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Keywords:

  • actin;
  • cytoskeleton;
  • integrin;
  • platelet;
  • vinculin

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Addendum
  8. Acknowledgements
  9. Disclosure of Conflict of Interests
  10. References

Summary. Background: Vinculin links integrins to the cell cytoskeleton by virtue of its binding to proteins such as talin and F-actin. It has been implicated in the transmission of mechanical forces from the extracellular matrix to the cytoskeleton of migrating cells. Vinculin’s function in platelets is unknown. Objective: To determine whether vinculin is required for the functions of platelets and their major integrin, αIIbβ3. Methods: The murine vinculin gene (Vcl) was deleted in the megakaryocyte/platelet lineage by breeding Vcl fl/fl mice with Pf4–Cre mice. Platelet and integrin functions were studied in vivo and ex vivo. Results: Vinculin was undetectable in platelets from Vcl fl/fl Cre+ mice, as determined by immunoblotting and fluorescence microscopy. Vinculin-deficient megakaryocytes exhibited increased membrane tethers in response to mechanical pulling on αIIbβ3 with laser tweezers, suggesting that vinculin helps to maintain membrane cytoskeleton integrity. Surprisingly, vinculin-deficient platelets displayed normal agonist-induced fibrinogen binding to αIIbβ3, aggregation, spreading, actin polymerization/organization, clot retraction and the ability to form a procoagulant surface. Furthermore, vinculin-deficient platelets adhered to immobilized fibrinogen or collagen normally, under both static and flow conditions. Tail bleeding times were prolonged in 59% of vinculin-deficient mice. However, these mice exhibited no spontaneous bleeding and they formed occlusive platelet thrombi comparable to those in wild-type littermates in response to carotid artery injury with FeCl3. Conclusion: Despite promoting membrane cytoskeleton integrity when mechanical force is applied to αIIbβ3, vinculin is not required for the traditional functions of αIIbβ3 or the platelet actin cytoskeleton.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Addendum
  8. Acknowledgements
  9. Disclosure of Conflict of Interests
  10. References

Vinculin is a 117-kDa protein that plays a role in the cellular responses to mechanical forces [1–3]. A less abundant splice variant, metavinculin, is present in certain cells, including platelets [4]. Both proteins contain N-terminal head (residues 1–835 in vinculin) and C-terminal tail (residues 896–1066) domains, connected by a flexible hinge region (residues 836–895) [5]. Vinculin is thought to be in a ‘closed’ head-to-tail conformation in the cytoplasm of unstimulated cells. Upon cell activation, the head and tail domains dissociate, exposing binding sites for a variety of ligands implicated in cytoskeletal rearrangements, including talin, vasodilator-stimulated phosphoprotein, paxillin, F-actin and phosphatidylinositol 4,5-bisphosphate [1,5–7]. A recurrent theme in the literature is the relationship between vinculin, integrins and actin within adhesion complexes, mediated in part by interactions of the vinculin head and tail domains with talin and F-actin, respectively [7,8].

Vinculin has been studied primarily in nucleated cells. Vinculin (Vcl) knockout F9 embryonic carcinoma cells exhibit a round shape with fewer, less stable lamellipodia and smaller focal adhesions than wild-type F9 cells [9,10]. The knockout cells also show decreased adhesion and increased motility on matrix ligands, such as fibronectin [11]. In contrast, vinculin overexpression in mouse 3T3 fibroblasts leads to stronger adhesion and less motility [9]. As global deletion of murine Vcl causes embryonic lethality by embryonic day 10 because of heart and brain defects [12], investigators have begun to study the effects of vinculin depletion in specific cell types, using CreloxP technology. For example, Vcl deletion in cardiac myocytes disrupts adherens junctions in the myocardium, causing death by 3 months of age by either sudden death or dilated cardiomyopathy with progressive heart failure [13].

In contrast to most cell types, platelets are anucleate and non-migratory, and their main physiologic function is to promote hemostasis by forming thrombi at sites of vessel injury. When platelets are allowed to spread on extracellular matrices via integrin αIIbβ3, vinculin can be found at the cell periphery and within patches at the termini of F-actin cables [14,15]. As the precise role of vinculin in platelets is unknown, we deleted vinculin selectively in the megakaryocyte/platelet lineage by mating vinculin-floxed (Vcl fl/fl) mice [13] with Platelet factor 4 (Pf4)–Cre mice [16]. Vcl fl/fl Cre+ mice were viable and healthy, and exhibited normal platelet counts, but their platelets were devoid of vinculin, affording us an unprecedented opportunity to determine what functions vinculin might be required for in these cells. Contrary to our expectations, we demonstrate here that vinculin is not required for most platelet responses in hemostasis and thrombosis, including those dependent on αIIbβ3 and the actin cytoskeleton.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Addendum
  8. Acknowledgements
  9. Disclosure of Conflict of Interests
  10. References

Generation and genotyping of mice conditionally deficient in vinculin

Mice homozygous for the vinculin floxed allele on a mixed C57BL/6–Sv129 genetic background were crossed with Pf4–Cre mice on a C57BL/6 background. The construction of the vinculin floxed mice has been detailed previously [13]. Genotypes were determined by polymerase chain reaction (PCR) with primers VinF (5′-AAATGGGTTTTGCTCACCTG-3′) and VinR (5′-TGGAGTCATCTCTCCAGCCT-3′), which amplified 562 bp from the wild-type allele and 679 bp from the floxed allele [13,16]. Primers specific for the Pf4 promoter (5′-CCCATACAGCACACCTTTTG-3′) and Cre cDNA (5′-TGCACAGTCAGCAGGTT-3′) produced a 450-bp Cre recombinase product. All mouse protocols were approved by the Animal Care Program of the University of California.

Platelet function studies in vivo

Tail bleeding assays were performed by cutting 2–3 mm off the tail tip and immersing the tail in isotonic saline at 37 °C [17]. Bleeding times were taken as the time required for bleeding to cease for at least 60 s. Platelet thrombus formation was studied with a carotid artery FeCl3 injury model [18]. Each side of the adventitia of the left carotid artery was treated with 5% FeCl3-soaked filter paper for 3 min. Blood flow was monitored with a miniature ultrasound flow probe (T402 flowmeter; Transonic Systems, Ithaca, NY, USA) for at least 2 min before and 30 min after application of FeCl3. The operator was blinded to the genotype of the mice, and each experiment included vinculin-deficient mice and littermate controls.

Platelet function studies ex vivo

Blood was collected from tail veins in EDTA for complete blood counts (Hemavet 850FS Multi Species Hematology System; Drew Scientific, Waterbury, CT, USA). All experiments with mouse platelets included controls matched for age and sex. To obtain platelets for functional studies, blood was collected by cardiac puncture, anticoagulated with acid–citrate–dextrose anticoagulant, washed and finally resuspended in Walsh buffer (137 mm NaCl, 2.7 mm KCl, 1 mm MgCl2.6H2O, 3.3 mm NaH2PO4.H2O, 20 mm HEPES, pH 7.4, 0.1% glucose, 0.1% bovine serum albumin (BSA)], as previously described [19]. Cells were lysed in NP-40-containing buffer for western blotting of proteins [18].

Platelet adhesion to immobilized fibrinogen under static conditions was determined by acid phosphatase assay, and specific adhesion was expressed as a percentage of total platelets added to each microtiter well [19]. Platelet adhesion to immobilized fibrinogen (20 μg mL−1) or collagen (50 μg mL−1) in laminar flow was determined over a range of shear stresses, using a microfluidic device, as previously described [20,21]. Briefly, mouse blood was drawn by cardiac puncture into heparin-containing syringes (20 U mL−1), and platelet dense granules were labeled with 2 μm mepacrine [22]. After mounting of the microfluidic device on the mechanical stage of a Nikon Diaphot inverted fluorescence microscope, platelets were imaged using a green fluorescent protein filter set (excitation at 470 nm; emission at 525 nm), a × 63 (numerical aperture: 1.4) oil immersion objective lens, a × 0.42 video adapter, and a Sony SX900 IEEE1394 camera (Sony Electronics, Park Ridge, NJ, USA) with a 1/2-inch (1280 × 960 pixel) CCD array.

Fibrinogen binding to agonist-stimulated platelets was determined by flow cytometry with 150 μg mL−1 Alexa-Fluor 647-labeled fibrinogen (Invitrogen, Carlsbad, CA, USA) [19]. Non-specific binding was determined in the presence of 5 mm EDTA, and specific binding was taken as total minus non-specific binding. In some experiments, F-actin-dependent, irreversible fibrinogen binding to agonist-stimulated platelets was determined as previously described [23]. αIIbβ3 expression was measured with a fluorescein isothiocyanate-conjugated anti-mouse αIIb antibody (anti-CD41; BD Biosciences, San Jose, CA, USA). Platelet surface P-selectin expression was measured with a biotin-conjugated anti-mouse P-selectin antibody (BD Biosciences) and phycoerythrin-conjugated streptavidin (Invitrogen, Carlsbad, CA, USA) [24]. For platelet aggregation and fibrin clot retraction [25] assays, blood was anticoagulated 9 : 1 with 3.8% sodium citrate and diluted 1 : 1 or 1 : 2.3 with Walsh buffer, respectively. After pooling of blood from four mice of each genotype, aggregation was measured in a Chrono-log aggregometer (Chrono-log, Havertown, PA, USA). The relative F-actin contents of unstimulated platelets and platelets stimulated with a protease-activated receptor 4 (PAR4) agonist peptide (AYPGKF) was determined by flow cytometry, using BODIPY–phallicidin (Invitrogen) [26]. The ability of platelets to develop a procoagulant surface was determined by stimulating washed platelets with 1 μm A23187 Ca2+-ionophore for 15 min at 37 °C, followed by flow cytometric measurement of factor (F)Va light chain binding to platelets and platelet-derived microparticles [27].

Platelet spreading on fibrinogen-coated coverslips and actin nodule formation during platelet spreading [28] were assessed by deconvolution microscopy [19]. The extent of spreading was quantified by measuring individual platelet surface areas, using image-pro plus software (Media Cybernetics, Silver Springs, MD, USA).

Vinculin and membrane–cytoskeletal interactions in megakaryocytes

Megakaryocytes were harvested from the femurs of adult mice, and expanded and separated as previously described [29]. Large, mature megakaryocytes were isolated by passing the culture through a discontinuous BSA density gradient. For immunofluorescence microscopy, megakaryocytes were fixed and stained with rhodamine–phalloidin to label F-actin, an antibody to label vinculin (Sigma-Aldrich, St. Louis, MO, USA) and a nuclear label (Hoechst; Molecular Probes, Carlsbad, CA, USA).

The ability of the plasma membranes of mature megakaryocytes to resist external mechanical pulling was assessed with fibrinogen-coated beads and laser tweezers [30]. Briefly, mouse fibrinogen (Sigma-Aldrich) was covalently coupled to 1.87-μm carboxylate-modified latex beads (Bangs Laboratories, Fishers, IN, USA). A mixture of megakaryocytes and beads in culture medium containing 1 mm MnCl2 (to activate integrins) was incubated for 5–10 min in a poly-(l-lysine)-coated flow microscope chamber. Beads were then laser-trapped near adherent megakaryocytes, and the microscope focus was adjusted so that bead and cell centers were approximately the same distance from the bottom surface. The position of the laser trap was then oscillated in a triangular waveform with a frequency of 1 Hz and constant peak-to-peak amplitude of 2 μm (0.2 pN nm−1 trap stiffness; loading rate, 800 pN s−1). Bead position was sensed and digitized at a rate of 2000 scans per second [30]. In this system, the less megakaryocyte plasma membranes are able to resist force, the greater their tendency to form long membrane tethers connected to the beads. Tether formation was calculated by pooling data from three independent experiments performed with cells of the same genotype.

Statistical analyses

Analyses of variance were conducted by Student’s t-test, except where noted. All error bars in figures represent the mean ± standard error of the mean.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Addendum
  8. Acknowledgements
  9. Disclosure of Conflict of Interests
  10. References

Selective depletion of vinculin from megakaryocytes and platelets

PCR analyses of genomic DNA isolated from ear biopsies showed that mice homozygous for the floxed vinculin allele (Vcl fl/fl) could be distinguished from those with a wild-type allele (Vcl +/+) (Fig. 1A), facilitating genetic analysis of mice mated with Pf4–Cre mice to generate Vcl fl/fl Cre+ mice in which Vcl exon 3 flanked by loxP sites had been excised from mature megakaryocytes and platelets. Cre-mediated excision should result in deletion of the Vcl gene, which produces both vinculin and metavinculin in platelets. Western blotting of various mouse tissues confirmed depletion of vinculin from Vcl fl/fl Cre+ platelets, but not from Vcl fl/fl Cre+ heart, kidney, spleen or liver (Fig. 1B,C). Western blots of megakaryocytes isolated from primary bone marrow cultures of Vcl fl/fl Cre+ mice showed a small amount of residual vinculin (Fig. 1C), which is probably the result of contamination with non-megakaryocytic cells and/or the presence of immature megakaryocytes not yet expressing Cre recombinase [16]. Vinculin was not detected in mature, large polyploid megakaryocytes by immunofluorescence microscopy (Fig. 1D). Although metavinculin should also be deleted in Vcl fl/fl Cre+ platelets, we could not detect it even in control Vcl fl/fl Cre platelets with the use of two different antibodies. Several proteins other than vinculin were expressed at normal levels in Vcl fl/fl Cre+ platelets, including FAK, paxillin, α-actinin, kindlin-3, Erk1/2, β-actin (Fig. 1E) and αIIbβ3 (flow cytometry data not shown). Thus, Vcl fl/fl Cre+ platelets provided us with an opportunity to study platelet function in the absence of vinculin.

image

Figure 1.  Genotyping and vinculin expression. (A) Genomic DNA isolated from ear biopsies of Vcl +/+, Vcl fl/+ and Vcl fl/fl mice were analyzed by polymerase chain reaction as described in Materials and Methods. (B) Vinculin expression in 20 μg of platelet lysates from two mice of each genotype was analyzed on immunoblots (IB). Note the absence of a detectable vinculin band in lysates from Vcl fl/fl Cre+ platelets. (C) Vinculin expression in 20 μg of lysates from various cells and tissues. (D) Vinculin and F-actin staining of mature, polyploid megakaryocytes. Cells were plated on fibrinogen for 45 min, and then fixed, permabilized and stained as indicated. The merged images included staining for vinculin (green) and F-actin (rhodamine–phalloidin, red). Note the absence of detectable vinculin in Vcl fl/fl Cre+ mouse megakaryocytes (× 40 objective; size bar = 15 μm). (E) Immunoblots showing the expression of various proteins in 20 μg of lysates from platelets of the indicated genotypes.

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Platelet functions in vivo

Vinculin-deficient, Vcl fl/fl Cre+ mice were healthy and did not exhibit spontaneous bleeding, and their blood counts were equivalent to those of Vcl fl/fl Cre littermates (Table 1). Tail bleeding times, which are dependent in part on platelet function [31], averaged 132 ± 10 s in control Vcl fl/fl Cre mice and 334 ± 26 s in Vcl fl/fl Cre+ mice. Fifty-nine per cent of the platelet vinculin-deficient mice had tail bleeding times that exceeded the highest value observed in Vcl fl/fl Cre littermate controls, and their mean bleeding time was significantly greater than that of the controls (< 0.0001), Fig. 2A). Thus, deficiency of platelet vinculin may cause a mild defect in hemostasis.

Table 1.   Blood cell parameters
 Vcl fl/fl CreVcl fl/fl Cre+Normal range
  1. Values are expressed as the mean ± standard error of the mean; n = 3 for both groups. WBC, white blood cells; Ne, neutrophils; Ly, lymphocytes; Mo, monocytes; Eo, esinophils; Ba, basophils; RBC, red blood cells; MCV, mean cell volume; MPV, mean platelet volume.

Leukocytes
 WBC (× 103)9.9 ± 0.113.4 ± 0.63.0–16.1
 Ne (× 103)1.2 ± 0.11.6 ± 0.10.3–3.3
 Ly (× 103)8.1 ± 0.211.2 ± 0.52.2–12.9
 Mo (× 103)0.5 ± 0.00.4 ± 0.00.1–0.9
 Eo (× 103)0.1 ± 0.00.1 ± 0.00.0–0.3
 Ba (× 103)0.0 ± 0.00.0 ± 0.00.0–0.2
Erythrocytes
 RBC (× 106)9.2 ± 0.19.8 ± 0.28.0–12.0
 Hemoglobin (g dL−1)13.3 ± 0.113.9 ± 0.212.8–16.4
 Hematocrit (%)48.0 ± 0.552.0 ± 0.738.0–59.3
 MCV (fL)52.2 ± 0.352.4 ± 0.543.0–54.3
Thrombocytes
 Platelets (× 103)602 ± 29578 ± 11473–1220
 MPV (fL)4.7 ± 0.04.8 ± 0.14.3–5.9
image

Figure 2.  Platelet function in vivo. (A) Tail bleeding times. Each triangle represents one mouse (Vcl fl/fl Cre, n = 30; Vcl fl/fl Cre+, n = 46). Time to cessation of bleeding after tail resection was recorded for up to 10 min, at which time bleeding was stopped by cauterization. Horizontal lines indicate the means. (B) Times to complete occlusion of carotid arteries after FeCl3 injury. Three mice of each genotype were studied. (C) Carotid artery blood flow profiles from representative mice. Blood flow was measured for at least 25 min.

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Another way to test platelet function in mice is to monitor platelet thrombus formation in response to carotid artery injury with FeCl3 [18]. In normal mice, this typically leads to complete carotid artery occlusion within 5–10 min of injury, a response that can be monitored with a flow probe [32]. In some mice with defects in αIIbβ3 signaling, occlusions may be transient or not occur at all [18]. The average time to carotid artery occlusion in vinculin-deficient mice was within the expected normal limits, similar to that of Vcl fl/fl Cre controls (Fig. 2B,C). Consequently, any dysfunction of vinculin-deficient platelets is not severe enough to protect mice from this type or degree of arterial injury.

Platelet functions ex vivo

As vinculin plays a role in integrin–cytoskeletal connections that enable nucleated cells to resist mechanical forces, including laminar shear stress [33–36], the adhesion of vinculin-deficient platelets to immobilized integrin ligands was examined under static and flow conditions. The adhesion of Vcl fl/fl Cre+ platelets to fibrinogen was similar to that of Vcl fl/fl Cre platelets under static conditions, whether or not platelets had been activated by ADP (Fig. 3A). Moreover, vinculin-deficient platelets adhered normally to fibrinogen (Fig. 3B) and collagen (Fig. 3C) under flow over a range of biologically relevant shear stresses. Thus, contrary to expectations based on studies in nucleated cells, vinculin appears to be dispensable for platelet adhesion to fibrinogen via αIIbβ3 [20,21] or to collagen via α2β1 [37].

image

Figure 3.  Platelet adhesion ex vivo. (A) Adhesion to fibrinogen under static conditions with or without 20 μm ADP as described in Materials and Methods. Data represent means ± standard errors of the mean of five independent experiments, each performed in triplicate. (B) Adhesion to fibrinogen under flow conditions, as described in Materials and Methods. The fibrinogen coating concentration was 20 μg mL−1, and adhesion was quantified after 1 min of flow at the indicated shear rates (n = 3). (C) Adhesion to collagen under flow conditions. The collagen coating concentration was 50 μg mL−1, and adhesion was quantified after 1 min of flow (n = 3).

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Inside-out signals initiated by platelet agonists convert αIIbβ3 from a low-affinity to a high-affinity state, enabling soluble fibrinogen binding and platelet aggregation [38]. When platelets were stimulated with ADP or a PAR4 thrombin receptor agonist peptide, AYPGFK, specific fibrinogen binding to Vcl fl/fl Cre+ platelets was not significantly different from binding to Vcl fl/fl Cre control platelets (Fig. 4A). Furthermore, under stirring conditions, Vcl fl/fl Cre+ platelets aggregated normally in response to ADP (0.5, 1 and 5 μm), AYPGKF (0.05 and 0.1 mm) or collagen (10 μg mL−1) (Fig. 4B).

image

Figure 4.  Fibrinogen binding and platelet aggregation. (A) Washed platelets were incubated for 20 min at room temperature with Alexa-Fluor 647-labeled fibrinogen with or without ADP or AYPGKF. Specific fibrinogen binding was determined by flow cytometry and depicted as mean fluorescence intensity (MFI) in arbitrary fluorescence units (n = 3). (B) Representative platelet aggregation tracings from Vcl fl/fl Cre and Vcl fl/fl Cre+ mice. Results are representative of at least three experiments.

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When platelets attach to immobilized fibrinogen, they undergo outside-in αIIbβ3 signaling and show a spreading response over 30–60 min that is characterized by the formation of F-actin nodules early in the process [28], progressive actin polymerization/reorganization, filopodial protrusion and lamellipodial extension [14]. Full platelet spreading requires cosignaling from αIIbβ3 and agonist receptors [26]. The spreading of Vcl fl/fl Cre+ platelets on fibrinogen was determined by quantification of individual platelet surface areas. The spreading of vinculin-deficient platelets was similar to that of Vcl fl/fl Cre control platelets, both in the absence and in the presence of ADP or AYPGKF (Fig. 5A). Moreover, actin nodules could be observed in both sets of platelets (Fig. 5B), and fully spread platelets displayed a rim distribution of F-actin and prominent F-actin cables (Fig. 5C). When stained for the focal adhesion protein Hic-5 [39], vinculin-deficient platelets even exhibited characteristic rim staining and patches at the termini of actin cables (Fig. 5C).

image

Figure 5.  Platelet spreading and cytoskeletal organization. (A) Platelet spreading. Washed platelets were plated onto fibrinogen-coated coverslips (100 μg mL−1) for 45 min at 37 °C with or without MnCl2, ADP or AYPGKF. Cells were fixed with 3.7% paraformaldehyde, permeabilized with 0.2% Triton X-100 and stained for αIIbβ3 with antibody 1B5. Platelet spreading was assessed by deconvolution microscopy and expressed as the mean platelet surface area. Data represent 500–800 platelets analyzed for each genotype. (B) Representative platelet images showing actin nodules. Washed platelets were plated on fibrinogen (100 μg mL−1) in the presence of 2 U mL−1 apyrase and 10 μm indomethacin for 45 min at 37 °C [28]. After fixation, permeabilization and staining with rhodamine-phalloidin to label F-actin, images were captured by deconvolution microscopy (scale bar = 10 μm). The area of interest in a single platelet (bordered in white) is expanded for better visualization of actin nodules, denoted by the white arrows. (C) Representative deconvolution images of platelets stained for vinculin (green) or Hic-5 (green) and F-actin (red) (scale bar = 10 μm).

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The above results do not exclude a role for vinculin in other PAR4-mediated and actin-dependent platelet responses. These include actin polymerization [40], fibrin clot retraction [41], and irreversible fibrinogen binding, a process that is blocked by cytochalasins and resistant to EDTA [23,42]. However, when F-actin content was monitored with BODIPY–phallicidin, vinculin-deficient platelets stimulated with AYPGFK exhibited an increase in F-actin content comparable to that in control platelets (Fig. 6A). Furthermore, vinculin-deficient platelets exhibited normal fibrin clot retraction (Fig. 6B) and irreversible fibrinogen binding (Fig. 6C).

image

Figure 6.  Analyses of platelet F-actin. (A) F-actin content. Washed platelets were incubated for 3 min with or without 1 mm AYPGKF. After fixation and permeabilization, platelets were stained with BODIPY–phallacidin with or without unlabeled excess phallicidin, and specific BODIPY–phallacidin was quantified by flow cytometry and taken as the relative platelet F-actin content (n = 3) [26]. (B) Fibrin clot retraction. Clotting proceeded for 2 h at 37 °C in platelet-rich plasma after addition of 9.4 U mL−1 thrombin and 1.9 mm CaCl2 (n = 3, each performed in duplicate). (C) Irreversible fibrinogen binding was analyzed by flow cytometry as previously described [23]. Note that irreversible binding (defined as binding not chased by EDTA) increased with time after platelet stimulation with AYPGKF but did not occur in the presence of cytochalasin E (cyto E) (n = 3). MFI, mean fluorescence intensity.

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Two additional platelet responses that may be partially dependent on αIIbβ3 and/or actin are granule secretion and the development of a procoagulant surface [43]. The former can be monitored indirectly by surface expression of P-selectin in response to the PAR4 thrombin receptor-activating peptide AYPGFK, and the latter by the binding of FVa light chain to the platelet procoagulant surface in response to the Ca2+-ionophore A23187 [27]. When measured by flow cytometry, vinculin deficiency had no effect on either of these platelet responses (Fig. 7A,B). Taken together, this set of studies indicates that vinculin is dispensable for ex vivo platelet responses that involve αIIbβ3, F-actin or both proteins.

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Figure 7.  Platelet α-granule secretion and procoagulant surface. Washed platelets were stimulated with the indicated agonists for 15 min at 37 °C. (A) α-Granule secretion was monitored by surface expression of P-selectin and detected by flow cytometry (n = 3). (B) After platelet stimulation with 1 μm A23187 + 2 μg mL−1 factor (F)Va light chain, the development of a procoagulant surface was assessed by measuring FVa light chain binding to platelets and platelet-derived microparticles with fluorescein isothiocyanate–V237 antibody [27]. These data represent the binding to platelets and microparticles combined (n = 3). MFI, mean fluorescence intensity.

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Vinculin and membrane–cytoskeletal interactions in megakaryocytes

Vinculin is required for plasma membrane cytoskeleton integrity, as determined by measurements of membrane ‘stiffness’ of mouse embryonic fibroblasts [44]. Recently, primary murine megakaryocytes were used in conjunction with laser tweezers and fibrinogen-coated beads to generate an external pulling force on αIIbβ3, leading to increased plasma membrane tethers in mice deficient in phosphatidylinositol-4-phosphate-5-kinase Iγ [45]. To determine whether loss of vinculin would result in a similar increase in membrane tethers, mature megakaryocytes from Vcl fl/fl Cre+ and Vcl fl/fl Cre mice were generated in culture, and laser tweezers were used to pull on fibrinogen-coated beads adherent to the cells. Under these conditions, megakaryocytes from Vcl fl/fl Cre+ mice displayed significantly more membrane tethers than megakaryocytes from the control mice (< 0.0003, anova; Fig. 8), suggesting that vinculin is required to maintain megakaryocyte membrane cytoskeleton integrity.

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Figure 8.  Membrane tether formation in response to a mechanical pulling force applied to αIIbβ3. Megakaryocytes were generated in culture from bone marrows of megakaryocyte/platelet vinculin-deficient mice (Vcl fl/fl Cre+) and two sets of control mice (C57/BL6 and Vcl fl/fl Cre). Experiments were performed by pulling with laser tweezers on adherent fibrinogen-coated beads, as described in Material and Methods. The numbers of fibrinogen-coated bead/membrane interactions analyzed for tether formation were 343 (C57/BL6), 473 (Vcl fl/fl Cre) and 663 (Vcl fl/fl Cre+). Data are expressed as the percentage of interactions resulting in membrane tethers [45]. The statistical analysis was performed using the χ2-test. NS, not significant.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Addendum
  8. Acknowledgements
  9. Disclosure of Conflict of Interests
  10. References

Integrins, including those expressed in platelets, are linked to the actin cytoskeleton both structurally and functionally [37,38,46]. With binding sites for actin and for integrin-associated proteins such as talin, vinculin is thought to regulate integrin–actin linkages, thereby influencing the adhesion and motility of nucleated cells [5,7,33,34,47]. Here, we sought to determine the function of vinculin in platelets by selectively deleting the murine vinculin gene in the megakaryocyte/platelet lineage. Vcl fl/fl Cre+ mice were viable, and their platelets and mature megakaryocytes were deficient in vinculin, as demonstrated by western blotting and fluorescence microscopy. Studies of these mice and their platelets enabled us to conclude the following. (i) Mice deficient in megakaryocyte/platelet vinculin display megakaryocytes with reduced membrane cytoskeleton integrity, as demonstrated by increased membrane tether formation in response to an external force imposed on αIIbβ3. (ii) Mice with vinculin-deficient megakaryocytes and platelets have normal platelet counts, and they do not bleed spontaneously. However, a majority of them exhibit prolonged tail bleeding times, suggesting a mild platelet function defect. (iii) On the other hand, platelet vinculin is not essential for the formation of occlusive thrombi in response to a carotid artery injury, and nor is it required for a variety of ex vivo platelet responses under static or flow conditions that involve bidirectional αIIbβ3 signaling. (iv) Most surprisingly, vinculin is not required for agonist-induced platelet functions that are dependent in part on αIIbβ3–actin linkages. These include actin polymerization and reorganization, fibrin clot retraction, irreversible fibrinogen binding, α-granule secretion, and the development of a procoagulant surface. Collectively, these results have forced us to re-examine assumptions about vinculin function in platelets that were based largely on studies in migratory nucleated cells. They suggest either that vinculin is not required for most platelet functions involving αIIbβ3 and F-actin, or that other proteins can substitute or compensate for vinculin in its absence.

In nucleated cells, vinculin is often localized at sites of cell contact with the extracellular matrix, where it functions as a scaffolding protein to couple adhesion receptors to the actin cytoskeleton [7,8,10]. Biophysical studies comparing vinculin-deficient and wild-type nucleated cells in culture have concluded that vinculin helps to stabilize the plasma membrane cytoskeleton [44,48]. Our experiments with laser tweezers demonstrated an increase in membrane tethers in vinculin-deficient megakaryocytes when a pulling force was applied to fibrinogen-coated beads adherent to these cells (Fig. 8). Similar results with megakaryocytes from phosphatidylinositol-4-phosphate-5-kinase Iγ knockout mice have implicated this enzyme in maintaining the integrity of the membrane cytoskeleton [45]. Interestingly, platelets can form long membrane tethers at very high shear stresses during von Willebrand factor-dependent platelet aggregation [49–51]. The role of vinculin in the generation of these types of membrane tethers and the relationship of these tethers to the ones that we have studied in megakaryocytes warrant further study.

Platelet counts were normal in Vin fl/fl Cre+ mice, but tail bleeding times were prolonged in a majority of them when compared to Vin fl/fl Cre littermates (Fig. 2A). This suggests that a deficiency of vinculin may cause a subtle defect in platelet function during hemostasis in a tail wound [31]. To further evaluate platelet function in vivo, FeCl3 was used to injure the carotid artery and to generate occlusive thrombi; this is one of several mouse arterial thrombosis models that are dependent on platelets and αIIbβ3 [52,53]. However, occlusive thrombi formed normally in this model in the absence of vinculin (Fig. 2B,C). It is always possible that a different form of arterial injury might have yielded a different result. Nonetheless, we can conclude that vinculin is not necessarily required for the formation of an occlusive platelet thrombus in vivo. There are several possible explanations for why tail bleeding times were affected by vinculin deficiency but carotid artery thrombus formation was not. These include differences in rheologic conditions as well as potential differences in the repertoire of platelet agonists generated after tail transection vs. chemical carotid artery injury [52,53]. The importance of environmental conditions in determining the requirements for vinculin in cellular responses is underscored by a recent study with nucleated cells, where vinculin deficiency enhanced cell motility on a two-dimensional collagen matrix but retarded cell invasion in a three-dimensional matrix [3]. Thus, the functions of platelet vinculin may be context-dependent.

We find it remarkable that vinculin-deficient platelets functioned normally in a wide variety of assays of platelet function ex vivo. Inside-out αIIbβ3 signaling, as measured by the specific binding of soluble fibrinogen to activated platelets, was similar in Vin fl/fl Cre+ and Vin fl/fl Cre backgrounds. Under normal circumstances, fibrinogen binding to platelets is initially reversible, in that bound fibrinogen can be chased with EDTA. However, fibrinogen binding becomes irreversible over time, a process variously ascribed to an increase in αIIbβ3 bond strength, αIIbβ3 clustering, and/or αIIbβ3 internalization. Significantly, irreversible fibrinogen binding can be prevented by pretreatment of platelets with cytochalasins, which inhibit actin polymerization [23,42]. However, we found that irreversible fibrinogen binding was unaffected by vinculin depletion (Fig. 6C), leading us to conclude that vinculin is not required for either reversible or irreversible fibrinogen binding to αIIbβ3.

Following fibrinogen binding to αIIbβ3, outside-in signals are triggered by enzymes that associate with the cytoplasmic tails of the integrin, and these signals collaborate with those generated through occupancy of various agonist receptors to generate a number of platelet responses, many involving actin polymerization and reorganization [38]. We found that vinculin-deficient platelets underwent actin polymerization normally in response to PAR4 thrombin receptor activation (Fig. 6A). This result is consistent with the observation in mouse F9 embryonic carcinoma cells that F-actin and vinculin contents are not correlated [54]. Also, platelet vinculin deficiency failed to impair platelet spreading, F-actin cable formation or fibrin clot retraction (Figs 5 and 6). Even platelet adhesion to fibrinogen and collagen under flow conditions was normal in vinculin-deficient platelets (Fig. 3). It is possible that other cytoskeletal proteins, such as talin [47], α-actinin [55] or kindlin-3 [56], may substitute for vinculin in promoting αIIbβ3-dependent and actin-dependent responses in platelets. Alternatively, expression of some cytoskeletal proteins may be upregulated in Vin fl/fl Cre+ platelets in order to compensate for the loss of vinculin, although we did not find this to be the case for several proteins examined (Fig. 1E). Our flow studies were conducted under conditions of low-to-intermediate levels of shear stress. We cannot rule out the possibility that the absence of vinculin may affect platelet responses under conditions of extremely high shear stresses, such as those that may be operative in highly stenotic blood vessels [50].

Finally, we speculate that the function of the actin cytoskeleton, and vinculin in particular, is subtly, if not fundamentally, different in platelets than in many types of nucleated cells. For example, certain nucleated cells migrate extensively during development or following injury, and this migration appears to be dependent on vinculin [10,57,58]. In contrast, whereas platelets roll, adhere, spread and aggregate following vascular injury, they are not known to engage in haptokinetic or ameboid migration. To understand better what vinculin is needed for in platelets, it may be informative to mate Vin fl/fl Cre+ mice with mice harboring heterozygous or homozygous deletions of other cytoskeletal proteins, to expose phenotypes in the offspring that are not obvious in the parents.

Addendum

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Addendum
  8. Acknowledgements
  9. Disclosure of Conflict of Interests
  10. References

J. V. Mitsios designed and performed research, analyzed data and wrote the article. N. Prévost designed research and analyzed data. A. Kasirer-Friede performed research and analyzed data. E. Gutierrez and A. Groisman designed research and analyzed data. C. S. Abrams, Y. Wang and R. I. Litvinov performed research and analyzed data. A. Zemljic-Harpf and R. S. Ross supplied essential mice. S. J. Shattil designed research, analyzed data and wrote the article.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Addendum
  8. Acknowledgements
  9. Disclosure of Conflict of Interests
  10. References

We thank R. Skoda (Basel University Hospital, Switzerland) for providing Pf4Cre mice, B. Coller (Rockefeller University, New York, NY, USA) for the 1B5 antibody, K. Burridge (University of North Carolina, Chapel Hill, NC, USA) for the α-actinin antibody and P. J. Sims (University of Rochester Medical Center, Rochester, NY, USA) for FVa and the V237 antibody. Research was supported by NIH grants HL56595, HL57900 and T32 HL086344-02 (to S. J. Shattil) and HL046345 (to R. S. Ross).

Disclosure of Conflict of Interests

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Addendum
  8. Acknowledgements
  9. Disclosure of Conflict of Interests
  10. References

The other authors state that they have no conflict of interest.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Addendum
  8. Acknowledgements
  9. Disclosure of Conflict of Interests
  10. References