Human plasma kallikrein: Effect on the induced platelet aggregation

THROMBOSIS RESEARCH 48; 81-87, 1987 0049-3848/87 $3.00 t .OO Printed in the USA.

Copyright

(c)

1987

Pergamon

HUMAN

C.M.F.

*Instl tuto S. Paulo, de Medicina.

PLASMA INDUCED

Ltd.

All

KALLIKREIN: PLATELET

CoracSo, Faculdade ** Departamento Caixa Postal 20372

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rights

reserved.

EFFECT ON THE AGGREGATION

Cassaro*, M.U. Sampaio**, D.F. Charnone* and C.A.M.

do and

(Received

Journals

N.Y. Sampaio**

Maeda*,

Universidade de de Medicina, Paul ista de Eioquimica, Escola CEP 04034 S.Paulo, SP, BRASIL

1.9.1986;

Accepted in revised form 7.7.1987 by Editor B. Vargaftig) in final form by Executive Editorial Office 17.8.1987)

ABSTRACT col lagen and aggregation induced by ADP, Platelet concentrations of human adrenaline is increased by low cause aggregation by plasma kallikrein, which does not aggregation induced by inhibits the itself and plasma, arachldonlc acid and Thrombofax in platelet rich thrombin in washed and the aggregation induced by plasma higher concentrations however, platelets. At by al I the kallikrein lnhi bits the aggregation induced agents tested with platelet rich plasma and also causes a decrease in the aggregation induced In whole blood.

INTRODUCTION PI asma kallikrein (E.G. 3.4.21.34) which participates In the early steps blood clotting (1). The hypothesis that provide the negative surface to initiate and It has been shown recently raised (21, can independently promote the activation

Is a serine proteinase of the contact phase of activated platelets could blood clotting has been that a platelet factor of Factor X (3).

Thrombin, which Is also a serine proteinase, can induce platelet aggregation displaying a physiopathological role In thrombogenesis (4). specific enzymes such as trypsin can Less also interfere with platelet physiology (5). Previous observations have shown that plasma kallikrein can decrease the Key

words:

kallikrein,

platelet

aggregation, 81

proteolysis

KALLIKREIN

82

EFFECT ON PLATELETS

Vol. 48, No. I

CAMP content induced to by of platelets which were aggregate (6); also plasma ADP this report states that kallikrein reinforces both waves of aggregation induced by ADP, but does not affect the ristocetin induced aggregation. High molecular weight kininogen was described in a-granules contents (7), and receptors for this glycoprotein were demonstrated in platelets (6). multiple The and thrombogenesis platelet aggregation. determine whether aggregatl on or different agents.

interactions

between components that kallikrein may play The objective of the present kallikrein has a direct effect participate on can aggregation SUggSSt

MATERIAL

AND

of clotting a role study was on platelet induced

in to by

METHODS

Blood obtained from healthy donors was collected in 0.4% rich citrate. Platelet plasma (PRP) was prepared by centrifugation of the blood at 4 minutes, 250x9 for at 20° C. Platelet poor plasma was obtained by centrifugation of the plasma at l,OOOxg 10 minutes, Washed for at 200 G. platelets were : PRP was adjusted prepared from PRP as follows to pH 6.4 with 0.15M citric acl d and suspended In Tyrode without solution calcium and magnesium. Following centrlfugation at l,OOOxg for 10 minutes the separated platelets were washed twice wlth Tyrode solution and the final suspension in the same solution contained 500,000 platelets per ml. ca. Active human plasma kallikrein was prepared by affinity in soybean-trypsin-inhibitor-Sepharose (12). The chromatography SDS-polyacrylamide exami ned enzyme preparation, by gel showed to be only a-kallikrein. Trypsin, ADP and electrophoresis, arachidonic acid were from Sigma (USA); collagen was from Hormon Laboratories (USA). thrombi n was from Roche Cheml e (Germany); Thrombofax was from Ortho (USA). Platelet aggregation, in 0.5 ml PRP, was monltored by the inducing agents (In transmittance increase; kallikreln and the PRP (10). Aggregation of 0.10 ml ) were added simultaneously to washed platelets was performed In the same manner. Aggregatlon in 1 ml whole blood was measured by the change in the impedance of to which kalllkrein (0.01 ml) and the Inducing agent (0.01 blood to 0.02 ml) was added (11). The final concentration of kallikrein specified in each experiment and the lowest concentration of IS enough to cause aggregatlon, was used in each the inducing agent, The concentration ranges were: IO-5 to IO-6 M ADP; 0.1-0.6 case. arachidonate; IO-6 to IO-7 M uglml collagen; go-300ug/ml sodium 0.022 NIH-units/ml (diluted 1~6); adrenaline; Thrombofax thrombin;

the mean6

0.4

ug/ml

Statistical value6 are (SEM).

trypsin.

analysis expressed

was a6

performed mean6

wlth and -+

the paired t-test error of standard

and the

Vol. 48, No. 1

83

KALLIKREIN EFFECT ON PLATELETS

RESULTS in concentrations up to 50 ug/ml added PI asma kallikrein, under to PRP, was able to produce platelet aggregatlon not M ADP caused aggregatlon. condltlons in which IO-5 to IO-6 Kallikrein also does not cause aggregation of washed platelets, under conditions in which the same concentrations of thrombin or trypsin were able to promote a fast rate of aggregation. When kallikreln (I .o-2.0 simultaneously with ADP there was rate of aggregation of the second (Fig. la), but the first aggregation

ug/ml 1 was a significant wave from 0.13 wave was not

added increase to 0.22 affected

to

PRP of the A T/mln .

The potentiation of the collagen Induced aggregatlon by kallikrein was demonstrable for both the latency and the rate of the second wave. The action of aggregation (68X increase) of kallikrein on aggregatlon induced by adrenaline was the significant only in decreasing the latency of the onset of the second wave of aggregation (table I). In some experiments, when adrenaline concentration was not high enough to initiate kalllkreln aggregation, was able to trigger the adrenaline effect.

TABLE Effect I atency

of of

kalllkrein (1.0 the collagen

1

ug/ml) Induced

on the rate aggregation

and on the in PRP.

Control

81 .2

(2

28.7)

0.42

(5

0.25)

Kallikreln

70.3

(+

21.3j*

0.25

(2

O.lEi)*

Latency of the 2nd wave of aggregatlon expressed in seconds. Collagen concentrations indicated in Methods. N=26. * Significantly lower than control in a t-paired test, p < 0.001 At a higher concentration of kallikrsin (IO ug/ml) rate of aggregation induced by both ADP and collagen In PRP decreased. In whole blood, even at the I ower kallikrein concentrations, an inhibition of the aggregation induced by or collagen (Fig. la and lb) was observed. induced

in by

PRP kalllkreln compounds which

also reduced the rate stimulate the synthesis

of of

aggregation thromboxane

the was ADP

a4

KALLIKREIN EFFECT ON PLATELETS

AZ, such platelets lnhiblted concentration on Platelet higher platelets

Vol. 48, No. 1

arachidonic acid or Thrombofax (Fig. 1~). With washed the aggregation Induced by thrombln was also partially by kalllkreln (Fig. 2~). Trypsln, in the same molar of kallikreln in PRP, falled to produce any effect aggregation Induced by ADP, but trypsln treatment, at concentrations, caused 1on of fast aggregat washed (5). as

FIG.

1

(cl

B

C

Relative kallikrein and whole acid (AA) thrombln

C

AA

the presence platelet aggregetlon in rate of (b) collagen, In (a) ADP and (1.0 ug/ml). (c) Aggregation in PRP for arachidonlc blood (E). and Thrombofax (TF), and In washed Platelets (TB). Control (Cl. N= 22 to 28.

of PRP for

DISCUSSION not Induce platelet kalllkrein Itself does The fact that to the restrictive addltlonal evidence aggregation provides an specific has a very action of this serlne proteinase. Thrombln which under physiologlcal actlon on platelets (4), but trypsln, shows a rather i ntense conditions does not act on platelets in vitro (5) probably due to the cleavage of a protein action structure involved ln’the activation of the platelets.

Vol. 48, No. 1

85

KALLIKREIN EFFECT ON PLATELETS

Our results are in agreement with the findings reported by of kallikrein on the second wave (61, on the effect Ohde et al. did not However, our results of aggregation i nduced by ADP. indicate an effect of kallikrein on the flrst wave of aggregation This difference can be due to the as reported by Ohde (6). quantlties of enzyme and inducing agents which we have used. On the other hand these authors dld not observe any effect of plasma was increased from 1.0 to kalllkreln when the ADP concentration for 3.0 uM. In this report, an ADP concentration was defined, each blood sample, to approximately the same rate of give aggregation. The evidence that kallikreln decreases the CAMP content of platelet could explain the sinergistlc effect on the action of other compounds able to cause platelet aggregation by this mechanism. The effects of kaliikrein can not be explained only by the eventual inhibiton of adenyl cyclase because in presence of arachidonic acid which causes the decrease of CAMP (121, kallikrein has an inhibitory activity. The proaggregant effect of arachidonic acid is also due to an increase of the synthesis of thromboxane A2, kallikrein and plasma may be preventing this action, indirectly by acting on some step of this pathway, or by blnding to arachidonic acid and decreasing the concentration of the precursor available for the thrombaxe A2 synthesis. The inhibitory action of high concentrations of kallikrein and on ADP col lagen can not be sxplai ned by any of those mechanisms, but it is similar to the action of another compound, PGE2, which decreases the CAMP content of platelet. Thls agent also potentiates aggregation at low concentration and shows an anti-aggregating action at higher concentrations (13). The actlon proteol ysis of permeability pattern response to different

of the

kallikrein platelet of this stimuli.

also be may membrane, structure, and

due to a thus changing by consequence

I imi ted the

its

The action of plasma kalikrein on whole blood, even at low concentrations, is always inhibitory; this experimental condition is rather different from PRP, for several other factors are present rn blood. Kallikrein is known to be able to release neutra I proteinases from neutrophlls (14), and these enzymes could change the responsiveness of the platelets. Other important condition in whole blood is that the number of platelets is lower than in PRP. The physiological role of kallikrein in thrombogenesis could be explained in the following manner: in early phase of thrombus formation, the platelet concentration increases in the region of lesion and a low amount of kallikrein is activated; platelet aggregation is then potentiated, and as the clotting proceeds, the concentration of kallikrein increases an inhibiton of thrombogenesi s is and observed, the activation of blood factors leads to the formatlon of the clot. In whole blood, in norma I conditions, the amounts of I ow activated kallikrein contribute to keep thrombogenesis inhibited.

KALLIKREIN

86

EFFECT ON PLATELETS

Vol. 48, No. 1

ACKNOWLEDGMENT

The authors are partial financial

grateful support

to FINEP, of this

CNPq work.

and

FAPESP

for

the

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KALLIKREIN EFFECT ON PLATELETS

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