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. 1999 Jan;103(2):229-38.
doi: 10.1172/JCI5487.

Beta3-integrin-deficient mice are a model for Glanzmann thrombasthenia showing placental defects and reduced survival

K M Hodivala-Dilke  1 , K P McHugh, D A Tsakiris, H Rayburn, D Crowley, M Ullman-Culleré, F P Ross, B S Coller, S Teitelbaum, R O Hynes
Affiliations

Beta3-integrin-deficient mice are a model for Glanzmann thrombasthenia showing placental defects and reduced survival

K M Hodivala-Dilke et al. J Clin Invest. 1999 Jan.

Abstract

beta3 integrins have been implicated in a wide variety of functions, including platelet aggregation and thrombosis (alphaIIbbeta3) and implantation, placentation, angiogenesis, bone remodeling, and tumor progression (alphavbeta3). The human bleeding disorder Glanzmann thrombasthenia (GT) can result from defects in the genes for either the alphaIIb or the beta3 subunit. In order to develop a mouse model of this disease and to further studies of hemostasis, thrombosis, and other suggested roles of beta3 integrins, we have generated a strain of beta3-null mice. The mice are viable and fertile, and show all the cardinal features of GT (defects in platelet aggregation and clot retraction, prolonged bleeding times, and cutaneous and gastrointestinal bleeding). Implantation appears to be unaffected, but placental defects do occur and lead to fetal mortality. Postnatal hemorrhage leads to anemia and reduced survival. These mice will allow analyses of the other suggested functions of beta3 integrins and we report that postnatal neovascularization of the retina appears to be beta3-integrin-independent, contrary to expectations from inhibition experiments.

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Figures

Figure 1
Figure 1
Targeting strategy, Southern blots and PCR analysis of ES cells and mice lacking the β3-integrin gene. (a) Structure of β3 targeting construct, wild-type β3 allele and targeted β3 allele. Exons are indicated as black boxes. The 1.7 kb pgk-neomycin-resistance cassette replaces a 1.4 kb HindIII fragment of the β3-gene including exons I and II. Pgk-thymidine kinase genes were used for negative selection. (b) Southern blot analysis of EcoRI, BstEII and NcoI-digested genomic DNA from a representative targeted clone (111). Probe A lay 5′ of the targeting construct and probe B lay inside it (a). (c) PCR analysis of wild-type, heterozygous and β3-null tail DNA used to screen offspring. Primer locations are indicated by half arrows in A and give products of 446 bp with primers 1 and 3 (wild-type) or 538 bp with primers 1 and 2 (mutant).
Figure 2
Figure 2
Integrin profiles of cells isolated from the β3-null mice. (a) Surface iodination of platelets and immunoprecipitation of integrins β3, β1, αIIb, αv, α5 and α6 from wild-type (+) and β3-null (–) mice. (b) Surface iodination of mouse embryo fibroblasts (MEFs) and immunoprecipitation of β3, β1, β5, αv, and α5. (c) Immunofluorescence staining of β3, β1, and αv-integrins on wild-type and β3-null MEFs. (d) Adhesion assays with wild-type and β3-null MEFs on fibronectin and vitronectin. Bar (c), 15 μm.
Figure 3
Figure 3
Survival of β3-null mice is compromised. Uteri from typical wild-type (a) and β3-null (b) females that had been bred to β3-null or wild-type males, respectively. Dark patches in (b) indicate bleeding in β3-null uterus. (c and d) Heterozygous E16 embryos in utero, in wild-type (c) or β3-null (d) mothers. Some pups die in β3-null mothers in utero, appearing pale, small and associated with a pale placenta (arrow). (e, f and h) H&E stained sections of placentae from β3-null females mated with wild-type males. H&E stained section of placenta from wild-type female mated with a β3-null male (g). Arrowhead indicates severe hemorrhage observed within the labyrinth (e) and under the Reichert's membrane (f). (g) Labyrinth of wild-type placenta; (h) Labyrinth of β3-null placenta where cell layers appear thick; this occurs in ∼25% of β3-null placentae. (i and j) E16 embryos, wild-type (i) and β3-null (j). Note hemorrhage in β3-null muzzle. Arrow indicates hemorrhage in the skin. (k, l, m, and n) After birth, some β3-null mice suffer hemorrhage in the skin. Petechiae around the mouth of a 1-day-old β3-null pup (k) and H&E-stained section of this region indicating bleeding under the epidermis (l). Purpura on the forelimb of a 1-day-old β3-null pup (m) and H&E-stained section of this region indicating bleeding within the dermis (n). (o and p) Gut from newborn pups. Wild-type (o) and β3-null pup with acute GI hemorrhage (p). Bar (e), 400 μm (f), 50 μm, and (g), 70 μm.
Figure 4
Figure 4
Blood cell counts and Bleeding assays. (a) Red blood cell counts are reduced in randomly selected 8–12-week-old β3-null mice. Means and standard errors of the means for wild-type, β3-het and β3-null animals are as follows: 8.9 ± 0.15, 9.0 ± 0.16 and 6.0 ± 0.3 (× 106/mm3), a significant difference (P < 0.005) between β3-null and others. (b) Tail bleeding times. β3-null mice all show bleeding times of at least 10 min (at which time bleeding was stopped by cauterization). This was significantly greater than that for either wild-type or β3-heterozygous mice (P < 0.005). (c–f) Localized Shwartzman reactions. Gross hemorrhage in wild-type (c), but not β3-null (d) skin after LPS and TNFα challenge. Histology of blood vessels in the skin after LPS and TNFα challenge in wild-type dermis (e) and β3-null dermis (f).
Figure 5
Figure 5
Functional assays of platelets in vitro show that the β3-null platelets do not aggregate (a), have reduced clot retraction (b), and greatly reduced fibrinogen uptake (c). A GT is a platelet lysate from an Arab patient with Glanzmann thrombasthenia, who has a defect in GPIIb and I-J GT is a platelet lysate from an Iraqi-Jewish GT patient with a defect in β3 (GPIIIa).
Figure 6
Figure 6
β3-null mice in crisis display a variety of secondary phenotypes. H&E-stained sections from wild-type (a, c, e, and g) and from β3-null mice found in crisis (b, d, f, and h). (a and b) Gastrointestinal bleeding can be observed in the lamina propria between the stomach epithelium (e) and muscularis mucosae (m). (c) White pulp (wp) and red pulp (rp) are clearly defined in wild-type spleen. Splenomegaly in anemic mice with extramedullary erythropoiesis (d). Centrilobular necrosis of the β3-null liver (f) is a consequence of severe anemia resulting in lack of well-oxygenated blood reaching the centrilobular vein (clv). Glomerular nephritis and resultant tubular necrosis are also secondary to anemia (h). Notice the dilated proximal tubules (dpt), enlarged glomerulus (g), and proteinaceous deposits (p) in the tubules. Normal proximal tubules are denoted, pt. Bar, 70 mm.
Figure 7
Figure 7
Neovascularization in the β3-null developing retina is not inhibited by the absence of β3-integrins. Blood vessels in wild-type (a and c) and β3-null (b and d) retinas were observed at P7. Whole flat-mount retinas at low power (a and b) and high-power views of branching from major blood vessels radiating from the optic disc (c and d). Bar (a), 800 μm; (c), 50 μm.
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