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Blood, 1 April 2004, Vol. 103, No. 7, pp. 2601-2609. Prepublished online as a Blood First Edition Paper on December 18, 2003; DOI 10.1182/blood-2003-09-3319.
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY GPIb-dependent platelet activation is dependent on Src kinases but not MAP kinase or cGMP-dependent kinaseFrom the Department of Pharmacology, University of Oxford, Oxford United Kingdom; Division of Medical Sciences, The Medical School, University of Birmingham, Edgbaston, Birmingham, United Kingdom; Institut für Pharmakologie und Toxikologie, Technische Universität (TU) München, München, Germany; Pfizer Global Research and Development, Sandwich, Kent, United Kingdom; and Cardiovascular Pharmacology, Pfizer Global Research and Development, Ann Arbor, MI.
Glycoprotein Ib-IX-V (GPIb-IX-V) mediates platelet tethering to von Willebrand factor (VWF), recruiting platelets into the thrombus, and activates integrin  IIb 3 through a pathway that is dependent on Src kinases. In addition, recent reports indicate that activation of IIb3 by VWF is dependent on protein kinase G (PKG) and mitogen-activated protein (MAP) kinases. The present study compares the importance of these signaling pathways in the activation of IIb3 by GPIb-IX-V. In contrast to a recent report, VWF did not promote an increase in cyclic guanosine monophosphate (cGMP), while agents that elevate cGMP, such as the nitrous oxide (NO) donor glyco–SNAP-1 (N-(-D-glucopyranosyl)-N2-acetyl-S-nitroso-D,L-penicillaminamide) or the type 5 phosphosdiesterase inhibitor, sildenafil, inhibited rather than promoted activation of IIb3 by GPIb-IX-V and blocked aggregate formation on collagen at an intermediate rate of shear (800 s-1). Additionally, sildenafil increased blood flow in a rabbit model of thrombus formation in vivo. A novel inhibitor of the MAP kinase pathway, which is active in plasma, PD184161, had no effect on aggregate formation on collagen under flow conditions, whereas a novel inhibitor of Src kinases, which is also active in plasma, PD173952, blocked this response. These results demonstrate a critical role for Src kinases but not MAP kinases in VWF-dependent platelet activation and demonstrate an inhibitory role for cGMP-elevating agents in regulating this process.
Glycoprotein Ib-IX-V (GPIb-IX-V), the receptor for von Willebrand factor (VWF), plays a critical role in thrombus formation in damaged blood vessels under high shear.1-4 A fast on-rate of association between VWF and GPIb-IX-V allows the adhesion protein to tether (or capture) rapidly flowing platelets into the developing thrombus. A fast off-rate of dissociation, however, means that platelets rapidly detach from VWF, unless integrins such as IIb3 or 21 are activated by intracellular signals, thereby enabling them to bind their ligands and mediate stable adhesion and thrombus growth. Several surface receptors are able to mediate integrin activation, including the collagen receptor GPVI, the G protein–coupled receptors for adenosine diphosphate (ADP) and thromboxanes, P2Y1, P2Y12, and thromboxane prostanoid (TP) receptors.5 Significantly, these receptors act in synergy to mediate integrin activation and thrombus growth.6-9
It is recognized that GPIb-IX-V is also able to stimulate activation of
There is increasing evidence for a role of Src kinases in signaling by GPIb-IX-V. Thus, activation of
Recently, Du and colleagues24,25 have proposed a distinct mechanism of
The present study was undertaken to compare the relative contributions of Src kinase and PKG/MAP kinase pathways in the activation of
Materials
Ristocetin and thrombin were from Sigma Chemical (Poole, United Kingdom). VWF (Haemate P) was from Behringwerke (Marburg, Germany). Lotrafiban was a gift from GlaxoSmithKline (Piscataway, NJ). PD173952, PD184161, and sildenafil were gifts from Pfizer Global Research and Development (Ann Arbor, MI; Sandwich, United Kingdom). Glyco–SNAP-1 (N-( Preparation of human platelet-rich plasma and washed platelets Platelet-rich plasma (PRP) was prepared from whole blood taken from drug-free donors into sodium citrate 3.8% (wt/vol). The ratio of anticoagulant to whole blood was 1:9. PRP was obtained by centrifugation at 200g for 20 minutes. To prepare washed platelets, whole blood was anticoagulated with acid-citrate-dextrose (ACD; 80 mM citric acid, 120 mM sodium citrate, 110 mM glucose). The ratio of anticoagulant to whole blood was 1:7. PRP was obtained by centrifugation at 200g for 20 minutes. Platelets were separated from PRP by centrifugation at 1000g for 10 minutes in the presence of prostacyclin (1 µM). Platelets were then washed twice in CGS (0.0129 M trisodium citrate, 0.03 M D-glucose, and 0.12 M NaCl; pH 6.5) containing 0.1% bovine serum albumin (BSA) in the presence of prostacyclin (1 µM). The platelet pellet was resuspended to the appropriate concentration in Ca2+-free modified Tyrodes-HEPES buffer (134 mM NaCl, 2.9 mM KCl, 12 mM NaHCO3, 0.34 mM Na2HPO4, 20 mM HEPES [N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid], 5 mM glucose, and 1 mM MgCl2, pH 7.3). Optical aggregation studies were carried out using a Chronolog dual channel light aggregometer (Chronolog, Havertown, PA). Platelets (2 x 108/mL) were added to an aggregometer tube at 37°C and stirred at 1200 rpm. Measurement of cGMP PRP or washed platelets (3 x 108 platelets/mL) were stirred at 37°C followed by addition of ristocetin/VWF, the NO donors glyco–SNAP-1, and sodium nitroprusside or the phosphodiesterase (PDE) type 5 inhibitor sildenafil. Reactions were stopped by the addition of an equal volume of ice-cold lysis buffer 12% (wt/vol) trichloroacetic acid. Lysates were analyzed for cGMP content using a cGMP direct Biotrak immunoassay kit (Amersham Biosciences) and are presented as the concentration of cGMP nmol/107 platelets. Measurement of [32P]phosphatidic acid PRP was centrifuged to produce a platelet pellet as described in "Preparation of human platelet-rich plasma and washed platelets" and resuspended in 1 mL of a modified phosphate-free Tyrodes buffer (134 mM NaCl, 2.9 mM KCl, 12 mM NaHCO3, 20 mM HEPES, 5 mM glucose, and 1 mM MgCl2; pH 6.5). The platelet suspension was labeled with [32P]orthophosphoric acid (0.5 mCi/mL [18.5 MBq/mL]) for 1 hour at 37°C. Remaining [32P]orthophosphoric acid was removed by washing platelets in 3 mL ACD and 24 mL modified Tyrodes-HEPES buffer followed by centrifugation at 1000g for 10 minutes. The pellet was then resuspended at 5 x 108 platelets/mL in platelet-poor plasma. Stimulations were terminated by the addition of 400 µL CHCl3/methanol, 1:1 (vol/vol) and phospholipids were extracted from the sample by centrifugation at 1000g for 5 minutes at 4°Cin the presence of 200 µL HCl/EDTA (ethylenediaminetetraacetic acid) (42% [vol/vol] 10 N HCl, 58 mM EDTA). Samples were separated by thin layer chromatography. Lipids were detected by autoradiography and analyzed by scintillation counting. Preparation of mouse platelets The generation of PKG null (-/-) mice by homologous recombination has been previously described.26 Male PKG null mice and litter-match controls (wild type; 4-5 weeks old) were anesthetized with ether, and whole blood was isolated by cardiac puncture into 1:10 volume ACD. PRP was prepared from whole blood by centrifugation at 200g for 7 minutes. Platelets were washed twice using a modified Tyrode-HEPES buffer and resuspended at 2 x 108 platelets/mL, as described for human platelets. For aggregation experiments platelets were pooled from 16 wild-type and 16 PKG null mice. Flow studies Human blood or mouse blood was isolated in sodium heparin (10 IU/mL) as previously described.27 Blood was perfused through glass microslides, 1 x 0.1-mm or 2 x 0.2-mm inner diameter (Camlab, Cambridge, United Kingdom), that had been coated with either 30 or 300 µg/mL type I collagen from equine tendon (Horm; Nycomed, Munich, Germany) before blocking with 2% BSA suspended in phosphate-buffered saline (PBS). A shear rate of 800 s-1 with a corresponding flow rate of 0.08 mL/minute in 1 x 0.1-mm microslides and 0.64 mL/minute in 2 x 0.2-mm microslides was generated by a syringe pump (Harvard Apparatus, Southnatick, MA). After 2 minutes perfusion with whole blood, Ca2+-free modified Tyrode-HEPES buffer was perfused for 8 minutes through 1 x 0.1-mm microslides and 3 minutes through 2 x 0.2-mm microslides at the same shear rate. Platelet thrombi that had formed on the surface of the collagen were visualized with an inverted stage videomicroscope system (DM IRB; Leica, Milton Keynes, United Kingdom). Percent surface coverage was quantified using Image-Pro Plus software (Media Cybernetics, Silver Spring, MD). Subsequently, adherent platelets were lysed in ice-cold nonidet P-40 (NP-40) lysis buffer (20 mM Tris [tris(hydroxymethyl)aminomethane], 300 mM NaCl, 2 mM EGTA [ethyleneglycotetraacetic acid], 2 mM EDTA, 2% [vol/vol] NP-40, 1 mM phenylmethylsulphonyl fluoride, 2 mM Na3VO4,10 µg/mL aprotinin, 1 µg/mL pepstatin A, pH 7.3). Proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) on 12% gels and transferred to polyvinylidene difluoride (PVDF) membranes using a semidry transfer system (Trans-blot SD; BioRad, Hercules, CA). Membranes were blocked by incubation in 10% wt/vol BSA in Tris-buffered saline/Tween 20 (TBS-T) for 1 hour to prevent nonspecific binding of antibodies. Phosphospecific p42/44 MAP kinase antibodies were incubated with the PVDF membranes for 1 hour. Membranes were washed in TBS-T following each incubation with antibodies and then analyzed using an enhanced chemiluminescence (ECL) detection system. Folts model The rabbit model of recurrent intravascular thrombus formation has been described in detail elsewhere28 and represents a modification of the canine model originally described by Folts et al.29 Briefly, male New Zealand White rabbits (2.0-2.5 kg, Porcellus) where anesthetized with a mixture of Hypnorm (Janssen Animal Health, Beerse, Belgium; containing fentanyl citrate 0.315 mg/mL plus fluanisone 10 mg/mL given 0.5 mL/kg intramuscularly) and Hypnovel (Roche, Welwyn Garden City, United Kingdom; containing midazolam 20 mg/mL given 2 mg/kg intraperitoneally). Anesthesia was maintained during the course of the experiment by an intravenous infusion of 3.5 to 5 mL/hour of a solution of Hypnorm (1:10 [vol/vol]) and Hypnoval (1:20 [vol/vol]) in sterile saline via a catheter placed in the left marginal ear vein, which was also used for drug administration. Through a median incision of the neck, the carotid arteries and trachea were exposed and carefully isolated from the surrounding tissue. Polyethylene catheters were inserted into the trachea to support artificial respiration and also into the abdominal aorta via a femoral artery for continuous blood pressure monitoring. A segment of the exposed left carotid artery was injured by gentle squeezing of the artery between a pair of rubber-covered forceps, and blood flow was subsequently partially restricted (to approximately 60% of baseline) using a rat renal artery Goldblatt clip. Carotid blood flow velocity was measured continuously by a Doppler flow probe positioned proximal to the constrictor. Induction of arterial damage and stenosis produced cyclic fluctuations of arterial blood flow (cyclic flow reductions [CFRs]). In all animals, heparin (250 IU/kg intravenously) was used so as to avoid the contribution of coagulation to thrombus formation and maintenance. In this model, CFRs are primarily due to recurrent cycles of formation and dislodgment of platelet-rich thrombi. CFRs were monitored in all animals for 30 minutes to establish a stable baseline and then in 4 rabbits. Vehicle, 0.01, and 0.03 mg/kg sildenafil were sequentially administered intravenously, and blood flow was monitored for each 20-minute treatment period. Heart rate and arterial blood pressure were monitored continuously throughout the experiment and CFRs were scored for rate and reversibility of flow reductions during each treatment period, using the following qualitative scale: 0 indicates complete reduction of flow without spontaneous reversal; 1, reduction of flow with spontaneous reversal; 2, change in rate of flow reduction with spontaneous reversal; and 3, cessation of CFRs. The effect of treatment on CFR score was evaluated by a 2-sided permutation test.30 A P value less than .05 defined significant differences between treatment periods. Analysis of data All experiments were performed at least 3 times and data are shown as means ± SEM. Statistical analysis was conducted using Student unpaired t test unless stated.
VWF activates PLC and IIb3 through a Src kinase–dependent pathway
Experiments were undertaken to investigate previous reports that GPIb-IX-V stimulates activation of
Recent reports have indicated that GPIb-IX-V stimulates activation of PLC VWF does not stimulate formation of cGMP in platelets
Experiments were undertaken to investigate previous reports that GPIb-IX-V stimulates formation of cGMP in platelets. These studies were performed in plasma using ristocetin and in washed platelets using ristocetin and VWF. The concentrations of VWF and ristocetin that were used in the experiments induced maximal
Elevation of cGMP inhibits activation of
Experiments were undertaken to investigate previous reports that agents that increase cGMP potentiate activation of Inhibitors of protein kinase G do not block phosphorylation and potentiate (rather than inhibit) the effect of cGMP-elevating agents in platelets A critical line of evidence for a role of cGMP in GPIb-IX-V signaling in the study by Li et al24 is the inhibitory effect of the PKG-blocking agents, KT5823 and Rp-8-pCPT-cGMPS, on aggregation induced by VWF. Li et al24 argued that the use of structurally distinct inhibitors of PKG served to minimize the possibility that effects were mediated by non–PKG-dependent mechanisms. However, the ability of KT5823 to block purified PKG has since been questioned,36 whereas Rp-8-pCPT-cGMPS and the structurally related PKG inhibitor, Rp-8-Br-PET-cGMPS, only work under certain conditions (see the accompanying article by Gambaryan et al, beginning on page 2593).37 The evidence presented by Li et al38 to demonstrate the effectiveness of KT5823 and Rp-8-pCPT-cGMPS in platelets was based on phosphorylation studies. Surprisingly, Li et al38 demonstrated that KT5823 and Rp-8-pCPT-cGMPS are unable to block phosphorylation of the recognized PKG substrate VASP by VWF-ristocetin or by cGMP-elevating agents. On the other hand, the PKG inhibitors blocked phosphorylation of p42/44 MAP kinases induced by VWF-ristocetin, cGMP-elevating agents, and membrane-permeable cGMP mimetics. These observations led the authors to conclude that VASP had been incorrectly assigned as a PKG substrate in platelets, whereas MAP kinases are regulated downstream of PKG.38 We have confirmed the observation that KT5823, Rp-8-pCPT-cGMPS, and Rp-8-Br-PET-cGMPS are unable to block phosphorylation of VASP induced by ristocetin-VWF (Figure 3Ai) or by cGMP-elevating agents (not shown) using the same period of incubation as that of Li et al38 to block aggregation. In contrast, however, we were unable to confirm the observation that VWF-ristocetin and cGMP-elevating agents, such as glyco–SNAP-1 and sildenafil, are able to induce phosphorylation of p42/44 MAP kinases in platelets (Figure 3Aii). Further, none of the PGK inhibitors are able to block phosphorylation of p42/44 MAP kinase by thrombin or phorbol ester in platelets (Figure 3Aii; data not shown). Taken together, these results demonstrate that cGMP does not lie upstream of the p42/44 MAP kinase pathway in platelets and therefore brings into question the effectiveness of the PKG inhibitors when used under the same experimental conditions as described by Li et al.38
Despite these observations, however, Rp-8-pCPT-cGMPS and Rp-8-Br-PET-cGMPS inhibited aggregation induced by ristocetin in plasma and to thrombin in washed platelets (Figure 3B; not shown). In contrast, KT5823 had no effect on the response to either stimulus (data not shown). Further, Rp-8-pCPT-cGMPS (0.5 mM) and Rp-8-Br-PET-cGMPS (0.5 mM) potentiated (rather than inhibited) the ability of the cGMP-elevating agents, glyco–SNAP-1, sildenafil, and sodium nitroprusside, to inhibit aggregation to ristocetin and thrombin (Figure 3B; not shown). These results demonstrate that the PKG inhibitors and cGMP elevators do not have opposing effects as would be expected if their actions were mediated at the level of PKG and thereby indicating that the PKG inhibitors may induce inhibition through a non–PKG-dependent mechanism. This possibility was tested directly through studies on mice that were deficient in PKG. The dose-response curve to thrombin was similar in the wild-type and PKG-/- platelets (not shown). Both Rp-8-pCPT-cGMPS (0.5 mM) and Rp-8-Br-PET-cGMPS (0.5 mM) partially inhibited aggregation to thrombin in wild-type and PKG-deficient platelets, confirming that they are able to block platelet activation through a PKG-independent pathway (Figure 3C). The accompanying manuscript also provides evidence for PKG-independent actions of these 2 inhibitors and other cGMP-based modulators.36,37
MAP kinase inhibition does not block activation of The role of the MAP kinase pathway in mediating aggregation to VWF-ristocetin and thrombin was investigated using a novel inhibitor of mitogen-induced extracellular kinase (MEK) kinases, PD184161 (5-bromo-2-[2-chloro-4-iodo-phenylamino]-N-cyclopropylmethoxy-3,4-difluoro-benzamide), which is biologically effective in plasma ("Inhibition of Src kinases, but not MAP kinases, blocks aggregate formation at high shear"). The structure of PD184161 is presented in Figure 4Ai. Confirmation that PD184161 inhibits activation of the MAP kinase pathway was shown using a phosphospecific antibody to p42/44 MAP kinases (Figure 4Aii). PD184161 causes complete inhibition of p42/44 MAP kinase activation at concentrations as low as 0.1 µM, but had no effect on GPIb-IX-V– or thrombin-mediated aggregation (Figure 4B).
Inhibition of Src kinases, but not MAP kinases, blocks aggregate formation at high shear
The results obtained in this study support a role for Src kinases but neither PKG or MAP kinases in the activation of
Agents that elevate cGMP inhibit thrombus formation at high shear Experiments were designed to investigate the effect of cGMP-elevating agents on thrombus formation on collagen in human platelets and in a rabbit model of thrombosis. Thrombus formation on collagen was not altered in the presence of sildenafil (1 µM) or glyco–SNAP-1 (10 µM), whereas the 2 agents in combination had a weak inhibitory effect that was similar to that seen with a higher concentration of sildenafil (Figure 6A). Importantly, under no experimental conditions was potentiation of thrombus formation observed in the presence of cGMP-elevating agents. Consistent with these observations, sildenafil increased the rate of flow in the Folts model of thrombosis (Figure 6B; Table 1). Further, at the doses tested, sildenafil did not alter blood flow in the uninjured vessel, supporting the contention that improved flow in the injured carotid is due to an action on platelet aggregate formation and not a direct vasorelaxant effect. These data suggest that an increase in cGMP inhibits thrombosis formation under high shear conditions both in vitro and in vivo.
The aim of this study was to compare the role of Src kinases, PKG, and MAP kinases in the activation of IIb3 by the GPIb-IX-V receptor complex and in thrombus formation. The results demonstrate a critical role for Src kinases, but neither PKG nor MAP kinases, in signaling by GPIb-IX-V. Indeed, the study has generated evidence in support of the more widely accepted view that PKG mediates platelet inhibition by VWF and thrombin.39-41
Several lines of evidence argue against a role for PKG in promoting activation of platelets by GPIb-IX-V. These include: (i) the inability of VWF to stimulate formation of cGMP; (ii) the observation that agents that elevate cGMP, such as glyco–SNAP-1, sodium nitroprusside, or sildenafil, inhibit rather than promote
The present study has provided evidence against a role for the p42/44 MAP kinase pathway in the GPIb-IX-V signaling cascade. This evidence includes the observation that a novel inhibitor of this pathway, which is biologically effective in plasma, PD184161, has minimal effect on the activation of An implicit argument in the work of Li et al24 is that threshold concentrations of thrombin signal through the same pathway as GPIb-IX-V. Although there is no doubt that GPIb-IX-V is a high-affinity binding site for thrombin, there is controversy as to whether it is also a signaling receptor for thrombin. In particular, activation of mouse platelets by thrombin is inhibited completely upon genetic ablation of protease-activated receptor 4, demonstrating that GPIb-IX-V is unable to mediate activation by thrombin in the absence of the G protein–coupled receptor.45 The role of the high-affinity binding site for thrombin on GPIb-IX-V may therefore be to bring the protease close to its G protein–coupled receptor. This model is consistent with the observation that the Src kinase inhibitor PD173952 is able to block aggregation by VWF-ristocetin, but not to thrombin.
The present study has further emphasized the importance of Src kinases in platelet activation. It is becoming increasingly recognized that Src kinases mediate activation of platelets by the major glycoprotein receptors, including GPVI, the integrins In conclusion, we have been unable to find support for a critical role of PKG and MAP kinases in the activation of platelets by GPIb-IX-V, but have provided further evidence for a role of Src family kinases in this pathway and in aggregate formation under flow conditions. The present study adds to the growing body of evidence for a critical role of Src kinases in the regulation of platelets by membrane glycoproteins and further emphasizes Src family kinases as important targets for development of novel antithrombotics. The present study has also identified 2 novel tools, PD173952 and PD184161, that are effective in plasma and can be used to establish the roles of Src and MAP kinases, respectively, in thrombus formation in blood.
We gratefully acknowledge the excellent support of Susan Waller in the Folts studies. We thank Alan Kraker, Pfizer Global Research and Development (Ann Arbor, MI) for critical discussions throughout the course of this work. The authors also thank Dr Ulrich Walter and colleagues for sharing data37 and for helpful discussion during the course of this work.
Submitted October 7, 2003; accepted November 19, 2003.
Prepublished online as Blood First Edition Paper, December 18, 2003; DOI 10.1182/blood-2003-09-3319.
Supported by the Biotechnology and Biological Services Research Council (BBSRC), the British Heart Foundation (BHF), and Pfizer Global Research and Development. S.P.W. holds a BHF Professorship. S.J.M. held a BBSRC "CASE" Award. J.M.A. holds a BHF Studentship.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Yotis Senis, Division of Medical Sciences, The Medical School, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom; e-mail: y.senis{at}bham.ac.uk.
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Abstract
Introduction
2 (PLC
