PROTEIN-C ACTIVATION AND FACTOR VA INACTIVATION ON HUMAN UMBILICAL VEIN ENDOTHELIAL-CELLS

The inactivation of factor Va was examined on primary cultures of huma n umbilical vein endothelial cells (HUVECs), either after addition of activated protein C (APC) or after addition of alpha-thrombin and prot ein C (PC) zymogen. Factor Va proteolysis was visualized by Western bl ot analysis using a monoclonal antibody (alpha HVa(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 sub strate 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 (3.5x10(5) cells per well) re sulted in a 75% inactivation of factor Va (20 nmol/L) within 10 minute s, with complete loss of cofactor activity within 2 hours. Measurement s 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 cleav age 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 t he HUVEC surface. Factor Va inactivation proceeded with initial cleava ge 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 mu mol/L), alpha-thrombin (1 nmol/L), and factor Va (20 nmol/L) were added to HUVECs an APC generation rate of 1.56+/-0.11x10 (-14) mol/min per cell was observed. With APC generated in situ, cleav age at Arg(506) on the HUVEC surface is followed by cleavage at Arg(30 6), generating M-r 75 000 and M-r 30 000 fragments, respectively. In a ddition, 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 97000 fragment, which is further processed by APC to generate an M-r 43 009 fragment. NH2-terminal sequence analysis of the M-r 97 000 fra gment revealed that the thrombin cleavage occurs in the COOH-terminus of the intact factor Va HC since both the intact KC 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 A PC 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 surf ace. This mechanism of inactivation of factor Va is similar to that ob served on synthetic phospholipid vesicles.