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-ANSNHC₄H₉), which permits quantitation of APC at 10 pmol/L. Addition of APC (5 nmol/L) to an adherent HUVEC monolayer (3.5×10⁵ 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 Arg⁵⁰⁶ and Arg³⁰⁶ in the presence and absence of the HUVEC monolayer indicated that the APC-dependent cleavage of the factor Va HC at Arg⁵⁰⁶ was accelerated in the presence of HUVECs, while cleavage at Arg³⁰⁶ was dependent on the presence of the HUVEC surface. Factor Va inactivation proceeded with initial cleavage of the factor Va HC at Arg⁵⁰⁶, generating an M_r 75 000 species. Further proteolysis at Arg³⁰⁶ 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 Arg⁵⁰⁶ on the HUVEC surface is followed by cleavage at Arg³⁰⁶, 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. NH₂-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 Arg⁵⁰⁶ followed by a second cleavage at Arg³⁰⁶. 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.