Integrin α2-Deficient Mice Develop Normally, Are Fertile, but Display Partially Defective Platelet Interaction with Collagen

The integrin α2-subunit was ablated in mice by targeted deletion of the ITGA2 gene. α2-Deficient animals develop normally, are fertile, and reproduce. Surprisingly, no obvious anatomical or histological differences were observed in mutant mice. Besides its significance in tissue morphogenesis, integrin α2β1 has been reported to play a major role in hemostasis by mediating platelet adhesion and activation on subendothelial collagen. To define its role in hemostasis, α2-deficient platelets were analyzed for their capacity to adhere to and aggregate in response to fibrillar or soluble collagen type I. We show that aggregation of α2-deficient platelets to fibrillar collagen is delayed but not reduced, whereas aggregation to enzymatically digested soluble collagen is abolished. Furthermore, α2-deficient platelets normally adhere to fibrillar collagen. However, in the presence of an antibody against GPVI (activating platelet collagen receptor), adhesion of α2-deficient but not wild type platelets is abrogated. These results demonstrate that integrin α2β1 significantly contributes to platelet adhesion to (fibrillar) collagen, which is further confirmed by the abolished adhesion of α2-deficient platelets to soluble collagen. Thus, α2β1 plays a supportive rather than an essential role in platelet-collagen interactions. These results are in agreement with the observation that α2β1-deficient animals suffer no bleeding anomalies. The integrin α2-subunit was ablated in mice by targeted deletion of the ITGA2 gene. α2-Deficient animals develop normally, are fertile, and reproduce. Surprisingly, no obvious anatomical or histological differences were observed in mutant mice. Besides its significance in tissue morphogenesis, integrin α2β1 has been reported to play a major role in hemostasis by mediating platelet adhesion and activation on subendothelial collagen. To define its role in hemostasis, α2-deficient platelets were analyzed for their capacity to adhere to and aggregate in response to fibrillar or soluble collagen type I. We show that aggregation of α2-deficient platelets to fibrillar collagen is delayed but not reduced, whereas aggregation to enzymatically digested soluble collagen is abolished. Furthermore, α2-deficient platelets normally adhere to fibrillar collagen. However, in the presence of an antibody against GPVI (activating platelet collagen receptor), adhesion of α2-deficient but not wild type platelets is abrogated. These results demonstrate that integrin α2β1 significantly contributes to platelet adhesion to (fibrillar) collagen, which is further confirmed by the abolished adhesion of α2-deficient platelets to soluble collagen. Thus, α2β1 plays a supportive rather than an essential role in platelet-collagen interactions. These results are in agreement with the observation that α2β1-deficient animals suffer no bleeding anomalies. Integrins are a large family of heterodimeric transmembrane receptors composed of noncovalently associated α- and β-subunits that function as receptors for extracellular matrix components and also bind to counter receptors on other cells (1.Hemler M.E. Annu. Rev. Immunol. 1990; 8: 365-400Crossref PubMed Google Scholar, 2.Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (8941) Google Scholar, 3.Humphries M.J. Biochem. Soc. Trans. 2000; 28: 311-339Crossref PubMed Google Scholar). Integrin receptors modulate critical cellular processes, including adhesion and spreading, migration, survival, gene expression, and differentiation. These processes are physiologically relevant to growth and development, angiogenesis, and hemostasis but may also be significant in pathological conditions such as tumor metastasis and thrombosis (4.van der Flier A. Sonnenberg A. Cell Tissue Res. 2001; 305: 285-298Crossref PubMed Scopus (793) Google Scholar, 5.De Arcangelis A. Georges-Labouesse E. Trends Genet. 2000; 16: 389-395Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, 6.Varner J.A. Cheresh D.A. Curr. Opin. Cell Biol. 1996; 8: 724-730Crossref PubMed Scopus (465) Google Scholar). The essential role of β1 integrins for development and differentiation was clearly demonstrated by the peri-implantation lethality of mouse embryos lacking β1 integrin (7.Fassler R. Meyer M. Genes Dev. 1995; 9: 1896-1908Crossref PubMed Scopus (603) Google Scholar). Four collagen-binding β1 integrin receptors have been identified, α1β1, α2β1, α10β1, and α11β1 (8.Heino J. Matrix Biol. 2000; 19: 319-323Crossref PubMed Scopus (238) Google Scholar), which interact with collagens via their individual I domains (9.Calderwood D.A. Tuckwell D.S. Eble J. Kuhn K. Humphries M.J. J. Biol. Chem. 1997; 272: 12311-12317Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 10.Knight C.G. Morton L.F. Peachey A.R. Tuckwell D.S. Farndale R.W. Barnes M.J. J. Biol. Chem. 2000; 275: 35-40Abstract Full Text Full Text PDF PubMed Scopus (528) Google Scholar, 11.Emsley J. Knight C.G. Farndale R.W. Barnes M.J. Liddington R.C. Cell. 2000; 101: 47-56Abstract Full Text Full Text PDF PubMed Scopus (818) Google Scholar, 12.Tulla M. Pentikainen O.T. Viitasalo T. Kapyla J. Impola U. Nykvist P. Nissinen L. Johnson M.S. Heino J. J. Biol. Chem. 2001; 276: 48206-48212Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). α2β1 integrin (VLA-2, platelet GPIaIIa) was thought to play a pivotal role in development, differentiation, and tissue morphogenesis. It is widely expressed, especially on cell types entering the final stages of differentiation (13.Zutter M.M. Santoro S.A. Am. J. Pathol. 1990; 137: 113-120PubMed Google Scholar, 14.Wu J.E. Santoro S.A. Dev. Dyn. 1994; 199: 292-314Crossref PubMed Scopus (69) Google Scholar). α2β1 receptors bind with high affinity to collagen I (15.Kern A. Eble J. Golbik R. Kuhn K. Eur. J. Biochem. 1993; 215: 151-159Crossref PubMed Scopus (178) Google Scholar) and also to collagens II–V (16.Kamata T. Takada Y. J. Biol. Chem. 1994; 269: 26006-26010Abstract Full Text PDF PubMed Google Scholar, 17.Nykvist P. Tu H. Ivaska J. Kapyla J. Pihlajaniemi T. Heino J. J. Biol. Chem. 2000; 275: 8255-8261Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar), and they mediate adhesion to laminins-1 and -5 (18.Pfaff M. Gohring W. Brown J.C. Timpl R. Eur. J. Biochem. 1994; 225: 975-984Crossref PubMed Scopus (86) Google Scholar, 19.Decline F. Rousselle P. J. Cell Sci. 2001; 114: 811-823Crossref PubMed Google Scholar). Contact with collagen of α2β1 on fibroblasts and epithelial cells induces synthesis and activation of several matrix metalloproteinases (20.Langholz O. Rockel D. Mauch C. Kozlowska E. Bank I. Krieg T. Eckes B. J. Cell Biol. 1995; 131: 1903-1915Crossref PubMed Scopus (376) Google Scholar, 21.Ravanti L. Heino J. Lopez-Otin C. Kahari V.M. J. Biol. Chem. 1999; 274: 2446-2455Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar, 22.Zigrino P. Drescher C. Mauch C. Eur. J. Cell Biol. 2001; 80: 68-77Crossref PubMed Scopus (96) Google Scholar) and is therefore thought to play an essential role in connective tissue remodeling and resurfacing of wounds. The α2 protein was initially isolated from platelets, where it is involved in the adhesion to subendothelial collagen at sites of vascular injury and thereby contributes to the formation of a hemostatic plug (23.Santoro S.A. Blood. 1999; 93: 3575-3577Crossref PubMed Google Scholar). However, the interaction between platelets and collagen is complex and can either occur indirectly via immobilized von Willebrand factor binding to platelet receptors glycoprotein (GP) 1The abbreviations used are: GPglycoproteinFITCfluorescein isothiocyanateESembryonic stemPRPplatelet-rich plasmaPBSphosphate-buffered salineCRPcollagen-related peptide 1The abbreviations used are: GPglycoproteinFITCfluorescein isothiocyanateESembryonic stemPRPplatelet-rich plasmaPBSphosphate-buffered salineCRPcollagen-related peptide Ib-V-IX and/or activated αIIbβ3 integrin (24.Savage B. Almus-Jacobs F. Ruggeri Z.M. Cell. 1998; 94: 657-666Abstract Full Text Full Text PDF PubMed Scopus (672) Google Scholar) or by direct interaction of collagen with specific receptors, including the Ig-like receptor GPVI (25.Moroi M. Jung S.M. Okuma M. Shinmyozu K. J. Clin. Invest. 1989; 84: 1440-1445Crossref PubMed Scopus (363) Google Scholar, 26.Clemetson J.M. Polgar J. Magnenat E. Wells T.N. Clemetson K.J. J. Biol. Chem. 1999; 274: 29019-29024Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar) and α2β1 integrin. GPVI is essential for this process as it mediates the activation of β1 and β3 integrins, which is a prerequisite for firm adhesion and thrombus growth (27.Nieswandt B. Brakebusch C. Bergmeier W. Schulte V. Bouvard D. Mokhtari-Nejad R. Lindhout T. Heemskerk J.W. Zirngibl H. Fassler R. EMBO J. 2001; 20: 2120-2130Crossref PubMed Scopus (435) Google Scholar). In contrast, the role of α2β1 in both platelet adhesion and activation on collagen has been controversially debated. Although previous studies (28.Nieuwenhuis H.K. Akkerman J.W. Houdijk W.P. Sixma J.J. Nature. 1985; 318: 470-472Crossref PubMed Scopus (388) Google Scholar, 29.Kehrel B. Balleisen L. Kokott R. Mesters R. Stenzinger W. Clemetson K.J. van de Loo J. Blood. 1988; 71: 1074-1078Crossref PubMed Google Scholar) emphasized an essential role of this integrin in platelet-collagen interactions and hemostasis, we have recently shown that mice lacking β1 integrins on their platelets display no major hemostatic defect. In vitro, however, β1-deficient platelets failed to interact with enzymatically digested collagen and displayed partial defects in their activation by and adhesion to native fibrillar collagen (27.Nieswandt B. Brakebusch C. Bergmeier W. Schulte V. Bouvard D. Mokhtari-Nejad R. Lindhout T. Heemskerk J.W. Zirngibl H. Fassler R. EMBO J. 2001; 20: 2120-2130Crossref PubMed Scopus (435) Google Scholar). In these studies, however, it could not be clarified definitively whether these defects were based on the absence of α2β1alone, because β1-deficient platelets also lack α5β1 and α6β1. glycoprotein fluorescein isothiocyanate embryonic stem platelet-rich plasma phosphate-buffered saline collagen-related peptide glycoprotein fluorescein isothiocyanate embryonic stem platelet-rich plasma phosphate-buffered saline collagen-related peptide To assess the function of the α2β1 receptor in vivo, particularly in hemostasis, we generated α2 integrin-deficient mice. In contrast to previous reports that suggested that homozygous deletion of the ITGA2gene results in embryonic lethality (5.De Arcangelis A. Georges-Labouesse E. Trends Genet. 2000; 16: 389-395Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, 30.Sheppard D. Matrix Biol. 2000; 19: 203-209Crossref PubMed Scopus (118) Google Scholar, 31.de Fougerolles A.R. Sprague A.G. Nickerson-Nutter C.L. Chi-Rosso G. Rennert P.D. Gardner H. Gotwals P.J. Lobb R.R. Koteliansky V.E. J. Clin. Invest. 2000; 105: 721-729Crossref PubMed Scopus (186) Google Scholar), these mice develop normally and reproduce. Strikingly, platelet counts and bleeding times are normal in α2 integrin-deficient mice. Although the interaction of α2-deficient platelets with soluble collagen is abrogated, they display only subtle defects in response to native fibrillar collagen. Fibrillar type I collagen from equine tendon (Horm, Nycomed, Munich, Germany), high molecular weight heparin, and soluble, non-fibrillar type I collagen from rat tail were from Sigma. The following antibodies were used: FITC anti-β1 integrin (Ha31/8), FITC anti-α2 integrin (Ha1/29), rat anti-α5 integrin (5H10-27), FITC anti-α6integrin (GoH3) (all from BD PharMingen). Polyclonal rabbit anti-mouse α2 integrin antibodies were kindly provided by U. Mayer (Manchester, UK). Rat anti-mouse β3 integrin (EDL4), GPIb-IX (p0p1), GPV (DOM1), CD9 (ULF1), and GPVI (JAQ1) have been described (32.Bergmeier W. Rackebrandt K. Schroder W. Zirngibl H. Nieswandt B. Blood. 2000; 95: 886-893Crossref PubMed Google Scholar, 33.Nieswandt B. Bergmeier W. Rackebrandt K. Gessner J.E. Zirngibl H. Blood. 2000; 96: 2520-2527Crossref PubMed Google Scholar, 34.Nieswandt B. Bergmeier W. Schulte V. Rackebrandt K. Gessner J.E. Zirngibl H. J. Biol. Chem. 2000; 275: 23998-24002Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). Fab fragments of JAQ1 were prepared as described (35.Nieswandt B. Schulte V. Bergmeier W. Mokhtari-Nejad R. Rackebrandt K. Cazenave J.P. Ohlmann P. Gachet C. Zirngibl H. J. Exp. Med. 2001; 193: 459-469Crossref PubMed Scopus (288) Google Scholar). FITC-conjugated rabbit anti-rat IgG (Dako) and horseradish peroxidase-conjugated swine anti-rabbit IgG were used as secondary reagents. A 439-bp cDNA fragment of mouse α2 integrin corresponding to exons 1–3 was used to screen a λ FIX II genomic library (Stratagene) of the 129SVJ mouse strain. A targeting vector was generated in pBluescript KS II (Stratagene) containing 3 kb of the promoter region, the first exon including the translation start, and 4 kb of the first intron. A HindIII and loxP site was placed 1 kb upstream of the first exon in a single StuI site, and a phosphoglycerate kinase-driven neomycin resistance (neor) cassette, flanked by loxP sites, was inserted 0.4 kb downstream of the exon into a single SacII site. Embryonic stem (ES) cells of the E14 subclone IB-10 were grown under standard conditions. ES cells were electroporated (Bio-Rad Gene Pulser II) with the SmaI-linearized targeting vector. G418-resistant clones, cut with HindIII, were analyzed by RFLP analysis using the external probe Ex for hybridization. Homologous recombination reduced the 19-kb wild type fragment to 7.5 kb, indicating cointegration of the single loxP site. Single copy integration was confirmed by reprobing with a neor probe (Int), resulting in a single 7.5-kb band. Deletion of the neor cassette was achieved by electroporating correctly targeted clones with the plasmid pIC Cre expressing Cre recombinase. Loss of the neor cassette reduced the 7.5-kb mutated fragment to 6 kb. Three individual clones then generated germ line transmitting chimeras as described (36.Smyth N. Vatansever H.S. Murray P. Meyer M. Frie C. Paulsson M. Edgar D. J. Cell Biol. 1999; 144: 151-160Crossref PubMed Scopus (408) Google Scholar). Mice homozygous for the loxP-flanked exon were bred to mice expressing Cre recombinase in the zygote, 2M. Hafner, manuscript in preparation. giving rise to offspring with a heterozygous deletion of the first exon of the ITGA2 gene. ITGA2 (+/−) mice were intercrossed to generate mice with a null mutation of the α2integrin. Platelets and tissue samples from ITGA2 (−/−) and wild type mice were homogenized in standard lysis buffer, and proteins were separated by SDS-PAGE under reducing conditions and transferred to nitrocellulose membranes. Following blocking, membranes were incubated with polyclonal rabbit antibodies against mouse α2 integrin. Bound antibodies were detected by horseradish peroxidase-conjugated anti-rabbit IgG and ECL (Amersham Biosciences). Individual animals were weighed on postnatal day 23. For determination of bleeding times, mice were anesthetized, and 5 mm of tail tip was amputated with a scalpel. Tails were then blotted with filter paper every 15 s until the paper was no longer bloodstained (37.Carmeliet P. Stassen J.M. Schoonjans L. Ream B. De van den Oord J.J. Mol M. Mulligan R.C. Collen D. J. Clin. Invest. 1993; 92: 2756-2760Crossref PubMed Scopus (343) Google Scholar). Mice were bled under ether anesthesia from the retro-orbital plexus. Blood was collected in a tube containing 10% (v/v) 7.5 units/ml heparin, and platelet-rich plasma (PRP) was obtained by centrifugation at 300 × gfor 10 min at room temperature. Before the start of the experiments, platelets were allowed to rest for 30 min in the presence of 0.02 units/ml of the ADP scavenger apyrase (adenosine 5′-triphosphate diphosphohydrolase), a concentration sufficient to prevent desensitization of platelet ADP receptors during storage. Platelets were kept at 37 °C throughout all experiments. For determination of platelet counts, blood (20 μl) was diluted 1:100 in Unopette kits (Becton Dickinson), and samples were allowed to settle for 20 min in an Improved Neubauer hemocytometer (Superior, Bad Mergentheim, Germany). Platelets were counted under a phase contrast microscope at ×400 magnification. For the analysis of platelet membrane glycoprotein expression, heparinized whole blood was diluted 1:30 with modified Tyrode's-HEPES buffer (137 mm NaCl, 2 mm KCl, 12 mm NaHCO3, 0.3 mm NaH2PO4, 5.5 mmglucose, 5 mm HEPES, pH 7.3) containing 0.35% bovine serum. Samples of 50 μl were stained with fluorophore-labeled monoclonal antibodies for 10 min at 37 °C and directly analyzed on a FACSCalibur (Becton Dickinson). Platelets were gated by forward scatter/side scatter characteristics. To determine platelet aggregation, light transmission was measured using PRP adjusted to a platelet concentration of 3 × 105 platelets/μl with Tyrode's buffer (detailed above) containing 0.35% bovine serum albumin. Transmission was recorded on a Fibrintimer 4-channel aggregometer (APACT Laborgeräte und Analysensysteme, Hamburg, Germany) over 10 min and expressed as arbitrary units relative to 100% transmission (adjusted with plasma). Fibrillar or soluble collagen (2 μg in 100 μl PBS per well) was immobilized on F96-MaxiSorp plates (Nunc, Wiesbaden, Germany) at 4 °C overnight. The plates were then blocked with 1 mg/ml bovine serum albumin in PBS for 3 h at 37 °C and washed with PBS. Washed platelets (107/well) in Tyrode's/albumin buffer containing 1 mm CaCl2 and 1 mm MgCl2 were incubated in the wells for up to 60 min. After three washing steps, adherent platelets were quantitated fluorimetrically as described (35.Nieswandt B. Schulte V. Bergmeier W. Mokhtari-Nejad R. Rackebrandt K. Cazenave J.P. Ohlmann P. Gachet C. Zirngibl H. J. Exp. Med. 2001; 193: 459-469Crossref PubMed Scopus (288) Google Scholar). A targeting vector was constructed in which the first exon of the ITGA2 gene along with the translation start were flanked by loxP sites to generate a mouse line that enables conditional inactivation of the ITGA2 gene (Fig. 1A). The vector was used to produce ES cell clones with a single homologous recombination event, as confirmed by Southern blot analysis (Fig. 1B). Deletion of the neor cassette was achieved by transiently transfecting these ES cell clones with a vector expressing Cre recombinase. Southern blot analysis of resulting G418-sensitive clones revealed 7 clones lacking the neor cassette and harboring the loxP-flanked first exon (Fig. 1B, Flox). Three individual clones were used to generate germ line chimeras. Germ line transmission in progeny of the chimeras was confirmed by Southern blot analysis of HindIII-digested tail DNA, and mice heterozygous for the mutation in the ITGA2 gene were intercrossed to produce mice homozygous for the loxP-flanked first exon (Fig. 1C, fl/fl). These animals (ITGA2flox) appeared normal, and Western blot analysis of several mouse organs confirmed that insertion of the loxP sites did not interfere with integrin α2 expression (Fig. 1D). To produce mice with a heterozygous ablation of the ITGA2gene, ITGA2flox animals were bred to mice expressing Cre recombinase in the zygote,2 leading to deletion of loxP-flanked regions. Deletion of the first exon was confirmed by Southern blot analysis, and heterozygous animals were intercrossed to produce α2 (−/−) mice (Fig. 1E). Homozygous α2-deficient mice are viable and show no striking phenotypical difference when compared with their heterozygous and wild type littermates. Complete loss of the α2-subunit was confirmed by Western blot analysis of proteins extracted from several mouse organs and from platelets (Fig. 1F). Litter sizes from breedings of heterozygotes are comparable with those of wild type animals, and genotyping of the viable offspring revealed normal Mendelian ratios, demonstrating that the loss of the integrin α2-subunit does not result in embryonic lethality. There was no significant difference in size or weight at birth nor at 3 weeks of postnatal life between α2 (−/−) mice (females, 13.1 ± 0.7 g; males, 15.1 ± 1.6 g) and wild type animals (females, 12.8 ± 1.8 g; males, 15.3 ± 1.7 g). Integrin α2 (−/−) mice are fertile, and intercrossing these mice produced normal litter sizes. Notably, the progeny of α2 (−/−) mice also developed normally, indicating that the α2 (−/−) females have no severe defects in placenta formation or lactation. No morphological or histological changes were obvious in these mice. That mice lacking α2β1 receptors were viable and fertile was surprising, given in vitro data suggesting that tissue morphogenesis could be impaired due to lack of proper adhesion, spreading, and migration. Further work will demonstrate whether subtle temporal alterations are present in these mice. Functional compensation by other collagen or laminin receptors, e.g. α1β1 or the discoidin domain receptors, may be an explanation for this subtle phenotype. Interestingly, ablation of both collagen receptors, α1β1 (38.Gardner H. Kreidberg J. Koteliansky V. Jaenisch R. Dev. Biol. 1996; 175: 301-313Crossref PubMed Scopus (225) Google Scholar) and α2β1, present mice with only subtle phenotypes, differing thereby from other integrin-deficient mice, most of which display severe defects (39.Fassler R. Georges-Labouesse E. Hirsch E. Curr. Opin. Cell Biol. 1996; 8: 641-646Crossref PubMed Scopus (109) Google Scholar, 40.Hynes R.O. Dev. Biol. 1996; 180: 402-412Crossref PubMed Scopus (250) Google Scholar). While integrin α2β1 has long been recognized as a platelet collagen receptor, its exact role in hemostasis has been controversial (41.Watson S. Berlanga O. Best D. Frampton J. Platelets. 2000; 11: 252-258Crossref PubMed Scopus (69) Google Scholar). To address this question, we analyzed platelets from α2-deficient mice. First, peripheral platelet counts were determined to assess platelet production in mutant mice. As shown in Fig. 2A, platelet counts were similar in control, α2 (+/−), and α2(−/−) mice. Flow cytometric analysis confirmed the absence of integrin α2-subunits on homozygous mutant platelets, whereas the expression levels in heterozygous platelets were reduced by ∼50% when compared with wild type (Table I). Interestingly, the levels of integrin β1 were reduced by ∼30% in α2 (−/−) and ∼15% in α2 (+/−) platelets as compared with controls, whereas the expression of α5 and α6 was significantly increased in mutant platelets (Table I). In contrast to β1 integrins, the expression levels of other membrane glycoproteins, such as integrin β3, GPVI, or the GPIb-V-IX complex, were not altered in mutant platelets (Table I). Western analyses of platelet lysates confirmed the absence of α2 (Fig. 1F) and normal expression of GPVI (not shown) in platelets from α2 (−/−) mice. These findings demonstrate that integrin α2 is not essential for megakaryocyte development and platelet production. However, its absence significantly alters the expression levels of other subunits of the β1 integrin family, suggesting that different α-subunits compete for association with β1 in platelets.Table ISurface expression of different glycoproteins on control, α2(+/−), and α2 (−/−) plateletsControlα2 (+/−)α2 (−/−)GPIa (α2)69.7 ± 8.334.6 ± 7.95.1 ± 1.3GPIc (α5)22.3 ± 6.128.7 ± 9.529.5 ± 9.8GPIc′ (α6)63.5 ± 4.782.3 ± 12.489.5 ± 6.2GPIIa (β1)200.3 ± 13.4169.1 ± 9.7142.2 ± 8.9GPIIIa (β3)251.4 ± 16.9247.8 ± 21.3255.4 ± 18.6GPVI63.1 ± 8.460.9 ± 5.961.7 ± 8.5GPIb411.7 ± 26.3432.2 ± 34.1412.8 ± 20.3GPV190.7 ± 13.9198.8 ± 11.6194.1 ± 6.5CD9743.3 ± 45.5748.9 ± 43.1737.8 ± 38.4The surface expression of the indicated glycoproteins was detected by flow cytometry on control and mutant platelets. Platelets were gated by FSC/SSC characteristics. Results are expressed as mean log fluorescence ± S.D. for seven mice per group. Open table in a new tab The surface expression of the indicated glycoproteins was detected by flow cytometry on control and mutant platelets. Platelets were gated by FSC/SSC characteristics. Results are expressed as mean log fluorescence ± S.D. for seven mice per group. Previous reports (28.Nieuwenhuis H.K. Akkerman J.W. Houdijk W.P. Sixma J.J. Nature. 1985; 318: 470-472Crossref PubMed Scopus (388) Google Scholar, 29.Kehrel B. Balleisen L. Kokott R. Mesters R. Stenzinger W. Clemetson K.J. van de Loo J. Blood. 1988; 71: 1074-1078Crossref PubMed Google Scholar) showed markedly increased bleeding in patients with reduced expression of α2β1integrin on platelets, suggesting a pivotal role of the integrin in hemostasis. In contrast to this hypothesis, we have shown recently (27.Nieswandt B. Brakebusch C. Bergmeier W. Schulte V. Bouvard D. Mokhtari-Nejad R. Lindhout T. Heemskerk J.W. Zirngibl H. Fassler R. EMBO J. 2001; 20: 2120-2130Crossref PubMed Scopus (435) Google Scholar) that bone marrow-chimeric mice with β1 integrin-deficient platelets display no increased bleeding tendency. To test directly these contrasting findings in a defined system, bleeding times were determined in α2 (−/−) mice. Strikingly, bleeding times were found comparable for α2 (−/−) and control mice (Fig. 2B), demonstrating that the lack of α2β1 integrin on platelets, and also on other cells of the cardiovascular system, has no major effect on normal hemostasis in mice. This finding confirms and extends the observations made in β1 integrin mutant mice but stands in sharp contrast to the reported severe bleeding in patients with reduced α2β1 levels on their platelets. The most likely explanation for this discrepancy is that these very few patients had additional defects in their platelets, although species-specific differences cannot be excluded. Several reports (28.Nieuwenhuis H.K. Akkerman J.W. Houdijk W.P. Sixma J.J. Nature. 1985; 318: 470-472Crossref PubMed Scopus (388) Google Scholar, 29.Kehrel B. Balleisen L. Kokott R. Mesters R. Stenzinger W. Clemetson K.J. van de Loo J. Blood. 1988; 71: 1074-1078Crossref PubMed Google Scholar, 42.Saelman E.U. Nieuwenhuis H.K. Hese K.M. de Groot P.G. Heijnen H.F. Sage E.H. Williams S. McKeown L. Gralnick H.R. Sixma J.J. Blood. 1994; 83: 1244-1250Crossref PubMed Google Scholar) suggested a central role of α2β1 integrin during collagen-induced platelet aggregation. In contrast to these findings, the analysis of integrin β1 (−/−) platelets demonstrated a supportive rather than an essential role of β1 integrins in platelet-collagen interactions (27.Nieswandt B. Brakebusch C. Bergmeier W. Schulte V. Bouvard D. Mokhtari-Nejad R. Lindhout T. Heemskerk J.W. Zirngibl H. Fassler R. EMBO J. 2001; 20: 2120-2130Crossref PubMed Scopus (435) Google Scholar). To define unequivocally the role of α2β1integrin in this process, we induced aggregation of control, α2 (+/−), and α2 (−/−) platelets using fibrillar type I collagen. Dose-response and maximum aggregation of mutant platelets did not differ from normal platelets (Fig. 3C). However, onset of aggregation was significantly delayed in α2 (−/−) platelets, and this was particularly evident at low collagen concentrations. Interestingly, no significant delay was observed in α2 (+/−) platelets (Fig. 3B). It is established that platelet activation by collagen strictly depends on functional GPVI (27.Nieswandt B. Brakebusch C. Bergmeier W. Schulte V. Bouvard D. Mokhtari-Nejad R. Lindhout T. Heemskerk J.W. Zirngibl H. Fassler R. EMBO J. 2001; 20: 2120-2130Crossref PubMed Scopus (435) Google Scholar, 35.Nieswandt B. Schulte V. Bergmeier W. Mokhtari-Nejad R. Rackebrandt K. Cazenave J.P. Ohlmann P. Gachet C. Zirngibl H. J. Exp. Med. 2001; 193: 459-469Crossref PubMed Scopus (288) Google Scholar, 43.Poole A. Gibbins J.M. Turner M. van Vugt M.J. van de Winkel J.G. Saito T. Tybulewicz V.L. Watson S.P. EMBO J. 1997; 16: 2333-2341Crossref PubMed Scopus (386) Google Scholar). To test GPVI function, activation of control and mutant platelets was induced by the GPVI-specific agonist collagen-related peptide (CRP) (44.Asselin J. Gibbins J.M. Achison M. Lee Y.H. Morton L.F. Farndale R.W. Barnes M.J. Watson S.P. Blood. 1997; 89: 1235-1242Crossref PubMed Google Scholar). As shown in Fig. 3D, CRP-induced aggregation occurred with the same dose-response characteristics in control and mutant platelets. Further studies showed that ADP- and thrombin-induced activation was not altered significantly in mutant platelets (not shown). These results clearly demonstrate that integrin α2 is not essential for platelet activation by collagen, although the process is slightly delayed in the absence of the integrin. A similar delay in collagen-induced aggregation has been observed on human platelets in the presence of α2β1-blocking antibodies (45.Coller B.S. Beer J.H. Scudder L.E. Steinberg M.H. Blood. 1989; 74: 182-192Crossref PubMed Google Scholar), in integrin β1-deficient mouse platelets (27.Nieswandt B. Brakebusch C. Bergmeier W. Schulte V. Bouvard D. Mokhtari-Nejad R. Lindhout T. Heemskerk J.W. Zirngibl H. Fassler R. EMBO J. 2001; 20: 2120-2130Crossref PubMed Scopus (435) Google Scholar), or in mouse platelets lacking GPV (46.Moog S. Mangin P. Lenain N. Strassel C. Ravanat C. Schuhler S. Freund M. Santer M. Kahn M. Nieswandt B. Gachet C. Cazenave J.P. Lanza F. Blood. 2001; 98: 1038-1046Crossref PubMed Scopus (104) Google Scholar). These defects most likely reflect a reduced stability of the initial platelet-collagen interaction due to the lack of collagen-binding sites on the cells. In vivo, secreted procollagen is proteolytically converted into collagen and assembled into insoluble, cross-striated fibrils (47.Kadler K.E. Holmes D.F. Trotter J.A. Chapman J.A. Biochem. J. 1996; 316: 1-11Crossref PubMed Scopus (1056) Google Scholar). In vitro, collagen fibrils can be partly digested by pepsin, which cleaves the molecule in the non-triple helical region, thereby releasing “soluble” collagen (48.Savage B. Ginsberg M.H. Ruggeri Z.M. Blood. 1999; 94: 2704-2715Crossref PubMed Google Scholar). In numerous studies, such preparations of soluble collagen have been used to characterize the interaction of individual platelet receptors with collagen (48.Savage B. Ginsberg M.H. Ruggeri Z.M. Blood. 1999; 94: 2704-2715Crossref PubMed Google Scholar, 49.Morton L.F. Peachey A.R. Zijenah L.S. Goodall A.H. Humphries M.J. Barnes M.J. Biochem. J. 1994; 299: 791-797Crossref PubMed Scopus (87) Google Scholar, 50.Siljander P. Lassila R. Arterioscler. Thromb. Vasc. Biol. 1999; 19: 3033-3043Crossref PubMed Scopus (40) Google Scholar). We have shown recently that aggregation in response to soluble collagen is abrogated in integrin β1 (−/−) platelets, but it remained unclear whether this defect was entirely based on the absence of α2β1 or, possibly, of other β1 integrins (27.Nieswandt B. Brakebusch C. Bergmeier W. Schulte V. Bouvard D. Mokhtari-Nejad R. Lindhout T. Heemskerk J.W. Zirngibl H. Fassler R. EMBO J. 2001; 20: 2120-2130Crossref PubMed Scopus (435) Google Scholar). To test this directly, we induced aggregation of control and α2 (−/−) platelets with increasing concentrations of soluble collagen. No aggregation of α2 (−/−) platelets occurred at concentrations of up to 500 μg/ml soluble collagen, whereas robust aggregation of control platelets was already seen at 5 μg/ml (Fig. 4A). The critical role of α2β1 integrin in this process was further confirmed by an ∼5-fold right shift of the dose-response curve of the α2 (+/−) platelets when compared with the control (Fig. 4B). Because platelet activation by collagen strictly depends on GPVI (25.Moroi M. Jung S.M. Okuma M. Shinmyozu K. J. Clin. Invest. 1989; 84: 1440-1445Crossref PubMed Scopus (363) Google Scholar, 34.Nieswandt B. Bergmeier W. Schulte V. Rackebrandt K. Gessner J.E. Zirngibl H. J. Biol. Chem. 2000; 275: 23998-24002Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, 43.Poole A. Gibbins J.M. Turner M. van Vugt M.J. van de Winkel J.G. Saito T. Tybulewicz V.L. Watson S.P. EMBO J. 1997; 16: 2333-2341Crossref PubMed Scopus (386) Google Scholar), this finding suggests that integrin α2β1 mediates the stabilization of GPVI-collagen interactions. This stabilization may be particularly important in the case of digested collagen which, in contrast to fibrillar collagen, lacks highly repetitive GPVI recognition sites. Platelet attachment to collagen is a complex process involving the synergistic action of different receptors and signaling pathways (41.Watson S. Berlanga O. Best D. Frampton J. Platelets. 2000; 11: 252-258Crossref PubMed Scopus (69) Google Scholar). We have shown recently that GPVI is essential in this process as GPVI-deficient platelets do not adhere to fibrillar or soluble collagen. In contrast, platelets lacking β1integrins display partial adhesion defects, but it was not clear whether these defects are due to the absence of α2β1 alone, as integrins α5β1 and α6β1are also not expressed on these platelets (27.Nieswandt B. Brakebusch C. Bergmeier W. Schulte V. Bouvard D. Mokhtari-Nejad R. Lindhout T. Heemskerk J.W. Zirngibl H. Fassler R. EMBO J. 2001; 20: 2120-2130Crossref PubMed Scopus (435) Google Scholar). To address this issue, adhesion of control, α2 (+/−), and α2(−/−) platelets to fibrillar as well as soluble collagen was tested in a static assay. Fibrillar collagen induced comparable adhesion of mutant and control platelets in a time-dependent manner (Fig. 5A), confirming that α2β1 is not essential for this process. However, blocking the major collagen-binding site on GPVI by JAQ1-Fab fragments (20 μg/ml (51.Schulte V. Snell D. Bergmeier W. Zirngibl H. Watson S.P. Nieswandt B. J. Biol. Chem. 2001; 276: 364-368Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar)) abolished adhesion of α2(−/−) platelets, whereas control platelets adhered to the same extent. Adhesion of α2 (+/−) platelets was markedly reduced and delayed in the presence of JAQ1 Fab fragments. These results demonstrate the existence of an α2-dependent adhesion mechanism on fibrillar collagen that becomes dominant when the major collagen-binding site on GPVI is blocked (Fig. 5A). A different picture emerged using soluble collagen, to which control platelets adhered in the absence, but not in the presence of JAQ1-Fab fragments, confirming the critical role of GPVI in this process. Strikingly, however, α2 (−/−) platelets did not adhere to soluble collagen in the presence or absence of JAQ1-Fab fragments, also demonstrating an essential role for α2β1 integrin in this interaction. The importance of α2β1 for adhesion to soluble collagen was further confirmed by the reduced and delayed adhesion of α2 (+/−) platelets (Fig. 5B). These results confirm the crucial role of GPVI in the interaction of platelets with collagen and demonstrate that α2β1 is essential for platelet adhesion to soluble collagen, whereas adhesion to fibrillar collagen is only dependent on the integrin when the major collagen-binding site on GPVI is blocked. Although in vivo collagens can be degraded in certain pathological situations, the majority of collagens is deposited in fibrillar form in normal vessel walls. Therefore, GPVI interaction with collagen at sites of vascular injury should not be dependent on integrin α2β1. Once the platelets are activated through GPVI, other adhesive receptors, most importantly integrin αIIbβ3, can mediate firm attachment and thrombus growth (24.Savage B. Almus-Jacobs F. Ruggeri Z.M. Cell. 1998; 94: 657-666Abstract Full Text Full Text PDF PubMed Scopus (672) Google Scholar, 27.Nieswandt B. Brakebusch C. Bergmeier W. Schulte V. Bouvard D. Mokhtari-Nejad R. Lindhout T. Heemskerk J.W. Zirngibl H. Fassler R. EMBO J. 2001; 20: 2120-2130Crossref PubMed Scopus (435) Google Scholar). In conclusion, we show that mice lacking α2β1 integrin receptors develop normally, are fertile, and exhibit surprisingly subtle alterations. More sophisticated analyses will be required to illustrate whether other subtle defects are present and which other receptor(s) may compensate for loss of α2β1 function. Possibly α2β1 integrins are not essential for development but may be needed for tissue repair, host defense, or other challenges that the adult organism has to meet. The analysis of α2-deficient platelets revealed a subtle rather than a major defect which is in line with recent studies on β1-deficient platelets. The mice described here will allow detailed studies on the involvement of integrin α2in thrombotic diseases where it has been proposed to play a major role (23.Santoro S.A. Blood. 1999; 93: 3575-3577Crossref PubMed Google Scholar). We thank Ulrike Mayer (Manchester, UK) for the generous gift of polyclonal anti-α2-antibodies; Kerstin Elias and Marion Reibetanz (Cologne) for excellent technical assistance; and Reinhard Fässler and Cord Brakebusch (Lund, Sweden) and Roswitha Nischt, Christoph Leuker, and Monzur Murshed (Cologne) for critical discussion.