Journal of Tongll Medical Universtty 19 (2): 102-104, 1999 N ~ ~ ~ ( ~ ] - ~ )

102

E f f e c t of S J A M P on ATP Release of Platelet" GUO Tao ( ~ itS), SHEN Di (~L i~), SONG Shanjun (.~1-1'~), WEI Wenning ( t ~ 5 ~ ) Institute of Hematology, Xiehe Hospaal, Tongfi Medtcal University, Wuhan 430022 Summary: The aggregation and ATP release of placelet of normal subjects were measured by platelet lumi-aggregometer. It was found that the aggregation curve induced by SJAMP at the concentration of 100 mg/L was a typical second phase aggregation. There existed a certain lag between platelet aggregation and secretion. The secretion actually began slightly after the second phase of aggregation, suggesting that the second phase aggregation induced by SJAMP is not dependent upon the release of contents of dense granule alone. If platelets were incubated with cyclo-oxygenase inhibitor, the second phase aggregation was inhibited and no ATP was released. The results indicated that the aggregation and release reaction induced by SJAMP were dependent upon the generation of prostaglandin endoperoxides and TXAz In normal subjects. The amount of ATP release was 0. 69=t=0.22 nmol/108 platelets as stimulated with SJAMP (100 rag/L). But the amount of ATP release were 1.60=1=0. 25 and 1. 374-0. 15 nmol/10 g platelets when platelets were stimulated with ADP (5 btmol/L) and collagen (5 rag/L). The amount of ATP release induced by SJAMP was slgmficantly lower than that of ADP and collagen. These findings indmated that SJAMP was a weaker agomst than ADP in terms of platelets release reaction. Key words: SJAMP; platelet aggregation; ATP release

Secretion of platelets is an agonist-induced response, which is of importance for the enhancement of platelet activation and for the various effects of secreted platelet constituents in other tissues. The secretable substances are stored in the dense granules, the a-granules and acid hydrolase-containing granules of platelets. Upon stimulation the contents of these granules are rapidly emptied into the surrounding media by exocytosis. This secretory step can be analyzed t h r o u g h measuring the secretary contents. The aim of this paper is to further understand the secretory mechanisms of platelets induced with S J A M P by using chrono-log model 600 platelet luml-aggregometer. 1

MATERIALS AND METHODS

1. 1

Reagents Collagen, luciferin/lumferase were obtained from C h r o n o - l o g , USA. A D P and A T P were products of Sigma, USA.

GUO Tao, male, born in 1968, Doctor in Charge " Thxs project was supported by grant from the National Nature Science Foundanon of China (No. 39370322).

S J A M P was from Tianjin Drug Institute. A S A was from the F o u r t h Pharmaceutical F a c t o r y , Wuhan. 1.2 Platelet Preparation H u m a n blood was taken by venipuncture from informed healthy volunteers and was put into 0. 1 m o l / L citrate (1 : 9, V / V ) . T h e blood was centrifuged for 10 min at 150 g at room temperature to separate the platelet-rich plasma ( P R P ) . The left blood was centrifuged for 15 min at 1200 g to obtain platelet-poor plasma ( P P P ). The platelet count of P R P was adjusted to 200 -- 3 0 0 • 109/L with PPP. Care were excised to ensure the accuracy of the counting results. 1.3 M e a s u r e m e n t of P l a t e l e t A g g r e g a t i o n The temperature of the aggregation module was set at 37 C and 450/~1 of P R P was added to aggregation module, with PPP serving as control. After having been warmed for 30 rain, a magnetm stirring bar was introduced. The stirring speed was adjusted to 1000 r / m i n . S J A M P (100 r a g / L ) , ADP (5 g m o l / L ) and collagen (5 m g / L ) were added to P R P , respectively. On the basis of the aggregation curve the maximum aggregation rate, maximum aggregation

GUO Tao et al. Effect of SJAMP on ATP Release of Platelet rate at 5 min were calculated. T h e results were expressed in percentage. 1. 4 Measurement of Platelet ATP Release A T P release from platelet granules was measured concurrently with aggregation by using a lumi-aggregometer. Before measurement, 50 ~tl luciferin/luciferase (40 g / L) was added to 4 5 0 / I L PRP. After warmlng, a magnetic stirring bar was introduced and aggregation agents were added. T h e aggregation and A T P release were measured at the same time. T h e A T P release was calculated with A T P standard by using the following formula : The amount of A T P release = (Fluorescence Recording of M e a s u r e m e n t / G a i n of Measurement) X (Gain of A T P S t a n d a r d / Fluorescence Recording of A T P S t a n d a r d ) • T h e results were expressed as nmol/ 108 platelets. 1.5 E f f e c t of ASA ASA were first pre-incubated with PRP for 30 rain. T h e aggregation and A T P release were concurrently measured by following the above-mentioned steps. 1.6 Statistical Analysis Results were expressed as ~'_+s and the mean values were compared by using variance analysis of rank test. An IBM computer with SAS software package was used for the statistical analysis. 2

103

fig. 1

The aggreagation and release curve of ht man platelets reduced hy SJAMP 2-2 The Effect of Cycio-Oxygenase Ir hibitor on the Aggregation and ATP Relea.~ of Platelet Induced by SJAMP (fig. 2) The fig. 2 showed that when ASA we first incubated with human P R P , the A T release cure was significantly inhibited ind caring that the A T P release was depender upon the pathway of cyclo-oxygenas metabolism.

RESULTS

2. 1 The Aggregation and ATP Release of Human Platelets Induced by SJAMP (fig. 1) As shown by the fig. 1, the aggregation curve was a typical second phase aggregation. The A T P release curve was also shown in the recording. At the end of recording curve, the drop of curve did not mirror the A T P re-absorption or metabolizing by plateIets, but gradual attenuation of fluorescence. The first phase of platelets aggregation induced by S J A M P did not accompany the detectable A T P release. But in the second phase of aggregation, A T P release could be measured, suggesting that A T P release was aggregation-dependent.

fig. 2 The aggregatlon and release curve whr ASA mcuhated with human PRP

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Journal of Tongji Medical University 19 (2): 102-104, 1999

2. 3 The Aggregation and ATP Release Induced by S J A M P , A D P and Collagen The table 1 shows the aggregation and ATP release by SJAMP 100 r a g / L , ADP 5

~tmol/L and collagen 5 mg/L. The results indicated that the ATP release induced by SJAMP was significantly lower than those by other aggregation agents ( P < 0 . 0 1 ) .

Table 1 ATP release and aggregation of normal PRP by different aggregation agents Agents

SJAMP ADP Collal~en

Concentration

n

100 mg/L 5 pmol/L 5 mg/L

10 5 5

ATP release (nmol/108platelet)

" P < 0 . 01 The Effect of C y c l o - O x y g e n a s e Inhibitor ( A S A ) on the ATP Release and Aggregation o f Platelets (table 2) 2. 4

Table 2 shows that ASA could significantly inhibit the ATP release induced by SJAMP and did not have second phase aggregation. There existed significant difference between the two maximum aggregation rates ( P < 0 . 0 1 ) . Table 2

Effect of ASA on platelet aggregation and ATP release by SJAMP

PRP

ATP release (nmol/108platelet)

MAR(0A)

0. 695:0. 22" 0

555:3. 31" 10. 255:6. 20

Normal PRP PRP incubated with ASA " P ~ 0 . 01

3

DISCUSION

The release reaction of platelets refers to the process of secretion of the granular contents of platelets induced by platelet aggregation agents. In human body there exists important relationship between the secretion reaction and the realization of platelet physiological functions, because the most of functions of platelet were realized by biological effects through the substances formed or released .by secretory reaction E~. The formation of large platelet aggregates depends on the formation of arachidonate metabolites and the release of platelet contents E2j. Thus, the secretory reaction of platelet is an important reaction of the realization of platelet physiological functions. In this paper we studied the platelet aggregation and ATP release induced by SJAMP by using lumi-aggregometer. Previous studies demonstrated that there was a increase in [3-TG and PF4 in

O. 69-{-0. 22" I. 60• 25 1.57+0. 15

MAR(~) 55-}-3. 31 60-{-4. 18 55.5+8

MARsm 47.38• 32 60+4. 18 53. 5-t-8

plasma when human platelets were induced by SJAMP, suggesting that SJAMP may have the effect of inducing release reaction of human platelets E33. In our study, we found that the platelet aggregation induced by SJAMP was a typical two phase aggregation and the release reaction was dependent upon the platelet aggregation. There existed a lag between platelet aggregation and release reactton. And the platelet release reaction could be inhibited by cyclo-oxygenase inhibitor ( A S A ) . The curve showed that second phase aggregation actually took place before release reaction, suggesting that the second phase aggregation induced by SJAMP was not only caused by the effect of substance released by platelet dense granules, but also by other substances activated before this event. Because cyclo-oxygenase inhibitor ( A S A ) can inhibit the second phase aggregation and ATP release, the t formation of arachidonate metabohtes (endoperoxides/thromboxane A2) is the key step in the transition of platelet first phase aggregation to second phase aggregation induced by SJAMP. It was also found that the amount of ATP release induced by SJAMP was significantly lower than that by ADP and collagen, indicating that SJAMP is a weaker aggregation agents than ADP in terms of platelet ATP release. From above, we are led to conclude that the release reaction induced by SJAMP is dependent upon the arachidonte metabolites. The ATP release is the results of further activation by positive-feedback loops, which is due to the formation of arachidonate metabolites. The secretion of platelet constituents from dense granules have synergistic effect on platelet aggregation and (Continued on page 107)