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Blood, 1 December 2003, Vol. 102, No. 12, pp. 4021-4027.
Prepublished online as a Blood First Edition Paper on July 31, 2003; DOI 10.1182/blood-2003-05-1391.
 Previous Article | Table of Contents | Next Article 
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Multiple integrin-ligand interactions synergize in shear-resistant platelet adhesion at sites of arterial injury in vivo
Sabine Grüner,
Miroslava Prostredna,
Valerie Schulte,
Thomas Krieg,
Beate Eckes,
Cord Brakebusch, and
Bernhard Nieswandt
From the Rudolf Virchow Center for Experimental Biomedicine, University of Würzburg, Würzburg, Germany; Department of Dermatology, University of Cologne, Cologne, Germany; Department of Molecular Medicine, Max-Planck-Institute for Biochemistry, Martinsried, Germany.
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Abstract
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Damage to the integrity of the vessel wall results in exposure of the subendothelial extracellular matrix (ECM), which triggers integrin-dependent adhesion and aggregation of platelets. The role of platelet  1 integrins in these processes remains mostly undefined. Here, we demonstrate by intravital fluorescence microscopy that platelet adhesion and thrombus growth on the exposed ECM of the injured carotid artery is not significantly altered in  2-null mice and even in mice with a Cre/loxP-mediated loss of all 1 integrins on their platelets. In contrast, inhibition of IIb3 integrin on platelets in wild-type mice blocked aggregate formation and reduced platelet adhesion by 60.0%. Strikingly, IIb3 inhibition had a comparable effect in 2-null mice, demonstrating that other receptors mediate shear-resistant adhesion in the absence of functional 21 and IIb3. These were identified to be 51 and/or 61 as IIb3 inhibition abrogated platelet adhesion in 1-null mice. We conclude that shear-resistant platelet adhesion on the injured vessel wall in vivo is a highly integrated process involving multiple integrin-ligand interactions, none of which by itself is essential. (Blood. 2003;102:4021-4027)
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Introduction
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At sites of vascular injury, the subendothelial extracellular matrix (ECM) is exposed to the flowing blood, which triggers adhesion and aggregation of platelets.1 This process is crucial to limit posttraumatic blood loss but may also lead to occlusion of diseased vessels and infarction of vital organs. Integrins are the major class of receptors that mediate firm adhesion and aggregation of platelets. Integrins are heterodimeric transmembrane receptors composed of an and a subunit.2,3 Platelets express integrins of the 1 and the 3 family that are present on the membrane in a low-affinity state. Once the platelets become activated the integrins shift to a high-affinity state and efficiently bind their ligands.2,4-6
The subendothelial ECM contains many different macromolecular constituents that are suitable substrates for integrin-mediated platelet adhesion including collagens, laminins, fibronectin, and von Willebrand factor (VWF). Collagen has a central role as it not only supports platelet adhesion but also strongly activates the cells.7,8 This activation is mediated by the low-affinity collagen receptor glycoprotein VI (GPVI).9 GPVI belongs to the immunoglobulin (Ig) superfamily10 and is noncovalently associated with the signal-transducing FcR chain.11,12 Under conditions of elevated shear, platelet adhesion on collagen strictly depends on the interaction of GPIb-V-IX with collagen-bound VWF13 and that of GPVI with collagen.5 During this process, ligation of GPVI5 (and GPIb14) leads to platelet activation and the shift of 1 and 3 integrins to a high-affinity state via "inside-out" signals, enabling the platelet to establish firm adhesion contacts that resist the shear forces in the bloodstream (shear-resistant adhesion) and subsequent thrombus growth. The importance of these initial processes in arterial thrombus formation has been established through the demonstration that platelet adhesion and thrombus formation on the injured arterial wall are largely inhibited in the absence of functional GPVI15,16 or GPIb.16,17 The molecular determinants of firm platelet adhesion on the ECM, however, have not been identified. This might be explained by the complexity of the ECM and the presence of multiple integrins on platelets that made it difficult to define the significance of individual integrins in this process.
The major platelet integrin, IIb3, binds multiple ligands including fibrinogen, VWF, and fibronectin and is essential for platelet aggregation but also contributes to platelet adhesion on collagen via VWF5,13 and fibronectin18 in vitro. The mandatory role of IIb3 in physiologic and pathophysiologic thrombus formation is well documented2,4,19 but its significance for the initial adhesion process on the ECM in vivo is not firmly established. The other 3 integrin on platelets, v3, is expressed at low levels and its role in platelet physiology is ill defined.20
Platelets express 3 different 1 integrins, namely 21 (collagen receptor), 51 (fibronectin receptor), and 61 (laminin receptor). Among them, 21 has been most intensively studied but its significance in the hemostatic and thrombotic process has been controversially debated (for review see Nieswandt and Watson21). A role for 21 in cardiovascular disease has been discussed for many years based on the results of clinical studies assessing a possible association of allelic differences in the 2 gene and different levels of 21 on platelets22 with an increased risk of myocardial infarction, diabetic retinopathy, and stroke. However, while many clinical studies found such an association, approximately the same number of studies did not.23-27 Furthermore, the pathologic significance of the other 2 1 integrins expressed on platelets, 51 and 61, with regard to hemostasis and thrombosis as well as their cellular regulation is poorly defined,19 although fibronectin and laminin are highly expressed in the vascular wall, suggesting an involvement of both integrins in platelet-vessel wall interactions.
Recently, several groups reported the generation of 2-integrin-deficient28,29 and conditional 1-integrin-deficient5 mice, the latter lacking 21, 51, and 61 on their platelets. Unexpectedly, these mice were found to have normal tail bleeding times, although their platelets display partial defects in their interaction with collagen in vitro. The mutant platelets can adhere and aggregate on fibrillar collagen under high-shear-flow conditions, presumably mediated by IIb3/VWF, but the newly formed aggregates detach from the collagen surface at a higher frequency than wild-type aggregates.30 Thus, 21 plays an important but not essential role in platelet adhesion on collagen in vitro. The significance of this interaction in vivo, where multiple integrin-ligand interactions may contribute to shear-resistant platelet deposition at sites of injury, has not been assessed.
In this study we used intravital fluorescence microscopy to examine the dynamic process of platelet accumulation at sites of vascular injury in the carotid artery of 2- and 1-deficient mice. Surprisingly, we found that platelet adhesion and thrombus growth on the exposed extracellular matrix of the arterial wall are largely unaffected in the absence of 21orall 1 integrins on platelets. In contrast, inhibition of IIb3 in wild-type platelets not only blocked aggregate formation but also reduced adhesion by 60.0%. Strikingly, the same effect was seen in 2-null mice, suggesting that receptors other than 21 and IIb3 mediate the reduced adhesion. These were identified to be 51 and/or 61, both of which are in a low-affinity state on resting platelets but efficiently bind their ligands upon platelet activation through GPVI.
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Materials and methods
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Animals
Mutant mice deficient in the 2 integrin subunit were produced as described.28 Both mutant and wild-type control animals were of 129/Sv x C57BL/6 genetic background and used at the age of 10 to 16 weeks.
Generation of mice with 1-null platelets. To produce mice carrying the 1-null allele in megakaryocytes, 1(fl/fl) mice31 were crossed with transgenic mice carrying the Mx-cre transgene (mx-cre+).32 Deletion of the 1 gene was induced in 4- to 5-week-old (1(fl/fl)/Mx-cre+) mice by 3 intraperitoneal injections of 250 µg polyinosinic-polycytidylic acid (pI-pC) at 2-day intervals. Control mice 1(fl/fl) received the same treatment and were derived from the same litters. For experiments, mice were used at least 2 weeks after pI-pC injection.
Generation of GPVI-deficient mice. To generate mice lacking GPVI, C57BL6/J mice were injected with 100 µg JAQ1 intraperitoneally. Animals were used for in vivo assessment of platelet adhesion/thrombus formation on day 5. As reported previously, GPVI was not detectable on the platelets of those mice by flow cytometry and Western blot analysis.33
Monoclonal antibodies and chemicals
Monoclonal antibodies (mAbs) against GPVI (JAQ1), integrin IIb3 (JON/A), and GPIb (p0p4) integrins 2 (LEN1) and 5 (BAR1) were generated as described.34-36 F(ab)2 fragments of JON/A were generated as described.33 Irrelevant control rat IgG and fluorescein isothiocyanate (FITC)-conjugated anti-1 integrin and 6 were obtained from Pharmingen (Hamburg, Germany). Mouse laminin and bovine fibronectin were from Sigma (Deisenhofen, Germany). Human VWF and collagen-related peptide (CRP) were kindly provided by G. Dickneite (Marburg, Germany) and S. P. Watson (Oxford, United Kingdom), respectively.
Flow cytometry
Heparinized whole blood was diluted 1:30 with modified Tyrode-HEPES buffer (134 mM NaCl, 0.34 mM Na2HPO4, 2.9 mM KCl, 12 mM NaHCO3, 20 mM HEPES [N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid], pH 7.0) containing 5 mM glucose, 0.35% bovine serum albumin (BSA), and 1 mM CaCl2. The samples were incubated with fluorophore-labeled antibodies for 10 minutes at room temperature and directly analyzed on a FACScalibur (Becton Dickinson, Heidelberg, Germany).
Preparation of platelets for intravital microscopy
Blood from wild-type or mutant mice was drawn from the retro-orbital plexus and collected in 1.5-mL polypropylene tubes containing 0.1-mL volume of 38 mM citric acid/75 mM trisodium citrate/100 mM dextrose. The blood was centrifuged at 250g for 10 minutes and platelet-rich plasma was gently transferred to a fresh tube and the centrifugation was repeated at 2000g for 10 minutes. The pellet was resuspended in modified Tyrode-HEPES buffer containing 0.35% BSA and 5 mM glucose. Isolated platelets were labeled with 5-carboxyfluorescein diacetate succinimidyl ester (DCF; 5 µg/mL for 2 minutes) and adjusted to a final concentration of 200 x 106 platelets/250 µL. Where indicated, platelets were preincubated with 50 µg/mL F(ab)2 fragments of anti-IIb3 (JON/A)35 for 5 minutes before infusion. Flow cytometric analysis with FITC-conjugated JON/A confirmed that more than 95% of surface IIb3 integrins were occupied under these conditions.
Intravital microscopy
Intravital microscopy of the injured carotid artery was performed essentially as described.16 Briefly, mice were anesthetized by intraperitoneal injection of ketamine/xylazine (ketamine 100 g/kg, Parke-Davis, Karlsruhe, Germany; xylazine 5 mg/kg, Bayer AG, Leverkusen, Germany). Polyethylene catheters (Portex, Hythe, England) were implanted into the right jugular vein and fluorescent platelets (200 x 106/250 µL) were infused intravenously. The right common carotid artery was dissected free and ligated vigorously near the carotid bifurcation for 30 seconds using a surgical filament to induce vascular injury. Prior to and following vascular injury, the fluorescent platelets were visualized in situ by in vivo video microscopy of the right common carotid artery using a Zeiss Axiotech microscope (x 20 water immersion objective; W x 20/0.5; Zeiss, Göttingen, Germany) with a 100-W mercury short arc photo optic lamp (HBO) for epi-illumination. Platelet adhesion was recorded for 5 minutes after the induction of injury and the videotaped images were evaluated using a computer-assisted image analysis program (Visitron, Munich, Germany). The number of adherent platelets was assessed by counting the cells that did not move or detach from the vascular wall for at least 10 seconds. In each mouse, 3 nonoverlapping fields (size, 100 µm x 100 µm) were analyzed for 30 seconds (2.5-3.0 minutes after injury) in a slow-motion modus. Clusters of 2 or more platelets were defined as microaggregates. The total number of adherent platelets or microaggregates at t = 3 minutes was calculated by the following formula that reflects concave shape of the vessel wall: vessel diameter (µm) x (circle constant) x 2 x sin-1 (amplitude of measured area, in µm) x length of measured area (µm) and is presented per mm2. All experiments performed on animals were approved by the German legislation on protection of animals.
Histology
For histologic examination, carotid arteries were perfusion-fixed in situ with 4% paraformaldehyde, pH 7.0. Thereafter, the vessels were excised, fixed in 0.1 M cacodylate-buffered Karnovsky solution (2.5% glutaraldehyde and 1% paraformaldehyde; overnight, room temperature) and then fixed in 1% osmium tetroxide (2 h) at pH 7.3. The samples were dehydrated in graded ethanols and embedded in the EmBed-812 epoxy resin (all reagents from Science Services, Munich, Germany). After 48 hours heat polymerization at 60°C, semithin (0.8 µm) sections were cut with a diamond knife (Diatome, Fort Washington, PA) on a Reichert Ultracut-S ultramicrotome (Leica-Reichert, Leica-Microsysteme, Vienna, Austria) and double stained with aqueous solutions of 1% toluidine blue and basic fuchsin (60°C, 1 minute).
Static adhesion
Static adhesion was performed with washed platelets in modified Tyrode buffer containing 0.35% BSA and Ca2+/Mg2+ (each at 1 mM) on 96-well plates (Nunc, Wiesbaden, Germany). The plates were coated with laminin (0.2 µg/well), fibronectin (0.2 µg/well), or VWF (0.4 µg/well) in phosphate-buffered saline (PBS) overnight at 4°C and then blocked with 5% BSA for 2 hours at 37°C. Resting or CRP-activated (0.2 µg/mL) platelets were allowed to adhere for 60 minutes and adhesion was quantitated colorimetrically as described.33 Where indicated, the experiments were performed in the presence of the function-blocking anti-IIb3 antibody, JON/A (50 µg/mL).
Statistical evaluation
Statistical analysis was performed using the unpaired Student t test.
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Results
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Platelet adhesion and thrombus growth on the injured arterial wall is unaltered in 2-null mice
To directly test the biologic significance of 21 in arterial thrombus formation we assessed platelet-vessel wall interactions following arterial injury in 2-null mice. Wild-type and 2-null mice had comparable platelet counts. Flow cytometric analysis confirmed the absence of 2 on mutant platelets, whereas expression levels of 5 and 6 were slightly increased.28 The 2 deficiency had no effect on expression levels of all other receptors tested including integrin 3 and the GPIb-V-IX complex (Figure 1A). In parallel, experiments were performed in mice with an antibody-induced GPVI deficiency.33
Platelets were purified from donor mice, fluorescently labeled, and injected into recipient mice of the same genotype. Vascular injury was induced by vigorous ligation of the carotid artery, which consistently causes disruption of the endothelial layer and frequently breaching of the internal elastic lamina followed by rapid platelet adhesion and aggregate formation at the site of injury.16 Unexpectedly, in vivo fluorescence microscopy revealed that the extent of platelet adhesion was indistinguishable between 2-null and wild-type mice (t = 3 minutes; n = 7 per group). Furthermore, in wild-type and 2-null mice, firmly adherent platelets recruited additional platelets from the circulation, leading to the formation of microaggregates that were similar in number in both groups of mice (Figure 1B-C). In contrast, platelet adhesion and aggregate formation was virtually abolished in GPVI-deficient mice, confirming previous results.16 In agreement with these early events, large platelet-rich thrombi were consistently found in the injured arteries of wild-type and 2-deficient mice after 20 minutes, whereas no thrombus had formed in GPVI-deficient mice (Figure 1D; n = 8 per group). These results demonstrated that although collagen is a major trigger for platelet adhesion and thrombus formation on the subendothelial matrix under high-shear conditions in vivo, 21 is not required for these processes.
Inhibition of integrin IIb3 reduces platelet adhesion on the ECM in wild-type and 2-null mice
The experiments described above suggested that other receptors can mediate shear-resistant platelet adhesion on the injured arterial wall in the absence of 21. Integrin IIb3, the major integrin on platelets, could mediate this adhesion as it binds to VWF and fibronectin, both of which are present in the ECM. To test the role of IIb3, wild-type mice received fluorescently labeled platelets preincubated with saturating concentrations (50 µg/mL) of F(ab)2 fragments of the blocking anti-IIb3 mAb JON/A35 prior to carotid injury. In vivo fluorescence microscopy revealed that platelet adhesion was reduced by 60.0% (1794 ± 118 vs 4495 ± 378/mm2; t = 3 minutes) upon IIb3 inhibition in wild-type mice (Figure 2). Furthermore, aggregate formation was blocked under these conditions, confirming the essential function of IIb3 in platelet aggregation in vivo. These results demonstrated that IIb3 plays an important role for shear-resistant platelet adhesion on the injured arterial wall in vivo but that other receptors significantly contribute to this process. To test whether the residual adhesion of IIb3-blocked platelets was mediated by 21, we inhibited IIb3 in 2-null mice. Very unexpectedly, however, inhibition of IIb3 resulted in a 56.7% reduction of platelet adhesion (1885 ± 176 vs 4355 ± 313/mm2; t = 3 minutes) and hence was similar to that observed in wild-type mice (Figure 2). As expected, aggregate formation was abolished under these conditions. These results demonstrated that platelets can firmly attach to the ECM under high-shear conditions in vivo independently of the 2 major integrins that mediate direct and indirect adhesion to collagen, 21 and IIb3, respectively.
Integrins 51 and 61 are involved in shear-resistant platelet adhesion on the ECM in vivo
Platelets express 2 additional integrins of the 1 family, 51 and 61, both of which bind ligands that are present in the ECM and could therefore mediate the reduced adhesion observed in IIb3-blocked 2-null platelets. This hypothesis was tested in mice with a Cre/loxP-mediated loss of 1 integrin on platelets.5 Flow cytometric analysis confirmed the absence of 1, 2, 5, and 6 integrin subunits on mutant platelets, whereas normal expression levels were found for all other tested receptors, including integrin 3 and the GPIb-V-IX complex (Figure 3A). Fluorescently labeled 1-null or wild-type platelets were injected into mice of the same genotype (1(fl/fl) cre+ vs 1(fl/fl) cre-, respectively) and carotid injury was induced as described above. Intravital videomicroscopy revealed that similar numbers of platelets adhered at the site of injury in control and mutant mice after 3 minutes (Figure 3B-C). Also, the time course and extent of microaggregate formation was not significantly different between wild-type and mutant mice and, in agreement with this observation, large platelet-rich thrombi were found in both groups of mice 20 minutes after the induction of injury (Figure 3D). Thus, platelet adhesion and thrombus formation on the subendothelial matrix in vivo occurs in the absence of 1 integrins. This strongly suggested that IIb3 alone is sufficient to mediate both platelet adhesion and thrombus growth in vivo in the absence of 1 integrins. This hypothesis was confirmed when IIb3 was inhibited in 1-null mice with JON/A F(ab)2 (50 µg/mL). Under these conditions, platelet adhesion was almost completely blocked (Figure 4). As expected, aggregate formation was absent in these mice. Altogether, these results demonstrated that both IIb3 and 1 integrins are independently able to mediate shear-resistant platelet adhesion on the subendothelial matrix under arterial flow conditions in vivo.
The affinity of platelet 51 and 61 for their ligands is regulated by GPVI
The finding that the IIb3 blockade almost completely inhibited platelet adhesion in 1-null but not 2-null mice demonstrated for the first time that integrins 51 and/or 61 can mediate shear-resistant platelet adhesion under arterial flow conditions in vivo. We have previously shown that GPVI-mediated integrin activation is an essential prerequisite for platelet adhesion to collagen in vitro5 and that platelet adhesion to the subendothelial matrix in vivo is virtually abolished in the absence of GPVI16 (Figure 1). These observations suggested that the affinity of 51 and 61 for their ligands is also regulated by GPVI-dependent mechanisms. To test this hypothesis, adhesion of wild-type and mutant platelets was studied on laminin, fibronectin, and, as a control, human VWF (HVWF) in vitro. Virtually no adhesion of resting platelets on laminin was observed for up to 1 hour. In contrast, when platelets were activated with the GPVI-specific agonist collagen-related peptide (CRP, 0.2 µg/mL), robust adhesion of wild-type and 2-null but not 1-null platelets occurred (Figure 5A), suggesting that 61 is essential to mediate platelet adhesion to laminin. A slightly different picture emerged when adhesion was studied on fibronectin. Virtually no adhesion of unstimulated platelets to this ligand was observed for up to 1 hour, whereas robust adhesion of wild-type, 2-null, and 1-null platelets occurred upon stimulation with CRP (Figure 5B). However, when IIb3 was blocked with JON/A (50 µg/mL), adhesion of wild-type and 2-null platelets was retained, whereas adhesion of 1-null platelets was almost completely abrogated. This finding suggests that the 2 major fibronectin-binding integrins on platelets are IIb3 and 51 and that they can independently mediate adhesion to this ligand. Furthermore, these results demonstrate that the affinity of both integrins for fibronectin is up-regulated by GPVI-dependent signaling. The normal function of IIb3 in wild-type and mutant platelets was confirmed when adhesion was studied on HVWF. It is important to note that hVWF does not efficiently interact with mouse GPIb.37 Therefore, GPIb-dependent IIb3 activation, which has been reported to induce adhesion of human platelets on HVWF,38 does not occur in this system. No adhesion of resting mouse platelets to this ligand was observed for up to 1 hour. However, when platelets were activated with CRP, strong adhesion of wild-type, 2-null, and 1-null platelets occurred. As expected, this adhesion was completely blocked in the presence of the anti-IIb3 mAb JON/A (50 µg/mL; Figure 5C).
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Discussion
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In the current study we assessed the significance of 21 in the dynamic process of platelet adhesion and thrombus formation on the subendothelial matrix by intravital fluorescence microscopy of the injured carotid artery of 2-null mice. We found that both platelet adhesion and thrombus formation are not significantly altered in the mutant mice compared with wild-type controls. This finding demonstrates that 21 is not essential for arterial thrombus formation in mice but it does not exclude a supportive role of the integrin in this process. Although the data obtained in mice cannot be directly extrapolated to the situation in humans, our results indicate that platelet 21 may not play a major role in the onset of acute ischemic syndromes, such as myocardial infarction or stroke. However, our findings do not rule out a possible role of the integrin in the pathogenesis of chronic vascular diseases such as atherogenesis, which could explain the correlation between high-21 levels and susceptibility to cardiovascular disease. A supportive rather than an essential role of 21 in thrombus formation in vivo is further suggested by the essentially normal tail bleeding times in 2-null mice,28,29 although this assay in mice has not been validated as a useful surrogate of normal hemostasis. Together, these observations show that 21 is dispensable for hemostasis and arterial thrombosis, at least in mice.
In addition to collagen, the subendothelial matrix contains multiple macromolecules that potentially provide an adhesive substrate for platelet integrins. Among these, VWF, immobilized on fibrillar collagen via its A3 domain, is thought to play a role in this process as it is a ligand for the dominant platelet integrin, IIb3.13 Our studies with the IIb3-blocking antibody, JON/A, demonstrate a major role of IIb3 in platelet adhesion on the ECM in vivo (Figures 2 and 4). This finding is in agreement with in vitro studies showing that IIb3-VWF interactions are sufficient to mediate shear-resistant platelet deposition on collagen in whole-blood perfusion experiments in the absence of functional 21.5,13 However, the newly formed aggregates are less stably attached to the collagen substrate compared with wild-type controls,30 suggesting that IIb3-VWF interactions are unable to fully substitute for the lack of 21 in this system. In vivo, the situation appears to be different as IIb3 not only binds to VWF but also to other ligands, including fibrinogen deposited on the surface of the damaged vessel and fibronectin, which is present in the ECM of the vessel wall. This may explain why no defect in adhesion and thrombus formation was detectable in 2-null mice. The pivotal role of IIb3 was revealed in 1-null mice, where platelet adhesion and thrombus formation was not reduced compared with controls but abrogated on inhibition of IIb3 (Figure 4). These results strongly suggest that IIb3 is the major integrin that supports shear-resistant platelet adhesion on the injured vessel wall in vivo, presumably by interacting with multiple ligands in the ECM.
The surprising finding that platelet adhesion and aggregation on the ECM was not significantly altered in 1-null mice also excludes an essential role of 51 and 61 in arterial thrombus formation, which is in line with normal tail bleeding times in those mice.5 However, the observation that inhibition of IIb3 abolished platelet adhesion in 1-null but not 2-null mice provides the first direct evidence that 51 and/or 61 can contribute to platelet attachment to the damaged vascular wall in vivo. We were unable to clarify whether 51, 61, or both were responsible for the observed adhesion in the absence of functional 21 and IIb3. It appears likely, however, that both integrins are involved in this process as both fibronectin and laminin are highly expressed in the vessel wall and become accessible to the flowing blood at sites of injury. Laminins are a family of structurally related glycoproteins that are tightly assembled with collagen type IV through the action of nidogen39 in the basement membrane. Therefore, laminins are among the first constituents of the ECM that platelets get in touch with at sites of endothelial denudation and they are closely associated with collagen. Although collagen type IV is a relatively weak platelet agonist compared with collagen types I, III, and VI,40 it is known to activate IIb3 through the GPVI/FcR-chain complex.41
Fibronectins are dimeric glycoproteins that are present in plasma (plasma fibronectin) and in tissue extracellular matrices (cellular fibronectin).42 At sites of vascular injury, platelets get in touch with both cellular and extravasated plasma fibronectin but the significance of either interaction is only partly understood. Initial studies in mice with a cre/loxP-mediated deletion of plasma fibronectin revealed no major hemostatic defect as shown by normal bleeding times, platelet aggregation, and clot retraction.43 However, recent studies with these mice in a model of arterial thrombosis demonstrated delayed thrombus formation and reduced thrombus stability, suggesting a role of plasma fibronectin in platelet-platelet interactions.44 Our results may extend the role of fibronectins also to the process of platelet adhesion and suggest that both fibronectin-binding integrins on platelets, IIb3 and 51, play a significant role in this process. In the vascular wall, cellular fibronectin is closely associated with collagens including types I and III, both of which are strong platelet agonists on GPVI, suggesting that GPVI-collagen interactions may facilitate platelet adhesion on fibronectin.
Our adhesion studies (Figure 5) suggest that 51 and 61 are in a low-affinity state on resting mouse platelets unable to efficiently mediate adhesion to fibronectin or laminin, respectively, and that ligation of GPVI shifts these integrins to a high-affinity state. This finding is in line with previous studies demonstrating a similar regulation of 21 in mouse platelets5 and suggests that both integrins, together with 21, may contribute to shear-resistant platelet adhesion at sites of arterial injury in a GPVI-dependent manner. However, these results stand in contrast to reports that human platelets adhere to fibronectin18 and laminin45 under static conditions in the absence of cellular stimulation, suggesting that both integrins may be constitutively in a high-affinity conformation on human platelets. One possible explanation for this apparent discrepancy might be species-specific differences in the affinity regulation of both integrins. However, at least the third 1 integrin on platelets, 21, has been shown to be expressed in a low-affinity state on resting human6,46 and mouse5 platelets and to shift to a high-affinity state in response to cellular stimulation in both species. Based on the assumption that the different 1 integrins on platelets may be regulated by similar mechanisms, species-specific differences in the regulation of 51 and 61 appear unlikely. Another possible explanation for the discrepant results might be different protocols used for platelet preparation or washing of the adhesion plates or differences in the laminin/fibronectin preparations used in the individual studies, but this is difficult to assess. Therefore, further studies will be required to clarify whether or not 51 and 61 are differently regulated in human and mouse platelets.
The results of the present study suggest that the affinity of 1 and 3 integrins on platelets is regulated by similar mechanisms. At sites of arterial injury, GPVI-collagen interactions are a major trigger of this activation process.15,16 However, since GPVI-deficient humans9,47 and mice33,48 display no major bleeding phenotype, it appears that other agonist receptors/signaling pathways can substitute for GPVI in mediating integrin activation in normal hemostasis. The G-protein-coupled receptors for adenosine diphosphate (ADP), thromboxanes, or thrombin are likely to play major roles in this process, but further studies will be required to confirm this hypothesis.
In summary, we have shown that platelet attachment and thrombus formation at sites of vascular injury in mice can occur independently of 21 or even all 1 integrins on platelets. On the other hand, 1 integrins can mediate shear-resistant platelet adhesion independently of IIb3, demonstrating that 1 and 3 integrins have largely redundant roles in this process. However, our findings do not rule out the possibility that individual integrins may have distinct functions that are of greater significance in other pathophysiologic processes. Studies with 2- and 1-null mice in models of systemic and local inflammation, tumor metastasis, and atherogenesis may help to answer this question. Besides mediating platelet adhesion and aggregation, platelet integrins trigger a variety of important functions like spreading, procoagulant activity, and clot retraction through "outside-in" signaling. This is best documented for IIb3 (for review see Shattil49) but recent studies by Inoue et al50 suggest that 21 triggers similar functions in platelets as both integrins regulate a similar set of intracellular signaling molecules including Syk, SLP-76, FAK, and phospholipase C2 (PLC2). These observations indicate that the signaling pathways triggered by IIb3 and 21 in platelets are conserved and may also apply to 51, 61, and v3. Together with the data reported here, this suggests that the cooperation of multiple integrin-ligand interactions ensures a high degree of functional redundancy enabling effective and well-controlled platelet adhesion, spreading, and thrombus formation on different compositions of the subendothelial matrix. These may vary significantly between different regions in the vascular system and may also depend on the type and severity of the lesion.
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Acknowledgements
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We are grateful to Reinhard Fässler for critically reading the manuscript and for helpful discussions. We thank J. Schröder for help with histology and Martina Koch and Stefanie Hartmann for excellent technical assistance.
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Footnotes
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Submitted May 5, 2003;
accepted July 22, 2003.
Prepublished online as Blood First Edition Paper, July 31, 2003; DOI 10.1182/blood-2003-05-1391.
Supported by grant Ni556/4-1 (B.N.) and SFB 589 (B.E., T.K.) from the Deutsche Forschungsgemeinschaft (DFG). B.N. and C.B. are Heisenberg fellows of the DFG.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Bernhard Nieswandt, Rudolf Virchow Center for Experimental Biomedicine, University of Würzburg, Versbacher Str 9, 97078 Würzburg, Germany; e-mail: bernhard.nieswandt{at}virchow.uni-wuerzburg.de.
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References
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- Weiss HJ. Platelet physiology and abnormalities of platelet function (first of two parts). N Engl J Med. 1975;293: 531-541.[Medline]
[Order article via Infotrieve]
- Shattil SJ, Kashiwagi H, Pampori N. Integrin signaling: the platelet paradigm. Blood. 1998;91: 2645-2657.
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Top
Abstract
Introduction
), 
), or 
) platelets were allowed to adhere for 60 minutes under static conditions to laminin (A), fibronectin (B), or VWF (C) immobilized in microtiter plates. The experiments were performed in the presence of Mg2+/Ca2+ (1 mM each). Where indicated, platelets were preincubated with anti-