A Plasma Coagulation Assay for an Activated Protein C-independent Anticoagulant Activity of Protein S

1998 ◽  
Vol 80 (12) ◽  
pp. 930-935 ◽  
Author(s):  
Cornelis van ’t Veer ◽  
Joost Meijers ◽  
Rogier Bertina ◽  
Merel van Wijnen ◽  
Bonno Bouma
Keyword(s):  
Protein C ◽  
Anticoagulant Activity ◽  
Oral Anticoagulant Therapy ◽  
Activated Protein C ◽  
Protein S ◽  
Laboratory Diagnosis ◽  
Clotting Time ◽  
Functional Protein ◽  
Physiological Importance ◽  
The Mean

SummaryTo study the physiological importance of the activated protein C (APC)-independent anticoagulant activity of protein S, we developed an assay specific for this activity. The ability of protein S to prolong the clotting time in an APC-independent way was expressed as the ratio of the clotting time in a plasma sample divided by the clotting time in the presence of a polyclonal antibody against human protein S (both after 1:1 dilution in protein S-C4BP deficient plasma). The mean protein S-dependent anticoagulant ratio (PSdAR) was 1.53 ± 0.18 in 34 healthy controls, and was significantly lower in 16 heterozygous protein S deficient patients (PSdAR = 1.15 ± 0.09). In plasmas from patients under oral anticoagulant therapy the mean PSdAR was within the range of the control population (1.50 ± 0.18). The mean total protein S antigen level in these plasmas was 58%, suggesting a higher specific APC-independent anticoagulant activity of protein S in these patients than in normals.This functional protein S assay can be used for the laboratory diagnosis of protein S deficiency, and to study the mechanism of the APC-independent anticoagulant activity in plasma.

Prevention of thrombosis following deep arterial injury in rats by bovine activated protein C requiring co-administration of bovine protein S

2003 ◽  
Vol 90 (08) ◽  
pp. 227-234 ◽  
Author(s):  
Björn Dahlbäck ◽  
Björn Arnljots ◽  
Karl Malm
Keyword(s):  
Protein C ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Protein S ◽  
Arterial Injury ◽  
Control Group ◽  
Clotting Time ◽  
Antithrombotic Effect

SummaryThe antithrombotic effect of bovine activated protein C (bAPC) given with or without bovine protein S (bPS) was investigated in a rat model of deep arterial injury. A segment of the left common carotid artery was isolated between vascular clamps and opened longitudinally. An endarterectomy was performed and the arteriotomy was closed with a running suture, whereafter the vessel was reperfused by removing the clamps. The antithrombotic effect (vascular patency rates 31 minutes after reperfusion) and the arteriotomy bleeding were measured. Ten treatment groups each containing 10 rats and a control group of 20 animals were in a blind random fashion given intravenous bolus injections of increasing doses of activated protein C, with or without co-administration of protein S. The groups received either bAPC alone (0.8, 0.4, 0.2 or 0.1 mg/kg), bAPC (0.8, 0.4, 0.2, 0.1 or 0.05 mg/kg) combined with bPS (0.6 mg/kg), or bPS alone (0.6 mg/kg) whereas the control group received vehicle only. Administered alone, bAPC or bPS had no antithrombotic effect, regardless of dosage. In contrast, all groups that were treated with bAPC in combination with bPS demonstrated a significant antithrombotic effect, as compared to controls. Neither bAPC, bPS, nor the combination of bAPC and bPS increased the arteriotomy bleeding significantly compared to controls. In vitro clotting assays using bAPC or bPS alone yielded only minor prolongation of clotting time, whereas bAPC combined with bPS prolonged the clotting time considerably, demonstrating the dependence on the APC-cofactor activity of bPS for expression of anticoagulant activity by bAPC. In conclusion, our study shows the in vivo significance of protein S as a cofactor to activated protein C, and that potent anti-thrombotic effect can be achieved by low doses of bAPC combined with bPS, without producing hemorrhagic side effects.

Protein S Regulates Factor IXa in the Absence and Presence of Factor VIIIa Independently of Activated Protein C

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1197-1197
Author(s):  
Rinku Majumder ◽  
Rima Chattopadhyay ◽  
Tanusree Sengupta
Keyword(s):  
Protein C ◽  
Thrombin Generation ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Protein S ◽  
Activity Measurement ◽  
Clotting Time ◽  
Thrombotic Risk ◽  
Increased Risk

Abstract Abstract 1197 Coagulation is a finely tuned process. During thrombin formation, several anticoagulant reactions are initiated to prevent systematic activation of coagulation, and impairment of anticoagulant activity causes an increased risk of venous thrombosis. One such anticoagulant factor is protein S, deficiencies of which have been linked to venous and arterial thrombosis. While protein S has been studied for over three decades, the precise role this protein plays in attenuating the hemostatic response is far from clear. Protein S is a vitamin K-dependent plasma protein that functions in feedback regulation of thrombin generation. Protein S was initially identified as a cofactor for activated protein C (APC) but later it was observed that there is only a 3–10 fold increase in APC activity in the presence of protein S. Plasma coagulation assays in the absence of APC suggest that protein S may have other anticoagulant role(s). We report here an anticoagulant activity of Protein S mediated by inhibition of fIXa in the absence and presence of fVIIIa independent of APC. Although an APC-independent anticoagulant activity has been reported for protein S interacting with fVIIIa, no study has shown that the inhibitory effect of protein S is mediated through its interaction with fIXa, thus making our observations novel and significant. Moreover, previous studies that reported an interaction between fVIIIa and protein S were performed with low amounts of phospholipid, a condition that produces activity measurement artifacts due to the presence of active protein S multimers. We used both ex vivo (plasma studies) and in vitro methods at high phospholipid (100–200 micro molar) concentration to determine whether and how the intrinsic pathway of blood coagulation is regulated by protein S. We obtained the following results: 1) activated partial thromboplastin time (aPTT) assays with protein S-supplemented plasma confirmed that protein S prolongs clotting time, and a normal clotting time was restored with addition of anti-protein S antibody, 2) a modified aPPT assay with fIX-deficient plasma confirmed that protein S affects fIX-initiated clotting time, 3) thrombin generation assay through fIXa/fVIIIa pathway, initiated with a limiting amount of tissue factor (TF), was regulated by protein S, 4) in vitro studies with fIXa/fVIIIa and protein S in the presence of phosphatidylserine (PS) vesicles showed ∼40% and ∼65% inhibition in the activity of fIXa in the absence and presence of fVIIIa, respectively, and 5) protein S altered only the KM for fX activation by fIXa but altered both kcat and KM for fX activation by fIXa and fVIIIa. Our findings underscore the central role of protein S in regulation of coagulation. We anticipate these results will unravel important implications for the evaluation of thrombotic risk associated with protein S-deficiency. Disclosures: No relevant conflicts of interest to declare.

β2-Glycoprotein I Modulates the Anticoagulant Activity of Activated Protein C on the Phospholipid Surface

1996 ◽  
Vol 75 (01) ◽  
pp. 049-055 ◽  
Author(s):  
Tatsuyuki Mori ◽  
Hiroyuki Takeya ◽  
Junji Nishioka ◽  
Esteban C Gabazza ◽  
Koji Suzuki
Keyword(s):  
Protein C ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Protein S ◽  
Inhibitory Effect ◽  
Factor V ◽  
Clotting Time ◽  
Factor Va ◽  
Glycoprotein I ◽  
Prothrombinase Activity

SummaryThe objective of this study was to determine whether (β2-glycoprotein I (β2GPI) has procoagulant activity by inhibiting the anticoagulant activity of activated protein C (APC). β2GPI inhibited significantly the APC-catalyzed inactivation of factor Va in an assay using factor V-deficient plasma and physiological levels of protein S and factor Va. This inhibitory effect was diminished by the addition of increasing concentrations of phospholipids, suggesting that β2GPI competitively inhibits the binding of APC to the phospholipid surface. β2GPI inhibited weakly factor Va- and phospholipid-dependent prothrombinase activity at concentrations similar to those to inhibit APC activity. The depletion of β2GPI from plasma led to only a slight shortening of the diluted Russell’s viper venom-dependent clotting time, but to a strong and significant potentiation of the anticoagulant activity of APC. These results suggest that under certain physiological conditions β2GPI has procoagulant property by inhibiting the phospholipid-dependent APC anticoagulant activity.

scholarly journals Plasma protein S deficiency in familial thrombotic disease

Blood ◽  
1984 ◽  
Vol 64 (6) ◽  
pp. 1297-1300 ◽  
Author(s):  
HP Schwarz ◽  
M Fischer ◽  
P Hopmeier ◽  
MA Batard ◽  
JH Griffin
Keyword(s):  
Plasma Protein ◽  
Protein C ◽  
Anticoagulant Activity ◽  
Oral Anticoagulant Therapy ◽  
Activated Protein C ◽  
Protein S ◽  
Factor X ◽  
Protein C Deficiency ◽  
Thrombotic Disease ◽  
Disease Protein

Abstract A family with a history of severe recurrent venous thromboembolic disease was studied to determine if a plasma protein deficiency could account for observed disease. Protein S levels in plasma were determined immunologically using the Laurell rocket technique. The propositus, his mother, his aunt, and his cousin who were clinically affected had 17% to 65% of the control levels of protein S antigen (normal range, 71% to 147%). Since three of these patients were receiving oral anticoagulant therapy, the ratios of protein S to prothrombin, factor X, and protein C in these patients were compared with values for a group of orally anticoagulated controls. These results suggested that protein S is half-normal in all family members with thrombotic disease. Other proteins known to be associated with familial thrombotic disease, including antithrombin III, plasminogen, fibrinogen, and protein C, were normal. Because plasma protein S serves as a cofactor for the anticoagulant activity of activated protein C and because protein C deficiency is associated with recurrent thrombotic disease, it is suggested that recurrent thrombotic disease in this family is the result of an inherited deficiency of protein S.

REGULATION OF THE ANTICOAGULANT ACTIVITY OF ACTIVATED PROTEIN C BY PROTEIN S AND PROTEIN S BINDING PROTEIN

1987 ◽  
Author(s):  
F J Walker
Keyword(s):  
Molecular Weight ◽  
Protein C ◽  
Copper Ions ◽  
Binding Protein ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Protein S ◽  
Single Chain ◽  
Functional Protein ◽  
Bovine Plasma

Protein S is a vitamin K-dependent protein that acts as a cofactor for the anticoagulant activity of activated protein C both in the proteolytic inactivation of factor V and VIII. Protein S is a single chain protein with a molecular weight of approximately 62 kDa. When the molecular weight of protein S in plasma was determined it was found to be much larger than the single chain protein. The molecular weight of functional protein S when measured by sedimentation equilibrium with the air-driven ultracentrifuge was observed to be between 115 and 130 kDa. In high salt or in the presence of copper ions this was observed to be reduced to approximately 62 kDa. Frontal analysis of plasma indicated that the functional protein by exist in as many as three molecular weight foras. Gel filtration of radiolabeled protein S also indicates heterogeneity in the molecular weight. In order to isolate the binding protein, bovine plasma was fractionated first on a column of immobilized iminodiacetic acid that had been equilibrated with copper ions. The proteins that eluted in the 0.6 M NaCl wash were passed over a column of protein S immobilized on agarose beads. A single protein was observed to elute from the protein S agarose at high salt. Fractionation of human plasma indicated the presence of several proteins. One major component isolated was C4-binding protein. A second major component has also been isolated that appears to correspond to protein S-binding protein that has been isolated from bovine plasma. When added to plasma depleted of both protein S and the binding protein, the binding protein was observed to enhance the anticoagulant activity of activated protein C only in the presence of protein S. Protein S-binding protein was also observed to enhance the rate of factor Va inactivation by activated protein C and protein S.

Determination of Plasma Protein S - The Protein Cofactor of Activated Protein C

1985 ◽  
Vol 53 (02) ◽  
pp. 268-272 ◽  
Author(s):  
R M Bertina ◽  
A van Wijngaarden ◽  
J Reinalda-Poot ◽  
S R Poort ◽  
V J J Bom
Keyword(s):  
Protein C ◽  
Binding Protein ◽  
Oral Anticoagulant Therapy ◽  
Activated Protein C ◽  
Protein S ◽  
Laboratory Diagnosis ◽  
Protein S Deficiency ◽  
S Deficiency ◽  
Immunologic Assays

SummaryProtein S, an important cofactor of activated protein C, and C4b-binding protein were purified from human plasma. Specific antibodies against the purified proteins were raised in rabbits and used for the development of immunologic assays for these proteins in plasma: an immunoradiometric assay for protein S (which measures both free protein S and protein S complexed with C4b-binding protein) and an electroimmunoassay for C4b- binding protein. Ranges for the concentrations of these proteins were established in healthy volunteers and patients using oral anticoagulant therapy. A slight decrease in protein S antigen was observed in patients with liver disease (0.78 ± 0.25 U/ml); no significant decrease in protein S was observed in patients with DIC (0.95 ± 0.25 U/ml).Criteria were developed for the laboratory diagnosis of an isolated protein S deficiency

Functional activity of protein S determined with use of protein C activated by venom activator.

1989 ◽  
Vol 35 (8) ◽  
pp. 1644-1648 ◽  
Author(s):  
I Kobayashi ◽  
N Amemiya ◽  
T Endo ◽  
K Okuyama ◽  
K Tamura ◽  
...  
Keyword(s):  
Protein C ◽  
Warfarin Therapy ◽  
Activated Protein C ◽  
Protein S ◽  
Normal Subjects ◽  
Clotting Time ◽  
Amidolytic Activity ◽  
Functional Protein ◽  
Protein S Activity ◽  
Cacl2 Solution

Abstract A new screening procedure, an easy and specific assay for determining functional Protein S activity, has been developed, with use of Protein C activated by venom activator (Protac). Purified Protein C (100% amidolytic activity) was activated by venom activator (6 units/mL). To a mixture of 100 microL of Protein S-deficient plasma, 20 microL of sample plasma, 100 microL of cephalin (Actin), and 20 microL of activated Protein C we added 100 microL of 25 mmol/L CaCl2 solution and measured the clotting time with a KC 10 coagulometer. The functional Protein S activity correlated well with the concentrations of Protein S antigen measured by enzyme immunoassay and the Laurell rocket technique (r = 0.810 and 0.850, respectively) in normal subjects, patients with myocardial infarction undergoing warfarin therapy, and patients with liver cirrhosis.

scholarly journals Plasma protein S deficiency in familial thrombotic disease

Blood ◽  
1984 ◽  
Vol 64 (6) ◽  
pp. 1297-1300 ◽  
Author(s):  
HP Schwarz ◽  
M Fischer ◽  
P Hopmeier ◽  
MA Batard ◽  
JH Griffin
Keyword(s):  
Plasma Protein ◽  
Protein C ◽  
Anticoagulant Activity ◽  
Oral Anticoagulant Therapy ◽  
Activated Protein C ◽  
Protein S ◽  
Factor X ◽  
Protein C Deficiency ◽  
Thrombotic Disease ◽  
Disease Protein

A family with a history of severe recurrent venous thromboembolic disease was studied to determine if a plasma protein deficiency could account for observed disease. Protein S levels in plasma were determined immunologically using the Laurell rocket technique. The propositus, his mother, his aunt, and his cousin who were clinically affected had 17% to 65% of the control levels of protein S antigen (normal range, 71% to 147%). Since three of these patients were receiving oral anticoagulant therapy, the ratios of protein S to prothrombin, factor X, and protein C in these patients were compared with values for a group of orally anticoagulated controls. These results suggested that protein S is half-normal in all family members with thrombotic disease. Other proteins known to be associated with familial thrombotic disease, including antithrombin III, plasminogen, fibrinogen, and protein C, were normal. Because plasma protein S serves as a cofactor for the anticoagulant activity of activated protein C and because protein C deficiency is associated with recurrent thrombotic disease, it is suggested that recurrent thrombotic disease in this family is the result of an inherited deficiency of protein S.

Platelet-mediated proteolytic down regulation of the anticoagulant activity of protein S in individuals with haematological malignancies

2012 ◽  
Vol 107 (03) ◽  
pp. 468-476 ◽  
Author(s):  
Ilze Dienava-Verdoold ◽  
Marina R. Marchetti ◽  
Liane C. J. te Boome ◽  
Laura Russo ◽  
Anna Falanga ◽  
...  
Keyword(s):  
Protein C ◽  
Normal Values ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Protein S ◽  
Intact Protein ◽  
The Impact ◽  
Independent Protein

SummaryThe natural anticoagulant protein S contains a so-called thrombin-sensitive region (TSR), which is susceptible to proteolytic cleavage. We have previously shown that a platelet-associated protease is able to cleave protein S under physiological plasma conditions in vitro. The aim of the present study was to investigate the relation between platelet-associated protein S cleaving activity and in vivo protein S cleavage, and to evaluate the impact of in vivo protein S cleavage on its anticoagulant activity. Protein S cleavage in healthy subjects and in thrombocytopenic and thrombocythaemic patients was evaluated by immunological techniques. Concentration of cleaved and intact protein S was correlated to levels of activated protein C (APC)-dependent and APC-independent protein S anticoagulant activity. In plasma from healthy volunteers 25% of protein S is cleaved in the TSR. While in plasma there was a clear positive correlation between levels of intact protein S and both APC-dependent and APC-independent protein S anticoagulant activities, these correlations were absent for cleaved protein S. Protein S cleavage was significantly increased in patients with essential thrombocythaemia (ET) and significantly reduced in patients with chemotherapy-induced thrombocytopenia. In ET patients on cytoreductive therapy, both platelet count and protein S cleavage returned to normal values. Accordingly, platelet transfusion restored cleavage of protein S to normal values in patients with chemotherapy-induced thrombocytopenia. In conclusion, proteases from platelets seem to contribute to the presence of cleaved protein S in the circulation and may enhance the coagulation response in vivo by down regulating the anticoagulant activity of protein S.

Inhibition of Activated Protein C Anticoagulant Activity by Prothrombin

Blood ◽  
1999 ◽  
Vol 94 (11) ◽  
pp. 3839-3846 ◽  
Author(s):  
Mikhail D. Smirnov ◽  
Omid Safa ◽  
Naomi L. Esmon ◽  
Charles T. Esmon
Keyword(s):  
Anticoagulant Therapy ◽  
Oral Anticoagulant ◽  
Protein C ◽  
Anticoagulant Activity ◽  
Oral Anticoagulant Therapy ◽  
Membrane Surface ◽  
Activated Protein C ◽  
Significant Risk ◽  
Thrombotic Risk ◽  
Factor Va

Abstract In this study, we test the hypothesis that prothrombin levels may modulate activated protein C (APC) anticoagulant activity. Prothrombin in purified systems or plasma dramatically inhibited the ability of APC to inactivate factor Va and to anticoagulate plasma. This was not due solely to competition for binding to the membrane surface, as prothrombin also inhibited factor Va inactivation by APC in the absence of a membrane surface. Compared with normal factor Va, inactivation of factor Va Leiden by APC was much less sensitive to prothrombin inhibition. This may account for the observation that the Leiden mutation has less of an effect on plasma-based clotting assays than would be predicted from the purified system. Reduction of protein C levels to 20% of normal constitutes a significant risk of thrombosis, yet these levels are observed in neonates and patients on oral anticoagulant therapy. In both situations, the correspondingly low prothrombin levels would result in an increased effectiveness of the remaining functional APC of ≈5-fold. Thus, while the protein C activation system is impaired by the reduction in protein C levels, the APC that is formed is a more effective anticoagulant, allowing protein C levels to be reduced without significant thrombotic risk. In situations where prothrombin is high and protein C levels are low, as in early stages of oral anticoagulant therapy, the reduction in protein C would result only in impaired function of the anticoagulant system, possibly explaining the tendency for warfarin-induced skin necrosis.

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