The contributions of the α₂β₁ integrin to vascular thrombosis in vivo

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)/F_c receptor γ (F_cγR) 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.

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 F_cγR 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.

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.

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.

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.

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 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.

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.

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.

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.

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.

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/F_cR 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 F_cγR-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 F_cγR-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 F_cγR-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).

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/F_cγR 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 F_cγR has provided unique and valuable tools to definitively characterize the independent and cooperative roles of the α₂β₁ integrin and the GPVI/F_cγR 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 F_cγR subunit of the GPVI/F_cγR 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/F_cγR 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/F_cγR 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.

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