Platelet Adhesion Via Glycoprotein IIb Integrin Is Critical for Atheroprogression and Focal Cerebral Ischemia
An In Vivo Study in Mice Lacking Glycoprotein IIb
Background— The platelet glycoprotein (GP) IIb/IIIa integrin binds to fibrinogen and thereby mediates platelet aggregation. Here, we addressed the role of GP IIb for platelet adhesion and determined the relevance of platelet GP IIb for the processes of atherosclerosis and cerebral ischemia-reperfusion (I/R) injury.
Methods and Results— GP IIb−/− mice were generated and bred with ApoE−/− animals to create GP IIb−/−ApoE−/− mice. Platelet adhesion to the mechanically injured or atherosclerotic vessel wall was monitored by in vivo video fluorescence microscopy. In the presence of GP IIb, vascular injury and early atherosclerosis induced platelet adhesion in the carotid artery (CA). In contrast, platelet adhesion was significantly reduced in the absence of GP IIb integrin (P<0.05). To address the contribution of platelet GP IIb to atheroprogression, we determined atherosclerotic lesion formation in the CA and aortic arch (AA) of GP IIb+/+ApoE−/− or GP IIb−/−ApoE−/− mice. Interestingly, the absence of GP IIb attenuated lesion formation in CA and AA, indicating that platelets, via GP IIb, contribute substantially to atherosclerosis. Next, we assessed the implication of GP IIb for cerebral I/R injury. We observed that after occlusion of the middle cerebral artery, the cerebral infarct size was drastically reduced in mice lacking GP IIb compared with wild-types.
Conclusions— These findings show for the first time in vivo that GP IIb not only mediates platelet aggregation but also triggers platelet adhesion to exposed extracellular matrices and dysfunctional endothelial cells. In a process strictly involving GP IIb, platelets, which are among the first blood cells to arrive at the scene of endothelial dysfunction, contribute essentially to atherosclerosis and cerebral I/R injury.
Received January 30, 2005; revision received May 6, 2005; accepted May 11, 2005.
Atherosclerosis is an inflammatory disease characterized by the recruitment of monocytes/macrophages to the vessel wall of large arteries.1,2 After rupture of the atherosclerotic plaque, exposure of the subendothelial matrix induces platelet adhesion and rapid platelet activation with ensuing recruitment of additional platelets into the growing thrombus.3–5 These processes eventually lead to thrombotic occlusion of diseased arteries with obstruction of blood flow and subsequent ischemia of vital organs, triggering heart attack and ischemic stroke, the most common causes of mortality in industrialized countries.
Endothelial disruption and exposure of extracellular matrices are not required for platelet–vessel wall interactions. In the presence of endothelial activation/dysfunction, platelets may also interact directly with the structurally intact endothelial cell surface.6–8 Such platelet–endothelial cell interactions have been reported to arise at various stages during atherogenesis. We9 and others10 have demonstrated that platelet–endothelial cell interactions are present in atherosclerosis-prone arteries at early stages of atherosclerosis, before the development of lesions. Conversely, in advanced atherosclerosis, thrombotic obstruction and subsequent reperfusion of large arteries initiates platelet adhesion to postischemic endothelial cells in the downstream microvascular network.6–8 Therefore, platelets play a multifactorial role in different phases of the complex process of atherosclerosis.
Principally, platelets are recruited to the diseased vessel wall in a multistep process. The initial platelet tethering stringently depends on the interaction of glycoprotein (GP) VI with subendothelial collagen5 and on that of GP Ib-V-IX with von Willebrand factor (vWF) bound to subendothelial collagen11 or to the surface of dysfunctional endothelial cells.10 In addition, P-selectin appears to play a prominent role for the initial loose contact between platelets and diseased vessel wall.7,12
Integrins are generally thought to be the major class of receptors that mediate firm platelet adhesion. Platelets express 2 different β3-integrins, αIIbβ3 and αvβ3 (vitronectin receptor), and 3 different β1-integrins, namely α2β1 (collagen receptor), α5β1 (fibronectin receptor), and α6β1 (laminin receptor).13 However, because of the presence of multiple integrins on the platelet surface, the contribution of individual integrin receptors to platelet adhesion is ill defined. The major platelet integrin is the αIIbβ3 integrin, the platelet fibrinogen receptor glycoprotein IIb/IIIa (GP IIb/IIIa). Activated GP IIb/IIIa mediates the immobilization of adhesive proteins, in particular of fibrinogen and vWF, on the surface of adherent platelets.13 Fibrinogen associated with GP IIb/IIIa serves as a substrate to which more platelets are recruited, resulting in platelet aggregation. Correspondingly, inhibition of GP IIb/IIIa has improved the treatment of unstable angina pectoris and myocardial infarction, and this effect has been attributed primarily to its central role for platelet aggregation. Conversely, there is growing in vitro evidence indicating that apart from mediating platelet aggregation, GP IIb/IIIa may also contribute to primary platelet adhesion to the exposed subendothelial matrices and possibly also to activated endothelial cells.14 However, the in vivo significance of GP IIb/IIIa for the process of primary platelet adhesion to the injured or inflamed vessel wall is in large part undefined.
Here, we use mice lacking GP IIb and show for the first time in vivo that apart from promoting platelet aggregation, GP IIb also contributes substantially to firm adhesion of platelets to the exposed subendothelium and to dysfunctional endothelial cells. Mice lacking GP IIb are protected from atherosclerosis and show increased resistance to focal cerebral ischemia.
To generate C57BL/6 mice lacking GP IIb (GP IIb−/− mice), a sequence encoding a GFP-Cre fusion protein was introduced into the first intron of the GP IIb locus by gene targeting as described previously.15 Age- and sex-matched wild-type littermates (C57BL/6–GP IIb+/+) served as controls. To generate GP IIb−/−ApoE−/− mice, we mated GP IIb−/− breeder males with ApoE−/− females (C57BL/6-ApoEtm1Unc; Jackson Laboratory, Bar Harbor, ME).
Genotyping of GP IIb−/− and GP IIb−/−ApoE−/− Mice
All mice were genotyped by polymerase chain reaction using primers that amplify 500 bp of the coding sequence for Cre in conjunction with primers (MWG Biotech) that amplify 435 bp of intron 1 of the gpIIb gene to discriminate GP IIb–null mutants from heterozygous GP IIb+/− or wild-type mice, respectively (Table 1). Primers specific for the ApoE gene were used to identify animals with inactivation of the ApoE locus (Table 1).
Assessment of Tail-Bleeding Time
Tail-bleeding time of GP IIb−/−ApoE−/− mice and GP IIb+/+ApoE−/− animals (n=8 per group) was assessed as described in detail previously.16
Blood was drawn from anesthetized GP IIb−/−ApoE−/− and ApoE−/− mice (n=8 per group) by cardiac puncture and diluted 1:10 with citrate. After recalcification of the blood sample, thromboplastin (Pentapharm) was added, and clot formation was monitored on a Rotem thrombelastometer (Abacus Diagnostics).
Characterization of GP IIb–Null Platelets by Flow Cytometry
Platelet-rich plasma was obtained from GP IIb−/− or GP IIb+/+ mice, stained with fluorophore-labeled anti–GP IIIa (2C9.G2, BD Pharmingen), anti–GP IIb (MWReg 30, BD Pharmingen), anti–GP Ibα (Xia.B2, Emfret Analytics), anti–GP VI (8H3, generated as described16), anti–P-selectin (RB40.34, BD Pharmingen) monoclonal antibodies (0.2 μg/μL), or isotype-matched control antibodies (BD Pharmingen) and directly analyzed on a FACS Calibur (Becton Dickinson).
Assessment of Platelet Adhesion to the Injured Carotid Artery by Intravital Microscopy
Platelets isolated from GP IIb−/− or wild-type mice were labeled with 5-carboxyfluorescein diacetate succinimidyl ester as previously reported and adjusted to a final concentration of 1.2×105 in 250 μL.5 Polyethylene catheters were introduced into the left jugular vein of anesthetized (isoflurane inhalation) recipient mice (n=4 to 6 per group) for intravenous application of fluorescence-tagged platelets. The carotid arteries (CAs) were then carefully exposed, and vascular injury was induced by ligation of the CA for 5 minutes as described previously.5,16 Platelet adhesion and aggregation before and after vascular injury were monitored in situ by intravital videofluorescence microscopy (IVM) as reported earlier.5,16 Adherent platelets were determined by counting single cells that did not detach from the surface within 15 seconds. Platelet aggregation is given as surface area (μm2) covered by platelet aggregates.
Evaluation of Platelet Adhesion and Plaque Formation in GP IIb–ApoE Double-Deficient Mice
To evaluate the role of platelet GP IIb in the process of atherosclerosis, 4-week-old GP IIb−/−ApoE−/− or GP IIb+/+ApoE−/− mice consumed a cholesterol diet (0.25% cholesterol; Altromin) for 8 or 12 weeks (n=9 per group). At the age of 12 or 16 weeks, the animals were anesthetized, both CAs were exposed, and IVM was performed to determine platelet adhesion to the atherosclerotic vessel wall. Subsequently, the animals were euthanized, and both CAs as well as the aortic arch (AA) were excised. The right CA and the AA of GP IIb−/−ApoE−/− or GP IIb+/+ApoE−/− mice were perfusion-fixed with 4% paraformaldehyde and stained with Sudan III to assess en face plaque extension as previously reported.9 The left CA was fixed with 4.5% formaldehyde and cut into 2-μm cross sections. The sections were stained with van Gieson’s elastica stain, and cross-sectional plaque area was determined on serial sections of the carotid bifurcation as previously described.9 In addition, to determine the role of GP IIb–dependent platelet adhesion for monocyte recruitment, carotid arteries of 16-week-old GP IIb−/−ApoE−/− or GP IIb+/+ApoE−/− mice (n=3 per group) were shock-frozen and evaluated for CD14 and CD11b mRNA expression by reverse transcription–polymerase chain reaction. CD14 and CD11b mRNA expression was semiquantitatively analyzed by densitometry. The β-actin mRNA expression was used as amplification control. The primer pairs are given in Table 2.
Assessment of Platelet Adhesion After Ischemia-Reperfusion
In a separate set of experiments, platelet–vessel wall interactions were assessed after mesenteric ischemia-reperfusion (I/R) as described.7 Briefly, GP IIb−/− (n=4) or wild-type (n=12) mice were anesthetized by inhalation of isoflurane and laparotomized, and syngeneic labeled platelets (1.2×105, GP IIb−/− or wild-type) were infused intravenously. Segmental mesenteric ischemia (60 minutes) was induced with subsequent reperfusion. Platelet–endothelial cell interactions in intestinal arterioles and venules were analyzed by IVM as reported.7
Role of Platelet GP IIb for Cerebral I/R Injury
To determine the potential contribution of GP IIb/IIIa–mediated platelet adhesion for cerebral I/R injury, transient focal cerebral ischemia was induced as described previously.17 In brief, male GP IIb−/− or wild-type mice (body weight, 18 to 22 g; n=6 per group) were anesthetized, and occlusion of the middle cerebral artery (MCA) was induced for 30 minutes by insertion of a silicone-coated 8-0 nylon monofilament via the internal CA. Thereafter, cerebral ischemia was terminated by removal of the intraluminal filament. Animals were deeply anesthetized and perfusion-fixed with 4% paraformaldehyde 24 hours after reperfusion. Brains were removed, postfixed in 4% paraformaldehyde, and embedded in paraffin. Fifteen 5-μm coronal serial sections comprising all infarcted brain structures were prepared. Infarct volume was calculated on the basis of the planimetrically assessed infarcted brain areas as previously described.17
Comparisons between group means were performed using the Mann-Whitney rank sum test. Data represent mean±SEM. A value of P<0.05 was regarded as significant.
Platelet Aggregation and Primary Platelet Adhesion at Sites of Vascular Injury Are Reduced in GP IIb–Null Mice
To address the differential role of GP IIb integrin in the processes of platelet adhesion and aggregation after endothelial denudation, we generated GP IIb–null mice as previously described.15 No GP IIb protein can be detected on bone marrow cells or platelets from GP IIb–null animals, whereas the expression of other platelet adhesion receptors, including GP Ibα, P-selectin, or GP VI, is not affected by the loss of GP IIb (not shown). First, GP IIb−/− mice were used to dissect the exact biological role of GP IIb in the sequels of thrombus formation. By IVM, we directly visualized the dynamic processes of platelet adhesion/aggregation after vascular injury.5 In wild-type mice, platelets rapidly adhered to the exposed subendothelium (662±62, 804±75, and 951±119 adherent platelets/mm2 vessel area 5 minutes, 30 minutes, and 60 minutes after injury, respectively; Figure 1, a–c). Within seconds, adherent platelets recruited additional circulating platelets, resulting in aggregate formation (Figure 1, a–c). In contrast, aggregate formation was virtually abolished in GP IIb−/− mice (Figure 1b), confirming the crucial role of GP IIb for platelet aggregation in vivo. Unexpectedly, however, the loss of GP IIb not only affected aggregation but also resulted in a substantial reduction in firm platelet adhesion (0±0, 108±42, and 43±33 adherent GP IIb–null platelets/mm2 5 minutes, 30 minutes, and 60 minutes after vascular injury, respectively; Figure 1, a and c). In fact, in GP IIb−/− animals, the number of adherent platelets was reduced by 87% and 95% compared with wild-type mice 30 and 60 minutes after vascular injury, respectively (P<0.05). These findings demonstrate for the first time in vivo that apart from mediating platelet aggregation, GP IIb/IIIa contributes essentially to primary platelet adhesion to the injured vessel wall under high-shear conditions.
Firm Platelet Adhesion to Dysfunctional Endothelial Cells Requires GP IIb
Next, we addressed the role of GP IIb/IIIa for firm platelet adhesion to dysfunctional endothelial cells in the setting of early and late atherosclerosis. We generated mice lacking both GP IIb and ApoE (Figure 2a). Homozygous GP IIb−/− ApoE−/− mice are viable and fertile. No GP IIb protein can be detected on bone marrow cells or platelets from ApoE−/−GP IIb−/− animals either by flow cytometry (Figure 2b) or by Western blot analysis using a monoclonal anti–GP IIb antibody (not shown). Platelets isolated from GP IIb−/−ApoE−/− mice reveal a significant reduction in the capability to bind fibrinogen and to aggregate in vitro (not shown). Likewise, clot formation time is prolonged in the absence of GP IIb, as assessed by thrombelastography (34±7 and 128±18 seconds in GP IIb+/+ApoE−/− and GP IIb−/−ApoE−/−, respectively; P<0.05; Figure 2c). Correspondingly, the animals resemble the phenotype of human Glanzmann thrombasthenia, with a prolonged bleeding time (Figure 2d) and bleeding disorders, including petechial bleedings (65% of GP IIb−/−ApoE−/− mice, 0% of GP IIb+/+ApoE−/− mice) and spontaneous intra-abdominal or intrapleural hemorrhage (53% of GP IIb−/−ApoE−/− mice, 0% of GP IIb+/+ApoE−/− mice). Correspondingly, the hemoglobin concentrations were significantly lower in GP IIb−/−ApoE−/− mutants compared with GP IIb+/+ApoE−/− animals. The complete laboratory findings are illustrated in Table 3.
To determine whether GP IIb regulates platelet adhesion to the endothelial surface during atherogenesis, 4-week-old GP IIb−/−ApoE−/− double-deficient or GP IIb+/+ApoE−/− control mice received a cholesterol diet for 8 or 12 weeks. Thereafter, we analyzed firm platelet adhesion at the carotid bifurcation using IVM. In GP IIb+/+ApoE−/− mice, platelet adhesion in the carotid bifurcation was substantially increased by the age of 12 weeks (67±26 adherent platelets/mm2; Figure 3a). At this age, ApoE-null mice show no or only minimal atherosclerotic lesions on histological examination, supporting previous findings that platelet adhesion occurs early in the process of atherosclerosis.9 A further increase in platelet adhesion occurred in 16-week-old GP IIb+/+ApoE−/− mice (458±137 adherent platelets/mm2; Figure 3a). Notably, firm platelet adhesion was virtually absent in mice lacking both ApoE and GP IIb, even at the age of 16 weeks. This clearly indicates that GP IIb/IIIa is absolutely mandatory to allow firm adhesion of platelets to the dysfunctional endothelial monolayer in the process of atherosclerosis. In contrast, other platelet adhesion receptors that promote platelet–endothelial cell interactions in vitro, including αvβ3 integrin,18 appear to play a minor role for platelet adhesion to the endothelium in vivo, because they are not sufficient to allow platelet adhesion in the absence of GP IIb.
Platelets Contribute to Atheroprogression Via GP IIb
Because platelets trigger a broad variety of immune responses in endothelial cells in vitro that might potentially contribute to the process of atherosclerosis,19,20 we next asked whether reduced platelet adhesion and aggregation in GP IIb–ApoE double-deficient mice might be paralleled by attenuated atherosclerotic lesion formation. To test this, GP IIb−/− ApoE−/− mice or GP IIb+/+ApoE−/− mice received a cholesterol diet for 8 or 12 weeks, and en face or cross-sectional plaque extension was evaluated in the AA and the CA. Plasma cholesterol concentrations and body weights did not differ between GP IIb−/−ApoE−/− or GP IIb+/+ApoE−/− mice (Table 3). Strikingly, the loss of GP IIb drastically reduced lesion formation in both the CA and the AA. Although substantial atherosclerotic lesion formation was detected in the AA of 12- and particularly 16-week-old ApoE−/− mice in the presence of GP IIb, targeted disruption of GP IIb induced a 20% reduction in en face aortic plaque extension (Figure 3b). Likewise, loss of GP IIb in ApoE−/− mice was associated with a 66% and 51% decrease in plaque formation in the CA at the age of 12 or 16 weeks, respectively (Figure 3c). Moreover, we observed a 74% and 54% reduction in cross-sectional carotid plaque area in 12- and 16-week-old GP IIb–null ApoE-null mice (Figure 3d).
Monocyte recruitment into the intima plays a central role in atherosclerotic lesion formation.21 Because platelets trigger monocyte adhesion to endothelial cells in vitro, we next determined whether loss of GP IIb might modulate monocyte accumulation during atherosclerotic plaque formation in vivo. In carotid arteries of GP IIb+/+ApoE−/− mice, we observed a substantial recruitment of monocytes, as assessed by immunohistochemistry (not shown) and by CD11b and CD14 mRNA expression (Figure 3, e and f). Interestingly, loss of GP IIb was paralleled by a considerable decrease in monocyte recruitment into the intima (Figure 3, e and f). Together, these findings demonstrate for the first time in vivo that platelet adhesion to the dysfunctional endothelium of ApoE−/− mice is mediated by GP IIb/IIIa and that loss of GP IIb attenuates the inflammatory process of atherosclerosis.
Platelet GP IIb Promotes Platelet Adhesion to Postischemic Microvascular Endothelial Cells
The major consequence of advanced atherosclerosis and atherothrombosis is ischemia of the downstream microvascular network. We and others have reported recently that microvascular platelet adhesion is a prominent phenomenon after I/R in various organs.7,8,22 Hence, using IVM, we next examined the relevance of GP IIb for platelet–endothelial cell interaction in the microcirculation during I/R of the mouse mesentery. As reported earlier, I/R promotes substantial platelet rolling (not shown) and platelet adhesion in both arterioles and venules (Figure 4a). In contrast, in GP IIb−/− mice, firm platelet adhesion to the postischemic endothelium was entirely absent, indicating that the platelet fibrinogen receptor is central to this process. Notably, platelet rolling was not affected by the loss of GP IIb (not shown).
GP IIb–Dependent Platelet Adhesion Contributes to Cerebral I/R Injury In Vivo
Although the contribution of platelets to large-artery atherothrombosis is generally accepted, the accumulation of platelets in the target microvascular beds in response to ischemia and their contribution to further injury has received little attention. Because microvascular platelet–endothelial cell interactions are a prominent phenomenon particularly after cerebral I/R,22 we next asked whether loss of GP IIb might be paralleled by a mitigation of cerebral I/R injury. Transient occlusion of the MCA (30 minutes) was induced in GP IIb−/− or wild-type animals as previously described.17 After 24 hours of reperfusion, the brains were removed, and the infarct volume was assessed by use of histomorphometry (Figure 4b). Focal cerebral I/R, which is associated with interaction of platelets with the vessel wall,22 produced substantial infarction in wild-type animals (50±9 mm3). In contrast, the loss of GP IIb induced a considerable reduction of cerebral infarct volume, by 46% compared with wild-type mice (infarct volume, 27±5 mm3; P<0.05).
Platelets play an important role in the pathogenesis of various pathological conditions, including I/R injury and cardiovascular diseases, such as atherothrombosis.3 Understanding the molecular requirements of platelet accumulation, therefore, has been a central goal to prevent and treat platelet-mediated tissue and vascular injury. Here, we show in vivo that the platelet integrin GP IIb/IIIa not only mediates platelet aggregation but also contributes substantially to firm platelet adhesion to subendothelial matrix proteins after endothelial denudation and to dysfunctional endothelial cells in the absence of exposure of extracellular matrices. Correspondingly, the loss of GP IIb profoundly reduced platelet-endothelium and platelet-subendothelium adhesion in vivo. In parallel, GP IIb deficiency diminished cerebral I/R injury and attenuated vascular remodeling during atherogenesis in mice.
Several macromolecules may potentially provide an adhesive substrate for platelet GP IIb/IIIa in vivo. After endothelial denudation, fibronectin23 and vWF immobilized on fibrillar collagen via its A3 domain act as potent ligands of platelet GP IIb/IIIa.11 Correspondingly, GP IIb/IIIa–vWF interactions have been demonstrated earlier to be sufficient to mediate shear-resistant platelet deposition on immobilized collagen in vitro.14 In contrast, the ligands expressed on dysfunctional endothelial cells are less well defined. However, we and others have demonstrated earlier that fibrinogen and vWF, both major ligands of GP IIb/IIIa, are present in substantial amounts on the surface of dysfunctional/activated endothelial cells,8,10,24 making them likely candidates for GP IIb/IIIa–dependent platelet-endothelium adhesion.
Ligand binding to GP IIb is stringently controlled by inside-out signals that modulate receptor conformation and clustering.13 Apart from soluble agonists (eg, ADP, thrombin), in particular the engagement of certain platelet adhesion receptors during initial platelet tethering, notably GP Ib-V-IX and GP VI, can trigger GP IIb activation.25 In fact, ligation of GP Ib-V-IX or GP VI during platelet tethering appears to be a prerequisite for subsequent firm platelet adhesion via GP IIb, because both platelet tethering and adhesion are attenuated by inhibition of GP Ib-V-IX or GP VI, respectively.5 Importantly, however, GP Ib-V-IX or GP VI per se is not sufficient to promote firm platelet adhesion, as is evidenced by the almost complete lack of platelet adhesion in GP IIb−/− mice. Likewise, other platelet integrins, such as α2β1, but also α5β1 and α6β1, appear to play a minor role for firm platelet adhesion in vivo, because they do not compensate for the loss of αIIbβ3. In line with this finding, platelet adhesion and aggregation are not significantly altered in mice lacking α2 or β1 integrin, respectively.26
Platelet adhesion, activation, and aggregation play a major role for the thrombembolic complications of advanced atherosclerosis.1,2 In addition, evidence suggesting that platelets might also contribute significantly to the inflammatory processes that initiate atherosclerotic lesion formation is accumulating. Activated platelets have been reported to circulate in the blood of patients with unstable atherosclerosis,27 but also in individuals with stable coronary artery disease.28 Moreover, we9 and others10 have demonstrated that platelets readily adhere to the structurally intact but dysfunctional endothelium early in atherosclerosis in ApoE−/− mice. Although these observations were suggestive, they did not directly prove a causative role of platelets in atherosclerotic lesion formation. However, we have reported that inhibition of platelet GP Ib-V-IX largely attenuates atherosclerotic lesion formation, providing the first direct evidence that platelets actually contribute to atherosclerotic plaque formation.9 Here, we have extended our previous findings and show in vivo that platelet adhesion to the dysfunctional endothelium of ApoE−/− mice strictly requires the platelet integrin GP IIb. Loss of GP IIb was paralleled by reduced monocyte recruitment into the CA and protected ApoE−/− mice from atherosclerotic plaque formation, supporting a critical role of platelets and platelet GP IIb integrin.
Interestingly, β3−/− animals have recently been reported to display accelerated atherosclerosis compared with β3+/+ littermates.29 The β3 integrin subfamily consists of 2 integrins, GP IIb/IIIa (αIIbβ3) and αvβ3. GP IIb/IIIa is expressed exclusively by megakaryocytes and platelets and mediates platelet adhesion, aggregation, and activation. In contrast, αvβ3 integrin is present on a variety of vascular cells, including monocytes, fibroblasts, endothelial cells, and smooth muscle cells, where it modulates multiple cellular functions, including proliferation, migration, and inflammation.29,30 Importantly, β3-null mutants lack both GP IIb/IIIa and αvβ3, resulting in enhanced atherosclerotic lesion formation.29 In contrast, we report here that the selective loss of platelet GP IIb/IIIa protects mice from atherosclerotic lesion formation. Together, these findings imply that GP IIb/IIIa deficiency attenuates atheroprogression, whereas the simultaneous loss of αvβ3 in β3-null mice appears to compensate for the lack of platelet GP IIb/IIIa, resulting in a net acceleration of atherosclerotic lesion formation.
The exact mechanisms underlying platelet-induced atherosclerotic lesion formation have not been fully defined as yet; however, activated platelets are known to release a variety of proinflammatory cytokines, including CD40L, interleukin-1β, and RANTES (regulated on activation, normal T cell expressed and secreted).31–33 In this manner, platelets might affect endothelial inflammation31,32 and trigger monocyte arrest and transendothelial migration,33 both crucial events in atherogenesis. In addition, activated platelets surface-express P-selectin, which promotes platelet interaction with leukocytes.34 Absence of platelet P-selectin in atherosclerosis-prone ApoE-deficient mice attenuates lesion formation.35 In addition, repeated infusions of activated wild-type but not P-selectin–deficient platelets have been demonstrated to exacerbate atherosclerosis,36 indicating that platelet P-selectin may in fact play an important role in platelet-mediated inflammatory processes. Interestingly, ligand binding to GP IIb triggers a cascade of events that culminate in the release of growth factors, adhesion receptors (including P-selectin), and proinflammatory cytokines/chemokines.13,19 Hence, engagement of GP IIb during platelet adhesion and aggregation at sites of endothelial dysfunction may per se contribute to the inflammatory response triggered by activated platelets.
Advanced atherosclerosis frequently promotes ischemic complications, including myocardial infarction and ischemic stroke, the major causes of morbidity and mortality in Europe and North America.1 Even prompt revascularization of the target vessel with restoration of blood flow may initiate a cascade of events that ultimately results in aggravation of tissue injury. Under these conditions, a prominent phenomenon documented in various vascular beds is platelet adhesion to the postischemic endothelium of the downstream microvascular network.7,8,22 Although the contribution of platelets to large-artery atherothrombosis, the major trigger of organ ischemia, is generally accepted, the molecular requirements for accumulation of platelets in the target microvascular beds in response to subsequent reperfusion and their contribution to further injury has received little attention. Here, we show in vivo that platelets adhere to postischemic arterioles and venules in a GP IIb–dependent manner. In contrast, loss of GP IIb did not affect initial platelet tethering/rolling, an event mediated primarily by the interaction between P-selectin and platelet P-selectin glycoprotein ligand 1.7,12 Importantly, GP IIb–dependent platelet adhesion contributes substantially to the pathophysiology of I/R–induced tissue injury. Correspondingly, we demonstrate here that mice lacking GP IIb are more resistant to transient focal cerebral ischemia compared with wild-type animals. Because the model of MCA occlusion used here is not associated with endothelial denudation,17 reduced cerebral I/R injury in GP IIb–null mice is likely to be largely a result of a reduction in microvascular platelet recruitment rather than loss of GP IIb–mediated platelet aggregation and thrombus formation arising from large-artery injury.
In conclusion, we have demonstrated in vivo that GP IIb regulates platelet adhesion both to exposed subendothelial extracellular matrices and to the dysfunctional endothelium. In this manner, GP IIb plays an important, previously unexpected multifactorial role at various stages during atherosclerosis. (1) Early in the process of atherosclerosis, platelets are recruited to the dysfunctional endothelium of large arteries and contribute to atheroprogression via GP IIb. (2) After rupture of manifest atherosclerotic plaques with ensuing exposure of extracellular matrices, GP IIb integrin mediates platelet aggregation and, unpredictably, also promotes primary platelet adhesion. (3) Finally, atherothrombosis and subsequent organ ischemia induce GP IIb–dependent platelet adhesion in the target microvasculature, an event that aggravates cerebral I/R injury.
We acknowledge the excellent technical assistance of Sandra Kerstan, Kirsten Langenbrink, Uta Mamrak, and Renate Hegenloh. This study was supported by the Graduiertenkolleg GRK 438 of the Deutsche Forschungsgemeinschaft. Drs Massberg and Gawaz received grants from the Deutsche Forschungsgemeinschaft (MA 2186/3-1) and the Wilhelm-Sander-Stiftung.
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Platelet adhesion and aggregation play an important role in arterial thrombosis, the acute complication that develops on the chronic lesions of advanced atherosclerosis, triggering myocardial infarction and stroke. Platelet activation during adhesion/aggregation results in the discharge of platelet granule contents, including cytokines/chemokines such as interleukin-1β, RANTES, and platelet factor 4. Although platelets trigger a variety of immune responses in vascular cells in vitro, the in vivo contribution of platelets to inflammatory vascular processes such as atherosclerosis and I/R injury remains undefined. Platelet aggregation, the major amplification step during thrombus formation, is mediated by the platelet integrin GP IIb/IIIa. Consequently, short-term inhibition of GP IIb/IIIa has improved the treatment of unstable angina pectoris and myocardial infarction. In our present study, we identified an additional role of platelet GP IIb. Using mice lacking GP IIb, we show in vivo that apart from promoting platelet aggregation, GP IIb may also contribute to the process of platelet adhesion to the vessel wall. Importantly, mice lacking GP IIb were protected from atherosclerosis and had increased resistance to focal cerebral ischemia. Therefore, apart from their acute antithrombotic effects, prolonged antiplatelet regimens may be an effective strategy in the prevention of I/R injury and atheroprogression, particularly in patients at high risk of cardiovascular events.
↵*The first 2 authors contributed equally to this work.