Targeted gene disruption of natural anticoagulant proteins in mice

Abstract

The blood coagulation system is a complicated cascade of reactions and feedback regulations that executes a rapid response to vascular injury, yet avoids occlusion of the vessel. There are several key components of this system in the regulation of blood clot propagation, such as antithrombin (AT), tissue factor pathway inhibitor (TFPI), thrombomodulin (TM) and protein C (PC), of which defect causes thromboembolic diseases. In recent years, targeted gene disruption technique by homologous recombination has been introduced to investigate the physiological roles of those natural anticoagulant molecules, not only in thrombogenesis but also in embyrogenesis. We have studied the natural anticoagulantion system in a decade, and recently established AT knockout mice as well as ryudocan (syndecan-4) knockout mice. Ryudocan is a cell surface heparan sulfate proteoglycan, which bears heparin-like glycosaminoglycan (heparan sulfate) cahins, originally cloned from rat microvascular endothelial cells. We have demonstrated that ryudocan deficiency impairs the control of coagulation in fetal vessels of the placenta in mice. We have also reported that complete antithrombin deficiency in mice results in embryonic lethality, with severe fibrin deposition in the myocardium and the liver, accompanied with extensive subcutaneous hemorrhage. In this presentation, recent advances in understanding roles of natural anticoagulant molecules through the researches of targeted gene-knockout mice, including our experiences in antithrombin deficient mice and ryudocan deficient mice, will be discussed.

Cell biology of embryonic migration

“…Reduced neural crest cell motility (Breau et al, 2006) β3 integrin Integrins Total Defects in platelet aggregation and clot retraction, hemmorhages, postnatal anemia, and reduced survival (Hodivala-Dilke, 1999) β8 integrin Integrins Total Embryonic or perinatal lethality with profound defects in vascular development, insufficient vascularization of the placenta and yolk sac (Zhu et al, 2002) VCAM-1 Integrins Total Embrynic lethality at E10-12, abnormal placental development (Gurtner et al, 1995) Syndecan-4 Extracellular matrix Total Blood vessel defects in the placenta; defects in angiogenesis and wound repair (Echtermeyer, 2001;Ishiguro, 2000;Kojima, 2002) N-syndecan Extracellular matrix Total Impaired neural migration in the developing brain (Hienola et al, 2006) Perlecan Extracellular matrix Total Defects in heart integrity, invasion of brain tissue into the overlaying ectoderm, severe defect in cartilage, cleft palates and death shortly after birth from respiratory failure. (Costell et al, 1999) Tenascin C Extracellular matrix Total NC cells fail to disperse laterally (Tucker, 2001) Laminin γ1 Extracellular matrix Total Death at E5.5, lack of basement membranes (Smyth et al, 1999) Laminin β2 Extracellular matrix Total Postnatal death between P15-30, neuromuscular junctions and glomerular defects (Noakes et al, 1995a;Noakes et al, 1995b;Patton et al, 1997) Laminin α2 Extracellular matrix Total Death by 5 weeks postnatal, severe muscular dystrophy and peripheral neurophathy (Miyagoe et al, 1997) Laminin α2 (dy/dy) Extracellular matrix Spontaneous Adult lethality, severe muscular dystrophy and peripheral nerve dysmyelination (Patton et al, 1999;Patton et al, 1997) Laminin α3 Extracellular matrix Total Death at P2-3, epithelial adhesion defect (Ryan et al, 1999) Laminin α4 Extracellular matrix Total Transient microvascular defect with hemorrhages and misalignment of neuromuscular junctions (Patton et al, 2001;Thyboll et al, 2002) Laminin α5 Extracellular matrix Total Death at E14-E17, with placental vessel, neural (Miner et al, 1998;Miner and Li, 2000) Protein Function Type Phenotype Reference tube, limb, and kidney defects Fibronectin Extracellular matrix Total Death before E14.5, shortened anterior-posterior axes, deformed neural tubes, and defects in mesodermally derived tissues.…”

Mechanisms of glycosaminoglycan activation of the serpins in hemostasis

“…It circulates at a concentration of 2.3 µmol L −1 and is capable of inhibiting several of the proteases in the coagulation cascade, but, based on rates of inhibition, its primary targets are FXa and thrombin. The importance of AT is demonstrated by the high association of deficiency with venous thrombosis [36], by the embryonic lethal phenotype in the mouse knockout model [37], and by the success of heparin therapy. The anticoagulant effect of natural heparin and the new synthetic heparins is mediated predominantly through the activation of AT.…”

Allosteric Activation of Antithrombin Critically Depends upon Hinge Region Extension

“…The serpin antithrombin (AT) 1 is capable of inhibiting most of the serine proteases generated in the blood coagulation cascade. Its central role is illustrated by the embryonic lethal phenotype of the AT knock-out mouse (1) and by the success of therapeutic heparin.…”

“…Its central role is illustrated by the embryonic lethal phenotype of the AT knock-out mouse (1) and by the success of therapeutic heparin. Heparin exerts its anticoagulant effect primarily through an interaction with AT, due to the presence of a specific pentasaccharide sequence found in one-third of heparin chains (2,3).…”