Prothrombin is activated on vascular endothelial cells by factor Xa and calcium.

Abstract

Vascular endothelial cells derived from adult bovine aorta (ABAE) treated with factor Xa and calcium were found to activate prothrombin. In contrast, nonvascular cells (human foreskin fibroblasts, bovine corneal endothelial cells, or human fetal lung cells) had either no or very little effect on prothrombin activation. In the presence of 6 x 105 ABAE cells, 20 ng of factor Xa converted 90 ,.g of prothrombin into 80 units of thrombin after 45 min at 37C. Exogenous factor V was not required for prothrombin activation, but thrombin generation was enhanced 2-to 4-fold by the addition of factor V (500-2,500 ng/ml). Treatment of ABAE cells with anti-bovine factor V IgG markedly inhibited prothrombin activation by factor Xa and calcium. In cells grown in serum-free medium for 3 months, the amount of factor V activity was equivalent to that found in cells grown with serum, which suggests that these cells probably synthesize factor V. Sparse ABAE cells increased prothrombin activation by factor Xa 6-fold compared to activation in confluent cells. Although previous thrombin treatment of ABAE cells did not enhance prothrombin activation, addition of dansyl arginine-4-ethyl piperidine amide markedly inhibited activation of 125I-labeled prothrombin by factor Xa, indicating that thrombin formation is necessary for optimal prothrombin activation. These data indicate that aortic endothelium may provide a physiologically important surface for activation of prothrombin as well as a mechanism for optimal formation of clots at sites of vascular injury.In an unperturbed state, vascular tissue is generally considered to be inert with regard to activation of blood clotting (1). (2,4,5). In separate experiments, ABAE cells were grown in serum-free medium (6, 7). In these studies, ABAE cells were grown without serum for two passages over a period of 3 months. In experiments in which disrupted cells were assayed, confluent ABAE cells were mechanically homogenized.Coagulation Proteins. All coagulation proteins used in these studies were purified from human plasma. Prothrombin and factor X were purified as described (8) and had specific activities of 34 units/mg and 160 units/mg, respectively. Factor V was provided by Joseph P. Miletich (Washington University, St. Louis, MO) and had a specific activity of 170 units/mg (9). Factor X was activated by RVV immobilized to Sepharose (10); the factor X-activating system consisted of factor X (0.03 ml of 2 mg/ml solution), washed RVV beads (0.03 ml), cephalint (0.015 ml of stock solution), and 100 mM CaCl2 (0.01 ml). This mixture was incubated at 37°C for 1 hr with frequent mixing. After centrifugation to sediment'the beads, the supernatant was assayed for factor Xa as described (11). In a clotting assay (11), factor Xa at 1 ng/ml yielded a clotting time of 38 sec. Factor V activity was measured using the method of Lewis and Ware (12). Protein was assayed by the method of Bradford (13).Prothrombinase Assay. Cultured adherent cells in 35-mm Petri dishes were washed twice with a buffer of ...

A coagulation pathway on bovine aortic segments leading to generation of Factor Xa and thrombin.

“…Bound Factor IX. can activate Factor X in the presence of VWF/Factor VIII (10) and Factor Xa can activate prothrombin when the cultured cells are subconfluent (14). Factor V synthesis by cultured endothelial cells has also been reported (44).…”

“…Cell-bound Factors IX and IXa retain their coagulant properties: cell-bound Factor IX can be activated by either the intrinsic or extrinsic pathways, and cell-bound Factor IXa can participate in Factor X activation (10). Factor Xa incubated with nonconfluent cultured endothelial cells can activate prothrombin (14). Conclusions from these studies rely on the assumption that cultured endothelial cells are a model for native endothelium.…”

Coagulant factor Xa inhibits prostacyclin formation in human endothelial cells. Role of factor V.

“…Several other observations are consistent with the presence and the important role of Factor V in endothelial cells. Bovine aortic endothelial cells have been shown 23 to synthesize Factor V. Prothrombin conversion by Factor Xa is accelerated on the surface of bovine endothelial cells, 24 and the process is blocked with an antibody against bovine Factor V, which suggests the presence of Factor V in the system. Preliminary studies 25 indicate that Factor Va binds to endothelial cells and accelerates the activation of protein C. This reaction is blocked by the same human monoclonal antibody used in this study.…”

Formation of factor Va by atherosclerotic rabbit aorta mediates factor Xa-catalyzed prothrombin activation.

“…Prothrombin and Factor V were iodinated using the lodogen technique (22). Standard clotting assays were used to measure Factor X activity (24), and Factor X was activated using Russell's Viper venom coupled to Sepharose (25). Normal rabbit plasma was obtained by venipuncture using an anticoagulant solution containing 3.8% sodium citrate, 10 mM benzamidine, and 0.1 mM PMSF.…”

“…The rationale for these experiments is that Factor X. cannot cleave 125I-prothrombin in the presence of a thrombin inhibitor unless Factor V has been activated. Factor X., aliquots of the incubation were removed at intervals, subjected to nonreduced SDS-PAGE, dried, and processed for autoradiography (25) to determine the extent of '23I-prothrombin cleavage.…”