Diminished Thrombogenic Responses by Deletion of the Podocalyxin Gene in Mouse Megakaryocytes

Abstract

Podocalyxin (Podxl) is a type I membrane sialoprotein of the CD34 family, originally described in the epithelial glomerular cells of the kidney (podocytes) in which it plays an important function. Podxl can also be found in megakaryocytes and platelets among other extrarenal places. The surface exposure of Podxl upon platelet activation suggested it could play some physiological role. To elucidate the function of Podxl in platelets, we generated mice with restricted ablation of the podxl gene in megakaryocytes using the Cre-LoxP gene targeting methodology. Mice with Podxl-null megakaryocytes did not show any apparent phenotypical change and their rates of growth, life span and fertility did not differ from the floxed controls. However, Podxl-null mice showed prolonged bleeding time and decreased platelet aggregation in response to physiological agonists. The number, size-distribution and polyploidy of Podxl-null megakaryocytes were similar to the floxed controls. Podxl-null platelets showed normal content of surface receptors and normal activation by agonists. However, the mice bearing Podxl-null platelets showed a significant retardation in the ferric chloride-induced occlusion of the carotid artery. Moreover, acute thrombosis induced by the i.v. injection of sublethal doses of collagen and phenylephrine produced a smaller fall in the number of circulating platelets in Podxl-null mice than in control mice. In addition, perfusion of uncoagulated blood from Podxl-null mice in parallel flow chamber showed reduced adhesion of platelets and formation of aggregates under high shear stress. It is concluded that platelet Podxl is involved in the control of hemostasis acting as a platelet co-stimulator, likely due to its pro-adhesive properties.

Role of CD34 family members in lumen formation in the developing kidney

“…Conditional Podxl2 flox/flox (hereafter referred to as EG flox/flox ) mice were mated with mice carrying conditional alleles of Podocalyxin (Podxl1 flox/flox ) [23] to generate EG flox/flox ; Podxl1 flox/flox mice. The doubly conditional mice were crossed to Six2-cre and Pax3-cre transgenic mice [24, 25] to obtain EG flox/flox ; Podxl1 flox/flox ; Six2-eGFP-cre and EG flox/flox ; Podxl1 flox/flox ; Pax3-cre mutant embryos.…”

Loss of endothelial barrier integrity in mice with conditional ablation of podocalyxin (Podxl) in endothelial cells

“…As previously reported (Pericacho et al, 2011), 100 µL of blood was collected from one of the jugular veins of anesthetized mice into a tube containing 10 µL of 0.129 mol/L sodium citrate. A mixture of collagen/epinephrine (150 ng collagen type I (Chronolog) and 15 ng epinephrine (SIGMA) per g of mice body weight) was then injected into the same vein and, after 2 minutes, blood was withdrawn from the other jugular vein as indicated above.…”

“…Recent reports have provided useful data on the role of extrarenal Podxl. Platelet Podxl is involved in the control of haemostasis (Alonso-Martín et al, 2010;Pericacho et al, 2011). In brain, it seems to play multiple roles in neural development (Nowakowski et al, 2010;Vitureira et al, 2010).…”

“…We have recently generated a Podxl "floxed" mouse line in which podxl alleles carry loxP sites flanking a DNA fragment comprising exons 5-7 (Nowakowski et al, 2010;Pericacho et al, 2011). In the present work, to determine whether Podxl plays a role in regulating EC functions, these mice were crossed with the Tie2-Cre mouse line that produces Cre recombinase in endothelial and some hematopoietic cells (Kisanuki et al, 2001).…”

Control of cell adhesion and migration by podocalyxin. Implication of Rac1 and Cdc42

“…Moreover, the restricted ablation of the podxl gene in megakaryocytes added further evidence in support of PODXL as a proadhesive molecule [21]. In high endothelial venules (HEV), PODXL binds L-selectin from circulating leucocytes and interact with integrins [22].…”