Blood Journal
Leading the way in experimental and clinical research in hematology

The contributions of the α2β1 integrin to vascular thrombosis in vivo

  1. Li He,
  2. Loretta K. Pappan,
  3. David G. Grenache,
  4. Zhengzhi Li,
  5. Douglas M. Tollefsen,
  6. Samuel A. Santoro, and
  7. Mary M. Zutter
  1. From the Department of Pathology and Immunology and the Department of Medicine, Washington University School of Medicine, St Louis, MO.

Abstract

The α2β1 integrin serves as a receptor for collagens, laminin, and several other nonmatrix ligands. Many studies have suggested that the α2β1 integrin is a critical mediator of platelet adhesion to collagen within the vessel wall after vascular injury and that the interactions of the platelet α2β1 integrin with subendothelial collagen after vascular injury are required for proper hemostasis. We have used the α2β1 integrin-deficient mouse to evaluate the contributions of the α2β1 integrin in 2 in vivo models of thrombosis. Studies using a model of endothelial injury to the carotid artery reveal that the α2β1 integrin plays a critical role in vascular thrombosis at the blood-vessel wall interface under flow conditions. In contrast, the α2β1 integrin is not required for the formation of thrombi and pulmonary emboli following intravascular injection of collagen. Our results are the first to document a critical in vivo role for the α2β1 integrin in thrombus formation at the vessel wall under conditions of shear following vascular injury. (Blood. 2003;102:3652-3657)

Introduction

The α2β1 integrin serves as a receptor for collagens, laminin, and several other nonmatrix ligands.1-3 The integrin has been extensively studied as a collagen receptor on platelets.4-7 Many studies have suggested that the α2β1 integrin is a critical mediator of platelet adhesion to collagen within the vessel wall after vascular injury and that the interactions of the platelet α2β1 integrin with subendothelial collagen after vascular injury are required for proper hemostasis. Collagen not only serves as an adhesive substrate, but in its fibrillar form also activates platelets leading to secretion of granule contents and platelet aggregation.4 The relative roles of the α2β1 integrin and the glycoprotein VI (GPVI)/Fc receptor γ (FcγR) chain complex, another platelet collagen receptor, in platelet adhesion, platelet activation, and platelet aggregation have been intensely debated.8-19 Experimental evidence documenting a role for the platelet α2β1 integrin in thrombus formation in vivo has been lacking.

The extent of α2β1 expression varies greatly among individuals and is determined by several linked polymorphisms within the α2-integrin subunit gene.20-22 A potentially important role for the α2β1 integrin is suggested by recent epidemiologic data. Some data reveal a direct correlation between the genetically determined surface density of platelet α2β1-integrin expression and the risk of thrombotic events. High-level expression of α2β1 integrin on platelets is an independent risk factor for nonfatal myocardial infarction in individuals younger than 62 years of age, for the development of diabetic retinopathy in patients with type 2 diabetes mellitus, and for stroke in the young.23-28 However, other studies failed to find a correlation between the level of expression of α2β1 integrin and the risk of thrombotic events. The mechanistic relationship inferred between the level of expression of α2β1 integrin and the development of vascular disease requires experimental evaluation in vivo using animal models.

To better define the role of the α2β1 integrin in vivo, we have recently developed a genetically engineered mouse in which expression of the α2β1 integrin has been completely eliminated.29 Mice deficient in the α2β1 integrin are viable and fertile and develop normally. Platelets from α2β1-deficient mice fail to adhere to collagen substrates under either static or flow conditions and they exhibit mildly impaired collagen-induced platelet aggregation. We now report that α2β1 integrin-deficient mice have delayed thrombus formation following carotid artery injury.

Materials and methods

Mice

The development of mice with complete genetic deletion of the α2-integrin subunit gene was previously described.29 Wild-type (α2+/+), heterozygous (α2+/-), and α2-deficient (α2-/-) mice were descendants of F2 intercrosses and were on a mixed C57Bl/6-129SvJ background. On average, the mice contained approximately 50% C57Bl/6 and 50% 129SvJ alleles. Heterozygous breeding pairs produced littermates that were wild type, heterozygous, or α2 deficient. Littermate controls were used in all experiments. The FcγR chain-deficient mice on a mixed C57Bl/6-129SvJ background were a generous gift from Dr Paul Allen of Washington University School of Medicine (St Louis, MO). Mice were weaned to a rodent diet at 21 days of age and used for experiments when 10 to 15 weeks of age.

Blood collection and blood counts

Mice were anesthetized with a ketamine and xylazine mixture, and blood was collected by puncture of the retro-orbital plexus with a Pasteur pipette and placed into EDTA (ethylenediaminetetraacetic acid)-coated microtainer tubes. Blood was analyzed for platelet count and hematocrit using a Mascot Hemavet 850 analyzer (CDC Technologies, Oxford, CT) set to a mouse cell program.

Arterial thrombosis model

Carotid artery thrombosis was induced as described previously.30 Briefly, male mice approximately 12 weeks of age were anesthetized with intraperitoneal sodium pentobarbital, secured in the supine position, and placed under a dissecting microscope. The right common carotid artery was isolated through a midline cervical incision, and an ultrasonic flow probe (Model 0.5 VB; Transonic Systems, Ithaca, NY) was applied. A 1.5-mW, 540-nm laser beam (Melles Griot, Carlsbad, CA) was applied to the artery from a distance of 6 cm. Rose bengal dye (Fisher Scientific, Fair Lawn, NJ), 50 mg/kg body weight, was then injected into the tail vein, and flow in the vessel was monitored until complete occlusion occurred.

Collagen-induced thrombosis

Studies of collagen-induced thrombosis were carried out as described by Smyth et al.31 Wild-type, α2β1 integrin-deficient, and FcRγ-deficient female mice were anesthetized by intraperitoneal injection of 100 to 150 μL of a mixture of ketamine and xylazine. Blood was collected into EDTA-coated microtainer tubes for determination of the baseline platelet count and hematocrit. Various amounts (25, 12, 6, and 3 μg) of collagen (equine tendon type I fibrillar collagen; Chrono-log, Havertown, PA) plus 1 μg epinephrine (Sigma, St Louis, MO) in phosphate-buffered saline (PBS), or PBS alone, were injected into the right jugular vein; 1 minute after injection a second blood sample was taken. Mice were humanely killed 3 minutes after injection and lungs were collected and placed in formalin.

Quantitation of pulmonary thrombosis

Lung sections stained with hematoxylin and eosin were digitally imaged using an Olympus Camedia C-3040 Zoom camera (Melville, NY). Five random × 20 fields were photographed for each specimen. Analysis of thrombus number for each mouse was made using Olympus Camedia Master 2.5 software, and then expressed as thrombi per square millimeter ± SEM.

Statistical analysis

Statistical significance was determined with the Student 2-tailed t test (http://faculty.vassar.edu/lowry/VassarStats.html). P values less than .05 was considered significant.

Histology

Tissues examined by light microscopy, including carotid artery segments and lungs, were fixed in 10% formalin and embedded in paraffin. Sections (4 μm) were stained with Harris hematoxylin and eosin.

Results

Carotid artery thrombosis

To determine the importance of the α2β1 integrin in acute thrombosis at the site of vascular injury we used a model of carotid artery occlusion. Mice deficient in the α2β1 integrin (8 males), their wild-type littermate controls (7 males), and their heterozygous littermates (6 males) were subjected to photochemical injury of the right common carotid artery. Carotid artery blood flow was monitored continuously throughout the experiment via a Doppler flow probe. The recordings of carotid blood flow from injured wild-type, heterozygous, or α2-deficient mice are presented in Figure 1A. The length of time to complete arterial occlusion following injury was 47 ± 9 minutes (mean ± SD) in the wild-type littermates (Figure 1B). In contrast, the time to arterial occlusion was 74 ± 20 minutes for the homozygous α2-deficient animals. Thus, the α2-deficient animals required almost twice the length of time to complete arterial occlusion following injury. The difference was statistically significant at P = .004. Comparison of the tracings of carotid artery blood flow in wild-type and heterozygous mice versus α2-deficient mice suggests that thrombus formation is initiated normally but that thrombi are less stable or are formed at a slower rate in the α2-deficient animals. The length of time to occlusion observed in the heterozygous α2-deficient littermates was 45 ± 11 minutes. This time to occlusion was not different from the time to complete occlusion observed in the wild-type mice. Thus, complete deficiency of the integrin was required for prolongation of the occlusion time following injury.

Figure 1.

Thrombotic occlusion of the carotid artery following photochemically induced endothelial injury. (A) The blood flow recordings of the α2+/+, α2+/-, and α2-/- mice. Rose bengal dye was injected at time = 0 minutes. (B) The length of time to complete arterial occlusion following photochemical injury was determined. The values represent the mean ± SD of α2+/+ (n = 7), α2+/- (n = 6), and α2-/- (n = 8). (C) Photomicrographs of transverse sections of the carotid arteries of α2+/+ mice and α2-/- mice excised immediately after complete thrombosis. Sections were stained with hematoxylin and eosin; original magnification, × 200.

Carotid arteries obtained immediately following complete occlusion were evaluated morphologically on routine hematoxylin and eosin-stained sections. As expected, the thrombi consisted of a mixture of platelets and fibrin (Figure 1C). There was no obvious histologic difference between the thrombi found in wild-type and α2-deficient mice.

Collagen-induced pulmonary embolism

The role of the α2β1 integrin was also evaluated in a model in which pulmonary embolism is induced by the intravenous injection of collagen. Baseline platelet counts were similar in wild-type (719 ± 124 × 103/μL) and α2-deficient (735 ± 144 × 103/μL) mice. The jugular vein of wild-type or α2 integrin-deficient female mice, 10 to 15 weeks of age, was injected with either saline or saline containing fibrillar collagen (25 μg) and epinephrine (1 μg). The absolute decrements in platelet count from baseline following injection of collagen (or saline) were determined. One minute after injection of 25 μg collagen the platelet count of wild-type mice (n = 21) decreased by 527 ± 97 × 103/μL or 74% ± 8% (Figure 2A). When α2-deficient mice (n = 21) were injected with fibrillar collagen, the platelet decrement was 589 ± 139 × 103/μL or 79.5% ± 8%. The decrease in platelet count between the α2-deficient mice and the wild-type littermate controls was not statistically significant (P > .05). Following injection with saline, the platelet count decrement for α2-deficient mice and wild-type mice was minimal, 87 ± 21 × 103/μLor 95 ± 39 × 103/μL, respectively.

Figure 2.

α2β1 integrin-independent collagen-induced pulmonary embolism. (A) The absolute decrement in the platelet count from baseline of α2+/+ or α2-/- mice one minute after injection of the indicated amount of collagen (or saline) plus epinephrine (1 μg) was determined. The values represent the mean ± SEM for each concentration of collagen injected (25 μg [n = 21, α2+/+; n = 21, α2-/-]), 12 μg [n = 6, α2+/+; n = 6, α2-/-], 6 μg [n = 7, α2+/+; n = 8, α2-/-], or 3 μg [n = 7, α2+/+; n = 7, α2-/-]). (B) The number of thrombi in the lungs at the time of killing 3 minutes after injection of the indicated amount of collagen was determined. Lung sections stained with hematoxylin and eosin were digitally imaged using an Olympus Camedia C3040 Zoom camera. The number of thrombi in × 20 fields was quantitated and expressed as thrombi per millimeter squared ± SEM for each concentration of collagen injected (25 μg [n = 27, α2+/+; n = 23, α2-/-], 12 μg [n = 6, α2+/+; n = 7, α2-/-], 6 μg [n = 7, α2+/+; n = 11, α2-/-], or 3 μg [n = 3, α2+/+; n = 3, α2-/-]). (C) Representative histologic sections of the lungs of α2+/+ mice injected with saline alone, or α2+/+, α2-/-, or FcγR-/- mice injected with 25 μg collagen. Shown at × 200 original magnification. Thrombi were not observed in α2+/+ mice injected with saline. In contrast, many thrombi were identified in the lungs of α2+/+ and α2-/- mice injected with collagen. Only rare thrombi were observed in FcγR-/- mice.

The number of thrombi in the lungs at the time of death 3 minutes after the injection was also quantitated (Figure 2B-C). Consistent with the decrements in platelet count, the numbers of intravascular thrombi in the lungs of wild-type and α2 integrin-deficient mice were 22 ± 1 thrombi/mm2 (n = 27) and 22 ± 1 thrombi/mm2 (n = 23), respectively, and were not different statistically (P > .5; Figure 2B-C). No thrombi were observed in the lungs of control mice injected with saline alone. Therefore, the presence of the α2β1 integrin is not required for formation of pulmonary emboli induced by intravascular injection of a high concentration of collagen (ie, 25 μg).

We previously reported that platelets from α2-null animals aggregated in response to high concentrations of type I collagen in a manner similar to platelets from wild-type littermates.29 In contrast, platelets from α2-null animals aggregated with a significantly prolonged lag phase and a decreased rate of platelet aggregation in response to low concentrations of collagen.29 We, therefore, evaluated whether the responses induced by lower doses of collagen in terms of decreased platelet count and formation of pulmonary emboli were different between wild-type and α2 integrin-deficient mice. As shown in Figure 2A-B, even at lower doses of collagen (3-12 μg) there was no difference in the platelet count decrement (Figure 2A) or in the formation of pulmonary emboli (Figure 2B) between α2-deficient and wild-type mice (P > .05 for 12 μg [n = 6, α2+/+; n = 6, α2-/-], 6 μg [n = 7, α2+/+; n = 8, α2-/-], or 3 μg [n = 3, α2+/+; n = 3, α2-/-] collagen).

It has been previously reported that mice deficient in the GPVI/FcR γ complex are protected from the formation of thrombi and pulmonary emboli following intravenous injection of fibrillar collagen.32 As a control for the negative findings regarding the role of the α2β1 integrin, we therefore examined the effect of intravenous injection of collagen on FcγR-deficient mice. Our results were in accord with the previous report. As shown in Figure 3A, the decrement in platelet count was only 133 ± 106 × 103/μL(n = 5) or 18% ± 15% in the FcγR-deficient mice. Healthy wild-type control mice decreased their platelet count by 483 ± 189 × 103/μL or 73% ± 3%. The difference was statistically significant at P = .0007. The number of pulmonary thrombi formed in the lungs of FcγR-deficient mice (5 ± 1 thrombi/mm2) following collagen injection was strikingly reduced when compared to wild-type mice (25 ± 1 thrombi/mm2) with P = .0001 (Figures 3B and 2C).

Figure 3.

FcγR chain-dependent collagen-induced pulmonary embolism. (A) The number of thrombi in the lungs of FcγR+/+ or FcγR-/- mice at the time of sacrifice 3 minutes after injection of 25 μg collagen was determined. Data are presented as mean ± SEM. (B) The absolute decrement in the platelet count from baseline of FcγR+/+ or FcγR-/- mice one minute after injection of 25 μg collagen was determined.

Discussion

Collagen serves as an adhesive substrate and in its fibrillar form activates platelets leading to secretion of granule contents and platelet aggregation.4-7 A number of studies have suggested that the α2β1 integrin is a critical mediator of platelet adhesion to subendothelial collagen and is required for effective hemostasis. The relative roles that the α2β1 integrin and the GPVI/FcγR complex play in mediating platelet adhesion and subsequent platelet activation, aggregation, and thrombus formation have been intensively debated.4-7,33 The sometimes conflicting results from various in vitro or ex vivo experimental systems that may differentially reflect the relative contributions of the 2 receptors have perhaps contributed to the confusion.

In the original version of the 2-step, 2-site model of collagen-induced platelet adhesion and activation, it was proposed that the α2β1 integrin supported the initial adhesion of platelets to vessel wall collagen.11 In a second step, a low-affinity signal transducing coreceptor for collagen, most likely GPVI, was subsequently engaged and mediated platelet activation and aggregation.8-19 Thus, the α2β1 integrin was thought to play a major role in adhesion, but was thought not to contribute signals to platelet activation or aggregation.12,13 More recent findings suggest that both the α2β1 integrin and GPVI may contribute to the overall process of platelet adhesion, activation, and aggregation, and that the relationship between the 2 receptors and their relative contributions may be more complex than initially envisioned.18 Furthermore, tethering of platelets to exposed subendothelium via a von Willebrand factor (VWF)-GPIb interaction precedes engagement of either of the 2 collagen receptors. Under some conditions tethering by VWF may be sufficient for a limited initiation of signaling through the GPVI complex.

The recent development of mice deficient in all β1 integrins or selectively deficient in the α2β1 integrin, GPVI, or the FcγR has provided unique and valuable tools to definitively characterize the independent and cooperative roles of the α2β1 integrin and the GPVI/FcγR complex in platelet biology and to address key unresolved issues regarding the role of the 2 receptors in platelet adhesion, activation, signaling, and vascular pathobiology.15-18,32,34-37 The α2-deficient mice have no obvious bleeding tendency.16,29 Using washed platelets purified from α2-deficient mice, we previously reported that the α2β1 integrin is required for platelet adhesion to collagen under both static and flow conditions. Prolongation of the lag phase of collagen-induced platelet aggregation was observed only at low concentrations of collagen.29

Our results differed in some ways from those of Holtkotter et al.16 Because their initial in vitro studies of platelet adhesion and aggregation using α2β1-deficient platelets under flow conditions in whole blood failed to show a collagen binding defect, they concluded that the α2β1 integrin was not required for platelet adhesion to collagen under flow conditions. However, Kuijpers et al17 more recently reported that α2β1-null platelets are, in fact, less adhesive to collagen and form loose platelet aggregates under flow conditions in vitro. All studies reported thus far are in accord that the α2β1 integrin is not essential for platelet aggregation as measured in vitro.

We have now used the α2β1 integrin-deficient mouse to tackle the question: what is the role of the α2β1 integrin in physiologic and pathophysiologic thrombus formation in vivo? We compared the responses of wild-type mice and α2β1 integrin-deficient mice in 2 models of in vivo thrombosis. The first model of photochemical injury to the carotid artery endothelium addressed the role of platelet adhesion and aggregation in thrombus formation at the blood-vessel wall interface following a defined endothelial injury.30,38-40 Absence of the α2β1 integrin significantly delays by almost 2-fold the time to complete vessel occlusion. The results obtained with the carotid artery endothelial injury model in vivo are consistent with the previously established role of the α2β1 integrin for adhesion under both static and flow conditions in vitro. In addition, the findings suggest that the absence or pharmacologic inhibition of the α2β1 integrin may be protective against the thrombotic complications of vascular disease. This conclusion is consistent with the previously demonstrated correlation between high-level expression of α2β1 integrin and an increased risk for thrombosis involving coronary and cerebral vessels.21-28 Based on some epidemiologic data, we expected to observe a gene dosage effect in mice heterozygous for α2β1-integrin deficiency. However, no gene dosage effect in the carotid injury model was observed. Only homozygous deficient animals were protected. These results are in line with the studies of Moshfegh and coworkers, who reported that homozygosity, but not heterozygosity, for the 807Thr/873Ala genotype was an independent risk factor for acute myocardial infarction.27 Our findings suggest that if pharmacologic inhibition of the α2β1 integrin is to be exploited clinically, a high degree of inhibition will be required for efficacy.

In contrast to the important role that the integrin plays in platelet adhesion and thrombus formation at the blood-vessel wall interface under flow conditions, the integrin does not appear to play a role in a model of pulmonary embolism following the intravascular injection of collagen to initiate thrombus formation. Although deficiency of α2β1 integrin was not protective, mice deficient in the FcγR subunit of the GPVI/FcγR complex were completely protected from the collagen-induced formation of pulmonary emboli. Nieswandt et al32 also recently reported that mice depleted of GPVI were completely protected from lethal collagen-induced pulmonary thromboemboli using a similar model. The results obtained in the intravascular collagen injection/pulmonary embolism model are reminiscent of the results of the in vitro platelet aggregation studies in their striking dependence on the presence of the GPVI/FcγR complex, but not on the α2β1 integrin. It is possible that the binding of VWF to collagen following its intravascular injection facilitates the tethering of platelets to collagen through the GPIb complex to an extent sufficient to initiate signaling via the GPVI complex.

In summary, the studies described in this report reveal distinctly different roles for the α2β1 integrin in 2 models of in vivo platelet thrombus formation. The carotid artery injury model in which the initial platelet-collagen interactions occur with collagen in the solid phase in the presence of shear forces at the flowing blood-vessel wall interface revealed an obligatory role for the α2β1 integrin. In contrast, in the model of pulmonary embolus formation following intravascular injection of collagen where the initial encounters of platelets with collagen occurred in suspension and in the absence of shear in a manner rather analogous to the conditions of collagen-induced platelet aggregation in vitro, no dependence on the α2β1 integrin was observed. As in the in vitro determination of collagen-induced platelet aggregation, GPVI was required. In the pulmonary embolism model significant shear forces are encountered only after platelet aggregate formation is complete at the time thrombi lodge in the pulmonary vasculature.

Several factors likely contribute to the observed differences in the role of the α2β1 integrin in the 2 models. First, it is likely that firm adhesion to collagen in the presence of shear requires the α2β1 integrin. This role of the integrin would be analogous to the role of the leukocyte β2 integrins in the leukocyte adhesion to the vascular endothelium under conditions of flow. Second, the unique signals identified by Inoue et al33 that are induced by ligation of the α2β1 integrin with solid-phase collagen but not by fluid-phase collagen may be essential for the development of a thrombus resistant to shear at the blood-vessel wall interface. Third, whereas some signals emanating from ligation of α2β1 integrin are independent of those emanating from the GPVI/FcγR complex, others such as the tyrosine phosphorylation of Src, Syk, and phospholipase Cγ2 (PLCγ2) are derived from both receptors.18 It seems likely that stable thrombus formation at the blood-vessel wall interface requires a more complete activation of these pathways that is dependent on the presence and engagement of the α2β1 integrin.

Acknowledgments

We would like to thank Mary Beth Flynn for secretarial assistance and Dr Paul Allen for providing the FcγR-deficient mice.

Footnotes

  • Reprints:

    Mary M. Zutter, Department of Pathology, Vanderbilt University School of Medicine, Pierce and 22nd Ave, 4800 B, The Vanderbilt Clinic, Nashville, TN 37232; e-mail: mary.zutter{at}vanderbilt.edu.
  • Prepublished online as Blood First Edition Paper, July 31, 2003; DOI 10.1182/blood-2003-04-1323.

  • Supported by National Institutes of Health grants RO1 HL55520, HL63446, and CA70275, and a grant from the Edward Mallinckrodt Jr Foundation. L.H. and L.K.P contributed equally to the manuscript.

  • 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.

  • Submitted April 28, 2003.
  • Accepted July 23, 2003.

References

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