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HUMAN PLATELET THROMBIN RECEPTORS Roles in Platelet Activation Frederick A. Ofosu, PhD, and Kwasi A. Nyarko, MSc
Platelets are essential participants in hemostasis and thrombosis. Platelets normally circulate in blood as discoid resting cells which become critical constituents of hemostatic plugs or arterial thrombi only after specific receptors on platelet membranes interact with their ligands (agonists) to initiate the reactions that lead to platelet activation. The well-characterized events associated with platelet activation include activation of membrane receptors (e.g., receptors for fibrinogen, von Willebrand's factor, and a-thrombin), shape change, granular secretion, cytoskeletal reassembly, platelet cohesion, and aggregation. Intracellular signals elaborated by activated platelets in response to the occupancy of platelet receptors by agonists and by interactions include transient increases in intracellular Ca2+levels, activation of phospholipase A, (PLA,) and phospholipase C (PLC), elaboration of secondary messengers, phosphorylation or dephosphorylation of cytoplasmic and membrane proteins, and inhibition of adenylate cyclase." l2, 28, 30, 33* 49, 56, 63 The plasma serine protease a-thrombin is the most potent physiologic platelet agonist; this enzyme also has other key roles in hemostasis, in the genesis of
This article was previously scheduled to appear in our April 2000 issue devoted to Blood Stasis and Thrombosis. We are including it here as a benefit to our readers.
From the Canadian Blood Services and Department of Pathology and Molecular Medicine, McMaster University, Hamilton (FAO); and the Department of Biomedical Science, Ontario Veterinary College, University of Guelph, Guelph (KAN), Ontario, Canada
HEMATOLOGY/ ONCOLOGY CLINICS OF NORTH AMERICA VOLUME 14 * NUMBER 5 OCTOBER 2000
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arterial thrombi, and in embryonic development, inflammation, wound l4 healing, and cell pr01iferation.l~~ RECEPTORS FOR a-THROMBIN ON HUMAN PLATELETS
Three platelet membrane receptors, namely glycoprotein Ib (GPIb),", and protease-activated receptors 1 and 4 (PAR-1 and PAR-4)26,29, 34, 35, 41, 67, 71 have been reported to be actual and potential binding sites for human a-thrombin on human platelets. Glycoprotein Ib is the most abundant of the three platelet glycoproteins reported to bind a-thrombin. There are approximately 25,000 GPIb molecules per platelet, and only 50 (i.e., 0.2%) of the total platelet GPIb molecules apparently complexed with glycoprotein (GP) IX (GPIX) bind a-thrombin with high affinity (K,= 0.3 n m ~ l ) 24, . ~72, If there is one a-thrombin binding site on each of the 50 high-affinity sites for a-thrombin (the most potent platelet agonist) on GPIb, then at 0.3 nmol a-thrombin (the I(d ), only 10% of the available a-thrombin would be bound to the high-affinity sites on GPIb. The reasons why only 50 GPIb molecules (out of the 25,000 per platelet) provide high-affinity binding sites for a-thrombin and why only 10% of these sites would be occupied by a-thrombin at 0.3 nmol/L have not yet been adequately explained. The other two potential platelet receptors for a-thrombin on human platelets, PAR-1 and PAR-4, are two of the four known members of the family of seven transmembrane G-protein-linked protease-activated 29, 31, 34, 35, 41, 42, 67, 71 A unique feature of these four proteaseactivated receptors is that their activation requires cleavage of each receptor by a protease at a unique site located in the amino-terminal extracellular domain of each receptor. Each receptor cleavage releases a peptide consisting of residues beginning with the original amino-terminal residue and ending with first of the two residues that constitute the cleavage site. The newly exposed amino-terminal domain of the cleaved protease-activated receptor still anchored to the cell membrane then binds to sites (as yet undefined) on the receptor to complete activation of the receptor. After PAR-1 was cloned,67PAR-1 was initially defined as a moderate-affinity receptor for a-thrombin on platelets with less than 2000 PAR-1 molecules per platelet with a I(d of 1 n m ~ l / L If. ~there is one a-thrombin binding site on each platelet PAR-1 molecule, then at 1 nmol a-thrombin (i.e., approximately the I(d ), essentially all the PAR-1 molecules on platelets would be able to bind to the a-thrombin. a-Thrombin binds to the hirudinlike domain of PAR-1 with high affinity (both in the presence and in the absence of CaClZa)to initiate cleavage 'ele;?, thereby releasing a 41-mer peptide, PAR-1 of this receptor at Arg/S 46, 51, 53, 71 This 41-mer peptide residues 1 through 41, from the is now known to activate platelets in its own right.ls,56 This cleavage of PAR-1 at Are/SeP unmasks the "true" or "tethered" ligand domain of PAR-1 which begins with the sequence Ser-Phe-Leu-Arg-Asn (SFLLRN); 24, 33,
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the tethered ligand domain of PAR-1 then binds to sites (that are not yet 67 Note that fully defined) on PAR-1 to propagate platelet a~tivation.’~, exogenous SFLLRN is a less potent platelet agonist than exogenous athrombin, because approximately 10 pmol/ L SFLLRN activates platelets 51, 67 This observation as effectively as 1 to 10 nrnol/L a-thr~mbin?~, suggests that continued occupancy of the hirudinlike domain of PAR-1 by a-thrombin contributes to PAR-1-dependent signaling in platelets. In contrast with PAR-1, human platelet PAR-4 lacks a high-affinity (hirudinlike) binding site for a-thrombin distal to the a-thrombin cleavage site (i.e., A1-V/Gly48).~~, 35, 71 Therefore, a-thrombin is unlikely to bind tightly (if at all) to PAR-4, either before or after PAR-4 cleavage. The absence of a hirudinlike domain for PAR-4 is probably why 30 nmol/L a-thrombin is apparently required to cleave PAR-4 to propagate activation of platelets when the PAR-1 of the platelets has been impaired by preincubation with a strong PAR-1 antagonist or polyclonal anti-PAR-1 1gG directed against amino acid residues spanning the PAR-1 cleavage site.34Therefore, it is unlikely that PAR-4 can be readily cleaved directly by the low (i.e., subnanomolar) concentrations of a-thrombin that are normally found in vivo. Confirmation of the view that less than 30 nmol/ L of a-thrombin does not at least initiate PAR-4 cleavage, however, requires methods for quantifying direct PAR-4 cleavage. Methods for quantifying PAR-4 cleavage and the subsequent release of PAR-4 residues 1 through 47 have not been reported. Like the tethered ligand of PAR-1, Gly-Tyr-Pro-Gly-Gln-Val (GYPGQV) (the first six residues of the PAR-4 tethered ligand domain) can, at 500 pmol/L, also activate human platelets.% Roles of Protease-Activated Receptor 1 Cleavage and Protease-activated Receptor 4 Cleavage in Human Platelet Activation
As noted previously PAR-1 has a hirudinlike domain distal to its thrombin cleavage site ( A r e/ SeF) whereas PAR-4 lacks such a hirudinlike domain distal to its cleavage site (Arg7/Glp8)?9,34, 67, 71 The presence of the hirudinlike domain on PAR-1 clearly facilitates a-thrombin binding to platelet PAR-1.41,42, 46, 50, 51, 53 a-Thrombin binding to this site, by the fibrinogen recognition exosite of the enzyme, probably contributes to the effective cleavage of PAR-1 observed after platelets are incubated with subnanomolar a-thrombin.41,67 The high affinity of a-thrombin for the hirudinlike domain of PAR-1 is also evident from observations that when washed human platelets are incubated with hirudin before 0.5 nmol/L a-thrombin, at least a tenfold molar excess of hirudin over athrombin is required for hirudin to abrogate platelet activation:’ Conversely a consequence of the absence of a hirudinlike domain in PAR-4 is the ineffective cleavage of platelet PAR-4 by a-thrombin, so that at least 30 nmol/ L a-thrombin is required for PAR-&dependent platelet a~tivation.~~ Cleavage of PAR-1 by a-thrombin causes the release of PAR-1 resi-
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dues 1 through 41.42,46,50* 51, 53 Cleavage of PAR-4 by a-thrombin probably also causes the release of PAR-4 residues 1 through 47 from the platel e t ~ ?35,~71, Exposure of the two new amino-terminal domains containing the tethered ligand domains is crucial for propagating PAR-1- and PAR&dependent platelet activation. Two lines of evidence support this view. (1) Potent antagonists of the PAR-1 tethered ligand domain effectively inhibit activation of human platelets in response to 1.0 nmol/L athrombin;" (2) Simultaneous incubation of platelets with antibodies directed against segments of PAR-1 and PAR-4 containing the a-thrombin cleavage sites of the two protease-activated receptors abrogates platelet activation by a-thrombin.34As noted previously, both tethered ligand domains (represented by the two 6-mer peptides given later) can independently effect platelet activation. The concentrations of SFLLRN (PAR1 tethered ligand) and GYPGQY (PAR-4 tethered ligand) required for optimal PAR-1- and PAR-Mependent platelet activation are, however, approximately four and five orders of magnitude higher, respectively, 31, 34, 35, 48, 51, 67, 71 The concentration of GYPthan those of a-thr~mbin.~, GQY required for optimal platelet activation is 50-fold higher than the required concentration of SFLLRN, suggesting that PAR-1 cleavage is normally the predominant pathway for propagating a-thrombindependent platelet activation. Primacy of Platelet Protease-Activated Receptor 1 as the Binding Site for a-Thrombin
The authors have recently compared PAR-1 and GPIb as potential binding sites for human a-thrombin on human platelets.4l That study used flow cytometry to quantify a-thrombin binding to washed human platelets and activation of the same platelets. Biotinylated, affinity-purified polyclonal rabbit anti-human a-thrombin IgG was added to paraformaldehyde-fixed periodic aliquots of platelets incubated with athrombin. Binding of human a-thrombin to the platelets in the periodic aliquots was quantified by flow cytometry." Monoclonal antibodies directed against CD62 (P-selectin) and CD63 (a platelet lysosomal protein) were used to quantify activation of the same aliquots of paraformalde", 51 The study compared four groups of platelet hyde-fixed suspensions, namely control platelets, GPIb-depleted platelets, platelets incubated with TM60 or LJ-IS10 (two anti-GPIb monocolonal antibodies reported to inhibit a-thrombin binding to platelets), and platelets incubated with ATAP 138 (an anti-PAR-1 monocolonal antibody directed against an epitope which contains the hirudinlike a-thrombin binding domain of PAR-1).5 The anti-PAR-1 monoclonal antibody, ATAP 138, had previously been shown to abrogate the responses of human platelets to subnanomolar human a-thr~mbin.~ Glycoprotein Ib-depleted platelets were obtained by incubating platelets with a protease from Serrutiu murcescens or the bacterial protease 0-sialoglyoprotein endopeptidase able specifically to remove GPIb from human platelets under the condi-
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tions employed. Because the physiologic concentration of Ca2+increases the affinity of platelets for binding a-thrornbin:l and previous studies defining GPIb as a binding site for a-thrombin were performed in the absence of CaC1,22,24, 72 the studies comparing GPIb and PAR-1 as potential binding sites for human a-thrombin were conducted both in the presence and in the absence of 2 mmol/ L CaC1,.4l The authors found that in the presence or absence of physiologic calcium, the anti-PAR-1 monoclonal IgG, ATAP 138, abrogated both binding of 1 nmol/L a-thrombin to human platelets and subsequent activation of the same platelets, as previously demonstrated by others? In contrast, neither of the anti-GPIb monoclonal antibodies nor GPIb depletion inhibited binding of a-thrombin washed to human platelets resuspended in a Tyrode's buffer containing albumin and 2 mmol/L CaC1,. Also, none of the three pretreatments of platelets aimed at impairing the functions of platelet GPIb inhibited activation of the platelets in response to a-thrombin. Significant involvement of GPIb as a binding site for a-thrombin could be demonstrated only in the absence of CaCl, and only when the incubation of platelets with a-thrombin was allowed to proceed for 30 minutes instead of 10 seconds. Both 1 nmol/L athrombin binding to human platelets and activation of the same platelets are readily demonstrable when platelets are incubated with a-thrombin for 10 seconds.41It seems, therefore, that in the absence of CaCb binding of the highly basic a-thrombin to the highly acidic extracellular region of GPIb essentially accounts for the hypothesized high-affinity interactions between a-thrombin and GPIb. Calcium would be expected to form salts with some of the highly acidic residues located within the extracellular domain of GPIb to reduce the net charge on GPIb. Others had also previously demonstrated, even in the absence of CaCl, that monocolonal antibodies that bind the proposed a-thrombin binding site on GPIb do not abrogate the responses of human platelets to subnanomolar concentrations of a-thrombin.", 72 Complete inhibition of a-thrombin binding to and activation of human platelets preincubated with ATAP 138 also suggests that 1.0 nmol/L a-thrombin does not bind to or readily cleave platelet PAR-4 to propagate platelet activation. Based on these observation^'^ and observations by others that ATAP 138 abrogates the responses of human platelets to less than 1 nmol/L awhereas anti-GPIb monoclonal antibodies do not abrogate the responses of human platelets to 1 nmol/L a-thrombin,", the authors have concluded that PAR-1 is the primary binding site for athrombin on human platelet^.^^ In summary, subnanomolar a-thrombin and approximately 30 nmol/ L a-thrombin can initiate PAR-l-dependent and PARMependent platelet activation. Initiation of PAR-1-and PAR-&dependent platelet activation critically depends on a-thrombin-mediated PAR-1 and PAR-4 cleavage, respectively, and the associated release of the tethered ligand domains of the two protease-activated receptors necessary to propagate platelet activation in response to a-thrombin. Platelet activa-
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tion can be quantified as one or more of transient increases in intracellular Ca2+.Platelet activation can also be quantified by: flow cytometric quantification of the percentage of platelets expressing CD62, CD63, or phosphatidylserine (or a combination thereof) externalized on the surface of activated platelets release of ADP, ATP, and serotonin from platelet granules platelet aggregation. It is apparent that subnanomolar concentrations of a-thrombin effectively initiate platelet PAR-1 cleavage, compared with the approximately 30 nmol/ L a-thrombin required to effect PAR-4 cleavage. Therefore, PAR-1 cleavage probably drives a-thrombin-mediated platelet activation, whereas PAR-4 cleavage may not be normally involved in athrombin-mediated activation in vivo. Given the lower abundance of the PAR-4 mRNA in human platelets (compared with the PAR-1 message), and that the PAR-4 tethered ligand is 50-fold higher than the PAR1 tethered ligand domain peptides required to activate the same human 51 PAR-4 is probably less important than PAR-1 for directing platelet~,3~,~~> platelet activation in vivo. It is worth noting, however, that fibrinogen may increase the concentration of a-thrombin required to activate platelets in vivo. In support of this view, more than 5.0 nmol/L a-thrombin is required to activate platelets resuspended in recalcified plasmas (containing a factor Xa inhibitor to prevent prothrombin activation) to the same extent as platelets resuspended in buffer to which 0.5 nmol/L or 1 nmol/L a-thrombin has been added." Platelet Membrane Protease and Propagation of Protease-Activated Receptor l-Platelet Activation
Incubation of platelets with SFLLRN invariably results in PAR-1 cleavage. Platelet PAR-1 cleavage in the course of platelet activation, and in response to SFLLRN, was abrogated by soybean trypsin inhibitor, an effective inhibitor of trypsin- and plasminlike enzymes. Abrogation of platelet PAR-1 cleavage in response to SFLLRN by soybean trypsin inhibitor was also associated with strong inhibition of platelet activat i ~ nThese . ~ ~ observations are consistent with a platelet protease cleaving PAR-1 in response to the addition of SFLLRN to propagate PAR-1dependent platelet activation. Significantly, PAR-1 cleavage (and release of PAR-1 residues 141),as well as platelet activation in response to athrombin, could also be abrogated by soybean trypsin inhibitor.5O.51 Thus, platelet PAR-1 cleavage in response to a-thrombin to the extent necessary to assure propagation of platelet activation requires PAR-1 cleavage by the platelet serine protease. The authors have preliminary evidence indicating that SFLLRN externalizes a platelet membrane protease that probably cleaves PAR-1 to release PAR-1 residues 1 through 41 to help propagate platelet activation in response to SFLLRN. This protease is detected on platelets using biotinylated soybean trypsin inhibitor and
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flow cytometry; this soybean trypsin inhibitor-reactive platelet protease is also externalized within 10 seconds on platelets incubated with PAR1 residues 1 through 41 (F. A. Ofosu et al, unpublished data). Given that the peptide consisting of PAR-1 residues 1 through 41 activates platelets,*8,50 both PAR-1-derived fragments (i.e./ the fragment of PAR beginning with the PAR-1 tethered ligand domain and PAR-1 residues 1 through 41) seem to act in concert to optimize the propagation of platelet activation in response to a-thrombin. The role of this protease in propagating PAR-1-dependent platelet activation helps explain a previous observation that simultaneous addition of 10 nmol/L a-thrombin and plasmin to recalcified platelet-rich plasma accelerates prothrombinase formation more effectively than addition of 10 nmol/L of a-thrombin alone. Plasmin alone does not enhance prothrombinase formation in platelet-rich plasma.4O Whether and when this platelet protease also cleaves PAR-4 to release PAR-4 residues 1 through 47 (thereby exposing the PAR-4 tethered ligand domain) to optimize platelet activation has not yet been established. Whether the peptide consisting of PAR-4 residues 1 through 47 can similarly activate human platelets is also not known. Possible Interactions Between Activated ProteaseActivated Receptor 1 and Glycoprotein Ib
Although 1 nmol/L a-thrombin does not bind to GPIb on human platelets resuspended in buffers containing 2 mmol/L CaC1, GPIb clearly influences the changes in intracellular Ca2+in response to subnanomolar a-thrombin.22The authors have found that GPI depletion of platelets (by preincubating platelets with 0-sialoglycoprotein endopeptidase) inhibits the rate of PAR-1 cleavage in response to a-thrombin. Specifically, following incubation of platelets with 1 nmol/ L a-thrombin for 10,60, and 180 seconds, GPIb depletion impaired PAR-1 cleavage by 76%, 35%, and 17%, respectively, in comparison with control platelets incubated for identical periods. Thus, inhibition of PAR-1 cleavage by GPIb depletion was most significant during the initial phase of incubating platelets with 1 nmol/L a-thrombin. Inhibition of a-thrombindependent PAR-1 cleavage by GPIb depletion insignificantly inhibited a-thrombin-dependent platelet activation, however. The authors have also observed that GPIb depletion significantly impairs platelet aggregation in response to PAR-1 residues 1 through 41. Thus, there seem to be functional interactions between GPIb and PAR-1 (probably through PAR1 residues 1 through 41) that optimize propagation of platelet activation in response to subnanomolar a-thrombin. THROMBIN-MEDIATED SIGNALING IN PLATELETS
Exposure of platelets to a-thrombin initiates a sequence of signaling events which ultimately serves to activate the platelets. &-Thrombin
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binds to its receptors on human platelets and activates them to initiate PLA, activation. Activated PLA, in turn, releases of arachidonate from platelet membranes to provide the initial substrate necessary for the synthesis of thromboxane A, TxA,.* a-Thrombin binding to platelets also initiates activation of Ca2+-dependent PLC (probably more than one PLC) and the associated production of the signaling molecules 41, 56* 65 Production diacylglycerol (DAG) and inositol triphosphate (IP3).30, of DAG leads to Ca2+-dependentactivation of protein kinase C (PKC), the cellular high-affinity receptor for DAG. The end results of these PLAT and PLC-dependent signaling events include platelet shape change, release of P-selectin and fibrinogen from platelet a-granules, release of ADP, ATP, serotonin, and other granular contents, externalization and activation of platelet GPIIb/IIIa and, ultimately, platelet aggregation. Both TxA, and ADP are significant platelet agonists in response to their binding to their specific receptors on human platelets. Thus, elaboration of these two platelet-derived agonists during platelet activation in response to subnanomolar a-thrombin probably enhances propagation of the signaling events that ultimately result in the release and expression of platelet granular contents, and the cohesion and aggregation of the platelets. Activation of the two major distinct platelet-signaling pathways (involving PLC and PLA, activation) in response to a-thrombin are accompanied by phosphorylation of several platelet proteins and phospholipids and elaboration of several signaling intermediates.t Many platelet proteins become phosphorylated within 15 seconds following a-thrombin addition to platelets,$ and tyrosine phosphorylation of platelet proteins occurs in several temporal “waves,” suggesting that the several protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs) act sequentially on their substrates. Dephosphorylation reactions catalyzed by phosphatases have also been associated with signaling.69Despite the considerable progress in identifying these phosphorylated proteins, however, the precise functions of many of these platelet proteins remain unclear. PROTEASE-ACTIVATED RECEPTOR 1 AND GLYCOPROTEIN Ilb/llla ACTIVATION
Glycoprotein IIb / IIIa, the Ca2+-dependentreceptor for fibrinogen on the surface of platelets and consisting of aIIband p3 subunits, is the platelet integrin thought to mediate the fibrinogen-dependent common pathway of platelet aggregation.’O.45, 5941, 70 This transmembrane receptor has a large extracellular domain with specific ligand recognition sequences, including the arginine-glycine-aspartate-serine(RGDS) sequence found in fibrinogen, von Willebrand’s factor, fibronectin, *References6, 12, 17, 28, 30, 39, 43, 49, 56, and 64-66. tReferences 7, 9, 15, 16, 20, 21, 23, 27, 32, 37-39, 44, 67, 69, and 73. *References 7, 9, 15, 16, 20, 21, 23, 27, 32, 37, 38, and 73.
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vitronectin, and thrombospondin. Each of these adhesive proteins is a ligand for the platelet integrin GPIIb / IIIa. The expression of the numbers and ligand-binding functions of the GPIIb/IIIa integrin on platelets can be modulated by outside-in, inside-out signaling and by limited proteolysis of GPIIb / IIIa. Approximately one half of the 100,000 GPIIb/ IIIa integrin molecules per platelet are constitutively expressed on platelet membranes; the balance, along with an increased capacity to bind fibrinogen, becomes evident only after GPIIb/ IIIa is activated by an inside-out signaling process. The resulting conformational modifications of the extracellular domains of GPIIb / IIIa facilitate the conversion of this integrin from a low-affinity to a high-affinity platelet membrane receptor for fibrinogen." 68, 70 Platelet activation initiated by ligand-receptor interactions, such as PAR-1 occupancy (at its hintdinlike domain) and subsequent PAR-1 cleavage by a-thrombin, the resulting outside-in signals generated, as well as the inside-out signals generated by binding of intracellular ADP, TxA, and serotonin to their platelet receptors, all contribute to the activation of GPIIb / IIIa. Only transitory GPIIb / IIIa activation is effected by ADP, platelet activating factor (PAF), SFLLRN, and adrenaline, whereas irreversible activation of this receptor is effected when platelets are incubated with 5 nmol/ L a-thrombin.6z,70 The transitory activation of GPIIb / IIIa by the weaker platelet agonists is reversible by prostaglandin I,. During the platelet release reaction, the intracellular fraction of GPIIb/ IIIa becomes translocated from its storage sites in the a-granules and elsewhere to the plasma membrane, thereby increasing the capacity of activated platelet GPIIb /IIIa to bind f i b r i n ~ g e n . ~ ~ Elastate, a-chymotrypsin, and cathepsin G activate platelet GPIIb / IIIa by a cleavage at the carboxyl-terminal domain of the am subunit heavy chain by inducing irreversible expression of fibrinogen binding sites on GPIIb/ IIIa.36,52, 55, 58 Proteolytic activation of GPIIb / IIIa apparently occurs independently of intracellular The platelet-derived protease which becomes externalized on platelet membranes when PAR-1 is activated by a-thrombin or SFLLRN may also modulate the functions of GPIIb/IIIa by a proteolytic mechanism. The reason for this view is that soybean trypsin inhibitor can abrogate platelet aggregation in response to SFLLRN or PAR-1 residues 1through 41 (F.A. Ofosu, K.A. Nyarko, et al, unpublished data). There is evidence that PAR-1 activation requires a functional GPIIb/ IIIa.19 Unlike GPIIb/ IIIa that can be activated by leukocyte elastase, this enzyme and two other leukocyte enzymes (namely cathepsin G and protease 3) cleave PAR-1 at sites within the PAR-1 extracellular domain and distinct from the thrombin cleavage site. Therefore, cleavage of PAR-1 by these three leukocyte proteases does not lead to platelet activation. Rather, these leukocyte proteases can abrogate or significantly reduce platelet responses to a-thrombin. To cleave PAR-1, however, 600 nmol/L of these leukocyte proteases were required.57Therefore, these leukocyte enzymes may not normally influence a-thrombin-mediated PAR-1 activation in vivo.
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Signals Attributable to Interactions Between Glycoprotein llb/illa Subunits allbP3and ProteaseActivated Receptor 1
Platelet aggregation requires platelet-platelet interactions that are in all likelihood mediated by a fibrinogen bridge binding to the activated conformer of GPIIb/II1a.lo,~ 5 ,47, 59, 6o As noted previously, incubation of platelets with 0.5 nmol/L a-thrombin results in PAR-1 cleavage and generation of intracellular signals which culminate in aggregation of the platelets. Activated PAR-1 couples to G-proteins able to effect PLC and, possible, PLA activation. Phospholipase C in turn hydrolyzes phosphatidylinositol-4,5-bisphosphate (PIPz)to generate IP, and DAG. The secondary intracellular messengers IP, and DAG ultimately enhance increases in intracellular Caz+ and activation of protein kinase C (PKC), respectively, in platelets. Activated PKC phosphorylates several platelet proteins, including PTKs. Phopholipidase Az liberates arachidonate from platelet membranes necessary for TxA, biosynthesis. Protein tyrosine kinases and protein phosphatases (PPs) are also activated in response to a-thrombin binding to platelet PAR-1 to cause PAR-1 activation. Interactions between fibrinogen and the activated GPIIb/IIIa conformer similarly result in the activation of PLC, PLA, PTK, and PPs and in the production of their respective rnes~engers.2~. 47 Thus, PAR-1 and GPIIb / IIIa activation results in the elaboration of the same intracellular signals within platelets. Activation of PKC increases both the number and the affinity of amp3 receptors for fibrinogen on platelets to enhance platelet aggregation. Activated PKC can directly activate PTKs, which in turn convert the nonactivated GPIIb/ IIIa conformer (closed integrin aIbp30) to the open or activated conformer (aIIbp,*).The open or activated conformer amp3. rapidly recloses in the absence of an appropriate ligand or its dephosphorylation by PTKs.= Depending on the degree of PKC activation, a significant fraction of the pool of internal (YIbp3 may become externalized, and the p3 chain of the internal pool of aIbp3becomes phosphorylated by PKC. This PKC-dependent phosphorylation of GPIIb/IIIa opposes the return of the activated conformer of GPIIb/ IIIa to the closed configuration. Dephosphorylation by serine-threonine protein phosphatases converts aIbpB.to its closed configuratio11.2~Thus, either aIIbp3or PAR-1 activation results in the generation of signals able to modulate platelet function, suggesting that activation of either aIbp3 or PAR-1 can ultimately result in platelet aggregation. Incubation of subnanomolar a-thrombin with platelets that have already bound fibrinogen decreases production of inositol phosphates and decreases the phosphorylation of 20-kd and 40-kd proteins relative to those observed when control platelets are incubated with a-thrombin.57These observations are consistent with interactions between a I & and PAR-1, probably at the signal-transduction level. Activation of PAR1 on the megakaryoblastic cells, CHRF-288, results in the cells' adhering and spreading on immobilized fibrinogen in a GPIIb / IIIa-dependent
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manner. In this instance, however, PAR-1 activation does not support GPIIb / IIIa-dependent binding of soluble fibrinogen or the binding of PAC-1, the monoclonal antibody directed against activated GPIIb/ IIIa.8 Despite their failure to bind soluble fibrinogen, PAR-1 activation on CHRF288 cells results in phosphoinositide hydrolysis, arachidonate metabolism, and phosphorylation of ~ 4 (a 422 kd mitogen-activated ~ ~ ~ protein kinase), PLA, and the Rac exchange protein Vav.8 The signals generated by PAR-1 activation supported amp3binding to immobilized fibrinogen, suggesting that amp3did not undergo the conformational change required to bind to soluble fibrinogen or to PAC-1 and that there was an increase in amp3 avidity rather than an increase in affinity.8 Increased avidity is thought to result from an accumulation of relatively low-affinity interactions between this integrin and its ligands. Thus, it is likely that changes in the lipid membrane composition (evident as increased externalization of platelet phosphatidylserine resulting from PAR-1 activation) increases the lateral mobility of within the platelet membrane, to cause accumulation of amp3at the interface. Thus, PAR-1 activation in response to a-thrombin modulates GPIIb/ IIIa functions, because PAR-1 activation initiates outside-in signals which facilitate increased expression of GPIIb / IIIa receptors on the platelet surface and also increases the avidity of GPIIb/IIIa for external ligands. Given that GPIIb/IIIa can be activated by limited proteolysis, it is possible that the platelet protease externalized on platelets during PAR1 activation also directly activates GPIIb/ IIIa. Additional studies are required to elucidate fully the functional interactions between platelet PAR-1 and resting and activated conformers of GPIIb/IIIa. References 1. Andersen H, Greenberg DL, Fujikawa K, et al: Protease-activated receptor 1 is the primary mediator of thrombin stimulated platelet-procoagulant activity. Proc Natl Acad Sci U S A 96:11189-11193, 1999 2. Andrade-Gordon P, Maryanoff BE, Derian CK, et al: Design, synthesis, and biological characterization of a peptide-mimetic antagonist for a tethered ligand receptor. Proc Natl Acad Sci U S A 96:12257-12262, 1999 3. Bematowicz MS, Klimase CE, Hart1 KS, et al: Development of potent thrombin receptor antagonist peptides. J Med Chem 39:48794887, 1996 4. Brass LF, Molino M: Protease-activated G protein-coupled receptor on human platelets and endothelial cells. Thromb Haemost 78:234-241, 1997 5. Brass LF, Vassallo RR Jr, Belmonte E, et a1 Structure and function of the human platelet thrombin receptor: Studies using monoclonal antibodies against a defined epitope with the receptor N-terminus. J Biol Chem 26713795-13798, 1992 6. Carter AN, Huang R, Sirosky A, et al: Phosphatidylinositol3,4,5-triphosphateis formed from phosphatidylinositol 4,5-biphosphate in thrombin-stimulated platelets. Biochem J 301:415-420, 1994 7. Cichowski K, Brugge JS, Brass LF: Thrombin receptor activation and integrin engagement stimulate tyrosine phosphorylation of proto-oncogene product p95vav, in platelets. J Biol Chem 271:7544-7550, 1996 8. Cichowski K, Orsini MJ, Brass LF: PAR-1 activation initiates integrin engagement and outside in signaling in megakaryoblastic CHRF-288 cells. Biochim Biophys Acta 1450:265276, 1999
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9. Clark EA, Shattil SJ, Brugge J S Regulation of protein tyrosine kinases in platelets. Trends in Biochemical Sciences 19:464-469, 1994 10. Coller BS Blockade of platelet-GPIIb /IIIa receptors as an antithrombotic strategy. Circulation 92:2373-2380, 1995 11. De Marco L, Mazzucato M, Masotti A, et al: Localization and characterization of an athrombin binding site on platelet glycoprotein Ib. J Biol Chem 269:6478-6484, 1994 12. Du X, Harris SJ, Tetaz TJ, et al: Association of a phospholipase A (14-3-3 protein) with platelet glycoprotein Ib-IX complex. J Biol Chem 269:18287-18290, 1994 13. Fenton JW 11: Thrombin bioregulatory functions. Advances in Clinical Enzymology 6:186-193, 1988 Ofosu FA, Brezniak DV, et al: Understanding thrombosis and hemostasis. 14. Fenton JW, Hematol Oncol Clin North Am 71107-1119, 1993 15. Ferrell JE, Martin GS: Platelet tyrosine-specific protein phosphorylation is regulated by thrombin. Mol Cell Biol 8:3603-3610, 1988 16. Ferrell JE, Martin GS: Thrombin stimulates the activities of multiple previously unidentified protein kinases in platelets. J Biol Chem 26420723-20729, 1998 17. Fitzgerald GA, Reilly IAG, Pedersen AK: The biochemical pharmacology of thromboxane synthesis and inhibition in man. Circulation 72:1194-1201, 1985 18. Furman MI, Liu L, Benoit SE, et al: The cleaved peptide of the thrombin receptor is a strong platelet agonist. Proc Natl Acad Sci U S A 95:3082-3087, 1992 19. Giesberts AN, van Willegen G, Lapetina E, et al: Regulation of platelets glycoprotein IIb/IIIa (integrin aIIbp3) function via the thrombin receptor. Biochem J 309:613420, 1995 20. Golden A, Brugge JS: Thrombin treatment induces rapid changes in tyrosine phosphorylation in platelets. Proc Natl Acad Sci U S A 86:901-905, 1989 21. Golden A, Brugge JS, Shattil SJ: Role of platelet glycoprotein IIb-IIIa in agonist-induced tyrosine phosphorylation of platelet proteins. J Cell Biol 111:3117-3127, 1989 22. Greco NJ, Tandan NN, Jamieson GA: Contributions of GpIb and the seven transmembrane domain receptor to increases in platelet cytoplasmic [Caz+], induced by athrombin. Biochemistry 35:906-914, 1996 23. Haimovich B, Lipfert L, Brugge JS, et al: Tyrosine phosphorylation and cytoskeletal reorganization in platelets are triggered by interaction of integrin receptors with their immobilized ligands. J Biol Chem 268:15868-15877, 1993 24. Harmon JT, Jamieson G A Activation of platelets by a-thrombin is a receptor mediated event. J Biol Chem 261:15928-15933, 1986 25. Hers I, Donath J, van Willigen G, et al: Differential involvement of tyrosine and serine/ threonine kinases in platelet integrin aIIbp3 exposure. Arterioscl Thromb Vasc Biol 183404414, 1998 26. Hoxie JA, Ahuja M, Belmonte E, et al: Internalization and recycling of activated thrombin receptors. J Biol Chem 268:13756-13763, 1993 27. Huang R, Kucera GL, Rittenhouse SE: Elevated cytosolic Ca2+activates phospholipase D in human platelets. J Biol Chem 266:1652-1655, 1991 28. Huang R, Sorisky A, Church WR, et al: Thrombin receptor-directed ligand accounts for activation of platelet phospholipase C and accumulation of phosphorylated phosphoinositides. J Biol Chem 266:18435-18438, 1991 29. Hung DT, Vu T-KH, Wheaton VI, et al: Cloned platelet-thrombin receptor is necessary for thrombin-induced platelet activation. J Clin Invest 89:1350-1353, 1992 30. Hung DT, Wong YH, Vu T-Kh, et al: Cloned thrombin receptor complexes to at least 2 distinct effectors to stimulate phosphoinositide hydrolysis and inhibit adenyl cyclase. J Biol Chem 26720831-20834, 1992 31. Ishihara H, Connolly AJ, Zeng D, et al: Protease-activated receptor 3 is a second thrombin receptor in humans. Nature 386502-506, 1997 32. Jackson SP, Schoenwaelder SM, Yuan Y, et al: Non-receptor protein tyrosine kinases and phosphatases in human platelets. Thromb Haemost 75:640-650, 1996 33. Jamieson G A Pathophysiology of platelet thrombin receptors. Thromb Haemost 78:242-246, 1997 34. Kahn ML, Nakanishi-Mitsuim M, Shapiro MJ, et al: Protease-activated receptors 1 and 4 mediate activation of human platelets by thrombin. J Clin Invest 103:879-887, 1999
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35. Kahn ML, Zhen Y-W, Huang W, et al: A dual thrombin receptor for platelet activation. Science 394690-694, 1998 36. Komecki E, Erlich YH, Egbring R, et al: Granulocyte-platelet interactions and platelet fibrinogen exposure. Am J Physiol255:H651-H658, 1998 37. Kramer RM, Roberts EF, Manetta JV,et al: Thrombin-induced phosphorylation and activation of Ca2+-sensitivecytosolic phospholipase A2 in human platelets. J Biol Chem 268:26796-26804, 1993 38. Kroll HM, Schafer AL Biochemical mechanisms of platelet activation. Blood 74:11811195, 1989 39. Lapetina E: Signal transduction induced by thrombin-activated platelets. FEBS Lett 268:400-404, 1990 40. Liu L, Freedman J, Homstein A, et al: Plasmin accelerates platelet-dependent prothrombinase assembly without activating the platelets. Br J Haematol 92:458-465, 1996 41. Liu L, Freedman J, Homstein A, et al: Binding of thrombin to the G-protein-linked receptor, and not to GPIb, precedes thrombin-mediated platelet activation. J Biol Chem 272:1997-2004, 1997 42. Liu L, Freedman J, Homstein A, et al: Thrombin binding to platelets and their activation in plasma. Br J Haematol88:592-600, 1994 43. McNichol A, Robson C A Thrombin receptor-activating peptide releases arachidonic acid from human platelets. J Pharmacol Exp Ther 281:861-867, 1997 44. Nieuwland R, van Willigen G, Akkerman J-WN: Different pathways for control of N a + / H +exchange via activation of thrombin receptor. Biochem J 29747-52, 1994 45. Niiya K, Hodson E, Bader R, et al: Increased surface expression of membrane glycoprotein IIb /IIIa complex induced by platelet activation: Relationship between binding of fibrinogen and platelet aggregation. Blood 70:475483, 1987 46. Norton KJ, Scarborough RM, Kutok JL, et al: Immunologic analysis of the cloned platelet thrombin receptor activation mechanism: Evidence supporting receptor cleavage, release of the N-terminal peptide and insertion of the tethered ligand into a protected environment. Blood 82:2125-2136, 1993 47. Nurden AT A review of the role of platelet membrane glycoproteins in the plateletvessel wall interaction. Baillieres Clin Haematol 6:653-690, 1993 48. Nystedt S, Emilsson K, Weahlestedt C, et al: Molecular cloning of a potential novel proteinase activated receptor. Proc Natl Acad Sci U S A 91:9208-9212, 1994 49. Offermanns S, Laugwitz KL, Spicher K, et al: Proc Natl Acad Sci U S A 91:504-508,1994 50. Ofosu FA, Dewar L, Fenton J W I1 et al: A trypsin-like platelet protease must release PAR-1 residues 1-41 and PAR-1 tethered ligand to optimize propagation of platelet activation [abstract]. Thromb Haemost (suppl):821, 1999 51. Ofosu FA, Freedman J, Dewar L, et al: A trypsin-like protease propagates platelet PAR-1 cleavage and platelet activation. Biochem J 336:283-285, 1998 52. Pidard D, Frelinger AL, Boullot C, et al: Activation of the fibrinogen receptor on human platelets exposed to a-chymotrypsin. Relationship to a major poteolytic cleavage at the carboxyl terminus of the membrane glycoprotein Ib heavy chain. Eur J Biochem 200:437477, 1991 53. Ramachandran R, Klufas AS, Molino M, et al: Release of the thrombin receptor (PAR1) N-terminus from the surface of human platelets activated by thrombin. Thromb Haemost 78:lllO-1124, 1997 54. Ray MJ, March N A Aprotinin reduces blood loss after cardiopulmonary bypass by direct inhibition of thrombin. Thromb Haemost 78:1021-1026, 1997 55. Renesto P, Si-Tahar M, Moniatte M, et al: Specific inhibition of thrombin-induced cell activation by neutrophil proteinases elastase, cathepsin G, and proteinase 3 Evidence for distinct cleavage sites within the amino terminal domain of the thrombin receptor. Blood 8931944-1953, 1997 56. Seiler SM, Michel A, Fenton JW I 1 Involvement of the ‘tethered-ligand’ receptor in thrombin inhibition of platelet adenyl cyclase. Biochem Biophys Res Commun 182:1296-1302, 1992 57. Sinigaglia F, Torti M, Ramaschi G, et al: The occupancy of glycoprotein IIb/IIIa complex modulates thrombin activation of human platelets. Biochim Biophys Acta 984:225-230, 1989
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58. Si-Tahar M, Pidard D, Bolloy V, et al: Human neutrophil elastase proteolytically through cleavage of the carboxyl terminus of the activates the platelet integrin a& am subunit heavy chain. J Biol Chem 272:11636-11647, 1997 59. Taylor DB, Derrick Jh4,Gartner TK Antibodies to GPIIba (300-312) inhibit fibrinogen binding, clot retraction, and platelet adhesion to multiple ligands. Proc SOCExp Biol Med 205:3543, 1994 60. Topol EJ: Prevention of cardiovascular ischemic complications with new platelet glycoprotein IIb/ma inhibitors. Am Heart J 130:666-672, 1995 61. van Willigen G, Akkennan JWN: Phosphorylation and the affinity state of platelet integrin mIIbp3. Platelets 8225-234, 1997 62. van Willegen G, Akkerman JWN: Regulation of glycoprotein IJb/IIIa exposure on platelets stimulated with thrombin. Blood 79:82-90, 1992 63. Vassallo RR Jr, Kieber-Emmons T, Chichowski K, et al: Structure function relationships in the activation of platelet thrombin receptors by receptor-derived peptides. J Biol Chem 26760814085, 1992 64. Vickers JD, Kinlough-Rathbone RL, Mustard JF: Accumulation of the inositol phosphates in thrombin-stimulated, washed rabbit platelets in the presence of lithium. Biochem J 2M399-405, 1984 65. Vickers JD,Packham MA, Kinlough-Rathbone RL: Differences between platelet-phosphoinositide metabolism stimulated by thrombin SFLLRN are not accounted for by interaction of thrombin with glycoprotein Ib. Am J Hematol54288-295, 1995 66. Vickers JD:Extraction of polyphosphoinositides from platelets. Analytical Biochemistry 224449456,1995 67. Vu T-KH, Hung DT, Wheaton VI, et al: Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell 64:10571068, 1991 68. Wencel-Drake JD, Plow EF, Kunicki TJ, et al: Localization of internal pools of membrane glycoproteins involved in platelet adhesive responses. Am J Pathol 124324334, 1986 69. Wera S, Hemmings BA Serine/threonine phosphatases. Biochem J 311:17-29, 1995 70. Woods V Jr, Wolff LE, Keller D M Resting platelets contain a substantial centrally located pool of glycoprotein IIb/IIIa complexes which may be accessible to some but not other extracellular proteins. J Biol Chem 261:15242-15251, 1986 71. Xu W-F, Andersen H, Whitmore TE, et al: Cloning and characterization of human protease-activated receptor 4. Proc Natl Acad Sci U S A 95:66424646, 1998 72. Yamamoto N, Greco NJ, Barnard MR, et al: Glycoprotein Ib (GPIb)-dependent and GPIb-independent pathways of thrombin-induced platelet activation. Blood 77A7401748,1991 73. Zhang J, Falck JR, Reddy KK, et al: Phosphatidylinositol(3,4,5)-triphosphatestimulates phosphorylation of pleckstrin in human platelets. J Biol Chem 270:22807-22810, 1995
Address reprint requests to Frederick A. Ofosu, PhD Department of Pathology and Molecular Medicine HSC - 3N26, McMaster University 1200 Main Street West Hamilton, ON L8N 325, Canada e-mail:[email protected]
0889-8588/00 $15.00
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HUMAN PLATELET THROMBIN RECEPTORS Roles in Platelet Activation Frederick A. Ofosu, PhD, and Kwasi A. Nyarko, MSc
Platelets are essential participants in hemostasis and thrombosis. Platelets normally circulate in blood as discoid resting cells which become critical constituents of hemostatic plugs or arterial thrombi only after specific receptors on platelet membranes interact with their ligands (agonists) to initiate the reactions that lead to platelet activation. The well-characterized events associated with platelet activation include activation of membrane receptors (e.g., receptors for fibrinogen, von Willebrand's factor, and a-thrombin), shape change, granular secretion, cytoskeletal reassembly, platelet cohesion, and aggregation. Intracellular signals elaborated by activated platelets in response to the occupancy of platelet receptors by agonists and by interactions include transient increases in intracellular Ca2+levels, activation of phospholipase A, (PLA,) and phospholipase C (PLC), elaboration of secondary messengers, phosphorylation or dephosphorylation of cytoplasmic and membrane proteins, and inhibition of adenylate cyclase." l2, 28, 30, 33* 49, 56, 63 The plasma serine protease a-thrombin is the most potent physiologic platelet agonist; this enzyme also has other key roles in hemostasis, in the genesis of
This article was previously scheduled to appear in our April 2000 issue devoted to Blood Stasis and Thrombosis. We are including it here as a benefit to our readers.
From the Canadian Blood Services and Department of Pathology and Molecular Medicine, McMaster University, Hamilton (FAO); and the Department of Biomedical Science, Ontario Veterinary College, University of Guelph, Guelph (KAN), Ontario, Canada
HEMATOLOGY/ ONCOLOGY CLINICS OF NORTH AMERICA VOLUME 14 * NUMBER 5 OCTOBER 2000
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arterial thrombi, and in embryonic development, inflammation, wound l4 healing, and cell pr01iferation.l~~ RECEPTORS FOR a-THROMBIN ON HUMAN PLATELETS
Three platelet membrane receptors, namely glycoprotein Ib (GPIb),", and protease-activated receptors 1 and 4 (PAR-1 and PAR-4)26,29, 34, 35, 41, 67, 71 have been reported to be actual and potential binding sites for human a-thrombin on human platelets. Glycoprotein Ib is the most abundant of the three platelet glycoproteins reported to bind a-thrombin. There are approximately 25,000 GPIb molecules per platelet, and only 50 (i.e., 0.2%) of the total platelet GPIb molecules apparently complexed with glycoprotein (GP) IX (GPIX) bind a-thrombin with high affinity (K,= 0.3 n m ~ l ) 24, . ~72, If there is one a-thrombin binding site on each of the 50 high-affinity sites for a-thrombin (the most potent platelet agonist) on GPIb, then at 0.3 nmol a-thrombin (the I(d ), only 10% of the available a-thrombin would be bound to the high-affinity sites on GPIb. The reasons why only 50 GPIb molecules (out of the 25,000 per platelet) provide high-affinity binding sites for a-thrombin and why only 10% of these sites would be occupied by a-thrombin at 0.3 nmol/L have not yet been adequately explained. The other two potential platelet receptors for a-thrombin on human platelets, PAR-1 and PAR-4, are two of the four known members of the family of seven transmembrane G-protein-linked protease-activated 29, 31, 34, 35, 41, 42, 67, 71 A unique feature of these four proteaseactivated receptors is that their activation requires cleavage of each receptor by a protease at a unique site located in the amino-terminal extracellular domain of each receptor. Each receptor cleavage releases a peptide consisting of residues beginning with the original amino-terminal residue and ending with first of the two residues that constitute the cleavage site. The newly exposed amino-terminal domain of the cleaved protease-activated receptor still anchored to the cell membrane then binds to sites (as yet undefined) on the receptor to complete activation of the receptor. After PAR-1 was cloned,67PAR-1 was initially defined as a moderate-affinity receptor for a-thrombin on platelets with less than 2000 PAR-1 molecules per platelet with a I(d of 1 n m ~ l / L If. ~there is one a-thrombin binding site on each platelet PAR-1 molecule, then at 1 nmol a-thrombin (i.e., approximately the I(d ), essentially all the PAR-1 molecules on platelets would be able to bind to the a-thrombin. a-Thrombin binds to the hirudinlike domain of PAR-1 with high affinity (both in the presence and in the absence of CaClZa)to initiate cleavage 'ele;?, thereby releasing a 41-mer peptide, PAR-1 of this receptor at Arg/S 46, 51, 53, 71 This 41-mer peptide residues 1 through 41, from the is now known to activate platelets in its own right.ls,56 This cleavage of PAR-1 at Are/SeP unmasks the "true" or "tethered" ligand domain of PAR-1 which begins with the sequence Ser-Phe-Leu-Arg-Asn (SFLLRN); 24, 33,
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the tethered ligand domain of PAR-1 then binds to sites (that are not yet 67 Note that fully defined) on PAR-1 to propagate platelet a~tivation.’~, exogenous SFLLRN is a less potent platelet agonist than exogenous athrombin, because approximately 10 pmol/ L SFLLRN activates platelets 51, 67 This observation as effectively as 1 to 10 nrnol/L a-thr~mbin?~, suggests that continued occupancy of the hirudinlike domain of PAR-1 by a-thrombin contributes to PAR-1-dependent signaling in platelets. In contrast with PAR-1, human platelet PAR-4 lacks a high-affinity (hirudinlike) binding site for a-thrombin distal to the a-thrombin cleavage site (i.e., A1-V/Gly48).~~, 35, 71 Therefore, a-thrombin is unlikely to bind tightly (if at all) to PAR-4, either before or after PAR-4 cleavage. The absence of a hirudinlike domain for PAR-4 is probably why 30 nmol/L a-thrombin is apparently required to cleave PAR-4 to propagate activation of platelets when the PAR-1 of the platelets has been impaired by preincubation with a strong PAR-1 antagonist or polyclonal anti-PAR-1 1gG directed against amino acid residues spanning the PAR-1 cleavage site.34Therefore, it is unlikely that PAR-4 can be readily cleaved directly by the low (i.e., subnanomolar) concentrations of a-thrombin that are normally found in vivo. Confirmation of the view that less than 30 nmol/ L of a-thrombin does not at least initiate PAR-4 cleavage, however, requires methods for quantifying direct PAR-4 cleavage. Methods for quantifying PAR-4 cleavage and the subsequent release of PAR-4 residues 1 through 47 have not been reported. Like the tethered ligand of PAR-1, Gly-Tyr-Pro-Gly-Gln-Val (GYPGQV) (the first six residues of the PAR-4 tethered ligand domain) can, at 500 pmol/L, also activate human platelets.% Roles of Protease-Activated Receptor 1 Cleavage and Protease-activated Receptor 4 Cleavage in Human Platelet Activation
As noted previously PAR-1 has a hirudinlike domain distal to its thrombin cleavage site ( A r e/ SeF) whereas PAR-4 lacks such a hirudinlike domain distal to its cleavage site (Arg7/Glp8)?9,34, 67, 71 The presence of the hirudinlike domain on PAR-1 clearly facilitates a-thrombin binding to platelet PAR-1.41,42, 46, 50, 51, 53 a-Thrombin binding to this site, by the fibrinogen recognition exosite of the enzyme, probably contributes to the effective cleavage of PAR-1 observed after platelets are incubated with subnanomolar a-thrombin.41,67 The high affinity of a-thrombin for the hirudinlike domain of PAR-1 is also evident from observations that when washed human platelets are incubated with hirudin before 0.5 nmol/L a-thrombin, at least a tenfold molar excess of hirudin over athrombin is required for hirudin to abrogate platelet activation:’ Conversely a consequence of the absence of a hirudinlike domain in PAR-4 is the ineffective cleavage of platelet PAR-4 by a-thrombin, so that at least 30 nmol/ L a-thrombin is required for PAR-&dependent platelet a~tivation.~~ Cleavage of PAR-1 by a-thrombin causes the release of PAR-1 resi-
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dues 1 through 41.42,46,50* 51, 53 Cleavage of PAR-4 by a-thrombin probably also causes the release of PAR-4 residues 1 through 47 from the platel e t ~ ?35,~71, Exposure of the two new amino-terminal domains containing the tethered ligand domains is crucial for propagating PAR-1- and PAR&dependent platelet activation. Two lines of evidence support this view. (1) Potent antagonists of the PAR-1 tethered ligand domain effectively inhibit activation of human platelets in response to 1.0 nmol/L athrombin;" (2) Simultaneous incubation of platelets with antibodies directed against segments of PAR-1 and PAR-4 containing the a-thrombin cleavage sites of the two protease-activated receptors abrogates platelet activation by a-thrombin.34As noted previously, both tethered ligand domains (represented by the two 6-mer peptides given later) can independently effect platelet activation. The concentrations of SFLLRN (PAR1 tethered ligand) and GYPGQY (PAR-4 tethered ligand) required for optimal PAR-1- and PAR-Mependent platelet activation are, however, approximately four and five orders of magnitude higher, respectively, 31, 34, 35, 48, 51, 67, 71 The concentration of GYPthan those of a-thr~mbin.~, GQY required for optimal platelet activation is 50-fold higher than the required concentration of SFLLRN, suggesting that PAR-1 cleavage is normally the predominant pathway for propagating a-thrombindependent platelet activation. Primacy of Platelet Protease-Activated Receptor 1 as the Binding Site for a-Thrombin
The authors have recently compared PAR-1 and GPIb as potential binding sites for human a-thrombin on human platelets.4l That study used flow cytometry to quantify a-thrombin binding to washed human platelets and activation of the same platelets. Biotinylated, affinity-purified polyclonal rabbit anti-human a-thrombin IgG was added to paraformaldehyde-fixed periodic aliquots of platelets incubated with athrombin. Binding of human a-thrombin to the platelets in the periodic aliquots was quantified by flow cytometry." Monoclonal antibodies directed against CD62 (P-selectin) and CD63 (a platelet lysosomal protein) were used to quantify activation of the same aliquots of paraformalde", 51 The study compared four groups of platelet hyde-fixed suspensions, namely control platelets, GPIb-depleted platelets, platelets incubated with TM60 or LJ-IS10 (two anti-GPIb monocolonal antibodies reported to inhibit a-thrombin binding to platelets), and platelets incubated with ATAP 138 (an anti-PAR-1 monocolonal antibody directed against an epitope which contains the hirudinlike a-thrombin binding domain of PAR-1).5 The anti-PAR-1 monoclonal antibody, ATAP 138, had previously been shown to abrogate the responses of human platelets to subnanomolar human a-thr~mbin.~ Glycoprotein Ib-depleted platelets were obtained by incubating platelets with a protease from Serrutiu murcescens or the bacterial protease 0-sialoglyoprotein endopeptidase able specifically to remove GPIb from human platelets under the condi-
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tions employed. Because the physiologic concentration of Ca2+increases the affinity of platelets for binding a-thrornbin:l and previous studies defining GPIb as a binding site for a-thrombin were performed in the absence of CaC1,22,24, 72 the studies comparing GPIb and PAR-1 as potential binding sites for human a-thrombin were conducted both in the presence and in the absence of 2 mmol/ L CaC1,.4l The authors found that in the presence or absence of physiologic calcium, the anti-PAR-1 monoclonal IgG, ATAP 138, abrogated both binding of 1 nmol/L a-thrombin to human platelets and subsequent activation of the same platelets, as previously demonstrated by others? In contrast, neither of the anti-GPIb monoclonal antibodies nor GPIb depletion inhibited binding of a-thrombin washed to human platelets resuspended in a Tyrode's buffer containing albumin and 2 mmol/L CaC1,. Also, none of the three pretreatments of platelets aimed at impairing the functions of platelet GPIb inhibited activation of the platelets in response to a-thrombin. Significant involvement of GPIb as a binding site for a-thrombin could be demonstrated only in the absence of CaCl, and only when the incubation of platelets with a-thrombin was allowed to proceed for 30 minutes instead of 10 seconds. Both 1 nmol/L athrombin binding to human platelets and activation of the same platelets are readily demonstrable when platelets are incubated with a-thrombin for 10 seconds.41It seems, therefore, that in the absence of CaCb binding of the highly basic a-thrombin to the highly acidic extracellular region of GPIb essentially accounts for the hypothesized high-affinity interactions between a-thrombin and GPIb. Calcium would be expected to form salts with some of the highly acidic residues located within the extracellular domain of GPIb to reduce the net charge on GPIb. Others had also previously demonstrated, even in the absence of CaCl, that monocolonal antibodies that bind the proposed a-thrombin binding site on GPIb do not abrogate the responses of human platelets to subnanomolar concentrations of a-thrombin.", 72 Complete inhibition of a-thrombin binding to and activation of human platelets preincubated with ATAP 138 also suggests that 1.0 nmol/L a-thrombin does not bind to or readily cleave platelet PAR-4 to propagate platelet activation. Based on these observation^'^ and observations by others that ATAP 138 abrogates the responses of human platelets to less than 1 nmol/L awhereas anti-GPIb monoclonal antibodies do not abrogate the responses of human platelets to 1 nmol/L a-thrombin,", the authors have concluded that PAR-1 is the primary binding site for athrombin on human platelet^.^^ In summary, subnanomolar a-thrombin and approximately 30 nmol/ L a-thrombin can initiate PAR-l-dependent and PARMependent platelet activation. Initiation of PAR-1-and PAR-&dependent platelet activation critically depends on a-thrombin-mediated PAR-1 and PAR-4 cleavage, respectively, and the associated release of the tethered ligand domains of the two protease-activated receptors necessary to propagate platelet activation in response to a-thrombin. Platelet activa-
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tion can be quantified as one or more of transient increases in intracellular Ca2+.Platelet activation can also be quantified by: flow cytometric quantification of the percentage of platelets expressing CD62, CD63, or phosphatidylserine (or a combination thereof) externalized on the surface of activated platelets release of ADP, ATP, and serotonin from platelet granules platelet aggregation. It is apparent that subnanomolar concentrations of a-thrombin effectively initiate platelet PAR-1 cleavage, compared with the approximately 30 nmol/ L a-thrombin required to effect PAR-4 cleavage. Therefore, PAR-1 cleavage probably drives a-thrombin-mediated platelet activation, whereas PAR-4 cleavage may not be normally involved in athrombin-mediated activation in vivo. Given the lower abundance of the PAR-4 mRNA in human platelets (compared with the PAR-1 message), and that the PAR-4 tethered ligand is 50-fold higher than the PAR1 tethered ligand domain peptides required to activate the same human 51 PAR-4 is probably less important than PAR-1 for directing platelet~,3~,~~> platelet activation in vivo. It is worth noting, however, that fibrinogen may increase the concentration of a-thrombin required to activate platelets in vivo. In support of this view, more than 5.0 nmol/L a-thrombin is required to activate platelets resuspended in recalcified plasmas (containing a factor Xa inhibitor to prevent prothrombin activation) to the same extent as platelets resuspended in buffer to which 0.5 nmol/L or 1 nmol/L a-thrombin has been added." Platelet Membrane Protease and Propagation of Protease-Activated Receptor l-Platelet Activation
Incubation of platelets with SFLLRN invariably results in PAR-1 cleavage. Platelet PAR-1 cleavage in the course of platelet activation, and in response to SFLLRN, was abrogated by soybean trypsin inhibitor, an effective inhibitor of trypsin- and plasminlike enzymes. Abrogation of platelet PAR-1 cleavage in response to SFLLRN by soybean trypsin inhibitor was also associated with strong inhibition of platelet activat i ~ nThese . ~ ~ observations are consistent with a platelet protease cleaving PAR-1 in response to the addition of SFLLRN to propagate PAR-1dependent platelet activation. Significantly, PAR-1 cleavage (and release of PAR-1 residues 141),as well as platelet activation in response to athrombin, could also be abrogated by soybean trypsin inhibitor.5O.51 Thus, platelet PAR-1 cleavage in response to a-thrombin to the extent necessary to assure propagation of platelet activation requires PAR-1 cleavage by the platelet serine protease. The authors have preliminary evidence indicating that SFLLRN externalizes a platelet membrane protease that probably cleaves PAR-1 to release PAR-1 residues 1 through 41 to help propagate platelet activation in response to SFLLRN. This protease is detected on platelets using biotinylated soybean trypsin inhibitor and
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flow cytometry; this soybean trypsin inhibitor-reactive platelet protease is also externalized within 10 seconds on platelets incubated with PAR1 residues 1 through 41 (F. A. Ofosu et al, unpublished data). Given that the peptide consisting of PAR-1 residues 1 through 41 activates platelets,*8,50 both PAR-1-derived fragments (i.e./ the fragment of PAR beginning with the PAR-1 tethered ligand domain and PAR-1 residues 1 through 41) seem to act in concert to optimize the propagation of platelet activation in response to a-thrombin. The role of this protease in propagating PAR-1-dependent platelet activation helps explain a previous observation that simultaneous addition of 10 nmol/L a-thrombin and plasmin to recalcified platelet-rich plasma accelerates prothrombinase formation more effectively than addition of 10 nmol/L of a-thrombin alone. Plasmin alone does not enhance prothrombinase formation in platelet-rich plasma.4O Whether and when this platelet protease also cleaves PAR-4 to release PAR-4 residues 1 through 47 (thereby exposing the PAR-4 tethered ligand domain) to optimize platelet activation has not yet been established. Whether the peptide consisting of PAR-4 residues 1 through 47 can similarly activate human platelets is also not known. Possible Interactions Between Activated ProteaseActivated Receptor 1 and Glycoprotein Ib
Although 1 nmol/L a-thrombin does not bind to GPIb on human platelets resuspended in buffers containing 2 mmol/L CaC1, GPIb clearly influences the changes in intracellular Ca2+in response to subnanomolar a-thrombin.22The authors have found that GPI depletion of platelets (by preincubating platelets with 0-sialoglycoprotein endopeptidase) inhibits the rate of PAR-1 cleavage in response to a-thrombin. Specifically, following incubation of platelets with 1 nmol/ L a-thrombin for 10,60, and 180 seconds, GPIb depletion impaired PAR-1 cleavage by 76%, 35%, and 17%, respectively, in comparison with control platelets incubated for identical periods. Thus, inhibition of PAR-1 cleavage by GPIb depletion was most significant during the initial phase of incubating platelets with 1 nmol/L a-thrombin. Inhibition of a-thrombindependent PAR-1 cleavage by GPIb depletion insignificantly inhibited a-thrombin-dependent platelet activation, however. The authors have also observed that GPIb depletion significantly impairs platelet aggregation in response to PAR-1 residues 1 through 41. Thus, there seem to be functional interactions between GPIb and PAR-1 (probably through PAR1 residues 1 through 41) that optimize propagation of platelet activation in response to subnanomolar a-thrombin. THROMBIN-MEDIATED SIGNALING IN PLATELETS
Exposure of platelets to a-thrombin initiates a sequence of signaling events which ultimately serves to activate the platelets. &-Thrombin
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binds to its receptors on human platelets and activates them to initiate PLA, activation. Activated PLA, in turn, releases of arachidonate from platelet membranes to provide the initial substrate necessary for the synthesis of thromboxane A, TxA,.* a-Thrombin binding to platelets also initiates activation of Ca2+-dependent PLC (probably more than one PLC) and the associated production of the signaling molecules 41, 56* 65 Production diacylglycerol (DAG) and inositol triphosphate (IP3).30, of DAG leads to Ca2+-dependentactivation of protein kinase C (PKC), the cellular high-affinity receptor for DAG. The end results of these PLAT and PLC-dependent signaling events include platelet shape change, release of P-selectin and fibrinogen from platelet a-granules, release of ADP, ATP, serotonin, and other granular contents, externalization and activation of platelet GPIIb/IIIa and, ultimately, platelet aggregation. Both TxA, and ADP are significant platelet agonists in response to their binding to their specific receptors on human platelets. Thus, elaboration of these two platelet-derived agonists during platelet activation in response to subnanomolar a-thrombin probably enhances propagation of the signaling events that ultimately result in the release and expression of platelet granular contents, and the cohesion and aggregation of the platelets. Activation of the two major distinct platelet-signaling pathways (involving PLC and PLA, activation) in response to a-thrombin are accompanied by phosphorylation of several platelet proteins and phospholipids and elaboration of several signaling intermediates.t Many platelet proteins become phosphorylated within 15 seconds following a-thrombin addition to platelets,$ and tyrosine phosphorylation of platelet proteins occurs in several temporal “waves,” suggesting that the several protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs) act sequentially on their substrates. Dephosphorylation reactions catalyzed by phosphatases have also been associated with signaling.69Despite the considerable progress in identifying these phosphorylated proteins, however, the precise functions of many of these platelet proteins remain unclear. PROTEASE-ACTIVATED RECEPTOR 1 AND GLYCOPROTEIN Ilb/llla ACTIVATION
Glycoprotein IIb / IIIa, the Ca2+-dependentreceptor for fibrinogen on the surface of platelets and consisting of aIIband p3 subunits, is the platelet integrin thought to mediate the fibrinogen-dependent common pathway of platelet aggregation.’O.45, 5941, 70 This transmembrane receptor has a large extracellular domain with specific ligand recognition sequences, including the arginine-glycine-aspartate-serine(RGDS) sequence found in fibrinogen, von Willebrand’s factor, fibronectin, *References6, 12, 17, 28, 30, 39, 43, 49, 56, and 64-66. tReferences 7, 9, 15, 16, 20, 21, 23, 27, 32, 37-39, 44, 67, 69, and 73. *References 7, 9, 15, 16, 20, 21, 23, 27, 32, 37, 38, and 73.
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vitronectin, and thrombospondin. Each of these adhesive proteins is a ligand for the platelet integrin GPIIb / IIIa. The expression of the numbers and ligand-binding functions of the GPIIb/IIIa integrin on platelets can be modulated by outside-in, inside-out signaling and by limited proteolysis of GPIIb / IIIa. Approximately one half of the 100,000 GPIIb/ IIIa integrin molecules per platelet are constitutively expressed on platelet membranes; the balance, along with an increased capacity to bind fibrinogen, becomes evident only after GPIIb/ IIIa is activated by an inside-out signaling process. The resulting conformational modifications of the extracellular domains of GPIIb / IIIa facilitate the conversion of this integrin from a low-affinity to a high-affinity platelet membrane receptor for fibrinogen." 68, 70 Platelet activation initiated by ligand-receptor interactions, such as PAR-1 occupancy (at its hintdinlike domain) and subsequent PAR-1 cleavage by a-thrombin, the resulting outside-in signals generated, as well as the inside-out signals generated by binding of intracellular ADP, TxA, and serotonin to their platelet receptors, all contribute to the activation of GPIIb / IIIa. Only transitory GPIIb / IIIa activation is effected by ADP, platelet activating factor (PAF), SFLLRN, and adrenaline, whereas irreversible activation of this receptor is effected when platelets are incubated with 5 nmol/ L a-thrombin.6z,70 The transitory activation of GPIIb / IIIa by the weaker platelet agonists is reversible by prostaglandin I,. During the platelet release reaction, the intracellular fraction of GPIIb/ IIIa becomes translocated from its storage sites in the a-granules and elsewhere to the plasma membrane, thereby increasing the capacity of activated platelet GPIIb /IIIa to bind f i b r i n ~ g e n . ~ ~ Elastate, a-chymotrypsin, and cathepsin G activate platelet GPIIb / IIIa by a cleavage at the carboxyl-terminal domain of the am subunit heavy chain by inducing irreversible expression of fibrinogen binding sites on GPIIb/ IIIa.36,52, 55, 58 Proteolytic activation of GPIIb / IIIa apparently occurs independently of intracellular The platelet-derived protease which becomes externalized on platelet membranes when PAR-1 is activated by a-thrombin or SFLLRN may also modulate the functions of GPIIb/IIIa by a proteolytic mechanism. The reason for this view is that soybean trypsin inhibitor can abrogate platelet aggregation in response to SFLLRN or PAR-1 residues 1through 41 (F.A. Ofosu, K.A. Nyarko, et al, unpublished data). There is evidence that PAR-1 activation requires a functional GPIIb/ IIIa.19 Unlike GPIIb/ IIIa that can be activated by leukocyte elastase, this enzyme and two other leukocyte enzymes (namely cathepsin G and protease 3) cleave PAR-1 at sites within the PAR-1 extracellular domain and distinct from the thrombin cleavage site. Therefore, cleavage of PAR-1 by these three leukocyte proteases does not lead to platelet activation. Rather, these leukocyte proteases can abrogate or significantly reduce platelet responses to a-thrombin. To cleave PAR-1, however, 600 nmol/L of these leukocyte proteases were required.57Therefore, these leukocyte enzymes may not normally influence a-thrombin-mediated PAR-1 activation in vivo.
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Signals Attributable to Interactions Between Glycoprotein llb/illa Subunits allbP3and ProteaseActivated Receptor 1
Platelet aggregation requires platelet-platelet interactions that are in all likelihood mediated by a fibrinogen bridge binding to the activated conformer of GPIIb/II1a.lo,~ 5 ,47, 59, 6o As noted previously, incubation of platelets with 0.5 nmol/L a-thrombin results in PAR-1 cleavage and generation of intracellular signals which culminate in aggregation of the platelets. Activated PAR-1 couples to G-proteins able to effect PLC and, possible, PLA activation. Phospholipase C in turn hydrolyzes phosphatidylinositol-4,5-bisphosphate (PIPz)to generate IP, and DAG. The secondary intracellular messengers IP, and DAG ultimately enhance increases in intracellular Caz+ and activation of protein kinase C (PKC), respectively, in platelets. Activated PKC phosphorylates several platelet proteins, including PTKs. Phopholipidase Az liberates arachidonate from platelet membranes necessary for TxA, biosynthesis. Protein tyrosine kinases and protein phosphatases (PPs) are also activated in response to a-thrombin binding to platelet PAR-1 to cause PAR-1 activation. Interactions between fibrinogen and the activated GPIIb/IIIa conformer similarly result in the activation of PLC, PLA, PTK, and PPs and in the production of their respective rnes~engers.2~. 47 Thus, PAR-1 and GPIIb / IIIa activation results in the elaboration of the same intracellular signals within platelets. Activation of PKC increases both the number and the affinity of amp3 receptors for fibrinogen on platelets to enhance platelet aggregation. Activated PKC can directly activate PTKs, which in turn convert the nonactivated GPIIb/ IIIa conformer (closed integrin aIbp30) to the open or activated conformer (aIIbp,*).The open or activated conformer amp3. rapidly recloses in the absence of an appropriate ligand or its dephosphorylation by PTKs.= Depending on the degree of PKC activation, a significant fraction of the pool of internal (YIbp3 may become externalized, and the p3 chain of the internal pool of aIbp3becomes phosphorylated by PKC. This PKC-dependent phosphorylation of GPIIb/IIIa opposes the return of the activated conformer of GPIIb/ IIIa to the closed configuration. Dephosphorylation by serine-threonine protein phosphatases converts aIbpB.to its closed configuratio11.2~Thus, either aIIbp3or PAR-1 activation results in the generation of signals able to modulate platelet function, suggesting that activation of either aIbp3 or PAR-1 can ultimately result in platelet aggregation. Incubation of subnanomolar a-thrombin with platelets that have already bound fibrinogen decreases production of inositol phosphates and decreases the phosphorylation of 20-kd and 40-kd proteins relative to those observed when control platelets are incubated with a-thrombin.57These observations are consistent with interactions between a I & and PAR-1, probably at the signal-transduction level. Activation of PAR1 on the megakaryoblastic cells, CHRF-288, results in the cells' adhering and spreading on immobilized fibrinogen in a GPIIb / IIIa-dependent
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manner. In this instance, however, PAR-1 activation does not support GPIIb / IIIa-dependent binding of soluble fibrinogen or the binding of PAC-1, the monoclonal antibody directed against activated GPIIb/ IIIa.8 Despite their failure to bind soluble fibrinogen, PAR-1 activation on CHRF288 cells results in phosphoinositide hydrolysis, arachidonate metabolism, and phosphorylation of ~ 4 (a 422 kd mitogen-activated ~ ~ ~ protein kinase), PLA, and the Rac exchange protein Vav.8 The signals generated by PAR-1 activation supported amp3binding to immobilized fibrinogen, suggesting that amp3did not undergo the conformational change required to bind to soluble fibrinogen or to PAC-1 and that there was an increase in amp3 avidity rather than an increase in affinity.8 Increased avidity is thought to result from an accumulation of relatively low-affinity interactions between this integrin and its ligands. Thus, it is likely that changes in the lipid membrane composition (evident as increased externalization of platelet phosphatidylserine resulting from PAR-1 activation) increases the lateral mobility of within the platelet membrane, to cause accumulation of amp3at the interface. Thus, PAR-1 activation in response to a-thrombin modulates GPIIb/ IIIa functions, because PAR-1 activation initiates outside-in signals which facilitate increased expression of GPIIb / IIIa receptors on the platelet surface and also increases the avidity of GPIIb/IIIa for external ligands. Given that GPIIb/IIIa can be activated by limited proteolysis, it is possible that the platelet protease externalized on platelets during PAR1 activation also directly activates GPIIb/ IIIa. Additional studies are required to elucidate fully the functional interactions between platelet PAR-1 and resting and activated conformers of GPIIb/IIIa. References 1. Andersen H, Greenberg DL, Fujikawa K, et al: Protease-activated receptor 1 is the primary mediator of thrombin stimulated platelet-procoagulant activity. Proc Natl Acad Sci U S A 96:11189-11193, 1999 2. Andrade-Gordon P, Maryanoff BE, Derian CK, et al: Design, synthesis, and biological characterization of a peptide-mimetic antagonist for a tethered ligand receptor. Proc Natl Acad Sci U S A 96:12257-12262, 1999 3. Bematowicz MS, Klimase CE, Hart1 KS, et al: Development of potent thrombin receptor antagonist peptides. J Med Chem 39:48794887, 1996 4. Brass LF, Molino M: Protease-activated G protein-coupled receptor on human platelets and endothelial cells. Thromb Haemost 78:234-241, 1997 5. Brass LF, Vassallo RR Jr, Belmonte E, et a1 Structure and function of the human platelet thrombin receptor: Studies using monoclonal antibodies against a defined epitope with the receptor N-terminus. J Biol Chem 26713795-13798, 1992 6. Carter AN, Huang R, Sirosky A, et al: Phosphatidylinositol3,4,5-triphosphateis formed from phosphatidylinositol 4,5-biphosphate in thrombin-stimulated platelets. Biochem J 301:415-420, 1994 7. Cichowski K, Brugge JS, Brass LF: Thrombin receptor activation and integrin engagement stimulate tyrosine phosphorylation of proto-oncogene product p95vav, in platelets. J Biol Chem 271:7544-7550, 1996 8. Cichowski K, Orsini MJ, Brass LF: PAR-1 activation initiates integrin engagement and outside in signaling in megakaryoblastic CHRF-288 cells. Biochim Biophys Acta 1450:265276, 1999
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9. Clark EA, Shattil SJ, Brugge J S Regulation of protein tyrosine kinases in platelets. Trends in Biochemical Sciences 19:464-469, 1994 10. Coller BS Blockade of platelet-GPIIb /IIIa receptors as an antithrombotic strategy. Circulation 92:2373-2380, 1995 11. De Marco L, Mazzucato M, Masotti A, et al: Localization and characterization of an athrombin binding site on platelet glycoprotein Ib. J Biol Chem 269:6478-6484, 1994 12. Du X, Harris SJ, Tetaz TJ, et al: Association of a phospholipase A (14-3-3 protein) with platelet glycoprotein Ib-IX complex. J Biol Chem 269:18287-18290, 1994 13. Fenton JW 11: Thrombin bioregulatory functions. Advances in Clinical Enzymology 6:186-193, 1988 Ofosu FA, Brezniak DV, et al: Understanding thrombosis and hemostasis. 14. Fenton JW, Hematol Oncol Clin North Am 71107-1119, 1993 15. Ferrell JE, Martin GS: Platelet tyrosine-specific protein phosphorylation is regulated by thrombin. Mol Cell Biol 8:3603-3610, 1988 16. Ferrell JE, Martin GS: Thrombin stimulates the activities of multiple previously unidentified protein kinases in platelets. J Biol Chem 26420723-20729, 1998 17. Fitzgerald GA, Reilly IAG, Pedersen AK: The biochemical pharmacology of thromboxane synthesis and inhibition in man. Circulation 72:1194-1201, 1985 18. Furman MI, Liu L, Benoit SE, et al: The cleaved peptide of the thrombin receptor is a strong platelet agonist. Proc Natl Acad Sci U S A 95:3082-3087, 1992 19. Giesberts AN, van Willegen G, Lapetina E, et al: Regulation of platelets glycoprotein IIb/IIIa (integrin aIIbp3) function via the thrombin receptor. Biochem J 309:613420, 1995 20. Golden A, Brugge JS: Thrombin treatment induces rapid changes in tyrosine phosphorylation in platelets. Proc Natl Acad Sci U S A 86:901-905, 1989 21. Golden A, Brugge JS, Shattil SJ: Role of platelet glycoprotein IIb-IIIa in agonist-induced tyrosine phosphorylation of platelet proteins. J Cell Biol 111:3117-3127, 1989 22. Greco NJ, Tandan NN, Jamieson GA: Contributions of GpIb and the seven transmembrane domain receptor to increases in platelet cytoplasmic [Caz+], induced by athrombin. Biochemistry 35:906-914, 1996 23. Haimovich B, Lipfert L, Brugge JS, et al: Tyrosine phosphorylation and cytoskeletal reorganization in platelets are triggered by interaction of integrin receptors with their immobilized ligands. J Biol Chem 268:15868-15877, 1993 24. Harmon JT, Jamieson G A Activation of platelets by a-thrombin is a receptor mediated event. J Biol Chem 261:15928-15933, 1986 25. Hers I, Donath J, van Willigen G, et al: Differential involvement of tyrosine and serine/ threonine kinases in platelet integrin aIIbp3 exposure. Arterioscl Thromb Vasc Biol 183404414, 1998 26. Hoxie JA, Ahuja M, Belmonte E, et al: Internalization and recycling of activated thrombin receptors. J Biol Chem 268:13756-13763, 1993 27. Huang R, Kucera GL, Rittenhouse SE: Elevated cytosolic Ca2+activates phospholipase D in human platelets. J Biol Chem 266:1652-1655, 1991 28. Huang R, Sorisky A, Church WR, et al: Thrombin receptor-directed ligand accounts for activation of platelet phospholipase C and accumulation of phosphorylated phosphoinositides. J Biol Chem 266:18435-18438, 1991 29. Hung DT, Vu T-KH, Wheaton VI, et al: Cloned platelet-thrombin receptor is necessary for thrombin-induced platelet activation. J Clin Invest 89:1350-1353, 1992 30. Hung DT, Wong YH, Vu T-Kh, et al: Cloned thrombin receptor complexes to at least 2 distinct effectors to stimulate phosphoinositide hydrolysis and inhibit adenyl cyclase. J Biol Chem 26720831-20834, 1992 31. Ishihara H, Connolly AJ, Zeng D, et al: Protease-activated receptor 3 is a second thrombin receptor in humans. Nature 386502-506, 1997 32. Jackson SP, Schoenwaelder SM, Yuan Y, et al: Non-receptor protein tyrosine kinases and phosphatases in human platelets. Thromb Haemost 75:640-650, 1996 33. Jamieson G A Pathophysiology of platelet thrombin receptors. Thromb Haemost 78:242-246, 1997 34. Kahn ML, Nakanishi-Mitsuim M, Shapiro MJ, et al: Protease-activated receptors 1 and 4 mediate activation of human platelets by thrombin. J Clin Invest 103:879-887, 1999
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35. Kahn ML, Zhen Y-W, Huang W, et al: A dual thrombin receptor for platelet activation. Science 394690-694, 1998 36. Komecki E, Erlich YH, Egbring R, et al: Granulocyte-platelet interactions and platelet fibrinogen exposure. Am J Physiol255:H651-H658, 1998 37. Kramer RM, Roberts EF, Manetta JV,et al: Thrombin-induced phosphorylation and activation of Ca2+-sensitivecytosolic phospholipase A2 in human platelets. J Biol Chem 268:26796-26804, 1993 38. Kroll HM, Schafer AL Biochemical mechanisms of platelet activation. Blood 74:11811195, 1989 39. Lapetina E: Signal transduction induced by thrombin-activated platelets. FEBS Lett 268:400-404, 1990 40. Liu L, Freedman J, Homstein A, et al: Plasmin accelerates platelet-dependent prothrombinase assembly without activating the platelets. Br J Haematol 92:458-465, 1996 41. Liu L, Freedman J, Homstein A, et al: Binding of thrombin to the G-protein-linked receptor, and not to GPIb, precedes thrombin-mediated platelet activation. J Biol Chem 272:1997-2004, 1997 42. Liu L, Freedman J, Homstein A, et al: Thrombin binding to platelets and their activation in plasma. Br J Haematol88:592-600, 1994 43. McNichol A, Robson C A Thrombin receptor-activating peptide releases arachidonic acid from human platelets. J Pharmacol Exp Ther 281:861-867, 1997 44. Nieuwland R, van Willigen G, Akkerman J-WN: Different pathways for control of N a + / H +exchange via activation of thrombin receptor. Biochem J 29747-52, 1994 45. Niiya K, Hodson E, Bader R, et al: Increased surface expression of membrane glycoprotein IIb /IIIa complex induced by platelet activation: Relationship between binding of fibrinogen and platelet aggregation. Blood 70:475483, 1987 46. Norton KJ, Scarborough RM, Kutok JL, et al: Immunologic analysis of the cloned platelet thrombin receptor activation mechanism: Evidence supporting receptor cleavage, release of the N-terminal peptide and insertion of the tethered ligand into a protected environment. Blood 82:2125-2136, 1993 47. Nurden AT A review of the role of platelet membrane glycoproteins in the plateletvessel wall interaction. Baillieres Clin Haematol 6:653-690, 1993 48. Nystedt S, Emilsson K, Weahlestedt C, et al: Molecular cloning of a potential novel proteinase activated receptor. Proc Natl Acad Sci U S A 91:9208-9212, 1994 49. Offermanns S, Laugwitz KL, Spicher K, et al: Proc Natl Acad Sci U S A 91:504-508,1994 50. Ofosu FA, Dewar L, Fenton J W I1 et al: A trypsin-like platelet protease must release PAR-1 residues 1-41 and PAR-1 tethered ligand to optimize propagation of platelet activation [abstract]. Thromb Haemost (suppl):821, 1999 51. Ofosu FA, Freedman J, Dewar L, et al: A trypsin-like protease propagates platelet PAR-1 cleavage and platelet activation. Biochem J 336:283-285, 1998 52. Pidard D, Frelinger AL, Boullot C, et al: Activation of the fibrinogen receptor on human platelets exposed to a-chymotrypsin. Relationship to a major poteolytic cleavage at the carboxyl terminus of the membrane glycoprotein Ib heavy chain. Eur J Biochem 200:437477, 1991 53. Ramachandran R, Klufas AS, Molino M, et al: Release of the thrombin receptor (PAR1) N-terminus from the surface of human platelets activated by thrombin. Thromb Haemost 78:lllO-1124, 1997 54. Ray MJ, March N A Aprotinin reduces blood loss after cardiopulmonary bypass by direct inhibition of thrombin. Thromb Haemost 78:1021-1026, 1997 55. Renesto P, Si-Tahar M, Moniatte M, et al: Specific inhibition of thrombin-induced cell activation by neutrophil proteinases elastase, cathepsin G, and proteinase 3 Evidence for distinct cleavage sites within the amino terminal domain of the thrombin receptor. Blood 8931944-1953, 1997 56. Seiler SM, Michel A, Fenton JW I 1 Involvement of the ‘tethered-ligand’ receptor in thrombin inhibition of platelet adenyl cyclase. Biochem Biophys Res Commun 182:1296-1302, 1992 57. Sinigaglia F, Torti M, Ramaschi G, et al: The occupancy of glycoprotein IIb/IIIa complex modulates thrombin activation of human platelets. Biochim Biophys Acta 984:225-230, 1989
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Address reprint requests to Frederick A. Ofosu, PhD Department of Pathology and Molecular Medicine HSC - 3N26, McMaster University 1200 Main Street West Hamilton, ON L8N 325, Canada e-mail:[email protected]