B. Wechowicat, T, Krajewski ACTIVATION OF BLOOO PLATELETS ANO PROSTAGLANDIN BIOSYNTHESIS
This article summarizes recent findings concerning the activation of mammalian platelets (adhesion, aggregation and secretion). Special attention is given to the metabolism of platelet arachldonic acid and production of prostaglandin end thromboxane Ao. Recent views on the role of erachidonic acid metabolites in the mechanisms of platelet aggregation, especially platelet-vessel wall Interactions, aro presented.
Introduction
Mammalian blood platelets are the fragments
bone marrow cells called megacaryocytes. They are nonnucleatod .smallest blood cells, approximately 2-3 u in diameter and about 7 in volume.
Blood platelet participate in haemostatis, arterial thrombo-sis, activation of plasma coagulation, maintenance of vascular integrity and may also contribute to atherogenesis and inflamma-tory process. Their physiological and pathological functions are related to their ability tc adhere, aggregate and release their granule contents.
The blood platelet normally circulates as a disc and conta-ins a number of different granules« mainly a-granules and very dense bodies. When stimulated, it changes the shape, ag-gregates and releases the granule contents. This action is ini-tiated by a variety of different agents including: adenosine di-phosphate, thrombin, collagen, serotonin, adrenalin snd thro-mboxane Ag.
Activation of blood platelets by different stimuli consists of morphological and functional change« which follow a membrane signal duo to the interaction of surface receptors with speci-fic substances what leads to a sequential display of certain me-asurable responses of these cell*. The primary stimulus for pla-telet aggregation appears to involve membrane glycoprotein re-ceptors and the first response is e change in cell shape, from a discoidal to a spherical form. This ia followed by the second response - aggregation, in which individual platelets form ag-gregates. This phase can be reversible.
A number of experiments demonstrated that fibrinogen binding to platelets ie important for platelet aggregation ( B e n n e t t , V i 1 a i r e [3]; G r a b e r , H a v i g e r [13]; M a r- q u e r i . P l o w [23]} M a r q u e r i , P l o w , E-d i n g t o n [22]). Fibrinogen receptor exposure on the pla-telet jperobrane and fibrinogen receptor interaction are the most critical events in platelet aggregation induced by many diffe-rent stimuli such an ADP, thrombin, adrenalin, collagen, ara- chidonic acid end prostaglandin endoperoxide analogues. The in vitro aggregation of platelets is presumably a reflection of their m a j o r functions, the formation of the primary haemostatic plug. The photometric measurement of platelet aggregation ( B o r n [4]) is by far most widely used parameter of in' vitro platelet function.
Some etlmuli induce a slight aggregation, called primary ag-gregation, followed in 1-2 minutes by a second wave of aggrega-tion celled secondary aggregaaggrega-tion. Parallely with the second wave of platelet aggregation the platelet secretion, i.e. plate-l e t r s plate-l « E s e reaction occurs. The next responses comprising cel-lular synthesis of prostaglandins and secretion of substances s to r e d in the dense bodies and «-granules are not clearly under-stood yet.
These events represent en ordered sequence of all repponces which are due to a common Intracellular messenger. The induced ceTbrane alterations lead to the same sequential biochemical rea- ction and result in e rise of free Ca ions in the cytoplasm ( D e t w i l e r et ai, [9j).The Ca plays a central role in platelet activation and «ay be a second messenger involved in the transmission of the elgnal from the plasma membrane to the
platelet. Ca2+ mobilized from Intracellular «tore*, most pro- bebly fro« the denee tubular system end membrane-bound sites ( B r a e a, S h a t t l l [5]). The increase of the free Ca2+ ion concentration glvee the typical morphological changes asso-ciated with the activation of contractile system: Co2* the common messenger for the pletelet response. The inhibitory effect of cyclic AMP on pletelet aggregation can be explained partly by a decrease of Ca-concentrations in the platelet cytoplasm (G o- r m a n et al. [l2]). On the other hand, cyclic AMP prevents ex-posure of the fibrinogen receptors on pletelet membrane (G r n- b e r , H a w l g e r [l3 j).
Arachldonlc acid metabolism
Activation of blood platelets induced by several physical and chemical stimuli la accompanied by the synthesis of p r o c t B - glandlns and proataglandin-like compounds which play on
impor-tant role in platelet function ( A l l y , H o r r o b i n [l]i G e r m a n [llli H a r l a n , H a r k a r ^18]j M a 1 in- » t e n 1 19 ] j M a r c u s [20]; M a r c u s [211; M o n - c a d a, A n e z o u a [25]; P i k e et al. [.291).
Proataglandina are not stored but repldly synthatized end released from cells following approplste stimulation. Twenty car-bon polyunsaturated fatty acida esterified to mombrane phospho-lipids are their precursors. These fatty acids are either ob-tained directly from the diet or from elongation of the essen-tial fatty acid, linolelc acid (G a 1 1 1 et al, [ 1 0 ] 5 . In platelets end endothelial cell membranes the twenty corfoon C ; 20 ; t 4 fatty acid with four double bonds, ©icosotetraenoic acid (arachldonlc ecid) la the precursor of the prostaglandins
contai-ning two double bonds.
The development» in ths prostaglandin field in the last few years have substantially enlarged our knowledge of the platelet - vessel wall interactions, haemostasis and thrombosis. Hcwsver, the expansion of the variety of products derived from the enzy-matic oxygenation of arachidonic acid h33 become exceedingly com-plex.
There are two main oxygenation pathway* of arachidonlc acid in platelet - rhc first one, based on the cyclooxygenaee*which leads via tho cyclic endoporoxides PGG^ and PGHj to the prosta-glandins and thromboxane and - the eecond one the llpo- oxygenaee pethway which leads via the firet formed hydroperoxy fatty acid euch a3 12-L-hydroperoxy, 5,8,10,14-elcosetatraenoic acid (HPETE), to a number of futher transformation products in-cluding hydroxy fatty acid 12-L-hydroxy, 5,8,10,14-eicosatetrae- nolc acid (HETE) end to recently diecovered series of compounds - the leukotriens (N u g t e r e n [28]). If the cyclooxygena- se pathway Is blocked, for instance by drugs (aspirin), platelet arachidonlc acid may be metabolized in the llpooxygenase pathway and produce lerge quantities of HETE.
Blood platelets contain very little free arachidonlc acid and thus regulation of prostaglandin synthesis in platelets must oc-cur et the level of arachidonlc acid supply. Wien platelets are stimulated by aggregating agents, arachidonlc ncid i» liberated. Once libereted it Is rapidly metabolized via one of two pathways. Two pho3pholipas*s liberating arachidonlc acid from membrane pho-spholipids have been identified in platelet. Their activities are stimulated by most eggregating agente causing the Increeee of Ca2* ion concentration. Phospholipase ^ is a membrane-bound li-pase which cleaves arachidonlc acid from membrane phosphatidyl-choline and phosphatidylethanolamine ( V e r e t r a e t e [34]), B e l l et al. [ 2 ] provided evidence that the mechanism for arachidonlc acid release from stimulated platelets Involves not only phospholipaee A2 but a phosphatidylinoeitol specific pho- spholip«3e C liberating a digliceride from which in turn era- chidonic acid is released by a membrane-bound dlgllcerlde-llpaee.
In the cyclooxygenese pathway arachidonlc acid ie oxygenated by cyclooxygenese to labile cyclic prostaglandin endoperoxide PGGg with its subsequent reduction to PGHg. The cyclic endope- roxides are intermediates with a half-life of about 5 minutes (R a z et el. [30] which may be spontaneously converted to a 17-carbon compound 12-L-hydroxy, 5,8,10-heptadecatrienoic acid (HHT) with the release of malonyldlaldehyde (P o r t e r [31]). The majority of platelet endoperoxides le converted by the enzyme thromboxane synthetase to the thromboxane Ag , a highly unstable compound ( H a m m e r s t r o m , F a l a r d o e u [16]}
H a m b u r g et al. [15]. It 1* rapidly converted to biolo-gically Inactive and product, thromboxane B2 , formed nonenzyma- tlcaly by incorporation of a molecule of water into TjcA£. Throm-boxane A2 has a abort half-life, approximately 30 seconds in a- queous solution and about 5 minutes in plasma ( N e e d l e -m a n ot al. [ 27J).
The cyclic endoperoxidee may also undergo nonenzymatic con-version to small amounts of the stable prostaglandins, PGE2 , PGF2 but primarily PGD2 ( H a m b e r g et al. [ 1 5 ] ; M a l m s t e n [19]. Prostaglandin endoperoxldes and thromboxane A2 are ex-tremely potent platelet aggregating agents. The mechanism by which prostaglandin endoperoxldes cause platelet aggregation i3 not yet well understood. These compounds being unstable it is difficult to evaluate their mechanism of action. It was found that PGHg could directly cause aggregation of platelets and ex-pose fibrinogen receptors on gelfiltrated platelets; the occupa-tion of prostaglandin endoperoxide receptors on platelet surface is required for the interaction of fibrinogen and platelets (M o~ r l n e l l l et al, [26]). Although the mechanism of TxA2 action in platelet aggregation has not been full understood. It appears to be involved in the regulation of Intraplatelet cyc-lic AMP level. TxA2 does Inhibit cyccyc-lic AMP accumulation ( G o r m a n et al [12]).
Contrary to platelet, in vessel wall free arachldonic acid is rapidly metabolized to cyclic prostaglandin endoperoxldes end futher to prostacyclin which la the mejor metabolite of ors- chldonlc acid In the vessel wall cells such as endothelial cells ( G r y g l e w s k « [14]). Some amounts of PGH^ are converted to the stable protaglandlne, mainly PGEg.
Prostacyclin (PGXg) is a labile compound. It has a half li-fe of about 3 minutes in aqueous solution and slightly longer in plasma. It is hydrolysed to the stable but inactive breakdown product 6-keto PGFj in vitfo. In vivo significant amounts of other metabolites are also produced including dinor 4-keto«7,9, 13-trihydroxy-prosta-li,12, enoic acid and dlnor 4,13, dlketo-7,9- ~dlhydroxyprostan-l,l8-dioic acid ( P i k e et al. [29]).
Prostacyclin stimulates adenylate cyclase to platelets and is therefore a potent antiaggregetory agent ( G o r m a n [11]; G r y g l e w e k i [14],' M o n c a d e , V a n e £.24]; H
o-w 1 g o r et «1. [17]), PGI2 1# also an Inhibitor of fibri-nogen binding by platelets ( H e w l g e r *t al. [17]). This inhibitory effect seems to b* due to enhanced CAMP lavel which prevents exposure of fibrinogen receptor. The vessels produce much more PGI2 than other proatoglandia. Recently the produc-tion of thromboxane a2 by vessel wall was also observed ( A l l y , H o r o b i n [1]). The small amounts of TxAg produced by vessel wall may be Important for function regulation and play a critical role in vessel physiology and pathology.
The role of arachidonate metabolites in platelet-vessel wall Interaction
Prostacyclin produced by vessel wall is a vasodilator. It not o n ly prevents platelets from aggregation In platelet rich plasma but a ls o dissipates preformed platelet clots and circulating p l a t e l e t aggregates in v iv o . Due to its properties PGIg was used in clinical t r i a l s as an antithrombotic agent (G r y g 1 • w- 3 k i [. 14]),
A rach id on ate m e ta b o lite s , especially tho unstable metabolites TxA, and P G I2 modulate many of the complex platslet-vesssl wal reactions ( G o r m a n [11], H a r l a m , H a r k e r [18]i M o n c a d a , V a n e [ 2 4 ] ; M o n c a d a , A m e z o u e [25]). P l a t e l e t s are n orm a lly n on - re a ctlv e to intact vascular e- ndothelium . A t a s i t e of v e s s e l injury platelets adhere to su-bendothelium, aggregate and develop a procoagulant activity which catalyses the i n t r i n s i c blood co a g u la tio n pathway. Plate-lets are thought to a c c e le r a t e coagulation by thrombin genera-tion. Vessel injury i n i t i a t e s platelet adherence with plasma cofectcr (von Willebrands factor). Activated platelets release the active substances in clu d in g AOP, which together with pro-duced TxA^ and thrombin couse fu th e r p l a t e l e t ag gre ga tion and
release of granule contents. Upon s t im u la t io n blood platelets rot only release the v a s o c o n s t r a c t ile compounds - se ro t on in but also synthetize the vasoconstractile PGG2< PGHg, TxA2 and PGF2 through the arachidonic acid cascad e. The relaxing prostaglan-dins such as PGE2 and PGC>2 are a lso produced (F i g . 1 ).
MEMBRANE PI PC
---- T "---
phospholipase C DJ6LYCERI0E » diglycende lipase PHOSPHOLIPIOS PE phoipholipa»« A 2 l'po-oxyg*nasr HYDROPEl&XYEICOSA- TETRAENOIC ACID 5-HPETE ARACHIOONIC ACID ________I__________ p tro x tdasr HVDRO/VPCOSA TETRAENOIC ACID HETE d th y d ra i* l e u k o tÂi e NE A, (LTA/ ) ‘ / \ prostacyclin sythetast p6i2 cyclJo x yg*na»t ENOO PEROXIDE PGG, I / ptraxidas* ENDOPEROXtDE PGH, ---- [—2---I p g d2 p g e2 r « d u c l a s » I PGF2a hydrolysis glutathione hydrolysisS transferase + 5k et o PGF1o(
I
S tränst« t l t b a l t c^ -l t d* (IN BLOOD) thromboxane synthetase TXA, I £ I hydrolysis I I TXB-IN LEUKOCYTES 15 hydroxy-prostoglan din d tx yd rss* 6,15-t)IKETO PGF, IN ENDOTHELIAL CELLS m a i oNDt a l d eh y d e IMOAI HYOROXYHEPTA- DECATRIENOIC ACID IHTTi IN PLATELETSFig. 1. Archldonlc acid metabolism Metabolizm kwasu archldonowego
Thrombin converts fibrinogen to fibrin to stabilize the pla-telet mass. Prostacyclin synthetized by the vessel wall in re-sponse to thrombin limits thrombus formation by inhibiting futher platelet aggregation.
It has been postulated that normal haemostasis represents a balance between platelet TxAg formation and vessel well PGI2 pro-duction. The unbalance between the production of both the com-pounds could lead to thrombosis or bleeding ( P i k e et al, 129]).
PGI, and TxA2 were at first enthueiastlcally Introduced in many aspects of haemostasis and thrombosis ( M a r c u s [20,
they play an important but United role in the physiological mo-dulation of platelet-veesel wall interaction». If the platelet function is inhibited haemoatesia ia likely to be impaired. The complete inhibition of platelet proataglandin production by cy- clooxygenase inhibitora (aspirin) produced only • mild haemosta-tic defect what indicates that platelet arechidonlc acid metabo-lism is not essential for platelet plug formation (H a r 1 a m, H a r k e r [18]), The precise role of these arachidonlc acid metabolites in haemostasis and thrombosis remains to be fully de-fin e d .
Activation pathwaya in platelets
There are th re e d if f e r e n t pathways of platelet activation (V a r g a f t i g at si, [33]). ADP la conaldered to be a f i r s t-pathway of a g g re ga tio n . Early studies suggested that adp re le a se d from the stim u la te d platelets was responsible for tha fu t h e r form a tion of the aggregates. The role of ADP aa a me d ia t o r of agg reg ation caused by o th a r agent* euch aa collagen or thrombin l a supported by i t s dose-dependent releaea during p la t e l e t s t im u la t io n and by re d u ctio n of platelet aggregation In the presence of ADP scavenger. The presence of ADP la required for the exposure of p l a t e l e t surface fibrinogen receptore (B a n- n o t, V i 1 a i r e L 3 3). The stimulated platelets release ADP and fib rin o g e n . Subsequently ADP acts on platelet membrane to expose fib rin o g e n receptors. Fibrinogen than bindo to the exposed receptore what in turn results in platelet aggregation and futher activation.
The arachidonlc acid cascade in presently regarded ae a se-cond pathway for aggregation mediated by produced In platelet TxA_ (V a r g a f t 1 g et al. [33]; V e r s t r a e t a
[34]).
Since platelet activation by thrombin or collagen la neither suppressed by the exhaustion of AOP from the granules nor Inhi-bited by aspirin and other inhibitors of platelet proataglandin biosynthesis, the theory wa* put forward that another mediator of platelet activation is formed in these cells ( C a z e n a v e
e t a l . [ 6 ] ) . Thera «ay e x ie t en a lt e r n a t iv o pathway independent of ADP re la a e e and p ro sta g la n d in form ation in platelet. Platelet a c t iv a t in g f a c t o r ( PA F-a ceth« r) can th e re fo re account fo r tho
HgC-O - (CH2 ) n - C H 3 c h3- c -o - c h 0 11 ! 11 © 0 H2C - 0 - P - 0 - C H 2- C H 2 - N - (CH3 )3 PAF-acether l-0-alkyl-2~0-acethyl-2 an -glyceryl-3-phosphorylcholine
third pathway of platelet aggregation ( V a r g e f t i g et al. [33]). PAF-acether i s a low molecular lipid, namely l-O-ol- kyl-2-O-acethyl,2 sn-glyceryl-3-phoephorylcholine (C u s a k [7]; D e o o p o u l o a et al. [8]s S n y d o r [32] . It is the most potent platelet aggregating agent known to be also rsle- aeed from various cells and participating in the inflammatory process ( V a r g a f t i g et al. [.33]). It is eni of the moat powerful aggregating substances so far described, however, the mode of release of PAF-acether 8nd the mnchani^m of At3 ac-tion is still unknown.
In activation of platelets, synergism between the action of different agents should also be taken into account.
The work was supported by «Project R. XXX.13.
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Department of Biochemistry Institute of Biochemistry and Biophysics
University of Lodz
B. Wachowicz, T. Krajewski
AKTYW*.C0A KRWINEK PŁYTKOWYCH I SYNTEZA PROSTAGLANDYN
Przedstawiono aktualne p o g lę d y dotyczące aktywacji krwinek płytkowych ssaków (adhezja, agregacja, sekrecja ). Zwrócono uwagę na metabolizm kwasu arachidonowego w płytce i wytwarzania troinbo- ksanu A 2. Przedstawiono rolę tromboksanu A2 i prostacykliny w mechanizmach procesu agregacji, a przede wszystkim w interakcji płytki i ściany naczynia krwionoćnego.
