1A.1 Animal model of fetal loss in factor V Leiden carriers: pathogenic mechanism and therapeutic targets

Abstract

Recurrent pregnancy loss (RPL), defined as two or more spontaneous abortions affects approximately 5% of all women of reproductive age. Epidemiological studies suggest that inherited and acquired thrombophilia of the mother, such as that caused by the Leiden polymorphism in blood coagulation factor V, contributes to the pathogenesis of fetal loss, as well as other adverse pregnancy outcomes [1 3]. These studies also demonstrate a high study-to-study variability of association strength, suggesting the existence of as yet uncharacterized risk modifiers. The pathogenesis of thrombophilia-associated fetal loss remains to be established. Evaluation of placental pathologies has not revealed a clear correlation between thrombophilia status of the mother and the presence of villous infarcts, blood clots or fibrin deposits in the placenta [4,5], raising the question whether thrombotic processes are indeed causative in thrombophilia-associated fetal loss or constitute an epiphenomenon. On the other hand, clinical trials suggest that heparin anticoagulation may indeed mitigate pregnancy failure in prothrombotic women [6 9]. In view of the uncertain etiology of thrombophilia-associated pregnancy failure, and the lack of established criteria for a more precise risk stratification enabling identification of truly atrisk pregnancies, the prophylactic anticoagulation of asymptomatic women and the risk-to-benefit balance of anticoagulation therapy during pregnancy are subjects of intense debate [10 12]. Pregnancy failure in carriers of the factor V Leiden (FVL) polymorphism, which renders coagulation factor V refractory to inactivating proteolysis by activated protein C, has not been replicated in experimental animal models. Homozygous status for the FVL polymorphism elicits pronounced thrombophilia in mice, but is not associated with intrauterine fetal loss or impaired fecundity [13]. In contrast, observations in animals lacking the receptor components of the protein C anticoagulant pathway, thrombomodulin (TM) and the endothelial protein C receptor (EPCR), demonstrate that the integrity of this pathway within the placenta is essential for the maintenance of pregnancy [14 16]. TMor EPCR knockout mice die in utero due to a placental malfunction that results from a lack of these receptors on trophoblast cells constituting fetal lining of the placental vascular bed. Placental malfunction is triggered by the initiator of blood coagulation, tissue factor (TF), and at least in the case of TM is not associated with thrombosis, nor prevented by the absence of fibrinogen [15]. These observations led us to propose a “coagulotoxic” mechanism distinct from thrombosis, in which localized, tissue factor-dependent engagement of coagulation factor receptors (i.e. PAR 1, PAR2, and PAR4) expressed by trophoblast cells would cause deleterious effects on placental growth and morphogenesis. Importantly, fetal loss in these animal models occurs in the absence of maternal thrombophilia. The relevance of these knockout models for fetal loss in thrombophilic mothers, and the pathogenesis of fetal loss remain unclear. Here, we experimentally test the role of coagulation receptors PAR1, 2, and 4 in the fetal loss of TM / embryos; and develop a mouse model of pregnancy loss in FVL carriers, in which fetal loss is triggered by the interaction of maternal FVL carrier status and reduced ability of fetal trophoblast cells to sustain the protein C anticoagulant pathway. Interaction of the same risk factors across the blood-endothelial interface of the systemic vasculature does not precipitate thrombosis. Fetal loss in this animal model is caused by defective placental morphogenesis in the absence of placental thrombosis, infarction or perfusion defects, and can be prevented by treatment with low molecular weight heparin, by platelet depletion, or by elimination of the thrombin receptor PAR4 from maternal platelets. Therapeutic intervention studies suggest that the efficacy of low molecular weight heparin in preventing fetal loss does not correlate with its anti-coagulant activity. These findings validate a cause effect relation between FVL carrier status and pregnancy failure, show that fetal