Abstract
Epac1 (Exchange protein directly activated by cAMP 1) limits fluid loss from the circulation by tightening the endothelial barrier. We show here that Epac1−/− mice, but not Epac2−/− mice, have prolonged bleeding time, suggesting that Epac1 may limit fluid loss also by restraining bleeding. The Epac1−/− mice had deficient in vitro secondary hemostasis. Quantitative comprehensive proteomics analysis revealed that Epac1−/− mouse platelets (thrombocytes) had unbalanced expression of key components of the glycoprotein Ib-IX-V (GPIb-IX-V) complex, with decrease of GP1bβ and no change of GP1bα. This complex is critical for platelet adhesion under arterial shear conditions. Furthermore, Epac1−/− mice have reduced levels of plasma coagulation factors and fibrinogen, increased size of circulating platelets, increased megakaryocytes (the GP1bβ level was decreased also in Epac1−/− bone marrow) and higher abundance of reticulated platelets. Viscoelastic measurement of clotting function revealed Epac1−/− mice with a dysfunction in the clotting process, which corresponds to reduced plasma levels of coagulation factors like factor XIII and fibrinogen. We propose that the observed platelet phenotype is due to deficient Epac1 activity during megakaryopoiesis and thrombopoiesis, and that the defects in blood clotting for Epac1−/− is connected to secondary hemostasis.
Highlights
Damage to blood vessel walls initiates a series of responses to limit blood loss
We report here that Epac1−/− mice have increased bleeding time, impaired secondary hemostasis, moderately increased platelet size, increased number of reticulated platelets and significantly changed expressions of several proteins, including up-regulated αIIbβ3-associated Integrin linked protein kinase (ILK) and down-regulated plasma coagulation factors
As vasopressin is able to rapidly induce such exocytosis, we determined plasma von Willebrand factor (vWF) concentrations for both resting state mice and mice injected with the vasopressin analog dDVAP6, 30
Summary
Damage to blood vessel walls initiates a series of responses to limit blood loss. At the sites of vascular injury extracellular matrix (ECM) proteins become exposed to blood and react with specific platelet receptors to induce a multistep platelet adhesion process[1,2,3,4]. At the shear rates in small arteries and microvasculature, the adhesion depends crucially on the GPIb-IX-V platelet receptors interaction with ECM-bound von Willebrand factor (vWF)[2]. This interaction has a rapid on-off-rate causing platelets to roll along the damaged vessel wall, effectively slowing down the platelet flow rate. Epac[1] deficiency led to generation of megakaryocytes (MK) and platelets with severely reduced levels of platelet GP1bβ, a subunit of the GPIb-IX-V receptor This may affect the interaction of platelet GPIb-IX-V with the vessel wall at sites of vascular injury, and may be translated into defective platelet adhesion and impaired hemostasis under high shear conditions. The defects in blood clotting for Epac1−/− mice seems to be connected to secondary hemostasis
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