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Keywords:

  • α5β1;
  • αIIbβ3;
  • fibronectin;
  • platelet

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Experimental procedures
  5. Reagents
  6. Preparation of human platelets
  7. Preparation of murine platelets
  8. Adhesion assays
  9. Single platelet Ca2+ measurement
  10. Phosphorylation studies
  11. Analysis of data
  12. Results
  13. Spreading of human platelets on fibronectin-coated surfaces
  14. Identification of platelet integrins mediating adhesion to fibronectin
  15. Role of Ca2+ mobilization and PLCγ2 on platelet spreading on fibronectin
  16. αIIb-deficient mouse platelets adhere but do not spread on fibronectin
  17. Regulation of FAK and PLCγ2 activity in platelets on fibronectin
  18. Spreading of platelets on fibronectin in plasma
  19. Discussion
  20. Acknowledgements
  21. References

Summary. Background: Recent studies have shown that platelet adhesion and subsequent aggregation can occur in vivo in the absence of the two principal platelets adhesive ligands, von Willebrand factor and fibrinogen. These results highlight a possible role for fibronectin in supporting thrombus formation. Objective and methods: To evaluate the platelet integrins and subsequent activation pathways associated with fibronectin-dependent platelet adhesion utilizing both human and murine platelets. Results: Platelets can adhere to fibronectin via the integrin αIIbβ3, leading to formation of lamellipodia. This is mediated through an interaction with the tenth type III domain in fibronectin. Spreading on fibronectin promotes αIIbβ3-mediated Ca2+ mobilization and tyrosine phosphorylation of focal adhesion kinase and phospholipase C γ2. In contrast, studies with blocking antibodies and inline image mice demonstrate that α5β1 and αvβ3 support adhesion and promote formation of filopodia but not lamellipodia or tyrosine phosphorylation of these proteins. Further, neither α5β1 nor αvβ3 is able to induce formation of lamellipodia in the presence of platelets agonists, such as collagen-related-peptide (CRP). Conclusions: These observations demonstrate that integrins α5β1 and αvβ3 support platelet adhesion and the generation of filopodia but that, in contrast to the integrin αIIbβ3, are unable to promote formation of lamellipodia.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Experimental procedures
  5. Reagents
  6. Preparation of human platelets
  7. Preparation of murine platelets
  8. Adhesion assays
  9. Single platelet Ca2+ measurement
  10. Phosphorylation studies
  11. Analysis of data
  12. Results
  13. Spreading of human platelets on fibronectin-coated surfaces
  14. Identification of platelet integrins mediating adhesion to fibronectin
  15. Role of Ca2+ mobilization and PLCγ2 on platelet spreading on fibronectin
  16. αIIb-deficient mouse platelets adhere but do not spread on fibronectin
  17. Regulation of FAK and PLCγ2 activity in platelets on fibronectin
  18. Spreading of platelets on fibronectin in plasma
  19. Discussion
  20. Acknowledgements
  21. References

Platelet adhesion to the subendothelium of an injured vessel wall and subsequent aggregation are critical events in hemostasis and thrombosis. The interaction of platelets with adhesive proteins both in the extracellular matrix (ECM) and plasma provides signals that affect their morphology, activation and binding. Following initial platelet-collagen adhesion, a variety of receptor–ligand interactions synergistically promote homotypic platelet aggregation leading to thrombus formation [1–3]. A critical event in this process is the activation of platelet integrins, which increases their affinity for their respective adhesive ligands. The key integrin in thrombus formation is αIIbβ3, which is a receptor for a number of adhesive proteins including fibrinogen, von Willebrand factor (VWF) and fibronectin [2,4]. Engagement of αIIbβ3 promotes platelet–platelet interaction [5] and generates ‘outside-in’ signals that reinforce activation [6,7]. Until recently, VWF and fibrinogen were thought to be the principal ligands in vivo that engage αIIbβ3. However, Ni et al. have shown that mice lacking both of these adhesive ligands retain the ability to develop thrombi at sites of vascular injury in vivo, highlighting a role for alternative adhesive proteins in promoting thrombus formation [8]. In this context, it is pertinent to consider a role for fibronectin, a recognized ligand for αIIbβ3 that is present in high levels in plasma and which has been shown to promote thrombus growth and stability in injured arterioles [9,10].

Fibronectin is present in plasma, the subendothelium of the vessel wall, and is stored in low amounts in platelet α-granules [11]. This soluble dimeric protein is a mosaic of three different types of homologous repeating modules that have been designated type I, type II and type III. The Arg-Gly-Asp (RGD) sequence in FNIII domain 10 (10FIII) is crucial for interactions with its integrin receptors, namely αIIbβ3, αvβ3 and α5β1. In addition, other sequences outside of this RGD region are sometimes required for full adhesive activity [12,13]. This is exemplified by a set of residues in the FNIII domain 9 (9FIII) which have been shown to contribute to high affinity binding to α5β1 integrin in baby hamster kidney (BHK) cells [14]. This region has been termed the synergy site.

The initial binding of an integrin to an adhesive protein, such as fibronectin, stimulates recruitment of signaling proteins to the vicinity of the integrin cytoplasmic tails [15]. This initial phase of outside-in signaling contributes to further platelet activation. This is illustrated by αIIbβ3 signaling mediated via Src and Syk tyrosine kinases leading to activation of a number of downstream pathways including focal adhesion kinase (FAK), PI 3-kinase and phospholipase C γ2 (PLCγ2), which leads to an increase in intracellular Ca2+; together these events are crucial for platelet spreading [16–19]. The contribution made by other platelet integrins which are expressed in lower levels, such as the α5β1 and αvβ3 integrins, to activation has yet to be defined.

This study focuses on characterizing the potential role of the platelet integrin receptors for fibronectin, αIIbβ3, αvβ3 and α5β1, in mediating adhesion and activation. We demonstrate that platelet α5β1 and αvβ3 integrins are capable of supporting platelet adhesion, but that lamellipodia formation and intracellular Ca2+ mobilization requires αIIbβ3. Furthermore, we demonstrate that platelet adhesion and spreading is critically dependent upon the RGD-sequence containing 10FIII module of fibronectin, although the 9FIII region is required to produce equivalents levels of signaling to that induced by full-length fibronectin. These results demonstrate that the integrins αIIbβ3, αvβ3 and α5β1 are able to support platelet adhesion but that only the first of these is able to mediate formation of lamellipodia and mobilization of Ca2+.

Reagents

  1. Top of page
  2. Abstract
  3. Introduction
  4. Experimental procedures
  5. Reagents
  6. Preparation of human platelets
  7. Preparation of murine platelets
  8. Adhesion assays
  9. Single platelet Ca2+ measurement
  10. Phosphorylation studies
  11. Analysis of data
  12. Results
  13. Spreading of human platelets on fibronectin-coated surfaces
  14. Identification of platelet integrins mediating adhesion to fibronectin
  15. Role of Ca2+ mobilization and PLCγ2 on platelet spreading on fibronectin
  16. αIIb-deficient mouse platelets adhere but do not spread on fibronectin
  17. Regulation of FAK and PLCγ2 activity in platelets on fibronectin
  18. Spreading of platelets on fibronectin in plasma
  19. Discussion
  20. Acknowledgements
  21. References

The blocking anti-α5 (JBS5) monoclonal antibody (mAb) was purchased from Serotec (Oxford, UK). The blocking anti-β1 (P4C10) mAb was purchased from Chemicon (Temecula, CA, USA). Polyclonal rabbit anti-FAK (C-903) antibody (pAb) was from Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA. Anti-PLCγ2 pAb was a gift from Dr Tomlinson (DNAX, Palo Alto, CA, USA). The αvβ3IIbβ3 antagonists lotrafiban and abciximab were supplied by GlaxoSmithKline (King of Prussia, PA, USA) and Eli Lilly and Company, Ltd (Basingstoke, UK), respectively. AR-C67085 was a gift from AstraZeneca R & D Charnwood (Loughborough, UK). BAPTA-AM, A3P5P, apyrase, indomethacin, thrombin and ADP were from Sigma (Poole, UK). D-phenyl-alanyl-1-prolyl-1 arginine chloromethyl ketone (PPACK) was purchased from Calbiochem (La Jolla, CA, USA). Fibrinogen depleted of plasminogen, VWF and fibronectin was from Kordia Laboratory Supplies, Leiden, NL. Bovine plasma fibronectin was from Invitrogen (Carlsbad, CA, USA). The expression and purification of fibronectin type III protein domains has been previously described [14] and were obtained from the authors. The purity and Mr of the proteins was confirmed by electrospray mass spectroscopy (data not shown). Oregon Green bis-(o-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid (BAPTA 1)-AM was purchased from Molecular Probes (Cambridge Bioscience, Cambridge, UK). CRP was synthesized by Tana Laboratories (Houston, TX, USA). All other reagents were from Sigma (Poole, UK) or previously named sources [16,18,20].

Preparation of human platelets

  1. Top of page
  2. Abstract
  3. Introduction
  4. Experimental procedures
  5. Reagents
  6. Preparation of human platelets
  7. Preparation of murine platelets
  8. Adhesion assays
  9. Single platelet Ca2+ measurement
  10. Phosphorylation studies
  11. Analysis of data
  12. Results
  13. Spreading of human platelets on fibronectin-coated surfaces
  14. Identification of platelet integrins mediating adhesion to fibronectin
  15. Role of Ca2+ mobilization and PLCγ2 on platelet spreading on fibronectin
  16. αIIb-deficient mouse platelets adhere but do not spread on fibronectin
  17. Regulation of FAK and PLCγ2 activity in platelets on fibronectin
  18. Spreading of platelets on fibronectin in plasma
  19. Discussion
  20. Acknowledgements
  21. References

Human venous blood was drawn by venipuncture from healthy volunteers into either sodium citrate and acid/citrate/dextrose (ACD) or 50 μM PPACK anticoagulant as previously described [16,21]. Platelet-rich plasma (PRP) was prepared by centrifugation of whole blood at 160 g for 20 min. Platelet-poor plasma (PPP) was obtained by further centrifugation of the blood at 1100 g for 10 min. The final platelet count in the PRP was adjusted to the desired levels with platelet-poor plasma.

In selected experiments, platelets were isolated from PRP by centrifugation at 1100 g for 10 min in the presence of prostacyclin (0.1 μg mL−1). The pellet was resuspended in modified HEPES-Tyrodes buffer (in mM: 129 NaCl, 0.34 Na2HPO4, 2.9 KCl, 12 NaHCO3, 20 HEPES, 5 glucose, 1 MgCl2; pH 7.3) containing 0.1 μg mL−1 prostacyclin. The platelets were washed once via centrifugation (1100 g for 10 min) and resuspended at the desired concentration with HEPES-Tyrode buffer, containing physiological levels of fibrinogen (3 mg mL−1), fibronectin (300 μg mL−1) and VWF (10 μg mL−1) in selected experiments. In separate experiments, platelet suspensions were treated with 0.2 μg mL−1 CRP, 10 μM ADP, 1 and 0.06 U mL−1 thrombin, 10 μM BAPTA-AM, 5 mM Mn2+, 10 μM lotrafiban (LOT), 20 μg mL−1 JBS5 and P4C10, 2 U mL−1 apyrase or 1 mM A3P5P and 1 μM AR-C67085, or 10 μM indomethacin for 10–30 min before use in the assays. All experiments were performed in the absence of exogenously added Ca2+.

Preparation of murine platelets

  1. Top of page
  2. Abstract
  3. Introduction
  4. Experimental procedures
  5. Reagents
  6. Preparation of human platelets
  7. Preparation of murine platelets
  8. Adhesion assays
  9. Single platelet Ca2+ measurement
  10. Phosphorylation studies
  11. Analysis of data
  12. Results
  13. Spreading of human platelets on fibronectin-coated surfaces
  14. Identification of platelet integrins mediating adhesion to fibronectin
  15. Role of Ca2+ mobilization and PLCγ2 on platelet spreading on fibronectin
  16. αIIb-deficient mouse platelets adhere but do not spread on fibronectin
  17. Regulation of FAK and PLCγ2 activity in platelets on fibronectin
  18. Spreading of platelets on fibronectin in plasma
  19. Discussion
  20. Acknowledgements
  21. References

The generation of mice disrupted in the αIIb gene (inline image) is described in Emambokus et al. [22]. Mice deficient in PLCγ2 were generated as described [23]. Wild type littermates were used as controls. Murine blood was drawn from CO2 terminally anesthetized mice by cardiac puncture and taken into 100 µL of acid-citrate-dextrose. PRP was obtained by centrifugation at 300 g for 6 min PPP was then obtained by further centrifugation of the blood at 1000 g for 6 min and used to adjust the platelet concentration of PRP. In selected experiments, washed platelets were obtained via centrifugation of PRP at 1000 g in the presence of prostacyclin (0.1 μg mL−1) for 6 min. The pellet was resuspended in modified HEPES-Tyrode buffer to the desired platelet level.

Adhesion assays

  1. Top of page
  2. Abstract
  3. Introduction
  4. Experimental procedures
  5. Reagents
  6. Preparation of human platelets
  7. Preparation of murine platelets
  8. Adhesion assays
  9. Single platelet Ca2+ measurement
  10. Phosphorylation studies
  11. Analysis of data
  12. Results
  13. Spreading of human platelets on fibronectin-coated surfaces
  14. Identification of platelet integrins mediating adhesion to fibronectin
  15. Role of Ca2+ mobilization and PLCγ2 on platelet spreading on fibronectin
  16. αIIb-deficient mouse platelets adhere but do not spread on fibronectin
  17. Regulation of FAK and PLCγ2 activity in platelets on fibronectin
  18. Spreading of platelets on fibronectin in plasma
  19. Discussion
  20. Acknowledgements
  21. References

Cover slips were incubated with a suspension of fibrinogen, fibronectin, recombinant fibronectin proteins (200 μg mL−1) or denatured BSA (5 mg mL−1) overnight at 4 °C. Surfaces were washed twice with PBS and blocked with denatured BSA (5 mg mL−1) for 1 h at room temperature (RT). Surfaces were then subsequently washed twice with PBS before use in spreading assays. Platelets failed to bind or become activated to surfaces coated with denatured BSA.

For the spreading experiments, washed murine platelets (2 × 107 mL−1) and washed human platelets (2 × 107 mL−1) were incubated on coated coverslips at 37 °C for 45 min in a humid environment. After removal of unbound platelets by washing with PBS, adhered platelets were fixed, permeabilized, and stained by TRITC-conjugated phalloidin, as described previously [24]. Platelets were viewed on an inverted fluorescent microscope (Carl Zeiss MicroImaging, Inc., Herts, UK), and digital imaging was performed using Openlab software for Macintosh.

Platelet spreading (6 × 107 mL−1) was imaged in real time using Köhler illuminated Nomarski differential interference contrast optics with a Zeiss 63× oil immersion 1.40 NA axioplan lens on a Zeiss Axiovert 100 Microscope. Time-lapse events were captured by a QICAM Mono 10-bit camera (QImaging, Burnaby, BC, Canada) using Openlab software for Macintosh. To compute the surface area of spreading platelets, time-lapse images were manually outlined and quantitated by determining the number of pixels within each outline using a Java plugin for the Image J software package. Imaging a graticule under the same conditions allowed the conversion of pixels size to microns.

Single platelet Ca2+ measurement

  1. Top of page
  2. Abstract
  3. Introduction
  4. Experimental procedures
  5. Reagents
  6. Preparation of human platelets
  7. Preparation of murine platelets
  8. Adhesion assays
  9. Single platelet Ca2+ measurement
  10. Phosphorylation studies
  11. Analysis of data
  12. Results
  13. Spreading of human platelets on fibronectin-coated surfaces
  14. Identification of platelet integrins mediating adhesion to fibronectin
  15. Role of Ca2+ mobilization and PLCγ2 on platelet spreading on fibronectin
  16. αIIb-deficient mouse platelets adhere but do not spread on fibronectin
  17. Regulation of FAK and PLCγ2 activity in platelets on fibronectin
  18. Spreading of platelets on fibronectin in plasma
  19. Discussion
  20. Acknowledgements
  21. References

Washed human platelets at 4 × 108 mL−1 were loaded with the Ca2+ sensititve dye Oregon Green BAPTA 1-AM (15 μM) for 1 h at 30 °C. Loaded platelets were subsequently washed, resuspended at 2 × 108 mL−1 and left for a minimum of 30 min before experimentation. Platelets (1 × 107 mL−1) were allowed to sediment onto either a fibronectin- or BSA-coated coverslip over a period of 30 min. Fluorescence changes in single platelets were measured using a Nikon TE200 microscope, fitted with a Zeiss 75 W xenon source and an epifluorescence attachment with excitation and emission filter wheels containing a green filter set as previously described by Harper et al. [25]. A Hamamatsu Orca 1 CCD camera and AQM Orca 2001 software was used for image capture and subsequent analysis (Kinetic Imaging, Wirral, UK).

Phosphorylation studies

  1. Top of page
  2. Abstract
  3. Introduction
  4. Experimental procedures
  5. Reagents
  6. Preparation of human platelets
  7. Preparation of murine platelets
  8. Adhesion assays
  9. Single platelet Ca2+ measurement
  10. Phosphorylation studies
  11. Analysis of data
  12. Results
  13. Spreading of human platelets on fibronectin-coated surfaces
  14. Identification of platelet integrins mediating adhesion to fibronectin
  15. Role of Ca2+ mobilization and PLCγ2 on platelet spreading on fibronectin
  16. αIIb-deficient mouse platelets adhere but do not spread on fibronectin
  17. Regulation of FAK and PLCγ2 activity in platelets on fibronectin
  18. Spreading of platelets on fibronectin in plasma
  19. Discussion
  20. Acknowledgements
  21. References

For protein precipitation studies, six well plates were incubated overnight with an equal molar concentration (1 μM) solution of either fibronectin or purified fibronectin domains. Following the aforementioned washing and BSA blocking procedure, 500 µL washed platelets (4 × 108 mL−1) were incubated on six-well plates at 37 °C for 45 min in a humid environment. Unbound platelets were removed by two washes with PBS followed by aspiration, and adhered cells were solubilized with an equal volume of ice-cold immunoprecipitation buffer (2% v/v NP-40, 20 mM Tris, 300 mM NaCl, 2 mM EDTA, 2 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 2 mM Na3VO4, 10 µg mL−1 leupeptin, 10 µg mL−1 aprotinin, 1 µg mL−1 pepstatin A, pH 7.3). A sample of the suspension over BSA was taken and used as a control. After removing debris and preclearing, selected supernatants were incubated with 6 µL anti-FAK pAb or 2 µL anti-PLCγ2 pAb and 20 µL protein A–Sepharose at 4 °C overnight. Subsequent immunoprecipitation and Western blotting were carried out as previously described [16,18,24].

Analysis of data

  1. Top of page
  2. Abstract
  3. Introduction
  4. Experimental procedures
  5. Reagents
  6. Preparation of human platelets
  7. Preparation of murine platelets
  8. Adhesion assays
  9. Single platelet Ca2+ measurement
  10. Phosphorylation studies
  11. Analysis of data
  12. Results
  13. Spreading of human platelets on fibronectin-coated surfaces
  14. Identification of platelet integrins mediating adhesion to fibronectin
  15. Role of Ca2+ mobilization and PLCγ2 on platelet spreading on fibronectin
  16. αIIb-deficient mouse platelets adhere but do not spread on fibronectin
  17. Regulation of FAK and PLCγ2 activity in platelets on fibronectin
  18. Spreading of platelets on fibronectin in plasma
  19. Discussion
  20. Acknowledgements
  21. References

Experiments were carried out on at least three occasions, and images shown are representative data from one experiment. Where applicable, results are shown as mean ± SEM. Statistical significance of differences between means was determined by one-way anova. If means were shown to be significantly different, multiple comparisons by pairs were performed by the Tukey test. Probability values of P < 0.01 were selected to be statistically significant.

Spreading of human platelets on fibronectin-coated surfaces

  1. Top of page
  2. Abstract
  3. Introduction
  4. Experimental procedures
  5. Reagents
  6. Preparation of human platelets
  7. Preparation of murine platelets
  8. Adhesion assays
  9. Single platelet Ca2+ measurement
  10. Phosphorylation studies
  11. Analysis of data
  12. Results
  13. Spreading of human platelets on fibronectin-coated surfaces
  14. Identification of platelet integrins mediating adhesion to fibronectin
  15. Role of Ca2+ mobilization and PLCγ2 on platelet spreading on fibronectin
  16. αIIb-deficient mouse platelets adhere but do not spread on fibronectin
  17. Regulation of FAK and PLCγ2 activity in platelets on fibronectin
  18. Spreading of platelets on fibronectin in plasma
  19. Discussion
  20. Acknowledgements
  21. References

To determine the molecular basis of platelet binding to fibronectin, we gently pippetted washed human platelets over surface-immobilized proteins and subsequently measured adhesion and spreading through labeling of the actin cytoskeleton using rhodamine-labeled phalloidin. We initially confirmed that very few platelets adhere to a BSA-coated surface or undergo spreading (Fig. 1A). In contrast, platelets bind and spread on immobilized fibronectin in the absence of external stimulation (Fig. 1B). The majority of adhered platelets had spread fully after 45 min, and both filopodia and lamellipodia structures could be seen. Adhesion and spreading on fibronectin was not significantly altered in the presence of the cyclooxygenase inhibitor indomethacin (10 μM) and the P2Y1 and P2Y12 ADP receptor antagonists A3P5P (1 mM) and AR-C67085 (1 μM), suggesting that these changes are directly mediated through engagement of fibronectin receptors (Fig. 1C). External stimulation with ADP (10 μM) slightly enhanced platelet spreading and led to most of the platelets forming stress fibres (Fig. 1D).

image

Figure 1. Identification of platelet receptors in adhesion to fibronectin. Human washed platelets were layered on a (A) BSA- or (B) fibronectin-coated slide for 45 min at 37°C. In selected experiments, platelets were treated with (C) indomethacin (10 μM), A3P5P (1 mM) and AR-C67085 (1 μM) ; (D) ADP (10 μM). In separate experiments, washed platelets were layered on (E) 9–10FIII or (F) 10FIII. In selected experiments, platelets were treated with the αIIbβ3 antagonists (G) lotrafiban (10 μM) or (H) abciximab (3.5 μg mL-1) alone or (I) in combination with a function-blocking α5β1 complex mAb (20 μg mL-1 JBS5/P4C10), before being layered on fibronectin. Slides were then stained with rhodamine-phalloidin and imaged by fluorescence microscopy. One experiment representative of 3–6 is depicted. Bar = 10 μm.

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Experiments were designed to elucidate the molecular constituents mediating platelet adhesion to fibronectin utilizing truncated portions of fibronectin containing either the single 10FIII domain or the 9–10FIII domain pair. Previous work has shown that the RGD sequence in 10FIII is critical for integrin engagement, and that the synergy site in 9FIII domain potentiates this interaction [26]. Our results show that the 9–10FIII sequence, as well as the 10FIII sequence alone, support spreading to a similar extent induced by native fibronectin (Fig. 1E–F).

Identification of platelet integrins mediating adhesion to fibronectin

  1. Top of page
  2. Abstract
  3. Introduction
  4. Experimental procedures
  5. Reagents
  6. Preparation of human platelets
  7. Preparation of murine platelets
  8. Adhesion assays
  9. Single platelet Ca2+ measurement
  10. Phosphorylation studies
  11. Analysis of data
  12. Results
  13. Spreading of human platelets on fibronectin-coated surfaces
  14. Identification of platelet integrins mediating adhesion to fibronectin
  15. Role of Ca2+ mobilization and PLCγ2 on platelet spreading on fibronectin
  16. αIIb-deficient mouse platelets adhere but do not spread on fibronectin
  17. Regulation of FAK and PLCγ2 activity in platelets on fibronectin
  18. Spreading of platelets on fibronectin in plasma
  19. Discussion
  20. Acknowledgements
  21. References

We examined the role of platelet membrane integrins in mediating adhesion to fibronectin using blocking antibodies and receptor antagonists. The integrin αIIbβ3 is the most abundant receptor on the platelet surface, with 60 000–80 000 copies per activated cell. αIIbβ3 has previously been shown to mediate platelet adhesion to fibrinogen-, VWF- and fibronectin-coated surfaces [2,4]. Blockade of αIIbβ3 with the antagonist lotrafiban, which also blocks the minor platelet integrin αvβ3[27], has no effect on the extent of adhesion to fibronectin (Table 1) but eliminates platelet formation of lamellipodia but not filopodia (Fig. 1G). Furthermore, prior stimulation with either the secondary mediator ADP, the GPVI-specific agonist CRP or the G-protein-coupled receptor agonist thrombin failed to potentiate αIIbβ3-blocked platelet spreading (Table 1). Similar results were observed in the presence of saturating concentrations of the αIIbβ3vβ3 antagonist abciximab (Fig. 1H). This αIIbβ3-independent adhesion of platelets to fibronectin was abrogated in the presence of antibodies against the α5β1 integrin in combination with lotrafiban (Fig. 1I). In contrast, α5β1-blockade alone had no effect on the adhesion or degree of spreading in the absence of an αIIbβ3 antagonist (Table 1). Platelet adhesive interactions were unaltered by the presence of an IgG control mAb (data not shown). Taken together, our data demonstrates that formation of lamellipodia on fibronectin is eliminated in the presence of αIIbβ3vβ3 blockade, and that while the α5β1 integrin can support platelet-fibronectin binding, it is insufficient to mediate platelet spreading.

Table 1.  Effects of αIIbβ3 and α5β1 antagonists on platelet adhesion to fibronectin
SubstrateTreatmentAdherent platelets per mm (×10)Platelet surface area (μm)
  1. Human washed platelets were layered on a BSA- or fibronectin-coated slide for 45 min. In selected experiments, platelets were treated the αIIbβ3vβ3 antagonist lotrafiban (10 μM LOT) alone or in combination with ADP (10 μM), CRP (0.2 μg mL-1), thrombin (1 U mL1), or with a function-blocking α5β1 complex mAb (20 μg mL-1 JBS5/P4C10), before being layered on fibronectin. The number and surface area of adherent platelets were recorded for five fields of view (7550 μm2). Values are reported as follows: adherent platelets = mean ±SEM of three experiments; platelet surface area = mean ± SEM of 50–75 cells.

BSA9 ± 2.814.4 ± 1.0
FN101 ± 8.743.7 ± 1.7
FNLOT107 ± 1014.7 ± 0.8
FNLOT +ADP110 ± 4.214.4 ± 0.9
FNLOT +CRP109 ± 4.114.1 ± 0.7
FNLOT +thrombin96 ± 7.115.0 ± 1.1
FNJBS5/P4C1099 ± 9.342.8 ± 1.8
FNLOT +JBS5/P4C108 ± 2.914.6 ± 0.8

We further examined platelet adhesion and spreading using Normarski differential interference contrast microscopy. This technology provides a clear resolution between lamellipodia and filopodia and also enables monitoring of adhesion and spreading in real time. Initial studies confirmed that platelets adhere extensively to fibronectin but not to BSA and demonstrated that lamellipodia but not filopodia formation is inhibited in the presence of the αIIbβ3vβ3 antagonist lotrafiban but that additional blockade of α5β1 is required to block adhesion (Fig. 2A-D). The time course of spreading was monitored by time-lapse video microscopy as illustrated in Fig. 2(E–F). Platelet binding to fibronectin is associated with a characteristic series of shape changes (Fig. 2E), as previously described for fibrinogen-coated surfaces [28]. Platelet rounding is followed by formation of filopodia and then lamellipodia resulting in an increase in size after 600 s from 11.6 ±0.7 μm2 to 43.7 ± 1.7 μm2 (Fig. 2G). The generation of stable filopodia and lamellipodia is drastically reduced by the presence of the αIIbβ3 antagonist lotrafiban, although dynamic extension and retraction of filopodia is observed as exemplified by the arrowhead at 245 s and 455 s, respectively, in Fig. 2. A quantitative assessment of the platelet surface area both in the absence and presence of lotrafiban over 20 min is shown in Fig. 2(G). The results demonstrate a uniform rate of spreading of platelets on fibronectin, which is complete within 600 s. In contrast, a smaller increase in surface area is seen in the presence of lotrafiban, which reaches a plateau in 200 s.

image

Figure 2. Real time imaging of platelet spreading on fibronectin. Human washed platelets were exposed to a (A) BSA- or (B) fibronectin-coated surface and observed in real time using Nomarski differential interference contrast microscopy. In selected experiments, platelets were treated with the (C) αIIbβ3 antagonist lotrafiban (10 μM) alone or (D) in combination with a function-blocking α5β1 complex mAb (20 μg mL-1 JBS5/P4C10), before being layered on fibronectin. A representative time course of a single platelet spreading on fibronectin in the (E) absence or (F) presence of the αIIbβ3 antagonist lotrafiban (10 μM) is shown. Note the filopodia formation and subsequent retraction as indicated by the arrowhead at 245 and 445 s, respectively. (G) The mean surface area of adherent platelets in the absence (filled boxes) or presence (open circles) of lotrafiban was quantified at the indicated time points using ImageJ as described under ‘Experimental Procedures’. One experiment representative of 3–6 is depicted, and values are mean ± SEM of 10–15 cells. Bar = 10 μm.

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Role of Ca2+ mobilization and PLCγ2 on platelet spreading on fibronectin

  1. Top of page
  2. Abstract
  3. Introduction
  4. Experimental procedures
  5. Reagents
  6. Preparation of human platelets
  7. Preparation of murine platelets
  8. Adhesion assays
  9. Single platelet Ca2+ measurement
  10. Phosphorylation studies
  11. Analysis of data
  12. Results
  13. Spreading of human platelets on fibronectin-coated surfaces
  14. Identification of platelet integrins mediating adhesion to fibronectin
  15. Role of Ca2+ mobilization and PLCγ2 on platelet spreading on fibronectin
  16. αIIb-deficient mouse platelets adhere but do not spread on fibronectin
  17. Regulation of FAK and PLCγ2 activity in platelets on fibronectin
  18. Spreading of platelets on fibronectin in plasma
  19. Discussion
  20. Acknowledgements
  21. References

To further assess the potential ability of the α5β1 integrin to transduce outside-in signaling, we examined the elevation of Ca2+ in single platelets that had bound to immobilized fibronectin in the absence and presence of the αIIbβ3 antagonist lotrafiban. Intracellular Ca2+ was measured in washed platelets loaded with the Ca2+ reporter dye Oregon Green BAPTA 1-AM utilizing dynamic video imaging. The majority of Oregon Green-loaded platelets moved continually on a BSA-coated surface, while the minority that settled did not exhibit an increase in Ca2+(Fig. 3A). More specifically, once the platelet arrived within the region of interest (as denoted by an arrowhead in Fig. 3), a basal Ca2+ level was observed with little fluctuation, as previously shown by Atkinson et al. [29]. In contrast, platelets readily formed stable adhesions on fibronectin and, after a variable lag phase of usually greater than 200 s, exhibited an increase in Ca2+ consisting of a series of oscillatory changes (Fig. 3B). This increase in Ca2+ was abolished in the presence of lotrafiban (Fig. 3C), demonstrating that while platelet activation and spreading via αIIbβ3 elicits an increase in intracellular Ca2+, platelet α5β1-mediated binding and dynamic synthesis of filopodia is independent of Ca2+ mobilization. Consistent with these findings, treatment of washed platelets with the intracellular Ca2+ chelating agent BAPTA-AM abrogated platelet lamellipodia formation but did not block extension and retraction of filopodia in the presence or absence of lotrafiban (data not shown).

image

Figure 3. Ca2+ elevation induced by a fibronectin surface is abolished by blockade of αIIbβ3 integrins. Washed human platelets loaded with the Ca2+-sensitive dye Oregon Green BAPTA 1-AM were imaged as they made contact with either a BSA- or fibronectin-coated slide. The Ca2+ traces are all from the same experiment and were analyzed using AQM Orca 2001 software. The graph shows the platelets arrive at the region of interest as denoted by an arrowhead and a basal level of Ca2+, then subsequent Ca2+ fluctuations are shown over a 20-min period of observation. The scale is in arbitrary units derived from the intensity of fluorescence emission. Traces shown are representative of 3 parallel experiments.

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In an attempt to further corroborate the notion that calcium mobilization is not required for filopodia formation, we examined adhesion of PLCγ2–/– murine platelets to fibronectin, bearing in mind that regulation of cytosolic calcium flux by fibrinogen has been shown to be dependent upon activation of this phospholipase [30]. Wild type mouse platelets extend filopodia and form partial lamellipodia on fibronectin in contrast to the full spreading response observed in human platelets (Fig. 4A). A similar difference between spreading of murine and human platelet on fibrinogen has previously been reported [17]. In comparison, the majority of PLCγ2–/– platelets produce filopodia but fail to form lamellipodia structures (Fig. 4B). This is illustrated by the reduction in platelet surface area between wild type and PLCγ2–/– platelets (Table 2). Furthermore, filopodia formation was still observed for PLCγ2–/– platelets in the presence of the αIIbβ3 antagonist lotrafiban (data not shown). It is noteworthy that treatment of PLCγ2–/– platelets with thrombin led to lamellipodia formation (as evidenced by an increase in surface area; Table 2), consistent with activation of phospholipase C-β (PLC-β) by the G-protein coupled receptor agonist [31].

image

Figure 4. PLCγ2 is required to promote platelet lamellipodia but not filopodia formation during adhesion to immobilized fibronectin. Purified (A) wild type murine platelets and (B) PLCγ2–/– murine platelets were exposed to a fibronectin-coated surface and observed in real time using Nomarski differential interference contrast microscopy. A representative time course of a single platelet spreading is shown. Bar = 10 μm.

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Table 2.  Effects of external stimulation on murine platelet surface area following adhesion to fibronectin
Mouse genotypeTreatmentPlatelet surface area (μm2)
  1. Wild type, PLCγ2–/– and αIIb–/– murine platelets were layered onto a fibronectin-coated slide for 45 min. In selected experiments, platelets were treated the αIIbβ3vβ3 antagonist lotrafiban (10 μM LOT), ADP (10 μM), CRP (0.2 μg mL-1) or thrombin (1 U mL−1) before being layered onto the slide. Values are reported as follows: platelet surface area = mean ± SEM of 75 cells.

WT15.1 ± 0.6
WTLOT6.0 ± 0.3
WTADP19.0 ± 0.6
WTCRP20.1 ± 0.9
WTthrombin20.2 ± 0.6
PLCγ2–/–9.2 ± 0.6
PLCγ2–/–thrombin19.6 ± 0.5
αIIb–/–5.9 ± 0.2
αIIb–/–ADP5.7 ± 0.3
αIIb–/–CRP5.8 ± 0.4
αIIb–/–thrombin5.9 ± 0.5

αIIb-deficient mouse platelets adhere but do not spread on fibronectin

  1. Top of page
  2. Abstract
  3. Introduction
  4. Experimental procedures
  5. Reagents
  6. Preparation of human platelets
  7. Preparation of murine platelets
  8. Adhesion assays
  9. Single platelet Ca2+ measurement
  10. Phosphorylation studies
  11. Analysis of data
  12. Results
  13. Spreading of human platelets on fibronectin-coated surfaces
  14. Identification of platelet integrins mediating adhesion to fibronectin
  15. Role of Ca2+ mobilization and PLCγ2 on platelet spreading on fibronectin
  16. αIIb-deficient mouse platelets adhere but do not spread on fibronectin
  17. Regulation of FAK and PLCγ2 activity in platelets on fibronectin
  18. Spreading of platelets on fibronectin in plasma
  19. Discussion
  20. Acknowledgements
  21. References

To further validate the aforementioned findings and determine the role of αvβ3 in these events, we monitored fibronectin-dependent binding of platelets from mice with a mutation that disrupts the GPIIb gene, resulting in the absence of the αIIbβ3 complex [22]. Purified wild type murine platelets adhere and spread on surface-immobilized intact fibronectin (Fig. 5A). These changes were not altered in the presence of indomethacin and the A3P5P and AR-C67085 antagonists, confirming that they are independent of the major secondary mediators (data not shown). As with human platelets, ADP further enhanced spreading of murine platelets and resulted in most of the platelets forming stress fibres (Fig. 5B). In contrast, platelet spreading was absent in inline image murine platelets (Fig. 5C), although these platelets retain the ability to bind to the fibronectin surface. Furthermore, stimulation with ADP, CRP or thrombin failed to induce formation of lamellipodia or stress fibers in the absence of the αIIbβ3 complex (Fig. 5D and Table 2). Together, these results demonstrate that platelet spreading is dependent upon the presence of the αIIbβ3 complex. However, in the absence or presence of external stimulation, platelets retain the ability to adhere to fibronectin in an αIIbβ3-independent manner, presumably through α5β1 and αvβ3 integrins.

image

Figure 5. Murine platelets lacking the αIIbβ3 complex fail to spread on fibronectin. Purified wild type murine platelets in the (A) absence or (B) presence of ADP (10 μM) and αIIb–/– murine platelets in the (C) absence or (D) presence of ADP (10 μM) were layered on a fibronectin-coated slide for 45 min at 37°C. Slides were then stained with rhodamine-phalloidin and imaged by fluorescence microscopy. One experiment representative of 3 is depicted. Bar = 10 μm.

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Further studies were undertaken to validate the ability of αvβ3 to support adhesion. For these studies, we used fibrinogen, which binds αIIbβ3 and αvβ3. In accordance with previous work, Fig. 6 (A) demonstrates that purified wild type murine platelets adhere to and spread on surface-immobilized fibrinogen [17]. The absence of the αIIbβ3 complex in the inline image murine platelets results in complete abrogation of platelet adhesion to fibrinogen (Fig. 6B). However, adhesion but not spreading on fibrinogen is restored in the inline image platelets upon cellular stimulation with Mn2+, which acts through the metal ion-dependent adhesion site (MIDAS) of the insertion I-like domain and promotes integrin activation in the absence of calcium (Fig. 6C) [32]. A smaller increase, relative to Mn2+ stimulation, in the number of cells undergoing adhesion of 59.1 ± 5.9% was seen in the presence of the weak agonist ADP. Importantly, lotrafiban (30 μM), which acts simultaneously as an αIIbβ3 and αvβ3 antagonist, eliminates binding of Mn2+ stimulated inline image murine platelets to the fibrinogen surface (Fig. 6D). These results demonstrate that the presence of the activated form of the αvβ3 integrin is sufficient to support platelet binding to fibrinogen, but that it is incapable of forming lamellipodia in the absence of the αIIbβ3 complex.

image

Figure 6. Exogenous stimulation of the platelet integrin αvβ3 supports binding but not spreading to fibrinogen in the absence of αIIbβ3. Purified (A) wild type murine platelets or (B) αIIb–/– murine platelets were layered on a fibrinogen-coated slide for 45 min at 37°C. αIIb–/– murine platelets were treated with 5 mM Mn2+ in the (C) absence or (D) presence of αIIbβ3vβ3 antagonist lotrafiban (30 μM). Slides were then stained with rhodamine-phalloidin and imaged by fluorescence microscopy. One experiment representative of 3 is depicted. Bar = 10 μm.

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Regulation of FAK and PLCγ2 activity in platelets on fibronectin

  1. Top of page
  2. Abstract
  3. Introduction
  4. Experimental procedures
  5. Reagents
  6. Preparation of human platelets
  7. Preparation of murine platelets
  8. Adhesion assays
  9. Single platelet Ca2+ measurement
  10. Phosphorylation studies
  11. Analysis of data
  12. Results
  13. Spreading of human platelets on fibronectin-coated surfaces
  14. Identification of platelet integrins mediating adhesion to fibronectin
  15. Role of Ca2+ mobilization and PLCγ2 on platelet spreading on fibronectin
  16. αIIb-deficient mouse platelets adhere but do not spread on fibronectin
  17. Regulation of FAK and PLCγ2 activity in platelets on fibronectin
  18. Spreading of platelets on fibronectin in plasma
  19. Discussion
  20. Acknowledgements
  21. References

It is well established that platelet spreading on fibrinogen mediates αIIbβ3-dependent tyrosine phosphorylation and that this is enhanced by actin polymerization [16,17]. Experiments were designed to determine whether the interaction of fibronectin with αIIbβ3 and α5β1 induces a similar set of signaling events. For these studies, purified platelets were allowed to attach and spread on fibronectin or recombinant proteins consisting of either the single 10FIII domain or the 9–10FIII domain pair. The actions of ADP and thromboxanes were eliminated in these experiments through the incorporation of specific inhibitors. Platelets suspended over BSA-coated dishes were used as a control. Phosphorylation was assessed in whole cell lysates and following immunoprecipitation of specific proteins by Western blotting with antiphosphotyrosine antibodies. Our data shows that platelet spreading on fibronectin was associated with an increase in tyrosine phosphorylation. Furthermore, 10FIII stimulates a similar pattern of tyrosine phosphorylation, which is further increased when this fragment is complexed with 9FIII (Fig. 7A). A similar set of observations was made for tyrosine phosphorylation of FAK and PLCγ2. Immunoprecipitation of these two proteins and Western blotting for tyrosine phosphorylation demonstrated that, while FAK and PLCγ2 are tyrosine phosphorylated by 10FIII and 9–10FIII (Fig. 7B–C), there is a decrease in magnitude of tyrosine phosphorylation in 10FIII samples of 58 ± 4% and 79 ± 7%, respectively, as assessed via densitometry. Tyrosine phosphorylation of FAK and PLCγ2 was abrogated in the presence of a concentration of lotrafiban (10 μM) that causes complete blockade of the αIIbβ3 complex but does not prevent binding to α5β1 (Fig. 7D). Taken together, our data indicate that αIIbβ3 binding to fibronectin promotes tyrosine phosphorylation independent of α5β1 integrin involvement.

image

Figure 7. Fibronectin induced tyrosine phosphorylation of platelets. Human washed platelets were placed in dishes coated with BSA or fibronectin (FN), 9–10FIII or 10FIII in the presence of apyrase and indomethacin and absence or presence of lotrafiban (10 μM LOT) for 45 min at 37°C. Dishes coated with fibronectin were washed twice to remove non-adherent cells. Platelets adherent to the immobilized proteins or in suspension over BSA were lyzed in ice-cold immunoprecipitation buffer and used directly for SDS-PAGE or subjected to immunoprecipitation for FAK or PLCγ2 and immunoblotted for tyrosine phosphorylated proteins. Samples were adjusted prior to immunoprecipitations so that there were similar amounts of protein in each group. Results are representative of 3–5 experiments.

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Spreading of platelets on fibronectin in plasma

  1. Top of page
  2. Abstract
  3. Introduction
  4. Experimental procedures
  5. Reagents
  6. Preparation of human platelets
  7. Preparation of murine platelets
  8. Adhesion assays
  9. Single platelet Ca2+ measurement
  10. Phosphorylation studies
  11. Analysis of data
  12. Results
  13. Spreading of human platelets on fibronectin-coated surfaces
  14. Identification of platelet integrins mediating adhesion to fibronectin
  15. Role of Ca2+ mobilization and PLCγ2 on platelet spreading on fibronectin
  16. αIIb-deficient mouse platelets adhere but do not spread on fibronectin
  17. Regulation of FAK and PLCγ2 activity in platelets on fibronectin
  18. Spreading of platelets on fibronectin in plasma
  19. Discussion
  20. Acknowledgements
  21. References

This study, in common with many others in the literature, has utilized washed platelets in order to examine the effects of proteins on their own and to facilitate measurement of phosphorylation. Physiologically however, adhesion and spreading occur in the presence of plasma proteins, which have the potential to influence these processes. In order to investigate this, we layered platelet-rich plasma over surface-immobilized fibronectin and compared the results to those obtained with washed platelets.

In contrast to studies using washed platelets, there was minimal adhesion to fibronectin or BSA in plasma, and the very few platelets that did adhere failed to undergo spreading (Fig. 8 A–B). Washed platelets resuspended in platelet-poor plasma also failed to attach or spread on the fibronectin surface demonstrating that the difference in the two conditions was not due to the method of platelet isolation (data not shown). Stimulation of platelet-rich-plasma with the GPVI-specific agonist collagen-related peptide (CRP, 0.2 μg mL−1) resulted in a dramatic increase in both the number of bound platelets and the degree of platelet spreading (Fig. 8C). These platelets exhibited a time-dependent increase in platelet surface area similar to that observed for washed platelets (data not shown). Similar results were observed using sodium citrate/ACD, PPACK, a selective thrombin inhibitor, or heparin (not shown) as anticoagulant (Table 3 and not shown). These results are in accordance with reports indicating that the affinity of integrins for fibronectin is up-regulated upon stimulation [33].

image

Figure 8. Platelets in plasma proteins require external stimulation to spread on fibronectin. Human platelets in PRP were layered onto a (A) BSA- or a fibronectin-coated slide for 45 min at 37°C in the (B) absence or (C) presence of the GPVI-specific agonist CRP (0.2 μg mL-1). In selected experiments, PRP was pretreated with the (D) αIIbβ3 antagonist lotrafiban (10 μM). Slides were then stained with rhodamine-phalloidin and imaged by fluorescence microscopy. In separate experiments, washed platelets were resuspended in buffer containing fibrinogen (3 mg mL−1), fibronectin (300 μg mL-1) and VWF (10 μg mL-1) in the (E) absence or (F) presence of CRP (0.2 μg mL-1) and imaged using Nomarski differential interference contrast microscopy. One experiment representative of 3 is depicted. Bar = 10 μm.

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Table 3.  Effects of external stimulation of platelets on platelet-rich-plasma adhesion to fibronectin
SubstrateAnticoagulantTreatmentAdherent platelets per mm (×10–2)
  1. Platelets (3 ×107 mL-1) in PRP anticoagulated with either sodium citrate and acid/citrate/dextrose (NaCit/ACD) or PPACK were layered on a BSA- or fibronectin-coated slide for 45 min in the absence or presence of CRP (0.2 μg mL-1). The number of adherent platelets was recorded for five fields of view (7550 μm2). Values are mean ±SEM of three experiments.

BSANaCit/ACD0.1 ± 0.3
FNNaCit/ACD0.8 ± 1.3
FNNaCit/ACDCRP46.6 ± 5.4
BSAPPACK0.2 ± 0.3
FNPPACK2.0 ± 0.9
FNPPACKCRP55.6 ± 3.5

CRP-induced platelet spreading on fibronectin was solely dependent upon the αIIbβ3 complex, as evidenced by the absence of spreading in the presence of the αIIbβ3 blocker lotrafiban (10 μM; Fig. 8D). However, CRP-stimulated platelets retained their ability to bind to the fibronectin surface in the presence of an αIIbβ3vβ3 antagonist, consistent with a role for α5β1 in mediating adhesion.

In an attempt to delineate the potential inhibitory role of soluble adhesive proteins present in plasma, which might account for the absence of adhesion in plasma by virtue of their ability to bind to platelet integrins in an active conformation (see Discussion), we resuspended human washed platelets in buffer containing physiological concentrations of the three major components of plasma: fibrinogen (3 mg mL−1), fibronectin (300 μg mL−1) and VWF (10 μg mL−1). Similar to studies using PRP, there was minimal adhesion of washed platelets to immobilized fibronectin in the presence of exogenously added plasma proteins (Fig. 8E). However, an increase in both the number of bound platelets and the degree of platelet spreading was observed upon stimulation with CRP (Fig. 8F). Platelet adhesive interactions were unaltered by the presence of similar levels of a non-adhesive protein (3.3 mg mL−1 BSA; data not shown).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Experimental procedures
  5. Reagents
  6. Preparation of human platelets
  7. Preparation of murine platelets
  8. Adhesion assays
  9. Single platelet Ca2+ measurement
  10. Phosphorylation studies
  11. Analysis of data
  12. Results
  13. Spreading of human platelets on fibronectin-coated surfaces
  14. Identification of platelet integrins mediating adhesion to fibronectin
  15. Role of Ca2+ mobilization and PLCγ2 on platelet spreading on fibronectin
  16. αIIb-deficient mouse platelets adhere but do not spread on fibronectin
  17. Regulation of FAK and PLCγ2 activity in platelets on fibronectin
  18. Spreading of platelets on fibronectin in plasma
  19. Discussion
  20. Acknowledgements
  21. References

In this study, we demonstrate that engagement of αIIbβ3 by fibronectin is sufficient to mediate platelet adhesion and subsequent development of stable filopodia and lamellipodia. This result is in accordance with the paradigm that ligand binding to αIIbβ3 triggers an ‘outside-in’ signaling cascade that supports platelet spreading [6,16,17]. Additionally, our data confirm the involvement of a second platelet surface receptor, α5β1, in mediating platelet-fibronectin adhesion [9]. In both cases, adhesion to immobilized fibronectin via αIIbβ3 or α5β1 was achieved in the absence of external stimulation of cellular activation and in the presence of inhibitors of the major platelet feedback agonists, ADP and thromboxane A2, suggesting that it is mediated through binding to the low affinity conformations of the two integrins or to a small population of ‘activated’ integrin. We favor the latter of these two explanations in view of the observation that plasma concentrations of the αIIbβ3 ligands, fibrinogen, VWF and fibronectin, block adhesion of washed platelets to immobilized ligands. These observations therefore demonstrate that fibronectin engagement of integrin αIIbβ3 in resting platelets leads to outside-in signaling independent of external stimuli [16,30] and that adhesion is reinforced through binding to α5β1[9,34].

Our data show that in the absence of the αIIbβ3 complex, α5β1 and αvβ3 are unable to support formation of lamellipodia. It is important to consider whether this reflects the inability of α5β1 and αvβ3 to initiate a signaling response or the low level of expression of the integrin giving rise to a weak intracellular signal. Upon contact with the fibronectin surface, platelets begin to extend filopodia. However, in the presence of an αIIbβ3 antagonist, these protrusions are unsuccessful in their attempt to anchor to the surface and are subsequently retracted, as demonstrated in Fig. 2(F). This dynamic extension/retraction process demonstrates that αIIbβ3-blocked platelets retain their ability to undergo cytoskeletal rearrangements, suggesting a limited degree of intracellular signaling. However, this is insufficient to generate lamellipodia, robust protein tyrosine phosphorylation or a rise in intracellular Ca2+.

The accepted model for integrin mediated signaling is based on integrin clustering and conformational changes leading to an increase in avidity/affinity for the ligand. In turn, ligand engagement initiates a complex network of signaling and structural cytoskeletal proteins leads to outside-in signaling, marked by an increase in tyrosine phosphorylation. In platelets, this process has been thoroughly documented for αIIbβ3-engagement by fibrinogen [2,6]. Our data demonstrate that formation of lamellipodia on fibronectin, or its active domains 10FIII or 9–10FIII, is associated with signaling events that have been attributed to αIIbβ3, including tyrosine phosphorylation of FAK and PLCγ2. Further, inhibitor studies confirmed that they are mediated through an αIIbβ3-dependent, but α5β1-independent pathway. The absence of tyrosine phosphorylation of PLCγ2 by α5β1 combined with the observation that PLCγ2–/– platelets retain the ability to form filopodia clearly demonstrates that the dynamic process of platelet filopodia formation is independent of activation of this phospholipase. This conclusion is corroborated by the lack of an increase in intracellular Ca2+ in the absence of αIIbβ3 engagement.

The data further support the notion that the information needed for platelets to adhere to fibronectin resides in the 120-kDa central cell-binding domain, which contains 9–10FIII [35]. Interestingly, while platelet spreading was unaltered by the presence of 9FIII, incorporation of this fragment with 10FIII yielded an increase in tyrosine phosphorylation as compared to 10FIII alone. This suggests that tyrosine phosphorylation is not rate-determining in mediating the extent of spreading and adhesion on fibronectin. The differences between 9FIII and 10FIII is thought to arise from differences in affinity of the ligands for the receptor, whereby 10FIII binds to the receptor with low affinity compared to 9–10FIII which binds to the receptor with high affinity [26,36]. Although this phenomenon might not have a direct effect on the pattern or degree of platelet spreading under static conditions, an increase in receptor affinity may have increased relevance in the physiological setting whereby platelet adhesion occurs in the presence of shear forces [37]. Along these lines, it has been suggested that enhanced platelet affinity for fibronectin is requisite for the formation of stable thrombi in vivo[10].

Under physiological conditions, platelet–ligand interactions occur in the presence of soluble plasma proteins. However, many of the studies in the literature have utilized washed platelets. Our data demonstrate that both PRP and washed platelets resuspended in purified plasma proteins fail to spread on fibronectin in the absence of external stimulation, suggesting that the presence of soluble proteins in the plasma has the ability to prevent platelets from binding and spreading. This is likely to be a consequence of the high concentrations of integrin-ligands present in the plasma [38], which effectively out compete integrin engagement of surface-immobilized proteins [39]. Support for this is provided by the observation that addition of soluble fibronectin, fibrinogen and VWF completely block adhesion of washed platelets on fibronectin. Importantly, modulation of the affinity conformation of integrins via GPVI-specific leads to adhesion and spreading of platelets in plasma, presumably through integrin activation and possibly an increase in the level of platelet surface glycoproteins which are present in intracellular granules [40,41]. Significantly, the difference between plasma and washed conditions is unlikely to be due to integrin activation during the preparation of washed platelets since adhesion was seen in the presence of ADP and thromboxane inhibitors or upon resuspension of washed platelets in purified plasma. We therefore propose that a small proportion of the integrins are in an active conformation, but are dynamically bound to soluble fibrinogen and fibronectin in plasma, thereby preventing interaction with immobilized ligand. The role of plasma proteins in influencing platelet–ligand interactions may provide a degree of clarification for differences in the adhesive properties of washed platelets that has been reported in the literature [16,24,30,33].

In summary, this study demonstrates that while α5β1,αvβ3 and αIIbβ3 are able to support adhesion of washed platelets to fibronectin, only αIIbβ3 is able to mediate formation of lamellipodia. This may reflect a difference in outside-in signaling from the integrin and/or the level of expression. The study also shows that αIIbβ3-dependent adhesion in plasma requires prior stimulation, demonstrating that plasma provides an anti-adhesive medium, which prevents adhesion in the absence of stimulating agonists such as GPVI.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Experimental procedures
  5. Reagents
  6. Preparation of human platelets
  7. Preparation of murine platelets
  8. Adhesion assays
  9. Single platelet Ca2+ measurement
  10. Phosphorylation studies
  11. Analysis of data
  12. Results
  13. Spreading of human platelets on fibronectin-coated surfaces
  14. Identification of platelet integrins mediating adhesion to fibronectin
  15. Role of Ca2+ mobilization and PLCγ2 on platelet spreading on fibronectin
  16. αIIb-deficient mouse platelets adhere but do not spread on fibronectin
  17. Regulation of FAK and PLCγ2 activity in platelets on fibronectin
  18. Spreading of platelets on fibronectin in plasma
  19. Discussion
  20. Acknowledgements
  21. References
  • 1
    Savage B, Almus-Jacobs F, Ruggeri ZM. Specific synergy of multiple substrate–receptor interactions in platelet thrombus formation under flow. Cell 1998; 94: 65766.
  • 2
    Ruggeri ZM. Platelets in atherothrombosis. Nat Med 2002; 8: 122734.DOI: 10.1038/nm1102-1227
  • 3
    Jackson SP, Nesbitt WS, Kulkarni S. Signaling events underlying thrombus formation. J Thromb Haemost 2003; 1: 160212.DOI: 10.1046/j.1538-7836.2003.00267.x
  • 4
    Gardner JM, Hynes RO. Interaction of fibronectin with its receptor on platelets. Cell 1985; 42: 43948.
  • 5
    Abulencia JP, Tien N, McCarty OJ, Plymire D, Mousa SA, Konstantopoulos K. Comparative antiplatelet efficacy of a novel, nonpeptide GPIIb/IIIa antagonist (XV454) and abciximab (c7E3) in flow models of thrombosis. Arterioscler Thromb Vasc Biol 2001; 21: 14956.
  • 6
    Shattil SJ, Kashiwagi H, Pampori N. Integrin signaling: the platelet paradigm. Blood 1998; 91: 264557.
  • 7
    Naik UP, Naik MU. Association of CIB with GPIIb/IIIa during outside-in signaling is required for platelet spreading on fibrinogen. Blood 2003; 102: 135562.
  • 8
    Ni H, Denis CV, Subbarao S, Degen JL, Sato TN, Hynes RO, Wagner DD. Persistence of platelet thrombus formation in arterioles of mice lacking both von Willebrand factor and fibrinogen. J Clin Invest 2000; 106: 38592.
  • 9
    Beumer S, Mj IJ, De Groot PG, Sixma JJ. Platelet adhesion to fibronectin in flow. dependence on surface concentration and shear rate, role of platelet membrane glycoproteins GP IIb/IIIa and VLA-5, and inhibition by heparin. Blood 1994; 84: 372433.
  • 10
    Ni H, Yuen PS, Papalia JM, Trevithick JE, Sakai T, Fassler R, Hynes RO, Wagner DD. Plasma fibronectin promotes thrombus growth and stability in injured arterioles. Proc Natl Acad Sci USA 2003; 100: 24159.DOI: 10.1073/pnas.2628067100
  • 11
    Magnusson MK, Mosher DF. Fibronectin. structure, assembly, and cardiovascular implications. Arterioscler Thromb Vasc Biol 1998; 18: 136370.
  • 12
    Bowditch RD, Halloran CE, Aota S, Obara M, Plow EF, Yamada KM, Ginsberg MH. Integrin alpha IIb beta 3 (platelet GPIIb-IIIa) recognizes multiple sites in fibronectin. J Biol Chem 1991; 266: 233238.
  • 13
    Altroff H, Van Der Walle CF, Asselin J, Fairless R, Campbell ID, Mardon HJ. The eighth FIII domain of human fibronectin promotes integrin alpha5beta1 binding via stabilization of the ninth FIII domain. J Biol Chem 2001; 276: 3888592.DOI: 10.1074/jbc.M105868200
  • 14
    Mardon HJ, Grant KE. The role of the ninth and tenth type III domains of human fibronectin in cell adhesion. FEBS Lett 1994; 340: 197201.DOI: 10.1016/0014-5793(94)80137-1
  • 15
    Tadokoro S, Shattil SJ, Eto K, Tai V, Liddington RC, De Pereda JM, Ginsberg MH, Calderwood DA. Talin binding to integrin beta tails: a final common step in integrin activation. Science 2003; 302: 1036.DOI: 10.1126/science.1086652
  • 16
    Wonerow P, Pearce AC, Vaux DJ, Watson SP. A critical role for phospholipase Cγ2 in αIIbβ3-mediated platelet spreading. J Biol Chem 2003; 278: 375209.DOI: 10.1074/jbc.M305077200
  • 17
    Obergfell A, Eto K, Mocsai A, Buensuceso C, Moores SL, Brugge JS, Lowell CA, Shattil SJ. Coordinate interactions of Csk Src, and Syk kinases with αIIbβ3 initiate integrin signaling to the cytoskeleton. J Cell Biol 2002; 157: 26575.DOI: 10.1083/jcb.200112113
  • 18
    Wonerow P, Obergfell A, Wilde JI, Bobe R, Asazuma N, Brdicka T, Leo A, Schraven B, Horejsi V, Shattil SJ, Watson SP. Differential role of glycolipid-enriched membrane domains in glycoprotein VI- and integrin-mediated phospholipase Cγ2 regulation in platelets. Biochem J 2002; 364: 75565.DOI: 10.1042/BJ20020128
  • 19
    Naik MU, Naik UP. Calcium-and integrin-binding protein regulates focal adhesion kinase activity during platelet spreading on immobilized fibrinogen. Blood 2003; 102: 362936.
  • 20
    Jarvis GE, Atkinson BT, Frampton J, Watson SP. Thrombin-induced conversion of fibrinogen to fibrin results in rapid platelet trapping which is not dependent on platelet activation or GPIb. Br J Pharmacol 2003; 138: 57483.DOI: 10.1038/sj.bjp.0705095
  • 21
    McCarty OJ, Abulencia JP, Mousa SA, Konstantopoulos K. Evaluation of platelet antagonists in in vitro flow models of thrombosis. Meth Mol Med 2004; 93: 2134.
  • 22
    Emambokus NR, Frampton J. The glycoprotein IIb molecule is expressed on early murine hematopoietic progenitors and regulates their numbers in sites of hematopoiesis. Immunity 2003; 19: 3345.DOI: 10.1016/S1074-7613(03)00173-0
  • 23
    Wang D, Feng J, Wen R, Marine JC, Sangster MY, Parganas E, Hoffmeyer A, Jackson CW, Cleveland JL, Murray PJ, Ihle JN. Phospholipase Cgamma2 is essential in the functions of B cell and several Fc receptors. Immunity 2000; 13: 2535.DOI: 10.1016/S1074-7613(00)00005-4
  • 24
    Inoue O, Suzuki-Inoue K, Dean WL, Frampton J, Watson SP. Integrin alpha2beta1 mediates outside-in regulation of platelet spreading on collagen through activation of Src kinases and PLCgamma2. J Cell Biol 2003; 160: 76980.DOI: 10.1083/jcb.200208043
  • 25
    Harper CV, Kirkman-Brown JC, Barratt CL, Publicover SJ. Encoding of progesterone stimulus intensity by intracellular [Ca2+] ([Ca2+]i) in human spermatozoa. Biochem J 2003; 372: 40717.DOI: 10.1042/BJ20021560
  • 26
    Hotchin NA, Kidd AG, Altroff H, Mardon HJ. Differential activation of focal adhesion kinase, Rho and Rac by the ninth and tenth FIII domains of fibronectin. J Cell Sci 1999; 112: 293746
  • 27
    Liu F, Craft RM, Morris SA, Carroll RC. Lotrafiban: an oral platelet glycoprotein IIb/IIIa blocker. Expert Opin Invest Drugs 2000; 9: 267387.
  • 28
    Yap CL, Hughan SC, Cranmer SL, Nesbitt WS, Rooney MM, Giuliano S, Kulkarni S, Dopheide SM, Yuan Y, Salem HH, Jackson SP. Synergistic adhesive interactions and signaling mechanisms operating between platelet glycoprotein Ib/IX and integrin αIIbβ3. Studies in human platelets ans transfected Chinese hamster ovary cells. J Biol Chem 2000; 275: 4137788.DOI: 10.1074/jbc.M005590200
  • 29
    Atkinson BT, Jarvis GE, Watson SP. Activation of GPVI by collagen is regulated by alpha2beta1 and secondary mediators. J Thromb Haemost 2003; 1: 127887.DOI: 10.1046/j.1538-7836.2003.00245.x
  • 30
    Goncalves I, Hughan SC, Schoenwaelder SM, Yap CL, Yuan Y, Jackson SP. Integrin αIIbβ3-dependent calcium signals regulate platelet–fibrinogen interactions under flow. Involvement of phospholipase C gamma 2. J Biol Chem 2003; 278: 3481222.DOI: 10.1074/jbc.M306504200
  • 31
    Brass LF, Molino M. Protease-activated G protein-coupled receptors on human platelets and endothelial cells. Thromb Haemost 1997; 78: 23441.
  • 32
    Mould AP, Barton SJ, Askari JA, Craig SE, Humphries MJ. Role of ADMIDAS Cation-binding Site in Ligand Recognition by Integrin α5β1. J Biol Chem 2003; 278: 516229.DOI: 10.1074/jbc.M306655200
  • 33
    Gruner S, Prostredna M, Schulte V, Krieg T, Eckes B, Brakebusch C, Nieswandt B. Multiple integrin–ligand interactions synergize in shear-resistant platelet adhesion at sites of arterial injury in vivo. Blood 2003; 102: 40217.
  • 34
    Piotrowicz RS, Orchekowski RP, Nugent DJ, Yamada KY, Kunicki TJ. Glycoprotein Ic-IIa functions as an activation-independent fibronectin receptor on human platelets. J Cell Biol 1988; 106: 135964.DOI: 10.1083/jcb.106.4.1359
  • 35
    Beumer S, Heijnen-Snyder GJ, Mj IJ, De Groot PG, Sixma JJ. Fibronectin in an extracellular matrix of cultured endothelial cells supports platelet adhesion via its ninth type III repeat: a comparison with platelet adhesion to isolated fibronectin. Arterioscler Thromb Vasc Biol 2000; 20: E1625.
  • 36
    Takagi J, Strokovich K, Springer TA, Walz T. Structure of integrin α5β1 in complex with fibronectin. Embo J 2003; 22: 460715.DOI: 10.1093/emboj/cdg445
  • 37
    Konstantopoulos K, McIntire LV. Effects of fluid dynamic forces on vascular cell adhesion. J Clin Invest 1997; 100 (Suppl 11): S1923.
  • 38
    Ni H, Freedman J. Platelets in hemostasis and thrombosis: role of integrins and their ligands. Transfus Apheresis Sci 2003; 28: 25764.DOI: 10.1016/S1473-0502(03)00044-2
  • 39
    Grinnell F, Phan TV. Platelet attachment and spreading on polystyrene surfaces: dependence on fibronectin and plasma concentration. Thromb Res 1985; 39: 16571.DOI: 10.1016/0049-3848(85)90104-5
  • 40
    Best D, Senis YA, Jarvis GE, Eagleton HJ, Roberts DJ, Saito T, Jung SM, Moroi M, Harrison P, Green FR, Watson SP. GPVI levels in platelets: relationship to platelet function at high shear. Blood 2003; 102: 28118.
  • 41
    Nieswandt B, Watson SP. Platelet–collagen interaction: is GPVI the central receptor? Blood 2003; 102: 44961.