Coextraction of Thrombomodulin and Tissue Factor from Human Placenta: Effects of Concanavalin A and Phospholipid Environment on Activity

Abstract

SummaryThrombomodulin and tissue factor activities have been coextracted from human placenta by several non-ionic detergents, n-octylglucoside and Triton X-100 being the most efficient ones. The n-octylglucoside placenta extract had a strong cofactor activity in the activation of human protein C by human a-thrombin. Treatment of the n-octylglucoside and Triton X-100 placenta extracts by phospholipases C and A2 revealed that an adequate phospholipid environment is necessary for maximal thrombomodulin activity, while it is well known that this is crucial for tissue factor activity. Soluble concanavalin A reversibly inhibited thrombomodulin and tissue factor activities to the same extent. Con-A-Sepharose affinity chromatography of the Triton X-100 placenta extract resulted in the same proportion (30%) of these two activities bound to the lectin, which were subsequently eluted in the same fractions by a linear gradient of α-methyl-D-glucoside. This observation suggests that thrombomodulin activity is associated to a glycoprotein component presenting the same degree of carbohydrate heterogeneity, involving α-D-mannosyl or α-D-glucosyl residues, as tissue factor apoprotein. Relipidation of fraction eluted by α-methyl-D-glucoside was essential to detect tissue factor activity, it was also necessary to recover full thrombomodulin activity. An antibody to human brain tissue factor apoprotein inhibited human placenta tissue factor activity, whereas thrombomodulin activity was unaffected, suggesting that these two cellular activities are related to distinct molecular entities sharing striking functional and structural similarities.

Thrombomodulin Activity Is Found in Tissue Thromboplastin Preparations from Placenta and from Lung but Not from Brain

“…It is also shown that an adequate procoagulant phospholipid en vironment is necessary for maximal throm bomodulin or tissue factor activity in these crude biological preparations. This confirms the conclusions of our recent study describ ing the co-extraction of thrombomodulin and tissue factor activities from human pla centa and their dependence on procoagulant phospholipids [8],…”

“…about 250 units/ vial) were obtained from the Centre Régional de Transfusion Sanguine, Strasbourg, France and used to purify human prothrombin to prepare a-thrombin according to a previously published method [8].…”

Antiphospholipid/antiprotein antibodies, hemostasis-related autoantibodies, and plasma homocysteine as risk factors for a first early pregnancy loss: a matched case-control study

“…IgG and IgM antibodies against tissue factor and against thrombomodulin were tested using home-made assays. Both glycoproteins were extracted from human placenta as described, [28][29][30] followed by an immunoaffinity chromatography step using an antitissue factor antibody (American Diagnostica, Greenwich, CT) or an antithrombomodulin antibody (Diagnostica Stago, Asnières, France). The same procedure as the one described for anti-␤2-glycoprotein I antibodies and anti-annexin V antibodies was then applied, the concentration of the purified human protein in the coating buffer always being at 10 g/mL.…”

Is heparin a placental anticoagulant in high-risk pregnancies?

“…These include the procoagulant proteins TF, VWF, factor VIII, 48 and tissue plasminogen activator inhibitor type 1. Inhibitory proteins of the cascade include TFPI1 and TFPI2, 49,50 thrombomodulin, 51,52 annexin V, 53 and tissue plasminogen activators such as urokinase. The placenta is theoretically vulnerable to thrombosis because TF is constitutively expressed, although as trophoblast cells differentiate, they adopt a thromboresistant phenotype.…”