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Arteriosclerosis, Thrombosis, and Vascular Biology. 2001;21:618-627

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2001;21:618.)
© 2001 American Heart Association, Inc.

Thrombosis

Platelet Adhesion Enhances the Glycoprotein VI–Dependent Procoagulant Response

Involvement of p38 MAP Kinase and Calpain

Pia Siljander; Richard W. Farndale; Marion A. H. Feijge; Paul Comfurius; Snjezana Kos; Edouard M. Bevers; Johan W. M. Heemskerk

From the Wihuri Research Institute (P.S.), and the Electron Microscopy Unit (P.S.), Institute of Biotechnology, University of Helsinki, Helsinki, Finland; the Departments of Biochemistry and Human Biology (P.S., M.A.H.F., P.C., S.K., E.M.B., J.W.M.H.), University of Maastricht, Maastricht, The Netherlands; and the Department of Biochemistry (P.S., R.W.F.), University of Cambridge, United Kingdom.

Correspondence to Pia Siljander, Department of Biochemistry, University of Cambridge, Cambridge CB2 1QW, UK. E-mail prms2{at}mole.bio.cam.ac.uk


*    Abstract
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Abstract—In the final stages of activation, platelets express coagulation-promoting activity by 2 simultaneous processes: exposure of aminophospholipids, eg, phosphatidylserine (PS), at the platelet surface, and formation of membrane blebs, which may be shed as microvesicles. Contact with collagen triggers both processes via platelet glycoprotein VI (GPVI). Here, we studied the capacity of 2 GPVI ligands, collagen-related peptide (CRP) and the snake venom protein convulxin (CVX), to elicit the procoagulant platelet response. In platelets in suspension, either ligand induced full aggregation and high Ca2+ signals but little microvesiculation or PS exposure. However, most of the platelets adhering to immobilized CRP or CVX had exposed PS and formed membrane blebs after a prolonged increase in cytosolic [Ca2+]i. Platelets adhering to fibrinogen responded similarly but only when exposed to soluble CRP or CVX. By scanning electron microscopic analysis, the bleb-forming platelets were detected as either round, spongelike structures with associated microparticles or as arrays of vesicular cell fragments. The phosphorylation of p38 mitogen-activated protein kinase (MAPK) elicited by CRP and CVX was enhanced in fibrinogen-adherent platelets compared with that in platelets in suspension. The p38 inhibitor SB203580 and the calpain protease inhibitor calpeptin reduced only the procoagulant bleb formation, having no effect on PS exposure. Inhibition of p38 also downregulated calpain activity. We conclude that the procoagulant response evoked by GPVI stimulation is potentiated by platelet adhesion. The sequential activation of p38 MAPK and calpain appears to regulate procoagulant membrane blebbing but not PS exposure.


Key Words: platelets • procoagulant activity • adhesion • glycoprotein VI • signal transduction


*    Introduction
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 up arrowAbstract
 *Introduction
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 down arrowResults
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Adhesion to collagen changes the cellular structure and function of platelets, and as a consequence, they transform into coagulation-promoting cells. Platelet procoagulant activity unites the processes of adhesion, activation, and coagulation and thus serves to localize hemostatic activity at sites of vascular injury. The procoagulant reaction of platelets consists of 2 processes. It involves loss of phospholipid asymmetry across the membrane, resulting in exposure of phosphatidylserine (PS) and phosphatidylethanolamine on the platelet outer membrane surface due to the action of a Ca2+-dependent phospholipid scramblase activity.1 The aminophospholipids act as assembly sites for tenase and prothrombinase complexes, thus enhancing the formation of thrombin in plasma many fold.2 3 Simultaneous with the phospholipid rearrangement, blebs are formed at the plasma membrane, which may be shed as procoagulant microvesicles.4 5 The latter process is reported to involve alterations in protein tyrosine phosphorylation6 7 and calpain-dependent degradation of cytoskeletal proteins.8 Microvesicle formation is analogous to membrane blebbing observed in other cells undergoing apoptosis, and cellular apoptotic markers have also been recently discovered in platelets.9 10 11 The physiological importance of the platelet procoagulant response is apparent from the bleeding tendency of patients with Scott syndrome, whose platelets are defective in surface exposure of PS and microvesicle formation.12

Recently, we have demonstrated that glycoprotein VI (GPVI) fulfills an important function by triggering the procoagulant response in collagen-adherent platelets.13 GPVI, a member of the immunoglobulin superfamily,14 is considered to be the main signaling receptor underlying collagen-induced platelet activation. Platelets from GPVI-deficient, hemostatically compromised patients are highly refractory to collagen but respond well to other agonists. On collagen stimulation, these platelets show greatly reduced tyrosine phosphorylation patterns, lacking, eg, tyrosine phosphorylation of the Fc receptor {gamma}-chain, Syk, and phospholipase C-{gamma}2.15 16 Experimental work with the GPVI-specific collagen-related peptide (CRP) has indicated that the same platelet proteins become tyrosine phosphorylated after GPVI stimulation17 and that in GPVI-deficient patients, these tyrosine phosphorylation events are abrogated.18 Another recently discovered ligand of GPVI, the protein convulxin (CVX) from the venom of the snake Crotalus durissus terrificus, induces similar tyrosine phosphorylation patterns in platelets.19 Such phosphorylation is required to achieve the high, phospholipase C–dependent, Ca2+ response required for PS exposure.6

Here, we have determined the requirements of these GPVI ligands to trigger the procoagulant response in platelets. Specifically, the effects of CRP and CVX were compared on platelets in suspension and platelets in contact with various adhesive surfaces. The PS-exposing, bleb-forming platelets were observed in detail by scanning electron microscopy, which revealed new morphological structures in adherent platelets. Pharmacological inhibitors were used to further dissect the pathways involved in exposure of PS and the formation of blebs and microvesicles. In particular, the role of the p38 forms of the mitogen-activated protein kinases (MAPKs), also known as stress-activated protein kinase 2 (SAPK2), appeared to be of interest, because their putative involvement in apoptosis and membrane blebbing has been considered in several studies.20 21 22


*    Methods
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 *Methods
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Materials
Plasma containing an anti-GPVI antibody from a patient with autoimmune thrombocytopenia was a kind gift of Dr H. Takayama (Kyoto University, Kyoto, Japan).23 IgG from this patient and from normal human plasma as a control was purified and used for preparation of Fab fragments, as described before.13 Monoclonal antibody (mAb) against phospho-p38 (No. 9211S) came from New England Biolabs, and the anti–{alpha}2-mAb 6F1 was a kind gift of Dr Barry S. Coller (Mount Sinai Hospital, New York, NY). Anti-phosphotyrosine mAb 4G10 was from Upstate Biotechnology. Horseradish peroxidase–linked anti-rabbit IgG (NA934), anti-mouse IgG (NA931), and ECL-Plus reagent were obtained from Amersham Pharmacia. Annexin V labeled with Oregon green 488 (OG488) isothiocyanate was obtained from Nexins Research. Prothrombin, thrombin, factor Va, and factor Xa were purified as described elsewhere.24 Chromogenic thrombin substrate S2238 was from Chromogenix, and fluorescent calpain substrate Succinyl-Leu-Leu-Val-Tyr 7-aminomethyl coumarin (Suc-LLVY-AMC) was from Calbiochem. SB203580 and calpeptin were from Alexis and Calbiochem-Novabiochem, respectively. CRP, NH2-Gly-Cys-hydroxyproline (Hyp)-(Gly-Pro-Hyp)10-Gly-Cys-Hyp-Gly-NH2, was synthesized and cross-linked as described before.18

CVX was purified from the venom of Crotalus durissus terrificus (Latoxan) basically as described elsewhere.25 In brief, the crude venom was applied to a Sephadex G-75 column (Pharmacia Biotech) and eluted with 150 mmol/L NaCl. The most active fractions in terms of inducing platelet aggregation were collected, applied to a 0.7x10-cm polystyrene QAE column from Bio-Rad, and eluted with a gradient of 120 to 300 mmol/L NaCl in 50 mmol/L Tris (pH 7.5). Active fractions were dialyzed against a buffer of 120 mmol/L NaCl in 50 mmol/L Tris (pH 7.5), analyzed, and then concentrated by using the same QAE column.

Preparation of Adhesive Surfaces
Degreased, round glass or Thermanox (Nunc) coverslips were incubated with either 10 µg/mL CRP (in 10 mmol/L acetic acid), 25 µg/mL purified CVX (in 50 mmol/L Tris, 120 mmol/L NaCl, pH 7.5), or 10 mg/mL fibrinogen (in 150 mmol/L NaCl) for 1 hour at 22°C in a humid chamber. After being coated, the coverslips were rinsed and blocked for 30 minutes with 10 mmol/L HEPES buffer, pH 7.4, containing 136 mmol/L NaCl, 5 mmol/L glucose, 2.7 mmol/L KCl, and 2 mmol/L MgCl2, to which was added 2.0% (wt/vol) bovine serum albumin (BSA). For use as a control, uncoated coverslips were incubated with the blocking buffer.

Platelet Isolation
Blood from healthy volunteers was collected into 1/6 volume of a mixture of 80 mmol/L sodium citrate, 52 mmol/L citric acid, and 183 mmol/L glucose. Subjects had not taken medication during the previous 2 weeks. Platelet-rich plasma (PRP) was prepared by centrifugation and supplemented with apyrase (0.2 U ADPase per mL). The PRP was subsequently processed by a washing procedure26 or, where indicated, by gel filtration.27 Platelet count was determined with a Coulter counter (Coulter Electronics).

Activation of Platelets in Suspension
Where applicable, the PRP was incubated with 3 µmol/L fura 2-pentaacetoxymethyl ester (Molecular Probes) for 45 minutes at 37°C.28 After being loaded with fura 2, platelets were centrifuged, washed once, and suspended in HEPES buffer containing 0.2% (wt/vol) BSA and apyrase (0.2 U ADPase per mL). Fura 2–loaded platelets (1x108/mL) were activated with the indicated amounts of CVX or CRP in the presence of 2 mmol/L CaCl2, and changes in [Ca2+]i were measured by ratio fluorometry.26 Aggregate formation was measured in unloaded platelets (2x108/mL), also in the presence of 2 mmol/L CaCl2, by turbidimetric aggregometry.26 For platelets in suspension (1x108/mL), exposure of PS and microvesicle formation were detected after 10 to 20 minutes of stimulation (no stirring) in the presence of 2 mmol/L CaCl2 and 0.5 µg/mL OG488-labeled annexin V by using flow cytometry. Where indicated, PS exposure and calcium changes were measured by Alexa-633 annexin V binding and fluo-3 labeling (7 µmol/L) of platelets (Molecular Probes). Samples were diluted with HEPES buffer, and 10 000 events were counted with an Epics XL-MCL fluorescence-activated cell sorter (Coulter Electronics).

Activation of Adherent Platelets
Platelets in HEPES buffer containing 2 mmol/L CaCl2 (0.5 to 1x108/mL, 500 µL) were incubated on CVX- or CRP-coated coverslips that were mounted either in 2-mL open incubation chambers or in multiwell plates. When fibrinogen-coated coverslips were used, platelets were first allowed to adhere to the fibrinogen for 20 minutes, after which unbound platelets were removed by flushing with HEPES buffer. Then 400 µL of CaCl2-containing HEPES buffer was added to the coverslips, followed by CVX (30 to 100 ng/mL) or CRP (0.5 to 5 µg/mL, all final concentrations) when required. Where indicated, SB203580, calpeptin, or dimethyl sulfoxide (DMSO) vehicle was added 5 minutes before the GPVI ligand. Platelets loaded with 100 µmol/L Suc-LLVY-AMC (1 hour at room temperature) were allowed to adhere to CVX-coated coverslips for 3 hours (37°C). Subsequently, the fluorescence of cleaved AMC was measured after lysis of the platelets with 0.1% (wt/vol) Triton X-100 at excitation and emission wavelengths of 380 and 440 nm, respectively.

Fluorescence Imaging Microscopy of Adherent Platelets
Coverslips coated with CVX, CRP, or fibrinogen that were mounted in the incubation chamber were incubated with platelets (fura 2 loaded or unloaded) and placed on the stage of an inverted microscope. A combined transmission phase-contrast and epifluorescence imaging system was used to record changes in platelet morphology and fluorescence. The system was equipped with 2 low-light-level intensified cameras recording fluorescence and (infrared) phase-contrast images that were connected to the microscope system and controlled by 2 UNIX-driven computer systems with Quanticell software (Visitech). Fluorescence was measured from adherent platelets in the focal plane of the coverslip at excitation and emission wavelengths as described before.6 In the case of fura 2, ratio images at 340/380-nm excitation were converted pixel by pixel to levels of [Ca2+]i by using standard calibration parameters previously determined for this optical setup. For phase-contrast recordings, platelets on coverslip were transilluminated with either white light or infrared light, when exact overlays with fluorescence images were needed. Bleb formation was clearly visible at magnifications of x2000.

To obtain high-resolution confocal images, Flow Multitest slides (8 wells) precoated with CVX were incubated with 25 µL of a suspension of platelets (1x108/mL) containing OG488-labeled annexin V (0.5 µg/mL) and CaCl2 (2 mmol/L), as described before.13 Fluorescence of platelets at the slide surface was observed with a single-photon confocal scanning microscope (Leica TCS-NT CLSM, based on a DMIRB inverted bright-field and epifluorescence light microscope), which was equipped with an argon laser. Light emission was collected at >505 nm, and images were captured after Kalman averaging for 16 lines.

Scanning Electron Microscopy
Adherent platelets on coated coverslips were fixed with phosphate-buffered saline containing 2.5% (wt/vol) glutaraldehyde for 2 hours, after which the samples were rinsed with phosphate-buffered saline. Fixed samples were dehydrated with an ethanol gradient, critical point–dried with CO2, and sputter-coated with platinum.27 Specimens were observed with a Zeiss 920 DSM scanning electron microscope at the Electron Microscopy Unit, Institute of Biotechnology, University of Helsinki (Helsinki, Finland).

Phosphorylation of p38 MAPK by Western Blotting and Densitometry
Plasma-free platelets (1x108/mL) were allowed to adhere to fibrinogen-coated 12-well dishes (Nunc). After 20 minutes of adhesion unbound platelets were washed away, and 5 minutes later, the remaining platelets were treated for 10 minutes with a low dose of CVX or CRP, as indicated. The platelets were then lysed with 200 µL of a buffer of 63.5 mmol/L Tris-HCl (pH 6.8), 2% (wt/vol) SDS, 10% (vol/vol) glycerol, 1 mmol/L orthovanadate, 1 mmol/L PMSF, 50 µg/mL leupeptin, and 1 mmol/L benzamidine. The buffer was transferred from well to well. Adherent platelets from 6 wells yielded 1 sample. Collected lysates were boiled for 10 minutes and centrifuged at 10 000g, after which the supernatants were dissolved in 5% (vol/vol) ß-mercaptoethanol and 0.1% (wt/vol) bromophenol blue.

Suspensions of activated platelets were centrifuged (2 minutes at 3000g) and immediately resuspended into 150 µL of lysis buffer, and samples containing 20 to 25 µg of protein were separated by SDS–polyacrylamide gel electrophoresis (PAGE; 7.5% acrylamide); the proteins were electrotransferred to a nitrocellulose membrane. Blots were stained with 0.5% (wt/vol) Ponceau S (Sigma) in 5% (vol/vol) acetic acid to check for uniformity of transfer and to quantify protein (see below) and then destained in Tris-buffered saline supplemented with 0.1% (wt/vol) Tween-20 (TBST). Blots were blocked with TBST containing 10% (wt/vol) BSA.29 The blots were then probed with anti–phospho-p38 used at 1:1000 dilution in TBST plus 5% BSA. Antibody binding was detected by using horseradish peroxidase–linked anti-rabbit IgG (diluted 1:10 000 in TBST plus 1% BSA) and developed with the ECL-Plus technique. Videodensitometry was used to quantify phospho-p38 MAPK in Western blots.29 To permit the comparison of samples from adherent platelets with those from platelets in suspension, the amount of platelet-derived material was normalized by videodensitometry of the platelet actin band, which was stained with Ponceau S on the Western blot.

Tyrosine Phosphorylation of Whole Platelets
Plasma-free platelets (300 µL of 1x108/mL) were allowed to adhere to fibrinogen- or BSA-coated 12-well dishes (Nunc) for 30 minutes. CRP or vehicle was added with 2 mmol/L Ca2+ for 20 minutes, and then the activation process was stopped by the addition of 5x Laemmli buffer without removing the nonadherent platelets to maintain equal platelet numbers throughout. Proteins were separated by SDS-PAGE, and the blots produced as above were probed with anti-phosphotyrosine mAb 4G10 (0.1 µg/mL) in TBST by using a horseradish peroxidase–linked anti-mouse IgG for detection by ECL.

Statistics
To test the significance of effects of pharmacological agents, a 1-way ANOVA was applied. To determine which groups differed from the control group, a 2-tailed t test was performed with adjustment of the {alpha} level according to the Bonferroni method for multiple comparisons.


*    Results
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 *Results
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Procoagulant Effect of GPVI Stimulation of Platelets in Suspension and in Contact With CRP or CVX
The procoagulant effects of the platelet-stimulating GPVI ligands CRP and CVX on isolated platelets in suspension (1 to 2x108/mL) were determined in the presence of 2 mmol/L CaCl2 to reach physiological free Ca2+ concentrations. As shown in Figure 1Down, both CRP (2.5 µg/mL) and CVX (50 ng/mL) induced rapid and complete aggregation of the platelets. Measurements with the aid of fura 2–loaded platelets showed both ligands to be potent agonists for increased [Ca2+]i. Flow cytometric analysis with fluorescently labeled annexin V as a PS-binding probe30 indicated, however, that these agents induced only minimal PS exposure in the suspended platelets (Figures 1CDown and 1FDown). Flow cytometry also showed that microvesicle formation was below the limit of detection under these conditions. Similar experiments that used Alexa-633 annexin V with fluo-3–loaded platelets indicated that PS-expressing platelets had higher [Ca2+]i (mean channel number 317±15 compared with 242±10 for annexin V–negative platelets). (Resting platelets showed values of 146±5, and ionomycin-stimulated platelets, 400±50.) Dose-response analysis demonstrated that 4-fold higher doses of CRP and CVX gave larger increases in [Ca2+]i, reaching micromolar levels, whereas the fractions of PS-exposing platelets increased to only 10% to 14% after 20 minutes of stimulation (Figures 1GDown and 1HDown). Typically with the high doses of agonists, the fractions of platelets with exposed PS decreased to {approx}1% when the platelet concentration in the suspension was lowered to 1x107/mL. Stirring of the platelets during activation with CVX (200 ng/mL) and in the presence of 0.1 mmol/L Arg-Gly-Asp-Ser had no effect on the percentages of annexin V–positive platelets (7±2% without stirring and 5±2% with stirring; mean±SEM, n=3 to 4). Stirring similarly had no effect on calcium measurements (data not shown).



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  		Figure 1. Activating effects of GPVI ligands on platelets in suspension in the presence of 2 mmol/L CaCl2. (A and D) Aggregation of platelets (2x108/mL) stimulated with CRP (2.5 µg/mL) or CVX (50 ng/mL). (B and E) Changes in [Ca2+]i in fura 2–loaded platelets (1x108/mL) stimulated with CRP or CVX. (C and F) Binding of OG488-labeled annexin V (0.5 µg/mL) to platelets stimulated with CRP or CVX for 10 minutes without stirring. Numbers indicate percentages of annexin V–positive platelets. The numbers of annexin V–positive platelets were not influenced by the presence of fibrinogen (2 mg/mL) in the platelet suspension. Data are representative of 3 or more independent experiments. (G) Platelets were activated in the presence of 2 mmol/L CaCl2 but with various doses of CRP (squares), CVX (circles), or fibrillar collagen (triangles). Maximal increases in [Ca2+]i in fura 2–loaded platelets. (H) Percentages of platelets binding OG488-labeled annexin V after 20 minutes of stimulation. As a positive control, platelets were stimulated with 5 µmol/L ionomycin (diamond). Data are mean values of 2 to 4 independent experiments.

On the other hand, when purified platelets were allowed to bind to surfaces coated with CRP or CVX, large increases in [Ca2+]i were measured, which in most cells were followed by surface exposure of PS, as detected by fluorescence videoimaging microscopy (Figure 2Down). The lag time between the moment of platelet contact with the surface and the initial rise in [Ca2+]i was shorter in the case of CVX (10±5 seconds, n=10 cells) than for CRP (35±11 seconds, n=9). After contact with the ligand for 20 minutes, most of the platelets were elevated in [Ca2+]i and had PS exposed (Table 1Down). Quantitative estimates of the increases in [Ca2+]i were made by applying a standard calibration procedure; for individual platelets on CRP and CVX, the rises amounted to 820±312 and 910±405 nmol/L, respectively (mean±SD, 28 to 35 cells). In either case, {approx}75% of the platelets showed a sustained rise in [Ca2+]i while the other cells had a spiking Ca2+ signal. Examination by phase-contrast microscopy indicated that almost all of the annexin V–binding platelets exhibited a bleb-forming morphology (data not shown, but see below). Thus, either of the GPVI ligands, CRP and CVX, was more potent in inducing a procoagulant response when immobilized on a surface than when added in soluble form to platelets in suspension.



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  		Figure 2. Calcium responses and annexin V binding of platelets in contact with immobilized CRP or CVX. Fura 2–loaded platelets were allowed to adhere to coverslips coated with CRP (A, B) or CVX (C, D) in the presence of 2 mmol/L CaCl2 and 0.5 µg/mL OG488-labeled annexin V while changes in fluorescence were continuously measured with an intensified camera. Traces represent changes in [Ca2+]i (A, C) or binding of OG488-labeled annexin V (B, D) of single but different platelets. Arrows indicate time points of platelet attachment to the surface. Data are representative of 5 independent experiments (>100 platelets). A.u. indicates arbitrary units.


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  		Table 1. Calcium and Procoagulant Responses of Platelets in Contact With Different GPVI Ligand-Coated Surfaces

We had previously established that the procoagulant activation of CRP-adhering platelets was fully dependent on GPVI by using Fab fragments derived from the IgG of a patient with autoantibodies against GPVI, whereas {alpha}2ß1-blockade with mAb 6F1 had little effect.13 By the same procedure, we determined the role of GPVI in inducing PS exposure after platelet binding to immobilized CVX. In the control condition, wherein Fab fragments were used from a normal subject, 78±5% (mean±SEM, 10 fields of view) of adherent platelets expressed PS, whereas the presence of 0.2 mg/mL Fab fragments from the autoimmune patient reduced the proportion of PS-expressing adherent platelets to 14±4% (mean±SEM from 8 fields of view), as measured by annexin V binding. The anti-GPVI Fab fragments had no effect on platelet adhesion measured under these conditions. Similarly, mAb 6F1 (2 µg/mL) did not influence platelet binding to CVX, but it did decrease the percentage of PS-exposing platelets to 64±4.0% (mean±SEM, 5 fields of view).

Morphological changes during platelet adhesion to immobilized CVX or CRP were visualized by real-time phase-contrast digital imaging. On contact, the platelets formed pseudopods and spread over the agonist-containing surface. After 3 to 5 minutes, the platelets started to round off and increase in transparency. Blebs became visible then, as well as small vesicles moving along the platelet outer surface by Brownian motion (Figures 3ADown and 3BDown). High-resolution scanning electron microscopy images of the morphology at this stage were obtained after fixation of the platelets with glutaraldehyde. Many of the platelets adhering to immobilized CRP or CVX featured a spongelike morphology, which was raised above the plane of adhesion, and were without pseudopods (Figures 3CDown through 3F). The size of the "sponges" ranged from 2 to 4 µm. In addition, small vesicular structures were often detected in the vicinity of these platelets. Typically, whereas platelets with pseudopods or platelets that were spread over the surface had a smooth appearance, that of the spongelike platelets was rougher. This feature may suggest that only the blebbing (procoagulant) platelets suffer from a loss of membrane components during the fixation procedure.



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  		Figure 3. Morphological changes of platelets in contact with immobilized CVX or CRP. Platelets adhered to CVX- or CRP-coated coverslips in the presence of 2 mmol/L CaCl2 at 22°C. Phase-contrast images of blebbing platelets after 20 minutes of adhesion to CVX (A) or CRP (B) (bar represents 5 µm). (C and D) Scanning electron photomicrographs of platelets adhering to CVX for 30 minutes at various magnifications (bars represent 10 and 2 µm, respectively). (E and F) Scanning electron photomicrographs of platelets adhering to CRP for 30 minutes at various magnifications (bars represent 10 and 2 µm, respectively).

Effect of Platelet Adhesion on GPVI-Induced Procoagulant Response
The relatively high procoagulant response of platelets bound to immobilized GPVI ligands prompted us to determine whether the responsiveness of the platelets is sensitized by the process of adhesion itself. Fibrinogen was chosen as a good adhesive surface, which is essentially unable to cause a procoagulant response.6 Fura 2–loaded platelets were added to fibrinogen-coated coverslips to allow firm attachment and spreading. After removal of unbound platelets, the effects of soluble CRP or CVX were examined in individual platelets in the presence of CaCl2 and OG488-labeled annexin V by simultaneous recording of the changes in fura 2 and OG488 fluorescence. When a low dose of CVX (25 ng/mL) was given to the fibrinogen-adherent platelets, ie, a dose unable to induce PS exposure and microvesiculation in platelets in suspension, almost all platelets responded with an immediate, large increase in [Ca2+]i. As in platelets adherent to CVX or CRP (Figure 2Up), addition of CVX to fibrinogen-adherent platelets caused, in most cases, a prolonged Ca2+ rise that after {approx}5 minutes’ lag time was followed by exposure of annexin V–binding sites (data not shown). In some platelets, the Ca2+ response consisted of repetitive spiking in [Ca2+]i, which was not followed by annexin V binding. Bleb formation was observed only in the annexin V–positive platelets. Stimulation of the fibrinogen-bound platelets with a low dose of CRP (1 µg/mL) gave similar responses. Fractions of platelets that remained high in [Ca2+]i and had their PS exposed increased with the concentration of added CRP or CVX (Table 2Down). Estimates of the rises in [Ca2+]i after 10 minutes of stimulation with CRP or CVX were 490±286 and 570±316 nmol/L (mean±SD, n=35 to 58), respectively. In agreement with earlier results,6 addition of {alpha}-thrombin resulted in only a little PS exposure, although it induced prominent albeit spiking increases in [Ca2+]i (Table 2Down).


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  		Table 2. Calcium and Procoagulant Responses of Fibrinogen-Adherent Platelets Stimulated With GPVI Ligands

Scanning electron microscopy was again used to monitor the CRP- and CVX-induced changes in morphology of the fibrinogen-adherent cells. As an example, Figure 4Down shows the effects of added CRP as a function of time. With time, fragmentation of the spread platelets was increasing, apparently starting from the fringes of a platelet. After 15 minutes of GPVI activation, some of the platelets still had a flat appearance with extensive fragmentation, while others had become rounded with a spongelike appearance. Stimulation with CVX instead of CRP caused similar morphological changes, but stimulation with {alpha}-thrombin resulted only in an increase of spread platelets (data not shown).



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  		Figure 4. Morphological changes induced by CRP in fibrinogen-adherent platelets. Platelets spread on a fibrinogen-coated coverslip (20 minutes) were incubated with 2 mmol/L CaCl2 and 5 µg/mL CRP for various times as indicated. Scanning electron photomicrographs of (A) control platelets not stimulated with CRP (bar=10 µm) and (B–D) platelets activated with CRP for 3.5, 7, and 15 minutes, respectively (bars=10 µm). (E, F) High-magnification images after 15 minutes of stimulation (bars=2 µm). Data are representative of 2 independent experiments (>10 images per experiment).

Involvement of p38 MAPK and Calpain in GPVI-Induced Procoagulant Response
In stress-activated endothelial cells, involvement of p38 MAPK activation in the formation of plasma membrane blebs has been proposed.31 In platelets activated with Ca2+ ionophores and other agents, cleavage of cytoskeletal proteins by calpain is thought to underlie the shedding of microvesicles.8 This prompted us to determine the involvement of p38 MAPK and calpain in the procoagulant platelet response evoked by GPVI ligands. To measure p38 MAPK activation, we probed Western blots from SDS-PAGE–separated platelet proteins with an antibody against activated, diphosphorylated p38 MAPK (pp38), which stained a band at {approx}42 kDa in platelets stimulated with CRP or CVX (Figure 5ADown). Densitometric analysis of the Ponceau S–stained actin band (Figure 5BDown) allowed the amount of platelet material from suspension and adhesion samples to be compared so that the relative degree of p38 phosphorylation could be determined.



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  		Figure 5. Effects of CRP and CVX on phosphorylation of p38 MAPK in fibrinogen-adherent and nonadherent platelets. Platelets were stimulated with either CRP (5 µg/mL) or CVX (100 ng/mL) in the presence of 2 mmol/L CaCl2 in suspension (1x108/mL) or alternatively, after 20 minutes of adhesion to immobilized fibrinogen, as described in Methods. (A) Phosphorylated p38 MAPK was detected in Western blots of separated proteins with anti–phospho-p38 active site–directed mAb. (B) Ponceau S staining of platelet-derived actin, determined as described in Methods in the same blots as A, indicating the amount of platelet protein in the samples. Bas indicates control samples from basal platelets.

Platelets in suspension showed little phosphorylation of p38 MAPK when no GPVI agonist was present, but both CRP and CVX caused substantial increases in p38 phosphorylation (see Figure 5Up). With 5 µg/mL CRP, we measured an {approx}4-fold increase in phosphorylation. However, the phosphorylation increased >8-fold in CRP-stimulated platelets that adhered to fibrinogen. With 25 ng/mL CVX, the corresponding increases for p38 phosphorylation were 8-fold in platelets in suspension and 26-fold in fibrinogen-adherent platelets.

In similar experiments, we measured the total tyrosine phosphorylation of whole-platelet lysates from fibrinogen-adherent and nonadherent platelets stimulated with increasing doses of CRP. We determined that the increase in total tyrosine phosphorylation was higher in adherent platelets than in platelets in suspension (Figure 6Down). Specific phosphotyrosine-containing bands from adherent platelets, notably those of {approx}100 and 70 kDa, were elevated {approx}4- and 10-fold over basal levels, whereas the same protein band from platelets activated in suspension was stimulated only 1.5- and 2.5-fold by the same level of CRP. In contrast, no difference was observed for the band of {approx}50 kDa.



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  		Figure 6. Effects of CRP on protein tyrosine phosphorylation in fibrinogen-adherent and nonadherent platelets. Platelets were stimulated with CRP (either 5 or 10 µg/mL) for 20 minutes in the presence of 2 mmol/L CaCl2 or in suspension in BSA- or fibrinogen-coated wells (1x108/mL) after allowing 30 minutes for adhesion, as described in Methods. Tyrosine phosphorylation was detected in Western blots as described.

To determine the involvement of p38 MAPK in the GPVI-induced procoagulant response, we used SB203580 as a specific inhibitor of the p38 ATP binding site.32 When added to fibrinogen-adherent platelets, SB203580 (2 µmol/L) caused an inhibition of {approx}50% in the CRP- or CVX-induced blebbing response, but was without effect on the PS exposure (Table 3Down). Higher doses of SB203580 caused lysis of the platelets and hence could not be used. The inhibitory effect of SB203580 remained the same throughout a 50-minute time course. The inclusion of aspirin and apyrase in conjunction with SB203580 had no effect on PS exposure and caused no further inhibition of blebbing than did SB203580 alone, showing that indirect effects of SB203580, ie, inhibition of thromboxane- or ADP-mediated activation pathways, were not responsible. In comparison, an inhibitor of the ERK1/2 MAPK pathway, PD09805933 (20 µmol/L), which also inhibits platelet cyclooxygenase,34 was without effect on both platelet blebbing and PS exposure (data not shown). When testing the effect of SB203580 on platelets adhering to CVX-coated coverslips, similar results were obtained: after 20 minutes of incubation, SB203580 (2 µmol/L) reduced the bleb formation from 67.3±5.3% to 39.4±7.4% of the adherent platelets without any effect on PS exposure (mean±SEM, n=3 to 4).


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  		Table 3. Involvement of p38 MAPK and Calpain in GPVI Ligand-Induced Bleb Formation and PS Exposure

The membrane-permeable inhibitor of calpain, calpeptin,35 also reduced bleb formation but not PS exposure in fibrinogen-bound platelets that were activated with CRP or CVX (Table 3Up). With both calpeptin and SB203580 present, a small, synergistic effect on inhibition of bleb forming was noted, although this was not statistically significant. When calpain activation was analyzed from the rate of cleavage of the intracellular fluorescent substrate Suc-LLVY-AMC, CVX-adherent platelets exhibited an increase in fluorescence on ligand contact, which could be reduced to 19.7±4.5% (mean±SEM, n=4) of the control level by calpeptin and to 69.8±2.6% by SB203580, suggesting that the mechanism of p38 MAPK involvement in platelet blebbing may lie upstream from calpain activation.


*    Discussion
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 *Discussion
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CRP and CVX, like fibrillar collagen, are potent platelet-aggregating agents.18 19 36 Experiments with GPVI-deficient platelets and IgG from patients with autoantibodies against GPVI have demonstrated that both CRP and CVX are potent stimulators of GPVI-mediated signaling pathways in platelets, sharing many features with collagen-induced signal transduction. An important event is the activation of phospholipase C-{gamma}2 mediated by the protein tyrosine kinase Syk, leading to the mobilization of Ca2+.15 16 Previously, we demonstrated that the procoagulant response induced by platelet-collagen adhesion is mostly mediated through GPVI.13 In the present study, both immobilized CRP and CVX evoked large and prolonged Ca2+ signals in adherent platelets, which preceded a procoagulant response demonstrated by PS exposure and bleb formation (Figures 2Up and 3Up and Table 1Up). As a procoagulant agonist, CVX appeared to be rather more active than CRP. This difference could depend on the higher binding affinity of CVX or the engagement of multiple receptors. The involvement of GPVI was reflected by the potent inhibitory effect of Fab fragments from anti-GPVI plasma on PS expression, though not on adhesion. Previous evidence showed that these Fab fragments inhibit platelet adhesion to CVX36 but only at shorter adhesion times (5 minutes) than used here.

Two binding sites for CVX have been identified on platelets by Scatchard analysis,37 only 1 of which competes with CRP binding, and thus, the anti-GPVI plasma may target only 1 of these sites, perhaps by blocking signaling but not adhesion. The interaction of CVX with platelets has previously been reported to involve both integrin {alpha}2ß1 as well as GPVI: blocking {alpha}2ß1 with mAb 6F1 caused inhibition of CVX-induced secretion and aggregation but not of Ca2+ signaling.36 Thus, {alpha}2ß1 might generate additional signals that could enhance the procoagulant response. In accord with this concept, we found modest inhibition of the procoagulant response with the integrin-specific antibody.

Activation of platelets in suspension with CRP or CVX elicited potent increases in [Ca2+]i and rapid formation of platelet aggregates, which indicates a major role for signaling from GPVI to phospholipase C{gamma}2. Signaling through GPVI is claimed to depend on receptor dimerization, as is true for other receptors of the immunoglobulin superfamily.14 The present results imply that on the 1 hand, both CRP (a polymeric, cross-linked triple-helical peptide) and CVX (a globular multimeric protein) can support GPVI dimerization and subsequent signaling, regardless of whether they are applied in solution or immobilized on a surface. On the other hand, despite the large [Ca2+]i signal, these ligands did not elicit procoagulant activity in platelets in suspension (Figure 1Up). Thus, despite the higher-order structure of the ligand, other signals in addition to high Ca2+ levels are required for the induction of PS exposure and bleb formation.

These procoagulant events appear to be triggered more effectively when the GPVI ligands are immobilized on a surface. This finding may indicate that either immobilized ligands have increased activity or that adhering platelets are more prone to these activation processes. Furthermore, low doses of CRP and CVX, being poor inducers of PS exposure in platelets in suspension, were much more active when applied to platelets that were spread on a fibrinogen surface (Figure 4Up and Table 2Up). Similarly, application of these agents to fibrinogen-adherent platelets led to increased prothrombinase activation (P.S. et al, unpublished results). An explanation for the difference in responsiveness of suspended and adherent platelets might be the higher total number of GPVI receptors in the suspensions, thereby causing partial depletion of the GPVI ligands. However, this possibility seems unlikely, because PS exposure appeared to decrease rather than increase after the platelet number was lowered. An alternative explanation is that adhesion-induced intracellular events may prime the cells for the GPVI-mediated procoagulant response. It is well known that platelet binding to fibrinogen via the integrin {alpha}IIbß3 receptor leads to extensive outside-in signaling and that platelet spreading on fibrinogen results in extensive tyrosine phosphorylation of focal adhesion kinase and other proteins.38 39 The present study does not discriminate between increased procoagulant activity simply as a consequence of adhesion and the existence of subsets of platelets more prone to adhere.

The GPVI ligands CRP and CVX potentiated p38 MAPK phosphorylation in fibrinogen-bound platelets compared with platelets in suspension (Figure 5Up), supporting the idea that adhesion primes the signaling events required for the procoagulant response. Furthermore, the CRP-stimulated tyrosine phosphorylation of several platelet proteins was also potentiated in platelets adhering to fibrinogen (Figure 6Up). As several studies have reported the role of mechanical stress in inducing MAPK activation, spreading per se rather than the engagement of specific receptors may induce the required signaling events. This concept is underscored by the inhibition of blebbing by preincubating platelets with cytochalasin D, an inhibitor of cytoskeletal rearrangement, or p38 activation in the absence of {alpha}IIbß3 (unpublished results; Siljander et al and Sundaresan et al). In addition, the inhibitory effect of SB203580 on platelet blebbing but not on PS exposure (Table 3Up) suggests that p38 MAPK signaling is involved in the process of microvesiculation but not in the regulation of phospholipid scrambling. It is plausible that platelet adhesion mediates signaling steps, eg, enhancing p38 activation, and that this protein kinase is involved in cytoskeletal dynamics. For instance, in stress-activated endothelial cells, early membrane blebbing (inhibitable by SB203580) was found to be dependent on p38 MAPK–mediated F-actin reorganization. It was suggested that p38 activates its substrates, MAPK-activated protein kinase 2/3, which in turn phosphorylates the 27-kDa heat-shock protein, acting as an actin polymerization modulator.31 We observed partial inhibition of blebbing with SB203580, an inhibitor of p38{alpha} (SAPK2a) and/or p38ß (SAPK2b).32 In T cells, the small GTP-binding protein Rac1 and the tyrosine kinase Syk synergistically activate another stress-dependent MAPK form, JNK/SAPK1,40 claimed to be activated in parallel with p38 in these cells. Furthermore, it was shown that integrin-mediated adhesion involving focal adhesion kinases is mandatory for the activation of p38 MAPK by growth factors.41 42 Similar considerations may apply to platelets, so that SB203580-inhibitable kinases are necessary but not sufficient for full activation of the blebbing response.

Finally, the observation that calpain activity was inhibited by p38 blockade suggests that p38 lies upstream of calpain in regulating morphological changes involving cytoskeletal reorganization. Altogether, the present findings with SB203580 and calpeptin underline previous evidence that both strands of the procoagulant response, ie, phospholipid scrambling (PS exposure) and blebbing (microvesiculation), are not necessarily tied together. Although all natural platelet agonists seem to induce PS exposure in combination with microvesiculation, reduced microvesicle formation with unchanged PS exposure has also been obtained with the compound 2,5-di-t-butyl-1,4-benzohydroquinone,43 or by inhibition of protein tyrosine phosphatases44 in addition to inhibition of calpain.10 11

The fundamental role of cytoskeletal changes in the procoagulant response is reflected by the extensive morphological changes observed in platelets adhering to CRP or CVX. Scanning electron microscopy of glutaraldehyde-fixed platelets allowed more detailed observation of the blebbing platelets than did earlier work with light microscopy, during which platelets in contact with collagen or CRP were described as gradually changing into balloon-shaped structures containing moving vesicles.6 13 Many of the platelets adhering to immobilized CRP or CVX had a spongelike appearance, with little direct contact between platelet and adhesive surface (Figure 3Up). Application of CRP or CVX to fibrinogen-spread platelets resulted in fragmented, smaller forms, which were still in contact with the surface (Figure 4Up). Similar structures with platelets "fraying at the edges" could also be observed in the absence of fixation by vital phase-contrast microscopy.45 It is likely that the formation of focal complexes by {alpha}IIbß3-mediated adhesion results in more extensive platelet-surface interactions than adhesion to immobilized CRP or CVX. Thus, fragmentation of the fibrinogen-bound platelets may be explained by an increased number of contact sites. Comparison of phase-contrast images of unfixed blebbing platelets and the scanning electron microscopic images of fixed, spongelike platelets suggests that the latter may represent cytoskeletal ghosts of the blebbing cells that have lost their phospholipid membrane surface during fixation and subsequent dehydration procedures. Because the rough, spongelike or fragmented structures were observed only under conditions that stimulated the procoagulant response, they seem to be a useful marker for this stage of platelet activation.

In summary, our results delineate the GPVI-induced platelet procoagulant response as 2 sets of reactions, ie, the exposure of coagulation-supporting PS and membrane blebbing. These responses are almost absent in nonadherent platelets, even after GPVI stimulation, but are strongly triggered either after adhesion of the platelets to CRP or CVX or by application of these ligands to platelets adherent to fibrinogen. We conclude that GPVI-evoked activation of both calpain and p38 MAPK is required for the cytoskeletal changes that underlie the dramatic alterations in platelet morphology apparent as bleb formation and membrane fragmentation, but not in PS exposure.


*    Acknowledgments
 
These investigations were supported by the Finnish Cultural Foundation and the Ella and Georg Ehrnrooth Foundation (P.S.), the Netherlands Foundation for Scientific Research (NWO 902-68-241), the British Heart Foundation, and the Medical Research Council, UK (R.W.F.).

Received July 12, 2000; accepted January 5, 2001.


*    References
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