Prog. Lipid Res. Vol. 31, No. 4, pp. 399-416, 1992 Printed in Great Britain.All rights reserved

0163-7827/92/$15.00 © 1992Pergamon Press Ltd

REGULATION A N D ROLE OF PHOSPHOINOSITIDE PHOSPHORYLATION IN H U M A N PLATELETS LISA M. THOMAS a n d BRUCE J. HOLUB Department of Nutritional Sciences, University of Guelph, Guelph, Ontario, N IG 2W I, Canada CONTENTS I. INTRODUCTION A. Phosphoinositides B. Platelet activation--general

399 399 400

II. MASS AND COMPOSITION OF PHOSPHOINOflTIDES IN H U M A N P L A ~ III. In Vitro ENZYMATICMEASUREMENTSOF PHOSPHOINOSITIDEK I N A ~ ACTIVITY IV. EVIDENCEOF PHO~PHOINOSITIDEPHOSPHORYLATIONIN INTACT HUMAN PLATELETS

A. Phosphoinositide intereonversion in unstimulated human platelets B. Enhanced formation of PI-4-P and PI-4,5-P2 in agonist-stimulated human platelets C. Formation of D-3 phosphorylated phosphoinositides in activated human platelets V. REGULATIONOF PHOSPHOINOSITIDEPHO~PHORYLAT1ONIN HUMAN PLATELETS

A. Regulation of PI-4-P and PI-4,5-P 2 formation 1. Protein kinase C 2. Cyclic AMP 3. Calcium 4. Guanine nucleotide binding proteins 5. N-3 fatty acids 6. Protein tyrosine kinases 7. Product inhibition 8. Thromboxane A2 formation B. Regulation of D-3 phosphorylated phophoinositide formation 1. Protein tyrosine kinases 2. Guanine nucleotide binding proteins 3. Protein kinase C 4. Fibrogen-receptor occupancy 5. Platelet-derived growth factor 6. Cyclic AMP VI. ROLE OF PHOSPHOINOSITIDEPHOSPHORYLATIONIN HUMAN PLATELETS

A. Role of PI-4-P and PI-4,5-P, formation 1. Source of second messengers 2. Cytoskeletal remodelling 3. Activation of PKC 4. Movement of calcium across membranes 5. Exocytosis B. Role of D-3 phosphorylated phosphoinositide formation VII. CONCLUSION ACKNOWLEDGEMENTS RE~RENCES

400 401 402

402 403 403 404

404 404 405 405 406 406 406 407 407 407 407 408 408 409 410 410 410

410 410 410 411 412 412 412 413 413 413

I. I N T R O D U C T I O N A . Phosphoinositides Interest in p h o s p h o i n o s i t i d e t u r n o v e r b e g a n as early as 1964 when H o k i n a n d H o k i n I p r o p o s e d the p h o s p h a t i d y l i n o s i t o l ( P I ) - p h o s p h a t i d i c acid (PA) cycle. Since then, interest in the role o f m e m b r a n e p h o s p h o i n o s i t i d e s in cellular signal t r a n s d u c t i o n has risen d r a m a t i c a l l y . M o s t o f the a t t e n t i o n has been focused on the role o f p h o s p h o i n o s i t i d e s in platelets a n d o t h e r cells as a reservoir o f second messengers such as i n o s i t o l - l , 4 , 5 - t r i s p h o s p h a t e (IP3) a n d diacylglycerol ( D G ) . 2'3 Little interest has been p a i d to the p h o s p h o r y l a t i o n o f P I to p h o s p h a t i d y l i n o s i t o l - 4 - p h o s p h a t e (PI-4-P) a n d p h o s p h a t i d y l i n o s i t o l - 4 , 5 - b i s p h o s p h a t e (PI-4,5-P2), except as necessary to replenish the s u p p l y o f PI-4,5-P2 for p h o s p h o d i esteratic cleavage to second messengers. H o w e v e r , recent studies have b e g u n to focus a t t e n t i o n on the p h o s p h o i n o s i t i d e kinases a n d the possible role a n d r e g u l a t i o n o f JPLR 3 I/4,.--E

399

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L.M. TrIOMASand B. J. HOLUn

phosphoinositide phosphorylation in cells.4's Very recently, novel phosphorylated phosphoinositides (phosphorylated at the D-3 position of the inositol ring) have been identified in cells, including platelets. These novel phosphoinositides have been suggested to function in cellular proliferation and transformation, but their role in platelets has yet to be determined. This review will address the recent advances in our understanding of phosphoinositide phosphorylation in human platelets. B. Platelet Activation--General

Blood platelets and their activation are intimately involved in haemostasis, both normal and abnormal. Upon activation by agonists, such as thrombin and collagen, a number of biochemical and physiological changes occur in platelets. The signal generated by the interaction of an agonist with its receptor on platelets is transduced across the plasma membrane by a guanine nucleotide binding protein (G-protein) resulting in the activation of phospholipase C. 6 This enzyme cleaves phosphoinositides producing DG and inositol phosphates. The DG and IP3 produced are believed to be second messengers within the cell for the activation of protein kinase C (PKC) and the release of intracellular calcium, respectively.2'3 The latter two events are thought to be responsible for eliciting the physiological changes in platelets, namely shape change, secretion and aggregation.7 The rise in intracellular calcium and activation of PKC have been implicated in the activation of phospholipase A2.s,9 This enzyme cleaves membrane phospholipids at the sn-2 position resulting in the release of fatty acids, predominantly arachidonic acid (AA). AA is converted to thromboxane A 2 (TxA2), a potent pro-aggregatory agent, which amplifies the initial agonist response by interacting with its own receptors on the platelet membrane, t° II. M A S S A N D C O M P O S I T I O N

OF PHOSPHOINOSITIDES

IN HUMAN PLATELETS

The phosphoinositides, like other phospholipids in platelets, are components of membranes. Whereas PI-4-P and PI-4,5-P2 have been found to be exclusively located in the plasma membrane of platelets, PI appears to reside in both the plasma membrane and endoplasmic reticulum, u Separate pools or compartmentalization of PI, PI-4-P and PI-4,5-P2 have been proposed based on studies with rabbit platelets, t2'~aIn human platelet studies, it was suggested that the phosphoinositides exist in a metabohcaUy homogeneous pool.~4,~S The mass of PI in resting human platelets has been shown to range between 17-19 nmol/10 9 platelets. ~6-t9PI-4-P and PI-4,5-P2 are present at much lower amounts (3 and 1 nmol/10 9 platelets, respectively), such that they represent approximately 15% (for PI-4-P) and 5% (for PI-4,5-P2) of the total phosphoinositides present in human platelets, t7,~s The formation of the conventional polyphosphoinositides PI-4-P and PI-4,5-P2 from the substrate PI are catalyzed by the enzymes PI 4-kinase and PI-4-P 5-kinase, respectively (see Fig. 1). Very recently, the discovery of PI 3-kinase activity in other cell types led several research groups to investigate the potential presence in platelets of phosphoinositides PI-3-P PI-3-P

4-kinase

"

.7 Pl'3'4"Pz . . . . . . .

PI 3-kinase

PI-4-P 3-kinase

-

Pl_4,S.p2 3-kinase

PI-4-P 5-kirmse

PI 4-kinase PI

Pl'3'4'5"P3

~

PI-4-P

~

"

PI-4,5-p

2

FIG. 1. Pathways for the formation of phosphoinositides in plat©lets.

Phosphoinositide phosphorylation

401

phosphorylated at the D-3 position on the inositol ring. The novel phosphoinositides, phosphatidylinositol-3-phosphate (PI-3-P), phosphatidylinositol-3,4-bisphosphate (PI-3,4P2) and phosphatidylinositol-3,4,5-trisphosphate (PI-3,4,5-P3) have been identified in 32p-labelled human platelets 2°-26as well as platelets labelled with [3H]inositol.2°' 2~In general, the levels of these D-3 phosphorylated phosphoinositides in platelets are very minor compared to the conventional phosphoinositides. 24 In 32p-labelled resting platelets, PI-3-P has been detected at levels < 2% of the total PIP isomers; 2~-2x26PI-3,4-P 2 was either not detected 23 or found at < 1 % of the total PIP2 isomers. 2°'21'26 The triphosphorylated phosphoinositide PI-3,4,5-P3 has been either undetected 23,25 in unstimulated platelets or found at < 1% of the level of PI-4,5-P2. 2L26 The fatty acid composition of platelet PI has been analyzed by several researchers. ]6'17'27-29The predominant fatty acids found are stearate and arachidonate, which account for approximately 80-90 mol% of the total fatty acids in PI (stearate, 40-45 tool%; arachidonate, 38-48 tool%). Other minor fatty acids present include palmitate (l-6mol%), oleate (6-13 mol%) and linoleate (0-6m01%). t6:7'27-29 Stearate resides almost exclusively in the sn- 1 position of platelet PI, while arachidonate occupies the sn-2 position. 29'3°The sn-1/sn-2 fatty acid molecular species of PI have been analyzed and the stearoyl/arachidonoyl species found to represent 70-90% of the total molecular species in PI. 29'3° Analysis of the fatty acid composition of the polyphosphoinositides, PI-4-P and PI-4,5-P2, show a predominance of stearate and arachidonate as well. ]7 Presumably, the fatty acid composition of D-3 phosphorylated phosphoinositides in platelets resembles that of PI and the conventional polyphosphoinositides; however, this has not been tested. In this regard, a similarity in the fatty acid composition has been found between other inositol phospholipids and PI-3,4,5-P 3 produced in vivo in rat cerebrum. 3t The similarity in the mol% (of total fatty acids) of stearate and arachidonate in PI and polyphosphoinositides in human platelets suggests the interconversion of the stearoyl/arachidonoyl species of these lipids) 7 III. I N V I T R O E N Z Y M A T I C M E A S U R E M E N T S KINASE ACTIVITY

OF PHOSPHOINOSITIDE

Early in vitro studies with human platelets demonstrated the presence of PI and/or PIP kinase activities by incubating either platelet membrane preparations or platelet homogenate with ATP, MgCI2 and substrate and monitoring product formation. 32-35 PI kinase activity, measured as PIP formation, was shown in these studies to have an absolute requirement for magnesium 34'35and was inhibited by the presence of calcium. 34 Removal of the non-ionic detergent, Triton X-100, from the incubation medium markedly diminished (by 90%) PI kinase activity in a platelet membrane preparation. 35 Cyclic AMP and other adenosine-containing compounds were shown to inhibit PI kinase activity in platelet homogenate. 3' This was thought to be due to the inhibition of the ATP binding site since the catalytic subunit of cyclic AMP-dependent protein kinase was not inhibitory to PI kinase activity. In another study, the catalytic subunit of cyclic AMP-dependent protein kinase was shown to actually stimulate phosphoinositide formation. 32 Membranes prepared from platelets have also been shown to phosphorylate the exogenously-added substrate, lysoPI. 35 LysoPI kinase activity was also dependent on magnesium and ATP, but less dependent on the presence of Triton X-100 compared to PI kinase activity. More recently, investigators have attempted to partially purify and characterize PI and PIP kinases from human platelets. 36'37About 80% of platelet PI kinase activity was found associated with the membrane fraction and 20% with the cytosolic fraction. For PIP kinase activity, 90% was associated with the platelet membrane fraction. 36 Extraction of the membrane fraction with KC1 released 98% of the membrane-associated PIP kinase activity, suggesting that most of the PIP kinase was loosely associated with the membrane. Conversely, there appears to be a tight association between PI kinase and the membrane since extraction with Triton X-100 was required to release the majority of the PI kinase activity (74%) from the membrane. 36 In this study 36, the Triton X-100 non-extractable

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L.M. THOMASand B. J. HOLUB

fraction of plateletmembranes, which has been shown to correspond to the cytoskeleton,38 also possessed PI kinasc activity.This amounted to about 16% of the membrane activity or approximately 13% of total platelet PI kinasc activity. The presence of at least 3 0 % of total platelet PI kinasc activity in the cytoskeletal fraction of platclets was suggested in an earlier study.3z Very recently, PI 4-kinase, PI 3-kinase and PI-4-P 5-kinase activities have been found associated with the cytoskelctal fraction of platelcts.39 In addition, thrombin stimulation greatly enhanced the activitiesof these enzymes in the cytoskeletal fraction. Increased enzyme activitiesmay have resulted from the activation of cytoskeletalassociated enzymes and/or from an increase in total enzymes associated with the cytoskclctons.39 T w o forms of membrane-associated and two forms of cytosolic-associated PI kinascs were isolated from human platclcts. The products of the two PI kinases from the membrane fraction were determined to bc PI-4-P, thereby suggesting the presence of PI 4-kinases.36 T w o forms of membrane-associated PIP kinasc were also obtained. By way of product analysis, one form of PIP kinasc was shown to bca PI-4-P 5-kinase.37The two forms of PI kinase isolated from the platelet membrane fraction appear to be distinct36, as do the two forms of membrane-associated PIP kinasc.37 In contrast, the two cytosolicassociated PI kinases appear to have no significant difference in biochemical characteristics.3~One type of membrane-associated PI kinasc partiallypurified from human platclcts was weakly stimulated by Triton X-100 while the other type was rather inhibited by this detergent.36 In contrast, both types were markedly inhibited by the presence of the ionic detergent, sodium cholate. The activitiesof the cytosolic PI kinases purified from platclcts were dramatically enhanced by sodium cholatc and only slightly stimulated by Triton X-100. 36 Membrane-associated PIP kinase activitieswcrc both enhanced by the presence of sodium cholate, but not by Triton X-100. 37 PIP kinase was also slightly inhibited by calcium in the presence of magnesium. 37 Regarding the effectsof Triton X-100, a very recent study on a partiallypurified platelct membrane PI kinase demonstrated that Triton X-100 was stimulatory to PI kinasc activity when present at low concentrations and inhibitory to this enzyme at higher concentrations.4° Furthermorc, the optimal Triton X-100 concentration was dependent on the PI concentration utilized. PI kinasc activity, however, was independent of Triton X-100 concentration when PI concentration was expressed as its micellar surfacic concentration instead of as its bulk concentration in solution. These authors suggested that the rate-limiting factor for in vitro platelet PI kinase activity was an intramiccllar reaction.4° Recently, the existence of a novel PIP kinase in platclcts was demonstrated. Platclct homogcnatc incubated with magnesium, A T P and the substrate PI-3-P produced PI-3,4-P2, demonstrating PI-3-P 4-kinase activity in platelcts.4t It was further determined that this PIP kinasc activity was present in both particulate and cytosolic fractions of platelets. Comparison of PI-3-P 4-kinase activitywith PI 4-kinase activityshowed distinctdifferences regarding detergent effects.Whereas PI 4-kinasc activitywas enhanced by detergent, PI-3-P 4-kinasc was dramatically inhibited by the presence of detergent.4'

IV. EVIDENCE OF PHOSPHOINOSITIDE PHOSPHORYLATION IN INTACT HUMAN PLATELETS A. PhosphoinoMtide Interconversion in Unstimulated Human Platelets

The phosphorylation of PI to PI-4,5-P2 in platelets occurs by two separate ATPdependent kinase reactions (see Fig. 1). PI-4,5-P2, in turn, is dephosphorylated in two steps to PI via phosphomonoesterase activities. 42 Evidence for the phosphorylation of PI to PI-4-P and PI-4,5-P 2 in unstimulated platelets has been obtained by incubation of platelets with 32p-labelled inorganic phosphate ([32P]P3.'2a4,'5 Radioactive Pi is readily incorporated into metabolic ATP within platelets which is then utilized in the phosphorylation of PI to polyphosphoinositides. In addition, incubation of [3H]inositol-labelled, electropermeabilized platelets with ATP also resulted in the phosphorylation of PI to PI-4-P and

Phosphoinositide phosphorylation

403

PI-4,5-P2 .43 The phosphorylation/dephosphorylation of phosphoinositides constitutes a futile cycle consuming metabolic ATP. Verhoeven et aL ~4 demonstrated a very high rate of turnover of the phosphomonoester groups of the phosphoinositides in unstimulated platelets. This futile cycling of phosphoinositides was shown to account for about 7% of the basal ATP consumption in resting platelets) 4 It is not known yet which pathway or pathways might be involved in the formation of D-3 phosphorylated phosphoinositides in platelets. PI 3-kinase activity in platelets has not yet been purified, but has been immunoprecipitated with protein tyrosine kinase. 44 A very low level of PI-3-P has been demonstrated in unstimulated platelets. 2~-23~6In addition, trace levels of PI-3,4-P 2 and PI-3,4,5-P3 have been detected in resting platelets. 2°~,26 Possible routes of synthesis of these latter two phosphoinositides are depicted in Fig. 1. PI-3,4-P2 and PI-3,4,5-P3 may be formed via PI 3-kinase(s) activity on the substrates PI-4-P and PI-4,5-P2, respectively.2°'2~In this regard, a purified PI 3-kinase was shown to phosphorylate PI, PI-4-P and PI-4,5-P2. 4 Alternatively, the enzymes PI-3-P 4-kinase 4t and PI-3,4-P 2 5-kinase may sequentially phosphorylate PI-3-P to form PI-3,4-P 2 and PI-3,4,5-P3, respectively. 24 Whether or not the D-3 phosphorylated phosphoinositides participate in a phosphorylation/dephosphorylation cycle in unstimulated platelets like the conventional phosphoinositides remains to be determined. The existence of a PI-3-P 3-phosphomonoesterase in NIH 3T3 cells has been documented. 45 In addition, neutrophils possess a 5-phosphomonoesterase activity towards PI-3,4,5-P 3 as well as 3-phosphomonoesterase and 4-phosphomonoesterase activities toward PI-3,4-P2. 46 B. Enhanced Formation of PI-4-P and PI-4,5-P2 in Agonist-Stimulated Human Platelets

Stimulation of human platelets with various agonists results in changes in the levels of the conventional polyphosphoinositides. It should be noted that these alterations are in the steady state levels, since the phosphomonoester groups of these phosphoinositides are in constant turnover as mentioned in the previous section. Early studies in thrombin-stimulated human platelets demonstrated a transient fall in the mass or [3H]glycerol-labelled level of PI-4,5-P2 at about 5-10 see, presumably due to phospholipase C degradation, which was followed at later stimulation times by a rise beyond basal levels, tT'ls The level of PI-4-P either decreased or remained unchanged. However, activation of platelets by thrombin in the presence of [32p]PI resulted in an increase in labelled PI-4-P as well as PI-4,5-P2, indicating phosphorylation of PI and PI-4-P, respectively.47 Similar to the thrombin-stimulated platelet data, decreases in PI-4-P and/or PI-4,5-P2 were followed by rises in both these polyphosphoinositides at later times of stimulation by the agonists collagen, 4s-52 U-46619, 5'-53 or the calcium i0nophore, A23187.53 A more recent study by Lassing and Lindberg 54 found increased phosphoinositide levels with very early, low-dose thrombin stimulation which preceded PI-4,5-P2 hydrolysis via phospholipase C. These increases in polyphosphoinositides are presumably due to an enhanced flux of PI to PI-4-P and PI-4,5-P2, although decreased phosphomonoesterase activities may also contribute to the enhancement of polyphosphoinositide levels. C. Formation of D-3 Phosphorylated Phosphoinositides in Activated Human Platelets

Based on 32p-labelling studies, the levels of D-3 phosphorylated phosphoinositides are very low ( < 2 % ) relative to the conventional phosphoinositides in resting platelets. 24 However, dramatic increases in these novel phosphoinositides have been observed upon platelet activation. ~°-26Stimulation of platelets with thrombin produced a substantial rise (as much as 10-fold) in PI-3,4-P 2 compared to basal levels.2°-25The increased PI-3,4-P 2, however, represents only a minor component of the total PIP2 isomers in stimulated platelets. Sultan et aL23 calculated that PI-3,4-P z, at its maximum level, represented 1 1% of the total PIP2 isomers. The novel triphosphorylated PI-3,4,5-P3 also increased significantly upon thrombin stimulation. 21'25~6Recently, the thrombin-induced formation of D-3 phosphorylated phosphoinositides was shown to be due to thrombin's proteolytic activity

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L.M. THOMASand B. J. HOLUB

rather than saturable thrombin binding. 55 The thrombin-induced rise in PI-3,4,5-P3 in platelets peaked early (30 sec2~ or 90 sec2s of stimulation) and then gradually declined, but remained significantly elevated at later times of activation. In contrast, PI-3,4-P2 was only slightly elevated with early thrombin stimulation and then reached its peak at 5 min of stimulation before gradually declining. 2~-23'25PI-3,4-P2 and PI-3,4,5-P3 remained significantly elevated above basal values with stimulation times as long as 10 or 20 min. 23'25 The TxA2 analogue, U-46619, was shown to stimulate PI-3,4-P2 and PI-3,4,5-P3 formation 2~. In contrast to the thrombin-induced changes in PI-3,4-P2 and PI-3,4,5-P3, the extremely low level of PI-3-P in resting platelets remained unchanged with platelet activation. 21,23.26 The increase in 32p-labelled D-3 phosphorylated phosphoinositides upon platelet activation may only indicate a rapid phosphorylation/dephosphorylation of the phosphate groups with no real increase in mass of these phosphoinositides. However, thrombin stimulation of [3H]inositol-labelled platelets also produced a dramatic rise in PI-3,4-P2, suggesting a net accumulation of this phosphoinositide in stimulated platelets.2°'21We have also observed a significant collagen-stimulated or U-46619-stimulated formation of [3H]inositol-labelled PI-3,4-P2 from undetectable levels in human platelets (D. C. Gaudette, R. C. Chapkin and B. J. Holub, unpublished observations). There are two possible routes of synthesis of D-3 phosphorylated phosphoinositides in stimulated platelets, as has already been discussed for resting platelets (see Fig. 1). Based on differences in the relative specific radioactivities of the phosphate groups in these phosphoinositides in thrombinstimulated platelets, Cunningham et al. 24 concluded that the predominant pathway for synthesis of these lipids was conversion of PI to PI-3-P to PI-3,4-P 2 to PI-3,4,5-P3. Recently, a PI-3-P 4-kinase activity has been described in plateletsfl The marked differences in the time-dependent increase in PI-3,4-P2 (peaking at 5 min) and PI-3,4,5-P3 (peaking at 30-90 sec) in thrombin-stimulated platelets suggests that different routes of synthesis may be operative for these two phosphoinositides. PI-3,4-P 2 may be synthesized via PI-3-P 4-kinase activity, while PI-3,4,5-P3 formation may occur via PI 3-kinase activity on the substrate PI-4,5-P2: Alternatively, if PI-3,4-P2 and PI-3,4,5-P3 were both formed via PI 3-kinase activity, then the different times of peak formation of these phosphoinositides may be due to differences in substrate affinity. In this regard, PI-4-P does not appear to be as good a substrate for purified PI 3-kinase as does PI-4,5-P2 .4 A third explanation for the differences in peak formation of PI-3,4-P2 and PI-3,4,5-P 3 may be that PI-3,4-P: is derived from the hydrolysis of PI-3,4,5-P3. Although not shown in platelets, a PI-3,4,5-P3 5-phosphomonoesterase activity was demonstrated in neutrophil membranes:6 V. REGULATION OF PHOSPHOINOSITIDE PHOSPHORYLATION IN HUMAN PLATELETS A. Regulation of P I - 4 - P and PI-4,5-P2 Formation 1. Protein Kinase C

Agonist-stimulation of platelets results in a receptor-mediated breakdown of PI-4,5-P2 and subsequent activation of PKC. Although the main PKC substrate in platelets (47 kDa protein) has been identified, the function of this protein in platelets has yet to be elucidated. However, other possible functions of PKC in platelets have been suggested. These studies have been aided by the use of phorbol esters which activate PKC independently of receptor involvement, and protein kinase inhibitors which inhibit PKC activity as measured by 47 kDa protein phosphorylation. Following the incubation of platelets with phorbol ester, the levels of 32p-labelled PI-4-P (especially) and PI-4,5-P2 increased markedly.2~'22'56-5s Concomitant with the rise in these phosphoinositides was a fall in PI, but no increase in phosphatidic acid (PA) was detected:6 The protein kinase inhibitors H-7 and staurosporine, which diminish PKC activation, attenuated the phorbol ester-induced rise in polyphosphoinositides:2'58 The DG analogue 1-oleoyl 2-acetylglycerol, which also stimulates PKC activation, produced a rise in platelet PI-4-P levels)9 These studies suggest that

Phosphoinositide phosphorylation

405

PKC activation via phorbol ester or DG analogue treatment in platelets enhances the phosphorylation of PI to PI-4-P and PI-4,5-P2. More recently, agonist-induced phosphoinositide phosphorylation was shown to be affected by an inhibitor of PKC. Increases in PI-4-P and PI-4,5-P2 in human platelets stimulated with the agonists collagen or U-46619 were blocked by the protein kinase inhibitor staurosporine 5~ at concentrations shown previously to inhibit 47 kDa protein phosphorylation in stimulated human platelets. 6° From this study, it can be concluded that PKC may positively regulate agonist-induced phosphoinositide phosphorylation. In this respect, Steen et al. 6m demonstrated that the activities of the phosphoinositide kinases/phosphomonoesterases were closely correlated with receptor-controlled phospholipase C activation. They suggested that phospholipase C reaction products/effects (e.g. PKC activation) may regulate phosphoinositide kinase/phosphomonoesterase activities. Very recently, a proteolytic fragment of PKC called kinase M, but not native PKC, was shown to possess PI-4-P kinase activity in vitro. 62 It was suggested that the activation and translocation of PKC to the membrane leading to calpain-induced PKC proteolysis produces kinase M which then serves to phosphorylate PI-4-P and restore the level of PI-4,5-P2 previously depleted by phospholipase C activation. 2. Cyclic A M P

An elevation of intracellular cyclic AMP and activation of cyclic AMP-dependent protein kinase in platelets is inhibitory to platelet activation. Interestingly, treatment of a2p-labelled platelets with agents known to increase intracellular cyclic AMP (such as prostacyclin and prostaglandin E~) or treatment with dibutyrl cyclic AMP resulted in an enhancement of labelled PI-4-P63,~4 and the phosphorylation of a 24 kDa protein. 63 A concomitant decrease in PI, but no change in PI-4,5-P2 or PA were also observed.~ In a platelet plasma membrane preparation incubated with 32p-labelled ATP, the catalytic subunit of cyclic AMP-dependent protein kinase stimulated phosphoinositide formation and the phosphorylation of a 24,000 Mr protein 32. These results suggest that a rise in platelet cyclic AMP levels leading to activation of cyclic AMP-dependent protein kinase produces a positive regulatory signal to PI-4-P (especially) and PI-4,5-P2 formation. Furthermore, the phosphorylation of a 24 kDa protein by cyclic AMP-dependent protein kinase may be responsible, directly or indirectly, for the enhanced phosphoinositide formation. 32'63 Phosphorylation of a 22-24 kDa protein in platelets by cyclic AMPdependent protein kinase has been reported to play an essential role in intracellular calcium regulation. 65'~ The effect of calcium on phosphoinositide phosphorylation is discussed in the next section. 3. Calcium

Available evidence indicates that the presence of calcium in platelets is inhibitory to phosphoinositide phosphorylation. In vitro PI kinase activity measured in a platelet homogenate was markedly inhibited by calcium. ~ Partially purified PIP kinase (PI-4-P 5-kinase) was also slightly inhibited by calcium. 37 In intact platelets, the chelation of intracellular calcium with the fluorescent indicator quin 2-AM produced a dose-related rise in PI-4-P. 64 Similarly, the calcium antagonist verapamil induced a rise in polyphosphoinositide level in unstimulated platelets. 67 The ADP-induced formation of PI-4-P and PI-4,5-P2 in platelets incubated in a low calcium medium was not evident when platelets were incubated in a medium containing a physiological calcium concentration. ¢ It appears, then, that phosphoinositide phosphorylation is negatively regulated by calcium and that this effect may occur directly at the phosphoinositide kinases. ~'37 The positive regulation of platelet phosphoinositide phosphorylation by PKC and cyclic AMP may be related to the alteration of intracellular calcium by these agents. Both the elevation of cyclic AMP and the activation of PKC stimulated the return of calcium to resting levels in activated platelets. 69.7°Three mechanisms may function to restore calcium

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L.M. THOMASand B. J. HOLUB

to basal levels following stimulation of platelets. These include the inhibition of calcium influx from the extracellular compartment, calcium efflux to the outside of the platelet via a calcium/ATPase pump and sequestration of calcium into intracellular storage organelles. 7~ Both the activation of PKC and a rise in cyclic AMP have been shown to cause the sequestration of calcium in platelets. 69'7°'72 In addition, Pollock et al. 73 demonstrated an enhanced calcium efflux by phorbol ester treatment, suggesting an effect of PKC on a plasma membrane calcium pump. 4. Guanine Nucleotide Binding Proteins

In other cellular systems, evidence suggests the presence of a stimulatory G-protein involved in PI-4-P kinase activation. GTP or GTP analogues (100/~M) enhanced the formation of PI-4,5-P2 via PI-4-P kinase in rat brain membranes. 74'75In contrast, PI-4-P formation was minimally or not stimulated by GTP or GTP analogues. In rat liver membranes, a small molecular weight (20-25 kDa) G-protein appears to activate purified PI-4-P kinase in vitro. 76"77 The possible involvement of a G-protein in phosphoinositide phosphorylation in platelets has not been extensively studied; only two reports have appeared in the literature to date. In permeabilized platelets incubated with ATP, 100/ZM GTP~,S appeared to be inhibitory to PI-4,5-P2 (especially) and PI-4-P formation, z1'43 However, decreasing the concentration of GTP analogue dramatically changed the effect on phosphoinositide levels. In permeabilized human platelets in the presence of 32p-labelled ATP, 0.1-10 #M GTP~ S enhanced the level of PI-4,5-P2 substantially, with minimal or no effect of low levels of GTP~S on PI-4-P. 2t This evidence suggests the possible existence of a concentration-dependent effect of GTP analogue on phosphoinositide phosphorylation in platelets, with positive and negative effects at low and high concentrations of GTP~ S, respectively. Alternatively, high levels of GTP analogue may increase PIP2-specific phospholipase C activity, thereby causing a decrease in PIPs and masking any stimulatory effect of GTP analogue on PIP2 formation. 5. N - 3 Fatty Acids

The n-3 fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) found in fish/fish oils have a dampening effect on platelet reactivity. 7s'79 This may contribute to the reported protective effects of n-3 fatty acids against cardiovascular disease. 8° Evidence indicates that n-3 fatty acids have a suppressive effect on phosphoinositide phosphorylation in collagen-stimulated human platelets. Fish oil consumption significantly reduced the collagen-stimulated accumulation of PIP 2 and concomitantly increased PIP levels, suggesting a dampening of PIP kinase activity. 8~This effect may have been mediated, in part, by a decreased formation of TxAz, since fish oil consumption is known to reduce TxA2 synthesis 82"s3and collagen-stimulated phosphoinositide phosphorylation appears to be dependent on TxA2 formation. 5] EPA and DHA found in fish oils may also reduce collagen-stimulated phosphoinositide phosphorylation by inhibiting the binding of TxA2 to its receptor on platelets. ~ Human platelets exposed to albumin-bound DHA, in vitro, showed a marked attenuation of the collagen-induced rise in PI-4-P and PI-4,5-P2. 5~ Albumin-bound EPA was without effect. The suppression of phosphoinositide formation by DHA in this system may also be explained, in part, by a lessened synthesis of TxA 2, since U-46619 (a TxA2 analogue)-induced phosphoinositide formation was unaffected by albumin-bound DHA. 5z The dampened PI-4-P and PI-4,5-P2 synthesis in collagen-stimulated platelets by n-3 fatty acids may partly account for the reduction in platelet reactivity seen in fish/fish oil consumers. 6. Protein Tyrosine Kinases

Protein tyrosine kinases have been found associated with PI 3-kinase activity in growth factor and oncogenically-transformed cells. 4,s The regulation of D-3 phosphorylated

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phosphoinositide generation by protein tyrosine kinases is discussed in a later section. Platelets possess a significant amount of pp60C"C-related tyrosine kinase activity,s5 The possible regulation of PI-4-P and PI-4,5-P2 formation by tyrosine kinases has been investigated in U-46619-stimulated human platelets. ~ Preincubation of platelets with genistein, a specific inhibitor of tyrosine kinases, s7 markedly inhibited U-46619-induced formation of polyphosphoinositides, PIP and PIP2 .~ More recently, we have separated the phosphoinositide isomers and determined that genistein or tyrphostin, another inhibitor of tyrosine kinases, as significantly inhibit U-46619-induced PI-4-P and PI-4,5-P2 formation (D. C. Gaudette, R. C. Chapkin and B. J. Holub, unpublished observations). These results suggest a positive regulation of these phosphoinositide formations by tyrosine kinases. Genistein also markedly inhibited (by 72%) PA formation, suggesting an attenuated phospholipase C response in these U-46619-stimulated platelets (D. C. Gaudette and B. J. Holub, unpublished observations). Dampened phospholipase C activity would lead to decreased PKC activation which may have contributed to the lessened phosphoinositide generation. Recently Grondin et al. 39 described the association of PI 4-kinase and PI-4-P 5-kinase activities with the cytoskeleton of platelets. Upon thrombin stimulation, the cytoskeletal activities of these enzymes increased dramatically. Also following thrombin stimulation, they observed a translocation of the tyrosine kinase ppr0 ¢.... to the cytoskeleton. They suggest a possible regulation of these enzymes by tyrosine kinase may occur in the cytoskeletal fraction. 39 7. Product Inhibition

PI-4,5-P2 appears to be inhibitory to PI-4-P kinase activity in rat brain, 89bovine retina9° and human red blood cells. 91 Although human platelet PI-4-P 5-kinase was recently partially purified, the effect of PI-4,5-P2 on this enzyme was not investigated. 37 It has been suggested that degradation of PI-4,5-P2 via phospholipase C activation may remove the product inhibition on PI-4-P kinase thus stimulating phosphoinositide phosphorylation, s9'9°In collagen-stimulated platelets in the presence of an inhibitor to TxA2 formation, there was a significant decrease in PI-4,5-P2 which was not followed by a rise in polyphosphoinositides. 5t This suggested that, in platelets, a decrease in PI-4,5-P: may not be an efficient stimulus for phosphoinositide kinase activity following collagen activation. Therefore, PI-4,5-P 2 may not be inhibitory to phosphoinositide generation in platelets. 8. Thromboxane A e Formation

TxA2 is formed in stimulated platelets from released arachidonic acid. Through the interaction with its own receptors, TxA2 amplifies the signal generated by the initial agonist response. The activation of platelets by certain agonists, such as collagen and ADP 7 and by calcium ionophore 53 is dependent on the formation of endogenous TxA2. This is reflected in various biochemical responses in stimulated platelets, including phosphoinositide phosphorylation. Collagen stimulation results in significant increases in the phosphoinositides, PI-4-P and PI-4,5-P2. Inhibition of TxA2 production completely blocked the collagen-induced phosphoinositide phosphorylation. 5~ Similarly, the formation of phosphoinositides induced by the calcium ionophore, A23187,was inhibited by prior treatment with an inhibitor to TxA2 synthesis.53 B. Regulation o f D-3 Phosphorylated Phosphoinositide Formation 1. Protein Tyrosine Kinases

The association of growth factor and oncoprotein receptors of the protein-tyrosine kinase family with PI 3-kinase suggests that products of this enzyme, namely PI-3,4-P2 and PI-3,4,5-P3, may be critical growth regulatory signals. 4'5 Platelets also contain a considerable quantity of ppr0c"C-related tyrosine kinase activity,s5 Indirect evidence has been

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documented for the regulation of PI 3-kinase by tyrosine kinases. It has been proposed that the activation of these tyrosine kinase-related receptors causes the recruitment of PI 3-kinase from the cytosolic compartment to the cytoplasmic domain of the receptor, in close proximity to the possible substrates, PI-4-P and PI-4,5-P2 .4 In this respect, the pp60 c.... in platelets has been located in dense bodies,92 as well as the plasma membrane and open canalicular system of platelets. 93 During the recruitment of PI 3-kinase, it was shown to be phosphorylated on tyrosine and serine residues. 4 Whether or not this phosphorylation is involved in the activation of PI 3-kinase is yet to be determined. In thrombin-stimulated platelets, PI 3-kinase was immunoprecipitated along with pp60 o's'c, suggesting an association between PI 3-kinase and a protein tyrosine kinase. 44 Very recently, Grondin et al. 39 found an association of PI 3-kinase with the cytoskeletal compartment in human platelets which increased substantially following thrombin stimulation. Interestingly, thrombin stimulation also promoted a translocation of pp60 c.... to the cytoskeletons. These authors suggest that regulation of PI 3-kinase by pp60¢srC-related tyrosine kinase may occur in the cytoskeletal domain. 39 More direct evidence for a regulation of D-3 phosphorylated phosphoinositide formation in platelets by tyrosine kinases has been obtained in our laboratory. Previously, we demonstrated an inhibition of PIP and PIP2 formation in U-46619-stimulated platelets by the tyrosine kinase inhibitor, genistein, s6 In this latter study, the phosphoinositide isomers were not separated. More recently, we have separated the isomers and determined that U-46619-induced formation of PI-3,4-P2 was completely suppressed by the tyrosine kinase inhibitors genistein and tyrphostin (D. C. Gaudette, R. C. Chapkin and B. J. Holub, unpublished observations). Whether this reflects an inhibition of PI 3-kinase activity (on the substrate PI-4-P) or an inhibition of PI-3-P 4-kinase activity has yet to be determined. 2. Guanine Nucleotide Binding Proteins

Permeabilized human platelets incubated with ATP and GTP~S showed marked formations of D-3 phosphorylated phosphoinositides. GTPyS, at a concentration of 10/~u, stimulated PI-3,4-P 2 and PI-3,4,5-P3 formations by over 3700% and 450% of basal levels, respectively.2~ Thrombin, on the other hand, enhanced D-3 phosphorylated phosphoinositide formation to a much lesser extent than GTP~S. The addition of GTP analogue to permeabilized platelets also substantially increased PI-3-P levels, whereas thrombin or U-46619 did not. 21 These results clearly suggest a role for a GTP-binding protein in PI 3-kinase activation in platelets. The rise in D-3 phosphorylated phosphoinositides in activated neutrophils also appears to be mediated by a G-protein .94 3. Protein Kinase C

Recent evidence in platelets suggests that PKC positively regulates the generation of D-3 phosphorylated phosphoinositides. Phorbol ester treatment of platelets stimulates the formation of PI-3,4-P: and PI-3,4,5-P3. 2~'22The phorbol ester-induced rise in PI-3,4-P2 was inhibited by a protein kinase C inhibitor, staurosporine. :2 Thrombin-induced D-3 phosphorylated phosphoinositide formation was also markedly attenuated by staurosporine 22 and by RFARK, a pseudosubstrate peptide which is a highly specific inhibitor of serine/threonine-directed PKC. 26 GTPyS-induced formation of PI-3,4-P: and PI-3,4,5-P3 was also markedly inhibited by RFARK. 26 In addition, a serine/threonine phosphatase inhibitor (okadaic acid) enhanced p47 protein phosphorylation and promoted PI-3,4-P 2 generation. 26 A comparison of phorbol ester-induced and thrombin or GTP~ S-induced formation of D-3 phosphorylated phosphoinositides reveals that the activation of PKC in itself is not sufficient to fully stimulate the formation of these novel phosphoinositides 2~. King et al. 26 hypothesized that this may be due to a diffuse activation of PKC by phorbol ester versus a more localized activation of PKC by thrombin or GTPyS. PKC activation, however,

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does appear to be critical for receptor-induced and post-receptor-induced formation of PI-3,4-P 2 and PI-3,4,5-P3. 26 Mitchell et al. 95 reported the formation of a complex between PI 3-kinase, a proteinserine/threonine kinase and an uncharacterized platelet membrane protein upon thrombin stimulation of platelets. They proposed that this complex formation, which occurred rapidly (30 sec), may initiate the generation of D-3 phosphorylated phosphoinositides. It was not determined whether the protein-serine/threonine kinase was PKC. However, King et al. 26 reported an unpublished observation that PI 3-kinase activity on the substrates PI and PI-4-P are co-localized with PKC in a sub-fraction derived from thrombin-stimulated platelets. Phosphotyrosine-containing proteins were not detected in the complex discovered by Mitchell et al. 95 The pseudosubstrate peptide, RFARK, was shown not to inhibit tyrosine phosphorylation in platelets. 26 This indicates that the diminished formation of D-3 phosphorylated phosphoinositides in thrombin or GTP?S-stimulated platelets by R F A R K does not involve inhibited tyrosine phosphorylation. This would suggest that PKC has a separate or additional role from the possible involvement of tyrosine phosphorylation in D-3 phosphorylated phosphoinositide generation. 26PKC may modulate agonist/receptor binding, receptor/G-protein coupling, G-protein function, G-protein/enzyme coupling and/or enzyme (e.g. PI 3-kinase) activity. The inhibition by R F A R K of PI 3-kinase and PI-4-P 3-kinase product accumulation in a Triton soluble fraction from platelets suggests a direct effect of PKC on 3-kinase activity. 26 4. F i b r i n o g e n - R e c e p t o r O c c u p a n c y

Fibrinogen is an adhesive protein stored in the at-granules of platelets which becomes secreted upon platelet activation. The binding of secreted fibrinogen to activated glycoprotein IIb-IIIa (GPIIb-IIIa) on the platelet surface results in platelet aggregation. 7 RGDS, a tetrapeptide derived from fibrinogen, binds to the fibrinogen receptor, GPIIb-IIIa, thereby competitively inhibiting the binding of fibrinogen. Treatment of platelets with RGDS prior to stimulation with agonist markedly inhibits fibrinogen binding to GPIIbIIIa and the resulting platelet aggregation. 9~ Recently, the thrombin-induced formation of the phosphoinositide isomer, PI-3,4-P 2, in human platelets was shown to be significantly inhibited by pretreatment with RGDS. ~ Results from our laboratory also show a marked reduction of PI-3,4-P 2 generation in U-46619-stimulated human platelets treated with the fibrinogen-receptor antagonist, RGDS (D. C. Gaudette, R. C. Chapkin and B. J. Holub, unpublished observations). In both the previous study 96 and our study, RGDS inhibited PI-3,4-P 2 generation to a maximum of about 60%. Platelets (from patients with Glanzmann's thrombasthenia) lacking the fibrinogen receptor, GPIIb-IIIa, failed to show hardly any detectable increase in PI-3,4-P 2 following thrombin stimulation. 9~ These results suggest an involvement of fibrinogen-receptor occupancy and the resulting aggregation in agonist-stimulated formation of the novel phosphoinositide, PI-3,4-P2. However, since PI-3,4-P 2 was only maximally inhibited by 60%, additional pathways appear to be involved in the synthesis of this novel phosphoinositide. Since in platelets PI-3,4-P 2 may be formed via 2 pathways, that is via PI 3-kinase activity on the substrate PI-4-P ~ or via PI-3-P 4-kinase activity4~ (see Fig. 1), one of these pathways could be dependent on fibrinogen-receptor occupancy and the other independent of this event. Interestingly, we also observed the inhibition by RGDS of about 50% of U-46619-induced generation of the PIP2 isomer, PI-4,5-P2. RGDS treatment, however, did not block PI-4-P formation (D. C. Gaudette, R. C. Chapkin and B. J. Holub, unpublished observations). Our results suggest that PIP kinase activation (both PI-4-P 5-kinase and PI-3-P 4-kinase) may be partly regulated by the interaction of fibrinogen with GPIIb-IIa. The inhibition of PI-3,4-P 2 formation by RGDS treatment does not appear to involve PKC activation or granule secretion since neither of these latter events are affected by inhibition of fibrinogen-receptor occupancy due to RGDS. 97'98 On the other hand, the

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inhibition of PI-3,4-P 2 formation in stimulated platelets via PKC inhibition (see previous section) may be due to the inhibited secretion of fibrinogen, since PKC activation is required for platelet secretion: °

5. Platelet-Derived Growth Factor Platelet-derived growth factor (PDGF), stored in platelet ~t-granules, is secreted following platelet activation. The possible involvement of PDGF in thrombin- or GTPy S-induced formation of D-3 phosphorylated phosphoinositides was recently investigated. PDGF added to platelets alone produced little increase in D-3 phosphorylated phosphoinositides, nor did it modulate the thrombin-induced formation of these novel phosphoinositides. 26 Secretion of PDGF by platelets does not appear to enhance D-3 phosphorylated phosphoinositide accumulation.

6. Cyclic A M P Human erythroleukemia (HEL) cells possess several platelet-like features. Stimulation of HEL cells with thrombin produced a rise in PI-3,4-P2, which was inhibited by pretreatment with a prostacyclin analogue. 99 Since prostacyclin stimulates cyclic AMP formation, these results suggest that cyclic AMP may inhibit the formation of D-3 phosphorylated phosphoinositides in this cell line. Whether or not cyclic AMP inhibits agonist-induced D-3 phosphorylated phosphoinositide production in platelets is yet to be determined. vI. ROLE OF PHOSPHOINOSITIDE PHOSPHORYLATION IN HUMAN PLATELETS

A. Role of PI-4-P and PI-4,5-P2 Formation 1. Source of Second Messengers Probably the best known role for the formation of PI-4-P and PI-4,5-P2 in stimulated platelets is to replenish the supply of these phosphoinositides for phosphodiesteratic cleavage to the second messengers, DG and IP3.2'3 It is not the intention of this review to go into detail regarding cellular signal transduction and second messenger generation. Several reviews are available describing this phenomena. 6,7,~°°-~°2

2. Cytoskeletal Remodelling When platelets are activated, they change from a discoid to a spherical shape and extend numerous pseudopodia.~°3 The contractile elements responsible for this shape change reside in the platelet cytoskeleton. The main contractile protein in platelets is actin. Actin exists in 3 forms; monomeric actin, filamentous actin, and actin bound to other proteins. In resting platelets,