α2-Macroglobulin Remains as Important as Antithrombin III for Thrombin Regulation in Cord Plasma in the Presence of Endothelial Cell Surfaces

Abstract

---r =--9 ------------ __1 ----7 ---' ------71-7 -- ABSTRACTInfants and children rarely develop thrombotic complications compared with adults, suggesting that there are protective mechanisms in place for the young. Because endothelial cell surfaces regulate thrombin formation and inhibition, we compared thrombin regulation by human umbilical vein endothelial cell surfaces exposed to defibrinated cord and adult plasmas. After activation by either 10% activated partial thromboplastin reagent (strong activator) or coagulant phospholipids (weak activator) the following were measured: free thrombin, thrombin bound to antithrombin I11 (ATIII), heparin cofactor 11, a,-macroglobulin (a2M), and prothrombin concentration. Free thrombin activity was expressed as remaining activity, after subtraction of thrombin-a2M activity. After 10% activated partial thromboplastin reagent, 100% of prothrombin was consumed and significant amounts of thrombin generated by 2 min. Cord plasma generated significantly less thrombin than adult plasma, reflecting the lower initial plasma concentration of prothrombin. Correspondingly, concentrations of thrombin inhibitor complexes were significantly greater in adult plasma than in cord plasma. After coagulant phospholipids, 50% of prothrombin was consumed and negligible thrombin activity measured for both adult and cord plasma. Similar amounts of thrombin inhibitor complexes were formed. ATIII was the predominant inhibitor of thrombin in adult plasma, whereas a,M was as important as ATIII in cord plasma for both activators. When cord plasma concentrations of ATIII were increased to adult values, the proportion complexed to a2M decreased. We conclude that on human umbilical vein endothelial cells, the capacity to generate thrombin is decreased in adult and cord plasmas. Furthermore, a2M is at least as important an inhibitor as ATIII in cord plasma, even in the presence of Thromboembolic complications rarely occur in fetuses and newborns, suggesting that protective mechanisms are in place in the young. Biologically, the ability to regulate thrombin formation and activity are of central importance to the prevention and formation of large-vessel thrombi. Regulation of thrombin under physiologic circumstances involves both plasma coagulation and inhibitors, as well as interactions with

Overexpression of the 78-kDa Glucose-regulated Protein/Immunoglobulin-binding Protein (GRP78/BiP) Inhibits Tissue Factor Procoagulant Activity

“…Thus, in the presence of factor VII-depleted plasma, free thrombin generation and prothrombin consumption were negligible for all cell lines over the 25-min time period (data not shown), a result consistent with the requirement of factor VIIa for TF-dependent thrombin generation (62)(63)(64)(65). To specifically examine TF PCA on cell surfaces, wildtype, vector-transfected, or GRP78/BiP-overexpressing cells were grown to confluency and factor Xa generation was measured over 30 min using the chromogenic substrate S-2765.…”

“…Factor VII levels in the deficient plasmas were Յ1%, according to the manufacturer. Prior to use in the thrombin generation assay, plasmas were defibrinated using arvin and total amidolytic activity of thrombin generated on cell surfaces was measured as previously described (62,63). Cell monolayers in 24-well plates were placed on a Thermolyne dri-bath set at 37°C.…”

“…Prothrombin, TAT, and thrombin-heparin cofactor II complexes were assayed using commercially available ELISA kits (Affinity Biologicals). Because thrombin bound to ␣ 2 macroglobulin (␣ 2 M) retains amidolytic activity against S-2238 (64), the contribution of thrombin-␣ 2 M to total thrombin activity was measured as previously described (63). Briefly, the amidolytic activity of total thrombin was measured as described above, except that the 25-l reaction mixture taken at each time point was incubated with 3.5 l of 0.15 M NaCl containing 0.25 units of standard heparin and 0.042 units of antithrombin (to inhibit any free thrombin) for 1 min on ice, followed by the addition of 475 l of 5 mM EDTA.…”

Alpha-2-Macroglobulin Inhibits the Anticoagulant Action of Activated Protein C in Cord and Adult Plasma

“…The observation that a2-M diminishes the effect of APC on feedback activation of thrombin is intriguing since it has been shown that a2-M accounts for a large amount of thrombin inhibition, especially in cord plasma [4]. Studies dealing with thrombin inhibition by a2-M determined thrombin/a2-M complex formation after a certain period of plasma incubation and did not assess the time course of thrombin generation and inhibition [23,24].…”

“…The capacity of cord plasma to generate thrombin is decreased to 30-50% of adult values [3]. In addition, alpha-2-macroglobulin (a2-M) has been suggested as a major anticoagulant protein in infancy and has been demon-strated to complex up to 49% of generated alpha-thrombin in cord plasma [4,5]. On the other hand, a2-M has been shown to bind activated protein C (APC) [6], one of the most important anticoagulant proteins.…”