Decreased Thrombotic Tendency in Mouse Models of the Bernard-Soulier Syndrome

【摘要】  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 GPIbß subunit.

Methods and Results- Complete deletion (GPIbß -/- ) or an intracellular truncation (GPIbß IC -/- ) 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 GPIbß IC -/-. 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 FeCl 3 -induced lesion of carotid arteries. In a model of laser-induced lesion of mesenteric arterioles, thrombosis was strongly reduced in GPIbß -/- mice, while a more modest effect was observed in GPIbß IC -/- 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.

Mouse models of Bernard-Soulier bleeding disorder were generated by targeting the GP GPIbß. These mice displayed, in addition to their defective hemostasis, decreased thrombotic responses in different models of arterial and systemic thrombosis.

【关键词】  GPIbVIX complex von Willebrand factor knockout thrombosis models hemostasis

Introduction

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 GPIb or GPIbß 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 GPIb 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 GPIbß gene (GPIbß -/- ) and a knock-in strain by interrupting the GPIbß intracellular domain (GPIbß IC -/- ). Thrombosis was studied in vitro in collagen perfusion assays and in several in vivo artery injury and thromboembolism models.

Materials and Methods

Generation of GPIbß -/- and GPIbß IC -/- Animals

A knockout construct (GPIbß -/- ) was generated by replacing the coding sequence with a neo cassette. A knock-in construct (GPIbß IC -/- ) 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. GPIbß -/-, GPIbß IC -/-, and GPIbß +/+ 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.

FeCl 3 -Induced Carotid Artery Thrombosis

FeCl 3 -induced arterial injury was performed according to published procedures. 12,13 The right common carotid artery was exposed to 20% FeCl 3 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.

Thromboembolism Model

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.

Statistical Analyses

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).

Results

Generation of Mouse Models of Bernard-Soulier Syndrome Targeting the GPIbß Subunit

Two targeting vectors were designed for inactivation of the GPIbß gene (GPIbß -/- ) and deletion of the GPIbß intracellular domain (GPIbß IC -/-; supplemental Figure I). GPIbß -/- was obtained by inserting a neo cassette into the second exon, thereby eliminating most of the coding sequence. GPIbß IC -/- 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 GPIbß in platelets from GPIbß -/- mice was confirmed by Western blotting using the mAb RAM.1 against the extracellular domain of mouse GPIbß 15 (supplemental Figure I). In GPIbß IC -/- cells, a GPIbß 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 GPIbß -/- and GPIbß IC -/- Mice

Analysis of the hematologic parameters of GPIbß -/- and GPIbß IC -/- revealed the characteristic decreased platelet counts and enlarged platelets of the Bernard-Soulier syndrome but normal leukocyte and erythrocyte counts ( Figure 1 A). Heterozygotes from both strains had normal platelet counts and morphology (data not shown).

Figure 1. Hematologic parameters and platelet phenotypes in GPIbß -/- and GPIbß IC -/- mice. A, Analysis of the hematologic parameters of GPIbß +/+, GPIbß -/- and GPIbß IC -/- mice. Platelets were counted manually and white blood cell (WBC) and red blood cell (RBC) using an automatic counter. Platelet counts were on average three times lower as compared with their wild-type littermates. B and C, GPIbß +/+, GPIbß -/- and GPIbß IC -/- washed platelets were studied by transmission electron microscopy. B, Representative GPIbß +/+ discoid platelets of normal size. GPIbß -/- and GPIbß IC -/- platelets displayed an enlarged size with an elliptic shape. C, High magnification illustrating an increase in the number of microtubules in GPIbß -/- and GPIbß IC -/-. D, The surface of -granules was similar in GPIbß -/- and GPIbß IC -/- as compared with control. The analysis in the number of microtubules showed an increase of two fold in platelets of GPIbß -/- and GPIbß IC -/- compared with control platelets. E, fluorescence-activated cell sorter (FACS) analysis of GPIbß and GPIb platelet surface expression with fluorescein-isothiocyanate (FITC)-tagged RAM.1 and RAM.6, respectively. GPIbß was absent from GPIbß -/- platelets and expressed at 20% of normal levels in GPIbß IC -/- mice. GPIb expression was strongly decreased in GPIbß -/- platelets (3% of GPIbß +/+ ) and found at 20% of normal levels in GPIbß IC -/- mice.

Transmission electronic microscopy (TEM) analysis revealed discoid platelets with an enlarged diameter (6±1 for GPIbß -/- and 6±2 µm for GPIbß IC -/- ) compared with control GPIbß +/+ (2±1 µm; Figure 1 B). Intracellularly, - 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 1 C).

Flow cytometry analysis showed that GPIbß -/- platelets lacked GPIbß expression and expressed little GPIb (3% of GPIbß +/+ cells). By contrast, in the GPIbß IC -/- mice, GPIbß, and GPIb represented close to 20% of normal levels ( Figure 1 D). Expression of integrin IIbß3 in GPIbß -/- and GPIbß IC -/- represented 170% as compared with GPIbß +/+ attriubutable to increased platelet size (data not shown).

Platelet counts were reduced by 73% and 71% in GPIbß -/- and GPIbß IC -/- mice, respectively, compared with wild-type littermates ( Figure 1 A). 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 GPIbß -/- mice bled for more than 20 minutes ( Figure 2 ). GPIbß IC -/- mice had a decreased bleeding tendency than GPIbß -/- with 52% of the mice bleeding after 20 minutes. Heterozygotes from the GPIbß -/- and GPIbß IC -/- strains had bleeding times similar to those of their respective +/+ genotypes (data not shown).

Figure 2. Tail bleeding times in GPIbß -/- and GPIbß IC -/- mice. Bleeding times were measured in GPIbß -/- and GPIbß IC -/- mice and their wild-type littermates (GPIbß +/+ ) by tail transection as described in the Methods. Each point corresponds to the time at which bleeding had ceased in individual mice or to a 20 minutes time when bleeding did not stop. In GPIbß -/- mice 76% of the animals still bled after 20 minutes compared with 18% in GPIbß +/+. GPIbß IC -/- mice had a lower bleeding tendency than GPIbß -/- mice with 52% of them still bleeding after 20 minutes. Statistical analyses were performed using a Log-rank test.

Decreased Thrombus Formation During Perfusion of GPIbß -/- and GPIbß IC -/- 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 GPIbß +/+, 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 3 D), with a slightly increased total surface coverage at 3000 s -1 ( Figure 3B and 3 E). A major defect was observed in GPIbß -/- and GPIbß IC -/- 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 3 B). Electron microscopy analysis revealed the rare attachment of individual GPIbß -/- platelets to the collagen fibers and attachment of a few GPIbß IC -/- platelets in strings along the fibers ( Figure 3 C). 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 GPIbß -/- blood which were greatly decreased in size as compared with the wild-type ( Figure 3 F). At this shear rate, adhesion and aggregate formation occurred in GPIbß IC -/- blood and was approximately 50 to 60% of that in wild-type blood ( Figure 3 E). 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.

Figure 3. Defective thrombus formation of GPIbß -/- and GPIbß IC -/- platelets during perfusion of blood over a collagen surface. Hirudin-anticoagulated whole blood from GPIbß -/- or GPIbß IC -/- mice or their wild-type littermates (GPIbß +/+ ) was perfused through glass microcapillaries coated with fibrillar type I collagen (2.5 mg/mL) at 3000 s -1 (A through C) or 1500 s -1 (D through F). A and D, Representative time course images of thrombus formation. Scale bar correspond to 6.5 µm. (B and E) Surface covered by platelets and thrombi expressed as the percentage of the total surface observed. Data are mean values (±SD). B, The number of animals was n=4 for GPIbß +/+, n=5 for GPIbß IC -/-, n=3 for GPIbß -/-. E, The number of animals was n=4 for GPIbß +/+, n=5 for GPIbß IC -/-, n=6 for GPIbß -/-. C and F, Scanning electron microscopy images after 2 minutes perfusion. High resolution images in the lower panel correspond to the boxed region. Scale bar correspond to 20 µm (upper panel) and 2 µm (lower panel). A major defect was observed for perfusion of GPIbß -/- or GPIbß IC -/- blood at 3000 s -1, where platelet adhesion was nearly abolished, resulting in a profound decrease in the total surface coverage at 2 minutes. At 1500 s -1, adhesion and aggregate formation were markedly decreased for GPIbß -/- platelets, but for GPIbß IC -/- platelets reached approximately 50 to 60% of levels in GPIbß +/+ blood. Statistical analyses were performed using a Mann-Whitney test.

Decreased Arterial Thrombosis in GPIbß -/- and GPIbß IC -/- 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 FeCl 3 ( Figure 4 ). In wild-type (n=8), stable occlusion occurred in 75% of the arteries, with permanent cessation of flow ( Figure 4 A). In GPIbß -/- (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 GPIbß IC -/- (n=8) animals where 62.5% of the arteries were patent (unstable or no occlusion) at the end of the 30 minutes period ( Figure 4 A). The time to first occlusion, either stable or transient, was increased in GPIbß -/- (1646±84 s) or GPIbß IC -/- (1242±169 s) as compared with their wild-type littermates (997±135 s and 631±68 s) ( Figure 4 B). Histological analysis revealed that the thrombi formed in GPIbß -/- and GPIbß IC -/- animals were less extensive than in the wild-type ( Figure 4 C) and ultrastructural analysis showed loose thrombi mainly composed of well demarcated platelets displaying less extensive shape change and little sign of degranulation ( Figure 4 D). This contrasted with the intricate appearance of wild-type aggregates which consisted of intertwined platelets having undergone degranulation.

Figure 4. Decreased tendency of GPIbß -/- and GPIbß IC -/- mice to form occlusive thrombi in a FeCl 3 -induced carotid artery injury model. Vessel wall injury was induced by applying a 20% FeCl 3 solution for 3 minutes and the blood flow was recorded with a Doppler flow probe. Vascular occlusion was defined as the cessation of blood flow for over 30 s, confirmed by visualization of the thrombus. A, At the end of a 30 minutes recording period, arteries were classified as those which did not occlude (open bar) those which developed stable occlusion (filled bar) and those in which the blood flow ceased but recommenced (gray bar; unstable occlusion). Data are presented as percentage of mice for each genotype (n=8). GPIbß -/- and GPIbß IC -/- mice displayed resistance to thrombosis as respectively 12.5% and 37.5% of vessels remained patent for 30 minutes, as compared with 75% in wild-type mice. B, The time to the first cessation of blood flow was determined and was delayed in GPIbß -/- (n=8) and GPIbß IC -/- (n=8) relative to their wild type littermates (n=8). Data are mean values (±SD). Statistical analyses used a Mann-Whitney test (C) Representative histological micrographs of longitudinal sections of carotid arteries. D, Transmission electron micrographs (TEM) of transversal sections of thrombi, in arteries removed 30 minutes after FeCl 3 injury. The inserts are low resolution micrographs in which the area chosen for enlargement is indicated by an arrowhead. A loose thrombus formed in GPIbß -/- and GPIbß IC -/- mice, appearing as layers of individual platelets, most of them showing minimal shape change and no degranulation. In contrast, the thrombi formed in wild-type mice were extensive and more compact, containing intertwined degranulated platelets.

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 5 B). In GPIbß -/-, 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 5 A). In GPIbß IC -/-, 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 5 B).

Figure 5. Resistance to thrombosis of GPIbß -/- and GPIbß IC -/- mice in a laser-induced mesenteric artery injury model. A laser-induced injury of the wall of mesenteric arterioles was produced in GPIbß -/- (A) and GPIbß IC -/- mice (B) to generate a superficial (left panels) (n=26 vessels) or severe lesion (right panels) (n=8 to 11 vessels for GPIbß +/+ and 5 to 6 vessels for GPIbß -/- and GPIbß IC -/-, respectively). Upper panels are representative photographs of the thrombus at maximal size (the direction of blood flow is indicated by an arrow). Lower panels are time courses of the thrombus surface area where the black and gray curves correspond respectively to wild type (+/+) and deficient (-/-) mice. The mean thrombus surface area was analyzed at 0.3 s intervals and the shading over the curve represents the SEM at each time point. A, Thrombus formation was almost absent in GPIbß -/- mice on superficial lesions and was strongly reduced on severe lesions. B, In GPIbß IC -/- mice, the thrombus was decreased in size on superficial lesions and was delayed on severe lesions but finally reached a size comparable to that of GPIbß +/+. Area under curve was used to perform statistical analyses.

Decreased Systemic Thromboembolism in GPIbß -/- and GPIbß IC -/- Mice

The GPIbß -/- and GPIbß IC -/- 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 ). GPIbß -/- and GPIbß IC -/- mice had a lower death rate, with 40% and 60% of the mice being still alive 30 minutes after collagen-adrenaline injection.

Figure 6. Resistance to thromboembolism in GPIbß -/- and GPIbß IC -/- mice. Systemic thromboembolism was induced by infusion of a collagen and adrenaline mixture. Results are expressed as the percentage of mice still alive (survival) as a function of time. GPIbß -/- (n=10) and GPIbß IC -/- (n=10) mice were protected against death when compared with GPIbß +/+ (n=10). Statistical analyses were performed using a Log-rank test.

Discussion

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 GPIbß gene reproduced a typical Bernard-Soulier phenotype and most of the defects observed in a previously described GPIbß deficient strain, 9 including a tripling of the platelet size, a 75% decrease in platelet number, a profound decrease in GPIb 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 -granules of increased size 9 which was attributed to upregulation of SEPT5 expression which gene is located 5' to the GPIbß 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 GPIbß IC -/- 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 GPIbß intracellular domain is required for efficient expression of the complex. In fact, the GPIbß IC -/- 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 GPIbß, is sufficient to support minimal hemostasis.

The GPIbß -/- and GPIbß IC -/- 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 GPIb knock out and missing the GPIb extracellular domain. 10 Compared with this strain, the GPIbß -/- and GPIbß IC -/- 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 GPIb 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 2ß1 and GPVI, which are the major receptors contributing to platelet adhesion and activation after direct interaction with collagen. GPVI-FcR deficiency induced by immunodepletion or after genetic ablation have established a critical role for GPVI in such perfusion assays over collagen 16-18 whereas 2ß1 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 2ß1 and GPVI can probably take place at these shears in mouse blood 10

In vivo results in a FeCl 3 carotid thrombosis model were consistent with the flow experiments demonstrating a lower incidence of vessel occlusion in both GPIbß -/- and GPIbß IC -/- 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 GPIb 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 GPIbß -/- strain. A more modest protection of GPIbß IC -/- contrasted with the carotid FeCl 3 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, FcR /GPVI deficiency has been reported to induce full or only minor protection against arterial thrombosis in different studies 17,20 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 FeCl 3 applied to arteries 21 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 GPIbß-deficient strain presented some similarities with results in vWF-deficient mice in a FeCl 3 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 mice 23 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, GPIbß-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

GPIbß -/- and GPIbß IC -/- 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 GPIbß -/- and GPIbß IC -/-, 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, GPIbß IC -/- 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 GPIbß IC -/- 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 agents 25-27 and recent results in GPIb 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 GPIbß -/- 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.

Acknowledgments

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.

Disclosures

None.

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作者单位：From INSERM U31 EFS-Alsace, ULP, Strasbourg, France.