The present results demonstrate that the tissue-restricted depletion of the TM gene in the endothelial cell lineage, as defined by tie2 expression, is compatible with early embryonic growth and morphogenesis beyond the critical developmental block encountered by mice with complete TM deficiency. Nevertheless, 40% of the animals lacking endothelial TM die in utero around day 10.5 pc, whereas the remaining animals are born alive and survive the postnatal period. The reduction of TM in vascular endothelium nevertheless results in a spontaneous and progressive coagulopathy at young age. Onset and progression of overt disease occur in the absence of pronounced inflammatory changes and can be entirely prevented by oral anticoagulation therapy, indicating that excessive coagulation activation and ensuing thrombosis are the predominant underlying mechanism of disease caused by the depletion of TM from vascular endothelium.
TM-inactivation restricted by tie2 promoter–dependent expression of Cre-recombinase bypassed the early midgestational developmental block encountered by completely TM-deficient embryos, but about 40% of TMLox embryos died at a later stage. The rescue of TMLox embryos from early midgestational lethality corroborates earlier results identifying nonendothelial placenta cells as the critical tissue sites requiring TM expression during development (16
embryos, in which TM expression is restricted to the placenta, are rescued from early lethality, but succumb to intrauterine hemorrhage at a later stage. The partial embryonic lethality of TMLox embryos differs in two aspects from the intrauterine hemorrhage observed in TM-nulltetraploid
): First, TM-nulltetraploid
embryos die between days 12.5 and 16.5 pc, whereas the partial embryonic lethality of TMLox embryos occurs around day 10.5 pc. Second, TM-nulltetraploid
embryos succumb to a characteristic hemorrhage within the embryo proper that was not observed in any of the TMLox embryos at day 10.5 pc or later stages. These differences indicate that the partial embryonic lethality of TMLox embryos is the consequence of a distinct defect not observed in TM-nulltetraploid
embryos. Given that the complete absence of TM from all cells except nonendothelial placental tissues is compatible with normal embryonic development (16
), the defect causing partial embryonic lethality in TMLox mice must reside in the placenta. Expression of tie2 in extraembryonic tissues has indeed been demonstrated in mice (30
) and in humans (31
). In the latter, the endovascular trophoblast, a highly specialized trophoblast subpopulation invading and breaching maternal blood vessels, has been identified as a tie2-expressing cell population (31
). Defects in the endovascular trophoblast are thought to impair pregnancy outcome in humans, e.g., in preeclampsia (33
). Experiments have been initiated to investigate whether tie2-dependent TM inactivation similarly occurs in mouse endovascular trophoblast cells of TMLox embryos, and to determine the mechanism leading to catastrophic placental failure. That only some, but not all, TMLox mice succumb to this defect suggests that excision of the conditional TM allele in tie2-expressing trophoblast might be incomplete, similar to the highly efficient, but incomplete (87–99%), deletion of floxed genes reported by others (34
). Indeed, residual TM expression was also observed in embryonic blood vessel endothelium of the yolk sac, as well as in a small number of brain and lung capillaries. Spurious TM expression in endothelial cells, together with the sustained expression in nonendothelial cells, likely contributes to some extent to the substantial in vivo PC activation remaining in thrombin-challenged TMLox mice. The latter could explain not only the much better survival of TMLox animals, compared with PC-knockout mice (27
), but also the absence of intrauterine hemorrhage observed in completely TM-deficient embryos (16
). On the other hand, TM expression by mononuclear cells is unlikely to result in significant generation of activated PC in wild-type or mutant mice, as (a) TM gene expression was observed in less than 0.1% of mononuclear cells, and (b) cell preparations enriched for monocytes did not result in detectable PC activation.
Animals bypassing the developmental impediment at day 10.5 pc are born healthy and lack a visible thrombotic disease until age 2–3 weeks. Nevertheless, virtually all of the mutant mice then develop a severe thrombosis, terminating in a lethal consumptive coagulopathy. The fate of TMLox mice resembles the catastrophic consequences of a disrupted TM/PC pathway in humans with PC or PS deficiency (11
). The hallmark of PC and PS deficiencies in humans are thrombotic occlusion of large peripheral veins and microvascular dermal thrombosis (11
). Other tissues, such as the liver, lung, and male genitals, are beginning to emerge as sides of primary thrombosis in these patients (37
). The high frequencies of priapism and thrombotic liver and lung injury in TMLox mice, including such catastrophic sequelae as Budd Chiari syndrome, emphysema, and pulmonary hypertension, demonstrate that these manifestations are a direct consequence of a disrupted TM/PC pathway. The disease progression in TMLox mice is characterized by a transition from a markedly prethrombotic state into overt thrombosis of progressive severity. Elevated TAT and D-Dimer plasma levels and consumption of fibrinogen and platelets demonstrate the presence of a hypercoagulable state already at age 3 weeks. Within a short time thereafter (5 weeks in males, 9 weeks in females), 80% of mice develop overt thrombotic disease. Cardiac hypertrophy is already apparent in 3-week-old TMLox mice, indicating preexisting increased vascular resistance in the pulmonary circulation.
The coincidence of overt thrombotic lesions with sexual maturation, the gender differences in the disease evolution, and the age- and gender-dependent differences of the hemostatic system in humans (42
) prompted us to investigate the effects of gonadal hormones on the onset and progression of phenotypically overt thrombosis. The unaltered disease course after gonadectomy of 18- to 19-day-old TMLox mice demonstrates that the sexual maturation and the associated hormonal changes do not trigger the transition from a severe prethrombotic or hypercoagulable state to overt thrombosis. Interestingly, gonadectomy eliminated the survival advantage of female mice, showing that gender-specific differences modulate disease progression through an undefined mechanism.
The efficiency of the warfarin treatment, together with the lack of evidence for excessive production of proinflammatory cytokines at early disease stages, implies that the underlying disease mechanism is almost exclusively caused by increased coagulation system activity. The prethrombotic phenotype in TMLox mice spontaneously progresses into a lethal consumptive coagulopathy in the absence of the complex cytokine changes observed in sepsis. Elevated IL-6 levels were observed only in older TMLox animals and might be related to excessive and widespread organ damage in these animals. Therefore, disruption of the TM/PC pathway per se is not sufficient to elicit alterations of proinflammatory cytokines, suggesting that the TM/PC pathway has no or only a minor role in controlling baseline levels of inflammatory cytokines.
Disruption of the TM/PC pathway in mice at the level of either TM or PC function elicits qualitatively distinct consequences that might reflect disparate physiological roles of individual pathway components. For example, atrial blood clots were observed only in mice lacking PC (27
) or carrying the factor V Leiden mutation (26
), but not in TM-nulltetraploid
) or TMLox mice. Furthermore, both PC-deficient (27
) and factor V Leiden (26
) mice displayed markedly increased fibrin deposition and intracerebral hemorrhage, whereas in mice with an impaired TM function, cerebral fibrin depositions and destruction of cerebral tissue are minor or absent (16
). Such distinct consequences of TM deficiency on the one hand, and PC deficiency or activated PC resistance on the other hand could be related to the preserved PC-independent anticoagulant function of TM (46
) and/or the TAFI-dependent differences in the fibrinolytic system activity (3
Mice with an endothelial specific disruption of the TM gene provide a unique mouse model for severe spontaneous thrombosis secondary to excess activation of the coagulation system that is caused by a single gene defect. The initial viability of mutant mice, the particular time course of disease onset at young age, the almost complete penetrance of overt thrombosis, involvement of the microvasculature as well as large venous and arterial vessels, and fatal bleeding due to coagulation factor consumption as the disease end point are unique features of this animal thrombosis model, setting it apart from previously described mice with genetically inactivated anticoagulant mechanisms (22
). These mice provide insight into the physiological role and importance of endothelial TM in vivo and constitute a valuable animal model for the evaluation of new therapeutic strategies for the treatment of inherited and acquired deficiencies of the TM/PC pathway and, more generally, for establishing the efficacy and safety of new anticoagulant therapies.