Kinetics of inactivation of membrane-bound factor Va by activated protein C. Protein S modulates factor Xa protection.

The anticoagulant activity of activated Protein C has been observed to be species specific. This could be due either to the inability of the bovine enzyme to recognize its substrate, Factor Va, in non-bovine plasmas, or the absence of cofactor-Protein S, a protein that has been shown to be necessary for the maximum expression of the anticoagulant activity of activated Protein C. Activated Protein C was found to be an effective inhibitor of Factor Xa-initiated clotting of bovine plasma, but without activity in either human or rabbit plasma. Human and rabbit plasma supplemented with bovine Protein S was sensitive to the anticoagulant activity of activated Protein C. Neither rabbit nor human plasma contained bovine activated Protein C cofactor activity as measured by the enhancement of bovine activated Protein C-catalyzed inactivation of Factor Va. However, bovine activated Protein C was able to inactivate both human and rabbit Factor Va. The inactivation of both of these proteins could be stimulated by the addition of bovine Protein S. These results indicate that the species specificity of bovine activated Protein C is due to the absence of a cofactor protein in non-bovine plasma that will interact with the bovine enzyme. Secondly, these findings further confirm that Protein S is required for the maximal expression of the anticoagulant activity of activated Protein C.

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ENDOTHELIUM AND PROTEIN S: SYNTHESIS, RELEASE AND REGULATION OF ANTICOAGULANT ACTIVITY

10.1055/s-0038-1642962 ◽

1987 ◽

Author(s):

Peter P Nawroth ◽

Jerry Brett ◽

Susan Steinberg ◽

Charles T Esmon ◽

David M Stern

Keyword(s):

Endothelial Cell ◽

Protein C ◽

Specific Binding ◽

Activated Protein C ◽

Protein S ◽

Intracellular Protein ◽

C Protein ◽

Factor Va ◽

Intracellular Vesicles ◽

Anticoagulant Function

The protein C-protein S pathway is closely linked to the vessel wall. In terms of protein C, endothelium has been shown to provide the receptor thrombomodulin, which promotes thrombin-mediated formation of activated protein C. Optimal anticoagulant function of activated protein C requires protein S and a cellular surface. Recent studies have indicated that endothelium can facilitate assembly of the activated protein C-protein S complex and that bovine endothelium expresses specific binding site(s) for protein S which promote its anticoagulant function. Expression of protein S binding sites is subject to down-regulation by Tumor Necrosis Factor (TNF) . Exposure of cultured bovine endothelium to TNF results in decreased 125I-protein s binding and attenuated rates of Factor Va inactivation after 2 hrs followed by negligible 125I-protein S binding and Factor Va inactivation by 10 hrs. These changes persist for over 48 hrs, in contrast to the more transient rise in endothelial cell tissue factor induced by TNF which returns to baseline by 24 hrs.In addition to providing binding sites for protein S, endothelium constitutively synthesizes and releases this vitamin K-dependent anticoagulant cofactor. Release of protein S is blocked by addition of warfarin, indicating that y-carboxylation facilitates the release of intracellular protein S. Morphologic studies, at the level of electron microscope, have shown protein S antigen to be present in cisternae of rough endoplasmic reticulum, the trans face of the golgi and a population of intracellular vesicles which appear to be distributed at the cellular periphery. By immunofluorescence, the distribution of protein S is distinct from that of von Willebrand Factor. The intracellular vesicles containing protein S constitute a storage pool potentially available for rapid release. Treatment of endothelium with norepinephrine results in release of protein S over the next 20 min. Release is half-maximal at a norepinephrine concentration of about 0.1 uM and is not observed with the biologically inactive entantiomer (+) norepinephrine. Norepinephrine-induced release of intracellular protein S can be blocked by prazosine (10-7 7 M), but not by propranolol (10-6 M) or yohimbine (10-5 M). These data are consistent with release of protein S being a receptor-mediated process dependent on an endothelial cell alpha 1 adrenergic receptor. Blockade of norepinephrine-induced release of protein S by pertussis toxin treatment of endothelium further defines the intracellular pathway of protein S and implicates regulatory G proteins in the stimulus-response coupling. Electron microscopic studies have shown that following exposure of endothelium to norepinephrine the intracellular vesicles containing protein S undergo exocytosis at the plasma membrane. These data define a new relationship between the autonomic nervous system and the coagulation mechanism.Protein S is clearly an endothelial cell-associated anticoagulant protein. A specific binding site on the endothelial cell surface can regulate its anticoagulant function on the vessel wall. Endothelial cell synthesis and release of protein S defines a new level of participation of endothelium in the protein C-protein S pathway.

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Cultured bovine aortic endothelial cells promote activated protein C-protein S-mediated inactivation of factor Va.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)36151-3 ◽

1986 ◽

Vol 261 (2) ◽

pp. 713-718

Author(s):

D M Stern ◽

P P Nawroth ◽

K Harris ◽

C T Esmon

Keyword(s):

Endothelial Cells ◽

Protein C ◽

Activated Protein C ◽

Protein S ◽

C Protein ◽

Aortic Endothelial Cells ◽

Factor Va ◽

Bovine Aortic Endothelial Cells ◽

Aortic Endothelial

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Effects of Protein S and Factor Xa on Peptide Bond Cleavages during Inactivation of Factor Va and Factor VaR506Qby Activated Protein C

Journal of Biological Chemistry ◽

10.1074/jbc.270.46.27852 ◽

1995 ◽

Vol 270 (46) ◽

pp. 27852-27858 ◽

Cited By ~ 155

Author(s):

Jan Rosing ◽

Lico Hoekema ◽

Gerry A. F. Nicolaes ◽

M. Christella L. G. D. Thomassen ◽

H. Coenraad Hemker ◽

...

Keyword(s):

Protein C ◽

Activated Protein C ◽

Factor Xa ◽

Protein S ◽

Peptide Bond ◽

Factor Va

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The roles of factor Va and protein S in formation of the activated protein C/protein S/factor Va inactivation complex

Journal of Thrombosis and Haemostasis ◽

10.1111/jth.14594 ◽

2019 ◽

Vol 17 (12) ◽

pp. 2056-2068 ◽

Cited By ~ 3

Author(s):

Magdalena Gierula ◽

Isabelle I. Salles‐Crawley ◽

Salvatore Santamaria ◽

Adrienn Teraz‐Orosz ◽

James T. B. Crawley ◽

...

Keyword(s):

Protein C ◽

Activated Protein C ◽

Protein S ◽

C Protein ◽

Factor Va ◽

S Factor

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Effects of Factor Xa and Protein S on the Individual Activated Protein C-mediated Cleavages of Coagulation Factor Va

Journal of Biological Chemistry ◽

10.1074/jbc.m606441200 ◽

2006 ◽

Vol 281 (42) ◽

pp. 31486-31494 ◽

Cited By ~ 22

Author(s):

Eva A. Norstrøm ◽

Sinh Tran ◽

Mårten Steen ◽

Björn Dahlbäck

Keyword(s):

Protein C ◽

Activated Protein C ◽

Factor Xa ◽

Coagulation Factor ◽

Protein S ◽

Factor Va ◽

The Individual

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Effects of Factor Xa and Protein S on the Individual Activated Protein C-mediated Cleavages of Coagulation Factor Va

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)84061-9 ◽

2006 ◽

Vol 281 (42) ◽

pp. 31486-31494

Author(s):

Eva A. Norstrøm ◽

Sinh Tran ◽

Mårten Steen ◽

Bjo¨rn Dahlba¨ck

Keyword(s):

Protein C ◽

Activated Protein C ◽

Factor Xa ◽

Coagulation Factor ◽

Protein S ◽

Factor Va ◽

The Individual

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A Novel Functional Assay of Protein C in Human Plasma and fts Comparison with Amidolytic and Anticoagulant Assays

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648377 ◽

1992 ◽

Vol 67 (01) ◽

pp. 046-049 ◽

Cited By ~ 10

Author(s):

H A Guglielmone ◽

M A Vides

Keyword(s):

Oral Anticoagulant ◽

Protein C ◽

Activated Protein C ◽

Protein S ◽

Normal Subjects ◽

Test Sample ◽

Functional Assay ◽

Fast Method ◽

Factor Va ◽

Protein C Activity

SummaryA simple and fast method for the quantitative determination of protein C activity in plasma is here described. The first step consists in the conversion of protein C in the test sample into activated protein C by means of an activator isolated from Southern Copperhead venom. Subsequently, the degradation of factor Va, in presence of protein C-deficient plasma, is measured by the prolongation of the prothrombin time which is proportional to the amount of protein C in the sample. The dose-response curve showed a linear relationship from 6 to 150% protein C activity and the inter- and intra-assay reproducibility was 3.5% and 5.6% respectively. In normal subjects, a mean of protein C level of 98 ± 15% of normal pooled plasma was found. Comparison with the anticoagulant assay in samples of patients with oral anticoagulant, liver cirrhosis, disseminated intravascular coagulation and severe preeclampsia revealed an excellent correlation (r = 0.94, p <0.001). Also, a similar correlation (r = 0.93, p <0.001) existed between amidolytic assay and the method here proposed for all the samples studied without including the oral anticoagulant group. These results allowed us to infer that this method evaluates the ability of protein C to interact with protein S, phospholipids, calcium ions and factor Va.

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