Decreased Thrombotic Tendency in Mouse Models of the Bernard-Soulier Syndrome
Objective— The platelet glycoprotein (GP)Ib-V-IX complex is a receptor required for normal hemostasis deficient in the Bernard-Soulier bleeding disorder. To evaluate the consequences of GPIb-V-IX deficiency in thrombosis we generated mouse models of the disease by targeting the GPIbβ subunit.
Methods and Results— Complete deletion (GPIbβ−/−) or an intracellular truncation (GPIbβΔIC−/−) reproduced typical and variant forms of Bernard-Soulier, with absent and partial (20%) expression of the complex on the platelet surface. Both strains exhibited thrombocytopenia and enlarged platelets with abnormal microtubular structures but normal granule composition. They exhibited prolonged tail bleeding times, which were less pronounced in GPIbβΔIC−/−. Decreased thrombus formation was observed after blood perfusion over a collagen coated surface at high shear. Resistance to vascular occlusion and an abnormal thrombus composition were observed in a model of FeCl3-induced lesion of carotid arteries. In a model of laser-induced lesion of mesenteric arterioles, thrombosis was strongly reduced in GPIbβ−/− mice, while a more modest effect was observed in GPIbβΔIC−/− animals. Finally, the two strains were protected against death in a model of systemic thromboembolism.
Conclusions— This study provides in vivo evidence of a decreased thrombotic tendency linked to defective platelet GPIb-V-IX in mouse models of Bernard-Soulier syndrome.
The glycoprotein (GP)Ib-V-IX complex is a specialized multi subunit receptor abundantly expressed at the surface of platelets which has a key role in normal hemostasis by ensuring reversible adhesion of platelets to exposed subendothelial collagen via von Willebrand factor (vWF).1,2 The GPIb-V-IX complex has also been shown to be responsible for intracellular signal transduction via interaction of intracellular domains with signaling molecules.3 An important role in the platelet morphology completes its main functions. These properties are clearly illustrated by the existence of a severe bleeding tendency in patients with the rare Bernard-Soulier syndrome who carry genetic defects of GPIb-V-IX.4 These patients also have decreased numbers of platelets of enlarged size owing to still unresolved mechanisms.
The extreme rarity of Bernard-Soulier has prevented correct assessment of the incidence of thrombotic events in this disease. One report described the occurrence of unstable angina in a Bernard-Soulier patient, suggesting that the absence of the complex does not fully protect against thrombosis.5 Indications of decreased thrombosis have also come from assays involving perfusion of Bernard-Soulier blood over thrombogenic surfaces such as exteriorized blood vessels or collagen.6,7 Mice deleted of the GPIbα or GPIbβ subunit have been reported to reproduce the bleeding and platelet morphological defects found in Bernard-Soulier patients, but there is as yet no information concerning their thrombotic tendency.8,9 Recently, transgenic mice missing the GPIbα extracellular domain, which display a partial Bernard Soulier phenotype, without the additional abnormalities in platelet morphology and count, have been reported with decreased arterial thrombosis.10
To evaluate the degree of protection against thrombosis in Bernard-Soulier syndrome we generated two mouse strains by genetic recombination: a knockout strain lacking receptor expression by inactivating the GPIbβ gene (GPIbβ−/−) and a knock-in strain by interrupting the GPIbβ intracellular domain (GPIbβΔIC−/−). Thrombosis was studied in vitro in collagen perfusion assays and in several in vivo artery injury and thromboembolism models.
Materials and Methods
Generation of GPIbβ−/− and GPIbβΔIC−/− Animals
A knockout construct (GPIbβ−/−) was generated by replacing the coding sequence with a neo cassette. A knock-in construct (GPIbβΔIC−/−) was obtained by introducing a stop codon after the transmembrane domain. The targeting vectors were electroporated into ES cells followed by injection into blastocytes and implantation into pseudopregnant females. GPIbβ−/−, GPIbβΔIC−/−, and GPIbβ+/+ mouse colonies were established by breeding heterozygotes. For detailed methods please see the supplemental materials (available online at http://atvb.ahajournals.org).
Platelet Preparation, Flow Cytometry, Western Blotting, and Electron Microscopy
Please see the supplemental materials for details
Bleeding Time Assays
The bleeding time was measured by severing a 3-mm segment from the distal end of the tail of 6- to 8-week-old mice. Please see the supplemental materials for details.
In Vitro Model of Thrombosis on Immobilized Collagen in a Flow System
Platelet adhesion under flow was studied as described previously.11 Whole blood anticoagulated with hirudin (100 U/mL) was perfused at 1500 s−1 or 3000 s−1 through a collagen-coated glass capillary and surface coverage was evaluated by off-line analysis. Some samples were prepared for scanning electron microscopy. Please see the supplemental materials for details.
FeCl3-Induced Carotid Artery Thrombosis
FeCl3-induced arterial injury was performed according to published procedures.12,13 The right common carotid artery was exposed to 20% FeCl3 for 3 minutes, rinsed with saline, and the blood flow was monitored for 30 minutes. The time to first occlusion and the numbers of arteries respectively patent and occluded at 30 minutes were recorded. Histological samples were prepared by perfusing the left ventricle with 3 mL of 4% paraformaldehyde in PBS, after which the artery was removed, incubated overnight in 4% paraformaldehyde solution, and embedded in paraffin. Samples for light and transmission electron microscopy (TEM) were prepared by fixing the arteries with 2.5% glutaraldehyde. Please see the supplemental materials for details.
Laser-Induced Mesenteric Artery Thrombosis
Laser-induced arterial thrombosis was studied as described by Nonne et al.11 Reproducible superficial lesions inducing reversible parietal thrombi or more extensive almost occlusive thrombi were produced by adjusting the firing time and laser intensity. Please see the supplemental materials for details.
The model of acute systemic vascular thromboembolism by infusion of a mixture of collagen (0.3 mg/kg) and adrenaline (60 μg/kg) has been described previously.14 Please see the supplemental materials for details.
Non parametric Mann–Whitney tests, area under curves, and Logrank tests were performed using GraphPad PrisM version 3.00 for Windows (GraphPad Software; ***P<0.0001, ** P<0.01, *P<0.05; ns P>0.05).
Generation of Mouse Models of Bernard-Soulier Syndrome Targeting the GPIbβ Subunit
Two targeting vectors were designed for inactivation of the GPIbβ gene (GPIbβ−/−) and deletion of the GPIbβ intracellular domain (GPIbβΔIC−/−; supplemental Figure I). GPIbβ−/− was obtained by inserting a neo cassette into the second exon, thereby eliminating most of the coding sequence. GPIbβΔIC−/− was generated by inserting a stop codon into the native sequence three amino acids after the transmembrane domain. After electroporation and karyotype analysis, one KO clone (194 ES) and two KI clones (59 ES and 22 ES) were selected for injection into blastocytes. Animals with germline transmission were obtained and the offspring from crossing of heterozygotes were screened for the +/+, +/−, and −/− genotypes. A Mendelian distribution and no overt developmental or morphological abnormalities were observed for the two mutations.
The lack of expression of GPIbβ in platelets from GPIbβ−/− mice was confirmed by Western blotting using the mAb RAM.1 against the extracellular domain of mouse GPIbβ15 (supplemental Figure I). In GPIbβΔIC−/− cells, a GPIbβ reactive band with a lower molecular weight (18 kDa) was revealed by RAM.1, confirming the deletion of this domain.
Hematologic Parameters, Platelet Properties and Bleeding Tendencies in GPIbβ−/− and GPIbβΔIC−/− Mice
Analysis of the hematologic parameters of GPIbβ−/− and GPIbβΔIC−/− revealed the characteristic decreased platelet counts and enlarged platelets of the Bernard-Soulier syndrome but normal leukocyte and erythrocyte counts (Figure 1A). Heterozygotes from both strains had normal platelet counts and morphology (data not shown).
Transmission electronic microscopy (TEM) analysis revealed discoid platelets with an enlarged diameter (6±1 for GPIbβ−/− and 6±2 μm for GPIbβΔIC −/−) compared with control GPIbβ+/+ (2±1 μm; Figure 1B). Intracellularly, α- granules were present at a normal density and size but a 2-fold increase in the number of microtubules per platelet was observed as compared with control platelets with however a normal equatorial localization (Figure 1C).
Flow cytometry analysis showed that GPIbβ−/− platelets lacked GPIbβ expression and expressed little GPIbα (3% of GPIbβ+/+ cells). By contrast, in the GPIbβΔIC−/− mice, GPIbβ, and GPIbα represented close to 20% of normal levels (Figure 1D). Expression of integrin αIIbβ3 in GPIbβ−/− and GPIbβΔIC−/− represented 170% as compared with GPIbβ+/+ attriubutable to increased platelet size (data not shown).
Platelet counts were reduced by 73% and 71% in GPIbβ−/− and GPIbβΔIC−/− mice, respectively, compared with wild-type littermates (Figure 1A). Heterozygotes had normal platelet counts and morphology (data not shown).
In wild-type animals, 82% had bleeding times shorter than 12 minutes, whereas 76% of the GPIbβ−/− mice bled for more than 20 minutes (Figure 2). GPIbβΔIC−/− mice had a decreased bleeding tendency than GPIbβ−/− with 52% of the mice bleeding after 20 minutes. Heterozygotes from the GPIbβ−/− and GPIbβΔIC−/− strains had bleeding times similar to those of their respective +/+ genotypes (data not shown).
Decreased Thrombus Formation During Perfusion of GPIbβ−/− and GPIbβΔIC−/− Blood Over a Collagen Surface at High Shear Rates
The thrombotic tendency was first explored in blood perfusion assays over immobilized collagen (Figure 3). In GPIbβ+/+, single adherent platelets progressively formed aggregates which were individually larger at 3000 s−1 than at 1500 s−1 after 2 minutes (Figure 3A and 3D), with a slightly increased total surface coverage at 3000 s−1 (Figure 3B and 3E). A major defect was observed in GPIbβ−/− and GPIbβΔIC−/− blood at 3000 s−1, where platelet adhesion was nearly abolished with respective decreases of 98 and 83% in the total surface coverage and no evidence of aggregate formation at 2 minutes (Figure 3B). Electron microscopy analysis revealed the rare attachment of individual GPIbβ−/− platelets to the collagen fibers and attachment of a few GPIbβΔIC−/− platelets in strings along the fibers (Figure 3C). These cells were however almost incapable of attracting circulating platelets to form an aggregate. At 1500 s−1 some adhesion and aggregates were observed in GPIbβ−/− blood which were greatly decreased in size as compared with the wild-type (Figure 3F). At this shear rate, adhesion and aggregate formation occurred in GPIbβΔIC−/− blood and was approximately 50 to 60% of that in wild-type blood (Figure 3E). Therefore, the absence of the GPIb-IX complex or a decrease in surface expression, following truncation of its intracellular domain, resulted in a decreased tendency to thrombus formation in vitro under high shear conditions.
Decreased Arterial Thrombosis in GPIbβ−/− and GPIbβΔIC−/− Mice
Thrombotic tendency was then evaluated in vivo in two arterial models. The first was a carotid artery model where injury was induced by application of FeCl3 (Figure 4). In wild-type (n=8), stable occlusion occurred in 75% of the arteries, with permanent cessation of flow (Figure 4A). In GPIbβ−/− (n=8), stable occlusion occurred in only 12.5% of the arteries, whereas 25% exhibited unstable occlusion and 62.5% did not occlude. A decrease in stable occlusion was also observed in GPIbβΔIC−/− (n=8) animals where 62.5% of the arteries were patent (unstable or no occlusion) at the end of the 30 minutes period (Figure 4A). The time to first occlusion, either stable or transient, was increased in GPIbβ−/− (1646±84 s) or GPIbβΔIC−/− (1242±169 s) as compared with their wild-type littermates (997±135 s and 631±68 s) (Figure 4B). Histological analysis revealed that the thrombi formed in GPIbβ−/− and GPIbβΔIC−/− animals were less extensive than in the wild-type (Figure 4C) and ultrastructural analysis showed loose thrombi mainly composed of well demarcated platelets displaying less extensive shape change and little sign of degranulation (Figure 4D). This contrasted with the intricate appearance of wild-type aggregates which consisted of intertwined platelets having undergone degranulation.
In a second model, lesions of mesenteric arterioles were induced with a laser beam so as to obtain desquamation of the endothelial layer or rupture of the underlying layers, respectively.11 In wild-type mice, a reversible parietal thrombus formed after superficial injury, while a larger almost occlusive thrombus was produced after severe injury (Figure 5A and 5B). In GPIbβ−/−, thrombus formation was almost absent on superficial lesions and on more severe lesions reached a size representing only 25% of that in the wild type at 160 s (Figure 5A). In GPIbβΔIC−/−, the maximum peak after superficial injury was decreased by 45% as compared with controls, while initial thrombus growth was delayed after more severe injury but reached at later times a size comparable to that of wild-type (Figure 5B).
Decreased Systemic Thromboembolism in GPIbβ−/− and GPIbβΔIC−/− Mice
The GPIbβ−/− and GPIbβΔIC−/− strains were finally evaluated in a model of platelet-dependent intravascular thrombosis induced by intravenous injection of a mixture of collagen (0.3 mg/kg) and adrenaline (60 μg/kg). In wild-type mice, collagen/adrenaline injection led to widespread pulmonary vascular thrombosis with 90% mortality ≈6 minutes after injection (Figure 6). GPIbβ−/− and GPIbβΔIC−/− mice had a lower death rate, with 40% and 60% of the mice being still alive 30 minutes after collagen-adrenaline injection.
The central objective of this study was to determine the thrombotic tendency in mouse models of the Bernard-Soulier syndrome. A knockout strain deleted of the GPIbβ gene reproduced a typical Bernard-Soulier phenotype and most of the defects observed in a previously described GPIbβ deficient strain,9 including a tripling of the platelet size, a 75% decrease in platelet number, a profound decrease in GPIbα expression, and an increased bleeding tendency. A difference was noted at the ultrastructural level with normal density and size of platelet granules. This contrasted with the report of α-granules of increased size9 which was attributed to upregulation of SEPT5 expression which gene is located 5′ to the GPIbβ gene and modulates exocytosis. This discrepancy is difficult to explain but does not appear to be attributable to the targeting strategy which is very similar in the two studies with insertion of a Neo cassette at the same 5′-restriction site, and only differed in the length of the 3′ untranslated region. In the present study, a novel finding was the observation of a doubling in the number of microtubule rings in the knock-out strain which retained an equatorial location and the capacity to maintain a discoid shape despite the enlarged platelet size.
The GPIbβΔIC−/− strain, which was originally developed to explore signaling functions of the receptor, happened to exhibit decreased level of GPIb-IX on the platelet surface. This suggests that the GPIbβ intracellular domain is required for efficient expression of the complex. In fact, the GPIbβΔIC−/− resembled the knockout for the ultrastructural defects and decreased number of circulating cells, and was classified as a variant form of Bernard-Soulier. Reduced bleeding complications were however observed compared with the knockout implying that an 80% decrease in GPIb, even with the added intracellular mutation of GPIbβ, is sufficient to support minimal hemostasis.
The GPIbβ−/− and GPIbβΔIC−/− models allowed us to assess the thrombotic tendency associated with Bernard-Soulier. Defective thrombus formation was documented in collagen flow assays at high shear rates known to depend on GPIb/VWF interaction.1,2 These defects are in line with those observed in earlier studies of Bernard-Soulier patients after blood perfusion over collagen or vascular matrices.6,7 More recently a similar defect in collagen flow studies has been reported in transgenic mice engineered on a GPIbα knock out and missing the GPIbα extracellular domain.10 Compared with this strain, the GPIbβ−/− and GPIbβΔIC−/− have the added defect of enlarged platelets which could result in increased drag-forces exerted on the platelets. Nevertheless these two strains behaved very similarly to the GPIbα transgenic mice in this flow assay.
Other receptors involved in platelet-collagen interaction have been evaluated in similar ex vivo perfusion systems, such as integrin α2β1 and GPVI, which are the major receptors contributing to platelet adhesion and activation after direct interaction with collagen. GPVI-FcRγ deficiency induced by immunodepletion or after genetic ablation have established a critical role for GPVI in such perfusion assays over collagen16–18 whereas α2β1 deficiency resulted in normal thrombus formation.19 GPVI-deficient platelets tethered normally, but failed to spread and extend aggregates, in contrast with the deficient initial tethering in GPIb-deficient mice. In the experiments presented here, platelet tethering and aggregation nevertheless occurred in GPIb-deficient mice under conditions of intermediate shear (1500 s−1), which in human blood have been described as being GPIb-dependent. Platelet capture and activation by α2β1 and GPVI can probably take place at these shears in mouse blood10
In vivo results in a FeCl3 carotid thrombosis model were consistent with the flow experiments demonstrating a lower incidence of vessel occlusion in both GPIbβ−/− and GPIbβΔIC−/− mice. Histology and ultrastructural analyses of the carotids showed that thrombus formation was not completely prevented but that thrombi consisted of less extensive loose aggregates. This suggested a defective propagation of activation through the platelet layers, resulting in decreased thrombus growth. Transgenic mice lacking the GPIbα extracellular domain evaluated in a similar carotid model appeared to be similarly protected against vessel occlusion.10 No information was presented on the presence and histology of the thrombi. Thrombosis was also severely impaired after laser-induced arterial injuries, especially in the GPIbβ−/− strain. A more modest protection of GPIbβΔIC−/− contrasted with the carotid FeCl3 model where both strains were similarly protected. Such variable responses in the same mouse strain depending on the nature of the vessel injury have now been observed in several mouse strains. For example, FcRγ/GPVI deficiency has been reported to induce full or only minor protection against arterial thrombosis in different studies17,20 depending on the type of injury and exposure to collagen and thrombin.10,17,21 The degree of collagen exposure and thrombin activation have been reported to vary depending on the concentration of FeCl3 applied to arteries21 and the sensitivity to thrombin blockade can change with the extent of laser injury in mesenteric arteries.11,17 In spite of these differences, which await standardization between laboratories, this study and the recent work by Konstantinides et al establish that the GPIb-VWF interaction is, contrary to the GPVI-collagen axis, critical to arterial thrombosis independently of the nature of the lesion.
Abnormal laser-induced arterial thrombosis in the GPIbβ-deficient strain presented some similarities with results in vWF-deficient mice in a FeCl3 mesenteric artery injury model.22 In the absence of this GPIb ligand a defect was also observed at the early stages of adhesion and in thrombus growth. Interestingly, thrombosis was still observed in vWF-deficient mice23 similar to the present detection of parietal thrombi in the carotid artery model. This analogous response further supports the hypothesis that mechanisms in addition to GPIb-vWF–dependent responses can support arterial thrombus formation.
Less expectedly, GPIbβ-deficient mice were also protected against systemic thromboembolism, after intra venous collagen-adrenaline injection. These results suggest that this model, which is widely thought to reflect platelet responses to soluble agonists, could also depend on GPIb/VWF-dependent responses. The mechanisms of platelet activation in this model are not entirely understood, but vWF-dependent activation could theoretically occur in the lung microcirculation where high shear conditions are encountered.23
GPIbβ−/− and GPIbβΔIC−/− present abnormalities of platelet morphology and count in addition to vWF-binding deficiencies. These additional defects could also contribute to decrease thrombus formation. Enlarged platelets are potentially more susceptible to stress and would have harder time to adhere. However this parameter is probably not sufficient to explain the whole defect as different thrombotic tendencies were observed in GPIbβ−/− and GPIbβΔIC−/−, despite a similar platelet size increase. Low platelet counts have also been linked to decreased thrombus formation in perfusion models.24 To evaluate the influence of thrombocytopenia in our models, GPIbβΔIC−/− mice were injected with TPO to increase platelet numbers. Despite doubling of the platelet count, comparable defects in thrombus formation were observed in collagen flow assays (data not shown). Similarly, doubling of the platelet count in splenectomized GPIbβΔIC−/− mice did not correct the defect in superficial laser-induced lesions (data not shown).
In conclusion, this study has provided in vivo demonstration of a decreased thrombotic tendency in mouse models of the Bernard-Soulier bleeding disorder. This antithrombotic protection linked to a GPIb defect is in line with in vitro and in vivo thrombosis studies using GPIb/VWF blocking agents25–27 and recent results in GPIbα transgenic mice.10 The finding is still difficult to extrapolate to man, especially in the face of a report of an acute coronary syndrome in a BSS patient.5 However, the GPIbβ−/− model should permit further evaluation of the role of this receptor in thrombosis under atherosclerotic conditions, particularly after crossing with APOE- or LDLR-deficient mice.
We thank Juliette Mulvihill for reviewing the English, Jean-Marie Garnier for assistance with the constructs, Dominique Cassel for platelet preparation, Dorothée Pierron for the bleeding time assays, and Institut Clinique de la Souris for assistance in developing the mouse strains.
Sources of Funding
Catherine Strassel was supported by a grant 2004.14 from Etablissement Français du Sang and Tovo David by a grant from Association de Recherche et de Développement en Médecine et en Santé Publique.
Original received July 27, 2006; final version accepted September 28, 2006.
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