in f i ve of the placebogroup and i n 12 of the treatment group), af ter 24 hr (i n 18 of the placebo group and in18 of the treatment group), and af ter 48 hr (i n four of the placebo group and in f i ve ofthe treatment group). T he study w as completed w i thi n 3 mo .

Study 2: T hi rteen normal subjects (I i males and 2 f emales) w ere studied using arandom ized, double-bl ind cross-over design. A l l subjects w ere tested i n the morning af tera l i ght f at-f ree break fast, and smok ing w as prohibi ted f or at l east 24 hr bef ore testi ng.The subjects w ere random ly al l ocated into i n i t i al aspi r i n or pl acebo groups, and 1 wkl ater the treatments w ere crossed over. A period of 1 wk w as selected to al l ow the ef f ectsof aspi rin to w ear of f in those subjects w ho w ere given aspi ri n f i rst and placebo second.B leeding times and platel et aggregation tests w i th connecti ve ti ssue were performedbef ore and 24 hr af ter treatment. T he study w as completed w i thin 2 w k using a singlebatch of connecti ve ti ssue suspension that w as f rozen in al iquots and thaw ed immediatelybefore use.

I n both studies al l subjects agreed not to take any other medi cat ion for at least a w eekbefore and dur ing the period of the study . N o subject had a history of bleedi ng. Four300-mg tablets of acety lsal i cy l i c acid w ere giv en in a si ngl e dose as the act i ve treatmentand four 300-mg tablets of sodium bi carbonate w ere giv en as the placebo.Platelet Studies

B lood w as col l ected in plasti c tubes contai ni ng 3.8% tr i sodium ci trate usi ng a pl ast i csy ringe and a 19-gauge needle. The blood w as mi xed w i th ci trate in a ratio of ni nepar ts of blood to one part of anti coagulant. T he blood was centri f uged at 300 g fo r10 mm at room temperature to obtai n pl atelet- ri ch plasma. The platelet-ri ch pl asma wasdispensed into plast i c tubes, and an al i quot w as retained for platelet counti ng. T heremaining blood was centri f uged at 2000 g f or 15 mm at room temperature to obtainplatelet-poor pl asma, w hich w as decanted i nto separate plast i c tubes. T he platelet countof the pl atel et-r i ch pl asma w as adj usted by di lution w i th autologous pl atelet-poor pl asmato obtain a count of 200,000/cu mm . Pl atel et aggregation was measured by a turbidimetri cmethod,27 28 using a Pay ton aggregometer.Bleeding Tim e

I n the f i rst study , the bleeding time w as perf ormed by a modi f i ed I vy method. T hetourni quet w as inf l ated to 40 mm H g, and three inci si ons (3 mm long and 3 mm deep)w ere made on the volar aspect of the f orearm . T he bleeding time was taken as themean of the three i ndi v idual measurements.

I n the second study , the bleeding t ime was perf ormed by the template method asdescribed by M i elk e et al .2 The tourni quet w as inf l ated to 40 mm H g, and tw o inci sions(9 mm l ong and 1 mm deep) w ere made on the volar aspect of the f orearm . T he bleedingtime was taken as the mean of the tw o indi v i dual measurements.



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BLEED ING TIME AND P LATELET CO NN EC TIVE TIS S UE 3 7 1

Prepar ati on of Connecti ve Tissue SuspensionConnectiv e ti ssue (Sigma Chem ical ) w as homogeni zed in a W ari ng B lendor using a

semim i cro contai ner that contained the blending assembly in an ice-f i l l ed cool ing jacket.F ive grams of connecti ve ti ssue w ere homogenized in 85 ml of 0.9% sal ine for 30 mm .T he suspensi on was centri f uged at 1740 g f or 15 mm at 4#{ 176} C ,nd the supernatant materialw as aspi rated, di v i ded into al iquots, stored at -20#{176}C, and thawed immediatel y beforeuse. The pl atel et-aggregating acti v i ty of the connecti ve t i ssue suspension remai ned stablefor a peri od of at least a month w hen tcsted in ser ial di lut ions in four normal control snot in the study , and new batches of suspensi on w ere prepared monthly .

Assessment of P latelet Aggr egation and Analysi s of Resul tsI n the f i rst study , the col lagen concentrat ion used in each exper iment w as that w hich

produced betw een 30% and 50% max imum aggregat ion in the same tw o control subjectsw ho w ere not in the study . Platelet aggregation w as assessed by measuri ng: (1) themax imum change in opt i cal densi ty uni ts, expressed as a percentage of the di f f erence i nopti cal densi ty betw een platelet-r i ch and pl atel et-poor plasma for each determ inati on(percentage aggregation), and (2) the t ime in seconds betw een the addi tion of connect i veti ssue and the beginning of the upw ard def lect ion of the curve (reaction time). The ef fectsof aspi rin and placebo on the platelet connecti v e ti ssue reaction w ere analy zed by com-par ing the absol ute values of these measurements in the treatment group w i th those inthe placebo group.

I n the second study , platelet aggregat ion w i th connecti v e ti ssue w as assessed bymeasuring: (1) the max imum change in opti cal densi ty uni ts, expressed as a percentageof the di f f erence in opti cal densi ty betw een pl atel et-r i ch and pl atelet-poor plasma foreach determ ination (percentage aggregation), (2) the slope of the curv e at i ts steepestpoi nt (slope) , and (3) the time in seconds betw een the addi tion of the connecti ve ti ssueand the begi nni ng of the upward def lect ion of the curve (reacti on time). Ser ial di l utionsof connecti ve ti ssue w ere prepared, and each sample of pl atel et-r i ch plasma w as testedat at l east f i ve di l uti ons of connecti ve ti ssue to obtai n a percentage aggregation over arange f rom greater than 75% to l ess than 25% on each occasion (Fig. 1). T he ef fects ofaspi rin and pl acebo on the platelet connecti ve ti ssue react ion were analyzed by calculat ingthe change in concentrat ion of connecti ve ti ssue that w as requi red to produce the samepercentage aggregation, slope, and reaction time as in the pretreatment sample. Thus,the concentrated connecti ve tissue suspension w as considered to have 100% act i v i ty , a1 :2 dilut ion 50% act i v i ty , a 1 :4 dilut ion 25% acti v i ty , and so on. For exampl e, as shown inFig. 1, if a 1 :14 d i lu t ion (7% activ ity) produced 75% aggregati on af ter pl acebo and a 1:2dilut ion (50% act i v i ty ) w as requi red to produce 75% aggregation af ter aspi r in, the changein concentrati on of connecti v e ti ssue w oul d be + 43 % . The resul ts of the change inbleedi ng t ime produced by aspirin or pl acebo w ere then pl otted against the change inconnect i ve ti ssue concentrati on requi red to produce 75%, 50%, and 25% aggregati on(F ig. 2), a slope of 1, 0.75 , 0.5, and 0.25 (Fig. 3), and a reacti on time of 60 and 90 sec(Fig. 4).

RESULTSStudy :r

The resul ts of the f i rst study are summari zed in Table 1. There w as nosigni f i cant di f f erence in the pretreatment values for the bleeding time or f orei ther of the tw o indices of the platelet connecti v e ti ssue reaction betw eenthe placebo and aspi r i n groups. In the placebo group, there w as no signi f i cantchange at 2, 24, or 48 hr in the bleeding time or in the platelet connecti veti ssue reaction. I n the aspi ri n group, there w as a signi f i cant increase in thebleeding time at 2 hr. A t 24 hr, f our subjects show ed a considerable pro-longation of the bleeding time, but there w as w ide indiv idual var iati on so that



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4 8 0 1 2A B leeding T ime (mm . )

40

4)Fig. 2. R elation betw een change in

bleeding tim e and change in collagenconc en tration ( # { 1 7 6 } / o )equired to produceaggregation of 75 #{176 }/otop), 50#{176}/om id-d le ), a nd 25 #{1 76 }/obottom ) after placebo(closed circles) and aspirin (opencircle s). N S , n ot sig nific an t, r, co rrela-tion coefficient.

37 2 H IR S H E T A L.

F ig. 1. P latelet reaction to increasing dilution of connective tissue (collagen)before and after placebo or aspirin in subject A .G . P latelet reaction has beenexpressed as percentage aggregation (top right), reaction tim e (bottom right),a nd slo pe (le ft).

2 4 6 8 10 12A B iseding I sne (m on.)

r . O1 2 / N 5 4

6 8 10 12A B is ding T ims (mm.)



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80

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12

12

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50

40

30

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0 - r -OOl /NS

B LEED ING TIME AND P LATELET CO NN EC TIVE TIS SU E 3 7 3

F ig . 3 . R e la tio n be tw e e n c ha ng e inb le e d in g tim e a nd c ha ng e in c o lla g e ncon cen tra t i o n ( # { 1 7 6 } / o )e qu ire d to p ro duc ea s lo p e o f 1 . 0 0 (to p ), 0 .7 5 (s e c o ndfro m to p ), 0 .5 0 (s e c o nd fro m bo tto m ),a nd 0 .2 5 (bo tto m ) a fte r p la c e bo (c lo s e dc irc le s ) a nd a s p irin (o p e n c irc le s ). N S ,n ot s ig nific an t; r, correla t ion coe f f i -c i en t .

. ,- a a {14 9} 2 A4B I .ed eng T im e (m i l l )-

- ...- .- .. 4- - .O42 NS 20(.2

1 # { 1 4 9 } a# { 149 } A 4B leed lng8T im e (m m.)

# { 1 7 6 } iI

# {1 4 9 } i A4 nT imSmIn )

t ! . { 149 } r r 2 4 8 8I A B leed ing T im e ( m m . )

I a 2ABle ed lngT ine (mln . )

30

200 r - O l O / NS

0 0 0

Ic 0 0a a 1 0 1 2A B leed ing T im . ( m m . )

F ig . 4 . R e la tio n b e tw e e n c ha ng e inb le e d in g tim e a nd c ha ng e in c o lla g e ncon cen tra t i o n (# {176}Io )e quire d to p ro duc ea re a c tio n tim e o f 6 0 s e c (to p ) a nd9 0 s e c (b o tto m ) a fte r p la c e bo (c lo s e dc irc le s ) a nd a s p irin (o p e n c irc le s ) .N S , n o t s ig n ific a n t; r, c o rre la tio n c o -eff icient .



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3 7 4 HIRS H ET AL.

Table 1 . B le ed in g Tim e a nd P la te le t C o nne ctiv e Tis su e R e ac tio nAfte r As p irin a nd P la c e bo (S tudy 1 )

T e s tP l a c e b o A s p irin

P re 2 24 4 8 P re 2 24 48B le ed in g tim e 4 .0 8 5 .0 0 4 .2 5 3 .5 0 4 .6 1 7 7 5 * 6 .8 3 4 .3 3

(m m ) (0 .0 3 ) (0 .0 0 ) (0 .3 5 ) (0 .0 0 ) (0 .2 9 ) (1 .0 3 ) (1 .2 7 ) ( 0 . 1 4 )A gg r e g a t i o n 4 6 .5 4 6 .0 4 1 .4 3 5 .3 4 2 .0 2 3 . 7 1 6 . 9 t 1 4 . 0 1

(# { 176} /o ) ( 3 . 2 7 ) (4 .3 2 ) (2 .9 2 ) (9 .8 2 ) ( 2 . 3 2 ) ( 4 . 0 0 ) (2.64) ( 3 . 7 8 )R e ac tio n tim e 6 3 6 9 6 4 6 1 5 8 7 3 t 7 9 * 6 3

( s e c ) (4 .1 ) (3 .6 ) (3 .0 ) (6 .7 ) (3 .9 ) (1 0 .3 ) (6 .7 ) (7 .6 )P re , 2 , 2 4 , a nd 4 8 re fe r to m e a s u re m e nts be fo re a nd 2 , 2 4 , a nd 4 8 h r a fte r tre a tm e nt w ith

e ith e r a s p irin o r p la c e bo . Va lu e s a re m e a n , w ith s ta nda rd e rro r in pa re nth e s e s .4 Va lu e a fte r tre a tm e n t s ig n ific a ntly d iffe re n t from tha t b e fo re tre a tm e n t (p a ire d t te s t),t p


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BLE ED IN G TIME AN D P LATELE T CO NN EC TIVE TIS S U E 3 7 5

Ta b le 2 . C ha ng e s in B le e d in g Tim e a nd P la te le t C o nn e c tive Tis s u e R e a c tio nAf t e r Aspirin a nd P la c e bo (S tudy 2 )

Tes t P lac eb o A s p i r i n p

B le e d ing tim e (m m ) + 0 . 0 1(1.08)

+ 4 . 0 9( 2 . 8 3 )

< 0 . 0 0 1

Ag gre ga tio n (# {1 76 }/o )7 5 - 0 . 3 1

( 3 . 8 4 )+ 4 0 . 5 8( 1 8 . 5 9 )

< 0 . 0 0 1

5 0 -0 .5 4( 4 . 1 6 )

+ 2 2 . 0 8( 1 3 . 3 3 )

< 0 . 0 0 1

2 5 -1 .1 5( 3 . 4 4 )

+ 1 2 . 7 7( 1 0 . 8 0 )

< 0 . 0 0 5

S l o p e1 . 0 0 + 0 . 7 7

( 2 . 7 4 )+ 2 2 . 3 8( 1 5 . 2 6 )


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3 7 6 HIRS H ET AL

these considerati ons are also relevant in the assessment of the anti thromboti cef fects of drugs that suppress platelet f uncti on.

The discrepancy betw een our tw o studies i s of i nterest because it i l lustratesthe importance of the general pr inciples outl i ned above in relati ng plateletfunction as assessed in v i tro to platelet functi on in v i vo. T he f i rst study hadthe f ol l ow ing shortcom ings. (1) A number of di f f erent connecti ve ti ssuesuspensions w ere used over the 3 mo of the study , al though al l w ere preparedf rom the same dried ex tract. T his resul ted in w ide var iati on in the plateletconnecti ve ti ssue reaction in the placebo group, despi te the fact that thestrength of the connecti ve ti ssue stimulus was standardi zed against plateletsf rom the same control subjects throughout the 3 mo of the study . (2) Theconnecti ve tissue reaction w as exam ined using only one strength of stimulusand was analyzed using only tw o indices of the platelet connecti ve ti ssuereaction. (3) The ef f ects of aspi ri n and placebo were compared using arandom ized, double-bl i nd design but w i th di f f erent subjects in each group.Thus, the drug ef fect may have been blurred by indiv idual variati ons. (4) Thebleeding time w as performed by the I vy method, w hich is less reproducibleand less sensi ti ve to aspi r i n than the modi f i ed template method used in thesecond study .

I n the second study , an attempt w as made to m inim ize these sources of erroras f ar as possible. T hus, the ef fect of the stimulus w as exam ined over a rangeof connecti v e ti ssue concentrati ons, and the strength of the stimulus wasr igidly standardi zed by using f rozen al i quots of the same connecti ve ti ssuepreparati on during the study , w hich w as l im i ted to a 2-w k per iod. T he ef f ectsof variati on of response betw een subjects w ere m inim ized by using each ashis own control . V ariati on of the response w i thin subjects w as m inim ized bycareful l y standardi zing f actors such as diet, exerci se, smok ing, and diurnalvariati on. I t w as only when these var iables w ere control l ed that a di rectcorrelati on w as found betw een the ef f ect of aspi r i n n the bleeding time andthe platelet connecti ve ti ssue reaction and then only w hen a relati vel y strongstimulus w as used. T he resul ts of our second study , therefore, support thehypothesis that the prolongation in the bleeding time produced by aspi ri n i scausal l y related to suppression of the platelet connecti ve ti ssue reaction.

REFERENCES1. H ov ig, T .: A ggregation of rabbi t blood

platel ets produced in v i tro by sal ine ex -tract of tendons. T hromb. D iath. H ae-morrh. 9:248, 1963.

2. -: Release of a platel et aggregatingsubstance (adenosine diphosphate) f rom rab-bi t blood platelets induced by sal ine ex -tract of tendons. T hromb. D iath. H aemorrh.9:264, 1963.

3. Spaet, T . H ., and Zucker, M . B .: M ech-anism of platelet pl ug f ormati on and roleof adenosine diphosphate. A m . J. Physiol.206:1267, 1964.

4. Z ucker, M . B ., and B orel l i , J.: Platelet

clumping produced by connect i ve ti ssue sus-pensions and by col lagen. Proc. Soc. Exp.B iol . M ed. 109:779, 1962.

5. M ustard, J. F.: H aemostasis and throm-bosis. Sem in. H ematol . 5:91, 1968.

6. H ov ig, T ., Row sel l , H . C ., D odds,W . J., Jorgensen, L ., and M ustard, J. F.:Experimental hemostasi s i n normal dogs andi n dogs w i th congeni tal disorders of bloodcoagulation. B lood 30 :636, 1967.

7. B ettex -Gal land, M ., and L uscher , E. F.:Ex tract ion of an actomyosin l i ke proteinf rom human thrombocy tes. N ature (L ond.)184:276, 1959.



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BLEED IN G T1 ME AN D P LATELE T C ON NEC TIVE TIS SUE 3 7 7

8. M ustard, J. F., and Packham , M . A .:Factors inf luencing platelet f unction, ad-hesion, release and aggregation. Pharmacol .Rev. 22 :97, 1970.

9. O B rien, J. R . : Ef fect of ant i -inf lamm a-tory agents on platelets. L ancet 1 :894, 1968.

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