Studies of the Mechanism for Enhanced Cell Surface Factor VIIa/Tissue Factor Activation of Factor X on Fibroblast Monolayers after Their Exposure to N-Ethylmaleimide

Dzung The Le; Samuel I Rapaport; L Vijaya Mohan Rao

doi:10.1055/s-0038-1648973

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 1994; 72(06): 848-855
DOI: 10.1055/s-0038-1648973

Original Article

Schattauer GmbH Stuttgart

Studies of the Mechanism for Enhanced Cell Surface Factor VIIa/Tissue Factor Activation of Factor X on Fibroblast Monolayers after Their Exposure to N-Ethylmaleimide

Dzung The Le

¹The Department of Pathology, University of California, San Diego, La Jolla, California, USA

,

Samuel I Rapaport

¹The Department of Pathology, University of California, San Diego, La Jolla, California, USA

²The Department of Medicine, University of California, San Diego, La Jolla, California, USA

,

L Vijaya Mohan Rao

³Department of Biochemistry, The University of Texas Health Center at Tyler, Tyler, Texas, USA

› Author Affiliations

Abstract

PDF Download

Summary

Fibroblast monolayers constitutively expressing surface membrane tissue factor (TF) were treated with 0.1 mM N-ethylmaleimide (NEM) for 1 min to inhibit aminophospholipid translocase activity without inducing general cell damage. This resulted in increased anionic phospholipid in the outer leaflet of the cell surface membrane as measured by the binding of 125I-annexin V and by the ability of the monolayers to support the generation of prothrombinase. Specific binding of 125I-rVIIa to TF on NEM-treated monolayers was increased 3- to 4-fold over control monolayers after only brief exposure to ¹²⁵I-rVIIa, but this difference progressively diminished with longer exposure times. A brief exposure of NEM-treated monolayers to rVIIa led to a maximum 3- to 4-fold enhancement of VIIa/TF catalytic activity towards factor X over control monolayers, but, in contrast to the binding studies, this 3- to 4-fold difference persisted despite increasing time of exposure to rVIIa. Adding prothrombin fragment 1 failed to diminish the enhanced VIIa/TF activation of factor X of NEM-treated monolayers. Moreover, adding annexin V, which was shown to abolish the ability of NEM to enhance factor X binding to the fibroblast monolayers, also failed to diminish the enhanced VIIa/TF activation of factor X. These data provide new evidence for a possible mechanism by which availability of anionic phospholipid in the outer layer of the cell membrane limits formation of functional VIIa/TF complexes on cell surfaces.

PDF (665 kb)

References

References
1 Nemerson Y. Tissue factor and hemostasis. Blood 1988; 71: 1-8
2 Drake TA, Morrissey JH, Edgington TS. Selective cellular expression of tissue factor in human tissues: Implications for disorders of hemostasis and thrombosis. Am J Path 1989; 134: 1087-97
3 Fleck RA, Rao LVM, Rapaport SI, Varki N. Localization of human tissue factor antigen by immunostaining with monospecific, polyclonal anti-human tissue factor antibody. Thromb Res 1990; 59: 421-37
4 Le DT, Rapaport SI, Rao LVM. Relations between factor Vila binding and expression of factor Vila/tissue factor catalytic activity on cell surfaces. J Biol Chem 1992; 267: 15447-54
5 Bach R, Rifkin DB. Expression of tissue factor procoagulant activity: regulation by cytosolic calcium. Proc Natl Acad Sci USA 1990; 87: 6995-9
6 McGee MP, Li LC, Xiong H. Diffusion control in blood coagulation. Activation of factor X by factors IXa/VIIIa assembled on human monocyte membranes. J Biol Chem 1992; 267: 24333-9
7 Andree HAM, Contino PB, Repke D, Gentry R, Nemerson Y. Transport rate limited catalysis on macroscopic surfaces: The activation of factor X in a continuous flow enzyme reactor. Biochem 1994; 33: 4368-74
8 Devaux PF. Static and dynamic lipid asymmetry in cell membranes. Biochem 1991; 30: 1163-73
9 Bevers EM, Tilly RHJ, Senden JMG, Comfurius P, Zwaal RFA. Exposure of endogenous phosphatidylserine at the outer surface of stimulated platelets is reversed by restoration of aminophospholipid translocase activity. Biochem 1989; 28: 2382-7
10 Kobayashi T, Arakawa Y. Transport of exogenous fluorescent phosphatidylserine analogue to the golgi apparatus in cultured fibroblasts. J Cell Biol 1991; 113: 235-44
11 Martin OC, Pagano RE. Transbilayer movement of fluorescent analogs of phosphatidylserine and phosphatidylethanolamine at the plasma membrane of cultured cells. J Biol Chem 1987; 262: 5890-8
12 Dachary-Prigent J, Freyssinet J, Pasquet J, Carron J, Nurder AT. Annexin V as a probe of aminophospholipid exposure and platelet membrane vesicula-tion: A flow cytometry study showing a role for free sulfhydryl groups. Blood 1993; 81: 2554-65
13 Bajaj SP, Rapaport SI, Prodanos C. A simplified procedure for purification of human prothrombin, factor IX and factor X. Prep Biochem 1981; 11: 397-412
14 Funakoshi T, Heimark RL, Hendrickson LE, McMullen BA, Fujikawa K. Human placental anticoagulant protein: Isolation and characterization. Biochem 1987; 26: 5572-8
15 Forman SD, Nemerson Y. Membrane-dependent coagulation reaction is independent of the concentration of phospholipid-bound substrate: fluid phase factor X regulates the extrinsic system. Proc Natl Acad Sci USA 1986; 83: 4675-9
16 Callander N, Rao LVM, Nordfang O, Sandset PM, Warn-Cramer B, Rapaport SI. Mechanisms of binding of recombinant extrinsic pathway inhibitor (rEPI) to cultured cell surfaces. Evidence that rEPI can bind to and inhibit factor VHa-tissue factor complexes in the absence of factor Xa. J Biol Chem 1992; 267: 876-82
17 Rao LVM, Tait JF, Hoang AD. Binding of annexin V to a human ovarian carcinoma cell line (OC-2008). Contrasting effects on cell surface factor Vila/tissue factor activity and prothrombinase activity. Thromb Res 1992; 67: 517-31
18 Reutelingsperger CPM, Kop JMM, Hornstra G, Hemker HC. Purification and characterization of a novel protein from bovine aorta that inhibits coagulation. Inhibition of the phospholipid-dependent factor Xa-catalyzed prothrombin activation through a high affinity binding of the anticoagulant to the phospholipids. EurJBiochem 1988; 172: 171-8
19 Tait JF, Gibson D, Fujikawa K. Phospholipid binding properties of human placental anticoagulant protein-I, a member of the lipocortin family. J Biol Chem 1989; 264: 7944-9
20 Connor J, Bucana C, Fidler IJ, Schroit AJ. Differentiation-dependent expression of phosphatidylserine in mammalian plasma membranes: Quantitative assessment of outer-leaflet lipid by prothrombinase complex formation. Proc Natl Acad Sci USA 1989; 86: 3184-3188
21 Kondo S, Noguchi M, Funakoshi T, Fujikawa K, Kisiel W. Inhibition of human factor VHa-tissue factor activity by placental anticoagulant protein. Thromb Res 1987; 48: 449-459
22 Gramzinski RA, Broze GJJr, Carson SD. Human fibroblast tissue factor is inhibited by lipoprotein-associated coagulation inhibitor and placental anticoagulant protein but not by apoprotein A-II. Blood 1989; 73: 983-989
23 Rao LVM. Tissue factor activity increase in fibroblasts treated with calcium ionophore is primarily due to formation of microvesicles. FASEB J 1994; 8: A1374
24 Krishnaswamy S, Field KA, Edgington TS, Morrissey JH, Mann KG. Role of the membrane surface in the activation of human coagulation factor X. J Biol Chem 1992; 267: 26110-26120
25 Ruf W, Rehemtulla A, Morrissey JH, Edgington TS. Phospholipid-independent and -dependent interactions required for tissue factor receptor and cofactor function. J Biol Chem 1991; 266: 2158-2166
26 Fiore M, Neuenschwander PF, Morrissey JH. The biochemical basis for the apparent defect of soluble mutant tissue factor in enhancing the proteolytic activities of factor Vila. J Biol Chem 1994; 269: 143-149
27 Carson SD, Perry GA, Pirruccello SJ. Fibroblast tissue factor: Calcium and ionophore induce shape changes, release of membrane vesicles and redistribution of tissue factor antigen in addition to increased procoagulant activity. Blood 1994; 84: 526-534
28 Bach R. Initiation of coagulation by tissue factor. CRC Crit Rev Biochem 1988; 23: 339-368
29 Sakai T, Lund-Hansen T, Paborsky L, Pedersen AH, Kisiel W. Binding of human factors VII and Vila to a human bladder carcinoma cell line (J82). J Biol Chem 1989; 264: 9980-9988