The human Bernard-Soulier syndrome is an autosomal recessive disorder of platelet dysfunction presenting with mild thrombocytopenia, circulating ''giant'' platelets and a bleeding phenotype. The bleeding in patients with the Bernard-Soulier syndrome is disproportionately more severe than suggested by the reduced platelet count and is explained by a defect in primary hemostasis owing to the absence of the platelet glycoprotein (GP) Ib-IX-V membrane receptor. However, the molecular basis for the giant platelet phenotype and thrombocytopenia have remained unresolved but assumed to be linked to an absent receptor complex. We have disrupted the gene encoding the ␣-subunit of mouse GP Ib-IX-V (GP Ib␣) and describe a murine model recapitulating the hallmark characteristics of the human Bernard-Soulier syndrome. The results demonstrate a direct link between expression of a GP Ib-IX-V complex and normal megakaryocytopoiesis and platelet morphogenesis. Moreover, using transgenic technology the murine Bernard-Soulier phenotype was rescued by expression of a human GP Ib␣ subunit on the surface of circulating mouse platelets. Thus, an in vivo model is defined for analysis of the human GP Ib-IX-V receptor and its role in the processes performed exclusively by megakaryocytes and platelets.

Platelet interaction with exposed adhesive ligands at sites of vascular injury is required to initiate a normal hemostatic response and may become a pathogenic factor in arterial diseases leading to thrombosis. We report a targeted disruption in a key receptor for collageninduced platelet activation, glycoprotein (GP) VI. The breeding of mice with heterozygous GP VI alleles produced the expected frequency of wild-type, heterozygous, and homozygous genotypes, indicating that these animals had no reproductive problems and normal viability. GP VI null platelets failed to aggregate in response to type I fibrillar collagen or convulxin, a snake venom protein and known platelet agonist of GP VI. Nevertheless, tail bleeding time measurements revealed no severe bleeding tendency as a consequence of GP VI deficiency. Ex vivo platelet thrombus formation on type I collagen fibrils was abolished using blood from either GP VI null or FcR-␥ null animals. Reflection interference contrast microscopy revealed that the lack of thrombus formation by GP VI null platelets could be linked to a defective platelet activation following normal initial tethering to the surface, visualized as lack of spreading and less stable adhesion. These results illustrate the role of GP VI in postadhesion events leading to the development of platelet thrombi on collagen fibrils. IntroductionPlatelet membrane receptors interact with surface-bound adhesive ligands and, as such, become essential for hemostasis and thrombosis. 1 There are numerous unique receptors interacting with different adhesive ligands suggesting that a large opportunity exists for functional redundancy in platelet adhesion. However, an emerging theme of platelet biology is the relevance of different membrane receptors in different areas of the vasculature. 2,3 A specific example is the exclusive role for the platelet glycoprotein (GP) Ib-IX-V complex and von Willebrand factor in areas of the vascular system where flow rates and high shear occur, such as in small arteries and arterioles. 4 Thus, defining the physiologic relevance of an individual receptor and its ligand is an important aspect for understanding participation of the platelet in hemostasis and thrombosis.Among adhesive ligands of the extravascular matrix, collagen is a significant component with a number of known collagen receptors on the platelet surface. 5,6 One of the more recently characterized collagen receptors is GP VI. 7 The molecular cloning of GP VI revealed it to be a member of the immunoglobulin superfamily of type I transmembrane proteins. [8][9][10] The surface expression of GP VI requires the concomitant expression of the ␥-subunit of the FcR receptor (FcR-␥) and their association is functionally relevant as collagen binding to GP VI results in platelet signaling via the immunoreceptor tyrosine-based activation motif (ITAM) located in the FcR-␥ subunit. 8,[11][12][13][14] As with many of the platelet receptors, the in vivo relevance of GP VI was established prior to its description and recognition as a p...

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The Wnt/␤-catenin signaling cascade activates genes that allow cells to adopt particular identities throughout development. In adult self-renewing tissues like intestine and blood, activation of the Wnt pathway maintains a progenitor phenotype, whereas forced inhibition of this pathway promotes differentiation. In the lung alveolus, type 2 epithelial cells (AT2) have been described as progenitors for the type 1 cell (AT1), but whether AT2 progenitors use the same signaling mechanisms to control differentiation as rapidly renewing tissues is not known. We show that adult AT2 cells do not exhibit constitutive ␤-catenin signaling in vivo, using the AXIN2

The platelet paradigm in hemostasis and thrombosis involves an initiation step that depends on platelet membrane receptors binding to ligands on a damaged or inflamed vascular surface. Once bound to the surface, platelets provide a unique microenvironment supporting the accumulation of more platelets and the elaboration of a fibrin-rich network produced by coagulation factors. The platelet-specific receptor glycoprotein (GP) Ib-IX, is critical in this process and initiates the formation of a platelet-rich thrombus by tethering the platelet to a thrombogenic surface. A role for platelets beyond the hemostasis/thrombosis paradigm is emerging with significant platelet contributions in both tumorigenesis and inflammation. We have established congenic (N10) mouse colonies (C57BL/6J) with dysfunctional GP Ib-IX receptors in our laboratory that allow us an opportunity to examine the relevance of platelet GP Ib-IX in syngeneic mouse models of experimental metastasis. Our results demonstrate platelet GP Ib-IX contributes to experimental metastasis because a functional absence of GP Ib-IX correlates with a 15-fold reduction in the number of lung metastatic foci using B16F10.1 melanoma cells. The results demonstrate that the extracellular domain of the ␣-subunit of GP Ib is the structurally relevant component of the GP Ib-IX complex contributing to metastasis. Our results support the hypothesis that platelet GP Ib-IX functions that support normal hemostasis or pathologic thrombosis also contribute to tumor malignancy.adhesion ͉ tumor ͉ hemostasis ͉ knockout ͉ melanoma C irculating blood platelets have an inherent adhesive potential long recognized as essential for hemostasis and thrombosis. Beyond the platelet paradigm for blood clotting and thrombosis, it is becoming apparent that the platelet's adhesive potential influences pathological events outside its role in hemostasis. Indeed, emerging hypotheses suggest the platelet paradigm in hemostasis and thrombosis, an accumulation of platelets and the elaboration of a fibrin matrix, may provide a mechanism for circulating tumor cells to metastasize. Experimental proof that platelets effect tumor metastasis was provided in models where an experimental lowering of circulating platelet counts reduced metastasis (1-3).Evidence suggests that carcinoma cells entering the circulation interact with both platelets and leukocytes to form tumor cell aggregates (1, 4). Data have suggested one mechanism linking the platelet to metastasis is a platelet ''cloak'' surrounding the tumor cell and protecting the tumor cell from immune surveillance (5, 6). Tumorigenesis has been linked to several molecules essential for blood coagulation and normal platelet function. These include thrombin, tissue factor, platelet P-selectin, fibrinogen, and lysophosphatidic acid (3,4,(7)(8)(9)(10)(11)(12)(13)(14)(15)(16). Together, these results suggest that platelets and their procoagulant activity support tumor metastasis, possibly by aiding tumor cells to lodge in the microvasculature and either extravasate...

Pulmonary fibrosis is a disease that results in loss of normal lung architecture, but the signaling events that drive tissue destruction are incompletely understood. Wnt/β-catenin signaling is important in normal lung development, but whether abnormal signaling occurs in lung fibrosis due to systemic sclerosis and the consequences of β-catenin signaling toward the fibrogenic phenotype remain poorly defined. In this study, we show nuclear β-catenin accumulation in fibroblastic foci from lungs of patients with systemic sclerosis-associated advanced pulmonary fibrosis. Forced activation of β-catenin signaling in three independently derived sources of normal human lung fibroblasts promotes proliferation and migratory activities but is not sufficient to activate classic markers of fibroblast activation, such as TGF-β, type 1 collagen, α-smooth muscle actin, and connective tissue growth factor. These findings indicate that activation of β-catenin signaling in pulmonary fibroblasts may be a common feature of lung fibrosis, contributing to fibroproliferative and migratory activities associated with the disease.

The platelet glycoprotein Ib-IX receptor binds surfacebound von Willebrand factor and supports platelet adhesion to damaged vascular surfaces. A limited number of mutations within the glycoprotein Ib-IX complex have been described that permit a structurally altered receptor to interact with soluble von Willebrand factor, and this is the molecular basis of platelet-type von Willebrand disease. We have developed and characterized a mouse model of platelet-type von Willebrand disease (G233V) and have confirmed a platelet phenotype mimicking the human disorder. The mice have a dramatic increase in splenic megakaryocytes and splenomegaly. Recent studies have demonstrated that hematopoetic cells can influence the differentiation of osteogenic cells. Thus, we examined the skeletal phenotype of mice expressing the G233V variant complex. At 6 months of age, G233V mice exhibit a high bone mass phenotype with an approximate doubling of trabecular bone volume in both the tibia and femur. Serum measures of bone resorption were significantly decreased in G233V animals. With decreased bone resorption, cortical thickness was increased, medullary area decreased, and consequently, the mechanical strength of the femur was significantly increased. Using ex vivo bone marrow cultures, osteoclast-specific staining in the G233V mutant marrow was diminished, whereas osteoblastogenesis was unaffected. These studies provide new insights into the relationship between the regulation of megakaryocytopoiesis and bone mass. (Am J

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