Abstract

Amniotic fluid (AF) may induce disseminated intravascular coagulation when it enters maternal circulation by breaching the placental-maternal circulation barrier. The precise mechanism of the procoagulant activity of AF is unclear, we speculate that AF cells have procoagulant activity due to the externalization of phosphatidylserine (PS). The present study aims to demonstrate that, in addition to tissue factor (TF), the PS that is externalized on AF cells is important for the procoagulant activity of AF. Ten AF samples from parturient women were analyzed and normal platelets, neutrophils, and lymphocytes were harvested as controls. Lactadherin, a glycoprotein, binds to membranes containing PS, inhibits prothrombinase activity, factor Xase activity, and tissue factor-factor VIIa activity by blocking PS-containing membrane binding. Thus lactadherin was utilized as a PS probe for flow cytometry and confocal microscopy to enable comparison of PS distribution with TF and intrinsic factor Xase complex formation. Procoagulant activity of AF cells was first measured in plasma with AF cells serving as thromboplastin. Activity of AF cells supporting intrinsic and extrinsic factor Xase complexes was measured in purified systems. Lactadherin, as an agent to block exposed PS, inhibited 85% of intrinsic and extrinsic factor Xase activity. Competition binding studies indicated that lactadherin competed for 55% of factor VIII binding sites. However, binding of factor VIII was completely inhibited by PS-containing vesicles and by mAb that recognize the factor VIII C2 domain indicating that all fVIII binding was mediated by the membrane-binding motif or an overlapping epitope. Confocal microscopy identified patches and a rim-pattern indicating a diffuse PS exposure. Lactadherin binding sites and TF distributed to discreet, but overlapping regions of the cells. These results indicate that PS exposure parallels procoagulant activity on AF cells and is required for at least 85% of intrinsic and extrinsic factor Xase activities. However, the topographical pattern of PS exposure differs from the pattern of TF and the pattern of binding site distribution for intrinsic factor Xase complexes. Thus, the results imply that intrinsic factor Xase and extrinsic factor Xase activity are localized to small cell regions where PS exposure coincides with TF and intrinsic factor Xase binding sites, respectively.

Disclosures: No relevant conflicts of interest to declare.

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