Protein C Activation and Factor Va Inactivation on Human Umbilical Vein Endothelial Cells

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Abstract
Abstract The inactivation of factor Va was examined on primary cultures of human umbilical vein endothelial cells (HUVECs), either after addition of activated protein C (APC) or after addition of α-thrombin and protein C (PC) zymogen. Factor Va proteolysis was visualized by Western blot analysis using a monoclonal antibody (αHVa HC No. 17) to the factor Va heavy chain (HC), and cofactor activity was followed both in a clotting assay using factor V–deficient plasma and by quantitation of prothrombinase function. APC generation was monitored using the substrate 6-(D-VPR)amino-1-naphthalenebutylsulfonamide (D-VPR-ANSNHC 4 H 9 ), which permits quantitation of APC at 10 pmol/L. Addition of APC (5 nmol/L) to an adherent HUVEC monolayer (3.5×10 5 cells per well) resulted in a 75% inactivation of factor Va (20 nmol/L) within 10 minutes, with complete loss of cofactor activity within 2 hours. Measurements of the rate of cleavage at Arg 506 and Arg 306 in the presence and absence of the HUVEC monolayer indicated that the APC-dependent cleavage of the factor Va HC at Arg 506 was accelerated in the presence of HUVECs, while cleavage at Arg 306 was dependent on the presence of the HUVEC surface. Factor Va inactivation proceeded with initial cleavage of the factor Va HC at Arg 506 , generating an M r 75 000 species. Further proteolysis at Arg 306 generated an M r 30 000 product. When protein C (0.5 μmol/L), α-thrombin (1 nmol/L), and factor Va (20 nmol/L) were added to HUVECs an APC generation rate of 1.56±0.11×10 −14 mol/min per cell was observed. With APC generated in situ, cleavage at Arg 506 on the HUVEC surface is followed by cleavage at Arg 306 , generating M r 75 000 and M r 30 000 fragments, respectively. In addition, the appearance of two novel products derived from the factor Va HC are observed when thrombin is present on the HUVEC surface: the HC is processed through limited thrombin proteolysis to generate an M r 97 000 fragment, which is further processed by APC to generate an M r 43 000 fragment. NH 2 -terminal sequence analysis of the M r 97 000 fragment revealed that the thrombin cleavage occurs in the COOH-terminus of the intact factor Va HC since both the intact HC as well as the M r 97 000 fragment have the same sequence. Our data demonstrate that the inactivation of factor Va on the HUVEC surface, initiated either by APC addition or PC activation, follows a mechanism whereby cleavage is observed first at Arg 506 followed by a second cleavage at Arg 306 . The latter cleavage is dependent on the availability of the HUVEC surface. This mechanism of inactivation of factor Va is similar to that observed on synthetic phospholipid vesicles.