Endothelium-specific loss of murine thrombomodulin disrupts the protein C anticoagulant pathway and causes juvenile-onset thrombosis (original) (raw)
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A targeted point mutation in thrombomodulin generates viable mice with a prethrombotic state
Journal of Clinical Investigation, 1998
The activity of the coagulation system is regulated, in part, by the interaction of thrombin with the endothelial cell receptor thrombomodulin with subsequent generation of activated protein C and suppression of thrombin production. Our previous investigation demonstrated that ablation of the thrombomodulin gene in mice causes embryonic lethality before the assembly of a functional cardiovascular system, indicating a critical role for the receptor in early development. In the current study, we show that a single amino acid substitution in thrombomodulin dissociates the developmental function of the receptor from its role as a regulator of blood coagulation. Homozygous mutant mice with severely reduced capacity to generate activated protein C or inhibit thrombin develop to term, and possess normal reproductive performance. The above animals exhibit increased fibrin deposition in selected organs, which implies tissue specific regulation of the coagulation system that is supported by further evidence from the examination of mice with defects in fibrinolysis. The thrombomodulin-deficient animals provide a murine model to examine known or identify unknown genetic and environmental factors that lead to the development of thrombosis. (
Thrombomodulin as a regulator of the anticoagulant pathway
Blood Coagulation & Fibrinolysis, 2012
Thrombomodulin is a cell surface-expressed glycoprotein that serves as a cofactor for thrombin-mediated activation of protein C (PC), an event further amplified by the endothelial cell PC receptor. The PC pathway is a major anticoagulant mechanism that downregulates thrombin formation and hedges thrombus formation. The objectives of this review were to review recent findings regarding thrombomodulin structure, its involvement in the regulation of hemostasis and further discuss the implication, if any, of the genetic polymorphisms in the thrombomodulin gene in the risk of development of thrombosis. We performed a literature search by using electronic bibliographic databases. Although the direct evaluation of risk situations associated with thrombomodulin mutations/ polymorphisms could be of clinical significance, it appears that mutations that affect the function of thrombomodulin are rarely associated with venous thromboembolism. However, several polymorphisms are reported to be associated with increased risk for arterial thrombosis. Additionally studies on knock out mice as well studies on humans bearing rare mutations suggest that thrombomodulin dysfunction may be implicated in the pathogenesis of myocardial infraction. Blood Coagul Fibrinolysis 23:1-10 ß 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins.
Characterization of a Mouse Model for Thrombomodulin Deficiency
Arteriosclerosis, Thrombosis, and Vascular Biology, 2001
Mutations in the gene encoding thrombomodulin (TM), a thrombin regulator, are suspected risk factors for venous and arterial thrombotic disease. We have previously described the generation of TM Pro/Pro mice carrying a TM gene mutation that disrupts the TM-dependent activation of protein C. Here, it is shown that inbred C57BL/6J TM Pro/Pro mice exhibit a hypercoagulable state and an increased susceptibility to thrombosis and sepsis. Platelet thrombus growth after FeCl 3 -induced acute endothelial injury was accelerated in mutant mice. Vascular stasis after permanent ligation of the carotid artery precipitated thrombosis in mutant but not in normal mice. Mutant mice showed increased mortality after exposure to high doses of endotoxin and demonstrated altered cytokine production in response to low-dose endotoxin. The severity of the hypercoagulable state and chronic microvascular thrombosis caused by the TM Pro mutation is profoundly influenced by mouse strain-specific genetic differences between C57BL/6 and 129SvPas mice. These data demonstrate that in mice, TM is a physiologically relevant regulator of platelet-and coagulation-driven large-vessel thrombosis and modifies the response to endotoxin-induced inflammation. The phenotypic penetrance of the TM Pro mutation is determined by as-yet-uncharacterized genetic modifiers of thrombosis other than TM. (Arterioscler Thromb Vasc Biol. 2001;21:1531-1537.)
AJP: Heart and Circulatory Physiology, 2013
Thrombomodulin (TM) is a 557-amino acid protein with a broad cell and tissue distribution consistent with its wide-ranging physiological roles. When expressed on the lumenal surface of vascular endothelial cells in both large vessels and capillaries, its primary function is to mediate endothelial thromboresistance. The complete integral membrane-bound protein form displays five distinct functional domains, although shorter soluble (functional) variants comprising the extracellular domains have also been reported in fluids such as serum and urine. TM-mediated binding of thrombin is known to enhance the specificity of the latter serine protease toward both protein C and thrombin activatable fibrinolysis inhibitor (TAFI), increasing their proteolytic activation rate by almost three orders of magnitude with concomitant anticoagulant, antifibrinolytic, and anti-inflammatory benefits to the vascular wall. Recent years have seen an abundance of research into the cellular mechanisms governi...
Arteriosclerosis, Thrombosis, and Vascular Biology, 2011
Methods and Results-Knockin mice that express human thrombomodulin from the murine thrombomodulin gene locus were generated. Compared with wild-type mice, human thrombomodulin knockin mice exhibited decreased protein C activation in the aorta (P < 0.01) and lung (P < 0.001). Activation of endogenous protein C following infusion of thrombin was decreased by 90% in knockin mice compared with wild-type mice (P < 0.05). Carotid artery thrombosis induced by photochemical injury occurred more rapidly in knockin mice (12 ± 3 minutes) than wild-type mice (31 ± 6 minutes; P < 0.05). No differences in serum cytokine levels were detected between knockin and wild-type mice after injection of endotoxin. When crossed with apolipoprotein E-deficient mice and fed a Western diet, knockin mice had a further decrease in protein C activation but did not exhibit increased atherosclerosis.
Journal of Clinical Investigation, 1998
Matings of mice heterozygous for a protein C (PC) deficient allele, produced by targeted PC gene inactivation, yielded the expected Mendelian distribution of PC genotypes. Pups with a total deficiency of PC (PC Ϫ / Ϫ), obtained at embryonic day (E) 17.5 and at birth, appeared to develop normally macroscopically, but possessed obvious signs of bleeding and thrombosis and did not survive beyond 24 h after delivery. Microscopic examination of tissues and blood vessels of E17.5 PC Ϫ / Ϫ mice revealed their normal development, but scattered microvascular thrombosis in the brain combined with focal necrosis in the liver was observed. In addition, bleeding was noted in the brain near sites of fibrin deposition. The severity of these pathologies was exaggerated in PC Ϫ / Ϫ neonates. Plasma clottable fibrinogen was not detectable in coagulation assays in PC Ϫ / Ϫ neonatal mice, suggestive of fibrinogen depletion and secondary consumptive coagulopathy. Thus, while total PC deficiency did not affect the anatomic development of the embryo, severe perinatal consumptive coagulopathy occurred in the brain and liver of PC Ϫ / Ϫ mice, suggesting that a total PC deficiency is inconsistent with short-term survival.
Production of cloned pigs expressing human thrombomodulin in endothelial cells
Xenotransplantation, 2012
For long-term xenograft survival, coagulation control is one of the remaining critical issues. Our attention has been directed toward human thrombomodulin (hTM), because it is expected to exhibit the following beneficial effects on coagulation control and cytoprotection: (i) to solve the problem of molecular incompatibility in protein C activation; (ii) to exert a role as a physiological regulator, only when thrombin is formed; (iii) to suppress direct prothrombinase activity; and (iv) to have anti-inflammatory properties. hTM gene was transfected into pig (Landrace/Yorkshire) fibroblasts using pCAGGS expression vector and pPGK-puro vector. After puromycin selection, only fibroblasts expressing a high level of hTM were collected by cell sorting and then applied to nuclear transfer. Following electroactivation and subsequent culture, a total of 1547 cleaved embryos were transferred to seven surrogate mother pigs. Two healthy cloned piglets expressing hTM were born, successfully grew to maturity and produced normal progeny. Immunohistochemical staining of organs from F1 generation pigs demonstrated hTM expression in endothelial cells as well as parenchymal cells. High expression was observed particularly in endothelial cells of kidney and liver. Aortic endothelial cells from cloned pigs were found to express hTM levels similar to human umbilical vein endothelial cells (HUVEC) and to make it possible to convert protein C into activated protein C. The blockade of human endothelial cell protein C receptor (hEPCR) significantly reduced APC production in HUVEC, but not in hTM-PAEC. Although no bleeding tendency was observed in hTM-cloned pigs, activated partial thromboplastin time (APTT) was slightly prolonged and soluble hTM was detected in pig plasma. hTM was expressed in platelets and mononuclear cells, but not in RBC. Cloned pigs expressing hTM in endothelial cells at a comparable level to HUVEC were produced. As complete suppression of antigen-antibody reaction in the graft is essential for accurate assessment of transgene related to coagulation control, production of genetically engineered pigs expressing hTM and complement regulatory protein based on galactosyltransferase knockout is desired.