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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:2765-2775.)
© 1997 American Heart Association, Inc.

Articles

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

Matthew F. Hockin; Michael Kalafatis; Marie Shatos; ; Kenneth G. Mann

From the College of Medicine, Department of Biochemistry, University of Vermont, Burlington.

Correspondence to Kenneth G. Mann, College of Medicine, Department of Biochemistry, University of Vermont, Burlington, VT 05405-0068. E-mail kmann{at}protein.med.uvm.edu

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 -thrombin and protein C (PC) zymogen. Factor Va proteolysis was visualized by Western blot analysis using a monoclonal antibody (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 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 (3.5x10⁵ 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), -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, 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.

Key Words: factor Va • protein C • endothelial cells • thrombomodulin

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