MONOCLONAL ANTIBODIES TO HUMAN PROTEIN S AND C4b-BINDING PROTEIN

1987 ◽  
Author(s):  
Martin Hessing ◽  
Joost C M Meijers ◽  
Jan A van Mourik ◽  
Bonno N Bouma
Keyword(s):  
Monoclonal Antibodies ◽  
Complex Formation ◽  
Binding Protein ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Protein S ◽  
Thrombin Cleavage ◽  
Prolonged Aptt ◽  
Acid Domain ◽  
A Minor

Protein S (PS) circulates in plasma both free and in reversible association with the complement component C4b-binding protein (C4bp). Only free PS is functional as a cofactor for activated protein C (APC). Cleavage of PS by thrombin at a site near the r-carboxyglutamic acid domain is associated with a loss of cofactor activity. This may be a control mechanism for the anticoagulant activity of APC. These observations led us to investigate the role of C4bp and thrombin in the regulation of PS. Complex formation between purified PS and C4bp was studied in plasma and in a system with purified components. 125I-labeled PS was first incubated with either C4bp or citrated plasma and then subjected to polyacrylamide gelelectrophoresis in the absence of SDS. The formation of the C4bp-PS complex in plasma and in the purified system was demonstrated by autoradiography. Crossed immuno-electrophoresis using an antiserum against PS was performed in the presence of 8 mM EDTA. Human citrated plasma showed two precipitin peaks. Free PS migrated rapidly in the first dimension, whereas the C4bp-PS complex was just anodal to the application slot. The addition of C4bp to either plasma or purified PS resulted in the disappearance of the free PS peak and an increase of the slower migrating peak. The effect of purified C4bp on the PS-cofactor function of APC was studied in citrated plasma. The prolongation of the APTT induced by the addition of APC could be inhibited by the addition of increasing amounts of C4bp. Monoclonal antibodies to PS and C4bp were prepared and characterized. The monoclonal antibodies to either PS or C4bp did not block the complex formation between and PS, as was demonstrated by dot blotting of C4bp with 125I-PS and agarose gelelectrophoresis followed by Western blotting. Three out of 7 monoclonal antibodies to PS did not detect PS after thrombin cleavage on an immunoblot after non-reduced SDS polyacrylamide gelelectrophoresis. These 3 antibodies gave a significant shortening of the prolonged APTT induced by the addition of APC to normal plasma, indicating that these monoclonals inhibited the cofactor function of PS. The other 4 monoclonals to PS that did detect PS after thrombin cleavage on an immunoblot, gave only a minor inhibition of the PS cofactor function.

Control of Thrombin Mediated Cleavage of Protein S

1986 ◽  
Vol 56 (02) ◽  
pp. 151-154 ◽  
Author(s):  
Christina A Mitchell ◽  
Lena Hau ◽  
Hatem H Salem
Keyword(s):  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Surface Protein ◽  
Protein S ◽  
Intact Protein ◽  
Control Mechanisms ◽  
Physiological Control ◽  
Endothelial Cell Surface ◽  
Thrombin Cleavage ◽  
Cofactor Activity

SummaryThrombin has been shown to cleave the vitamin K dependent cofactor protein S with subsequent loss of its cofactor activity. This study examines the control mechanisms for thrombin cleavage of protein S.The anticoagulant activity of activated protein C (APC) is enhanced fourteen fold by the addition of protein S. Thrombin cleaved protein S is seven fold less efficient than the native protein, and this loss of activity is due to reduced affinity of cleaved protein S for APC or the lipid surface compared to the intact protein.In the absence of Ca++, protein S is very sensitive to minimal concentrations of thrombin. As little as 1.5 nM thrombin results in complete cleavage of 20 nM protein S in 10 min and loss of cofactor activity. Ca++, in concentrations greater than 0.5 mM, will inhibit this cleavage and in the presence of physiological Ca++ concentrations, no cleavage of protein S could be demonstrated in spite of high concentrations of thrombin (up to 1 μM) and prolonged incubations (up to two hours). The endothelial surface protein thrombomodulin is very efficient in inhibiting the cleavage of protein S by thrombin suggesting that any thrombin formed on the endothelial cell surface is unlikely to cleave protein S, thus allowing the intact protein to act as a cofactor to APC.We conclude that the inhibitory effects of Ca++ and thrombomodulin on thrombin mediated cleavage of protein S imply that this event, by itself, is unlikely to represent a physiological control of the activity of protein S.

Coagulation, inflammation, and apoptosis: different roles for protein S and the protein S–C4b binding protein complex

Blood ◽  
2004 ◽  
Vol 103 (4) ◽  
pp. 1192-1201 ◽  
Author(s):  
Suely Meireles Rezende ◽  
Rachel Elizabeth Simmonds ◽  
David Anthony Lane
Keyword(s):  
Venous Thromboembolism ◽  
Complement Activation ◽  
Protein C ◽  
Binding Protein ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Protein S ◽  
Increased Risk ◽  
Established Role ◽  
Structure And Functions

AbstractProtein S (PS) has an established role as an important cofactor to activated protein C (APC) in the degradation of coagulation cofactors Va and VIIIa. This anticoagulant role is evident from the consequences of its deficiency, when there is an increased risk of venous thromboembolism. In human plasma, PS circulates approximately 40% as free PS (FPS) and 60% in complex with C4b-binding protein (C4BP). Formation of this complex results in loss of PS cofactor function, and C4BP can then modulate the anticoagulant activity of APC. It had long been predicted that the complex could act as a bridge between coagulation and inflammation due to the involvement of C4BP in regulating complement activation. This prediction was recently supported by the demonstration of binding of the PS-C4BP complex to apoptotic cells. This review aims to summarize recent findings on the structure and functions of PS, the basis and importance of its deficiency, its interaction with C4BP, and the possible physiologic and pathologic importance of the PS-C4BP interaction.

scholarly journals Inhibition of APC anticoagulant activity on oxidized phospholipid by anti–β2-glycoprotein I monoclonal antibodies

Blood ◽  
2005 ◽  
Vol 106 (5) ◽  
pp. 1629-1635 ◽  
Author(s):  
Omid Safa ◽  
Charles T. Esmon ◽  
Naomi L. Esmon
Keyword(s):  
Monoclonal Antibodies ◽  
Protein Interactions ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Membrane Vesicles ◽  
Protein S ◽  
Specific Protein ◽  
Protein Protein Interactions ◽  
Glycoprotein I ◽  
Antibody Complex

Abstract Activated protein C (APC) anticoagulant activity and the ability to be inhibited by auto-antibodies associated with thrombosis are strongly augmented by the presence of phosphatidylethanolamine (PE) and phospholipid oxidation. β2-glycoprotein I (β2-GPI) is a major antigen for antiphospholipid antibodies present in patients with the antiphospholipid syndrome. We therefore investigated whether anti–β2-GPI monoclonal antibodies (mAbs) could inhibit APC with similar membrane specificity. Five mouse mAbs that reacted with different epitopes on β2-GPI were examined. Each inhibited the PE-, phospholipid oxidation–dependent enhancement of APC anticoagulant activity and required antibody divalency. A chimeric APC that retains anticoagulant activity but is relatively unaffected by protein S, PE, or oxidation was not inhibited by the antibodies. In purified systems, anti–β2-GPI mAb inhibition of factor Va inactivation was greater in the presence of protein S and required β2-GPI. Surprisingly, although the mAbs did increase β2-GPI affinity for membranes, PE and oxidation had little influence on the affinity of the β2-GPI antibody complex for the membrane vesicles. We conclude that antibodies to β2-GPI inhibit APC function specifically and contribute to a hypercoaguable state by disrupting specific protein-protein interactions induced by oxidation of PE-containing membranes.

scholarly journals Low total protein S antigen but high protein S activity due to decreased C4b-binding protein in neonates

Blood ◽  
1988 ◽  
Vol 71 (3) ◽  
pp. 562-565
Author(s):  
HP Schwarz ◽  
W Muntean ◽  
H Watzke ◽  
B Richter ◽  
JH Griffin
Keyword(s):  
Vitamin K ◽  
Total Protein ◽  
Protein C ◽  
Binding Protein ◽  
Anticoagulant Activity ◽  
Active Form ◽  
Activated Protein C ◽  
Newborn Infants ◽  
Protein S ◽  
High Level

Protein S, a vitamin K-dependent cofactor for activated protein C, exists in normal adult plasma in a free anticoagulantly active form and in an inactive form complexed to C4b-binding protein. Immunologic and functional levels of protein S and C4b-binding protein in plasma were determined for 20 newborn infants and compared with adult normal pooled plasma. Total protein S antigen levels averaged 23%, similar to other vitamin K-dependent plasma proteins. However, the protein S anticoagulant activity was 74% of that of adult normal plasma. This apparent discrepancy of activity to antigen was shown to be due to low or undetectable levels of C4b-binding protein, which results in the presence of most if not all of protein S in its free and active form. The relatively high level of anticoagulantly active protein S in infants may enhance the potential of the protein C pathway, thereby minimizing risks of venous thrombosis in this group.

scholarly journals Low total protein S antigen but high protein S activity due to decreased C4b-binding protein in neonates

Blood ◽  
1988 ◽  
Vol 71 (3) ◽  
pp. 562-565 ◽  
Author(s):  
HP Schwarz ◽  
W Muntean ◽  
H Watzke ◽  
B Richter ◽  
JH Griffin
Keyword(s):  
Vitamin K ◽  
Total Protein ◽  
Protein C ◽  
Binding Protein ◽  
Anticoagulant Activity ◽  
Active Form ◽  
Activated Protein C ◽  
Newborn Infants ◽  
Protein S ◽  
High Level

Abstract Protein S, a vitamin K-dependent cofactor for activated protein C, exists in normal adult plasma in a free anticoagulantly active form and in an inactive form complexed to C4b-binding protein. Immunologic and functional levels of protein S and C4b-binding protein in plasma were determined for 20 newborn infants and compared with adult normal pooled plasma. Total protein S antigen levels averaged 23%, similar to other vitamin K-dependent plasma proteins. However, the protein S anticoagulant activity was 74% of that of adult normal plasma. This apparent discrepancy of activity to antigen was shown to be due to low or undetectable levels of C4b-binding protein, which results in the presence of most if not all of protein S in its free and active form. The relatively high level of anticoagulantly active protein S in infants may enhance the potential of the protein C pathway, thereby minimizing risks of venous thrombosis in this group.

BINDING SITE OF VITAMIN K-DEPENDENT PROTEIN S ON C4b-BINDING PROTEIN

1987 ◽  
Author(s):  
K Suzuki ◽  
J Nishioka ◽  
H Kusumoto ◽  
Y Deyashiki
Keyword(s):  
Monoclonal Antibodies ◽  
Binding Site ◽  
Disulfide Bonds ◽  
Protein C ◽  
Binding Protein ◽  
Gel Filtration ◽  
Activated Protein C ◽  
Protein S ◽  
Cofactor Activity ◽  
Carboxyl Terminal

Protein S, a cofactor for activated protein C, reversibly complexes with a regulatory complement component C4b-binding protein (C4bp) in plasma. In plasma of patients with congenital protein S deficiency, most protein S exists as a complex with C4bp, which has no cofactor activity. C4bp (Mw 550,000) is composed of approximately seven subunits with Mw 75,000 which are linked by disulfide bonds near the carboxy1-terminus. We report here about the complex formation between protein S and C4bp particularly on the binding site of protein S on C4bp molecule. Protein S and C4bp were purified from human plasma. Seventeen mouse monoclonal antibodies against C4bp were prepared. Chymotrypsin-digested C4bp was separated on gel filtration into a fragment with Mw 160,000 derived from the carboxyl-terminal core of the intact C4bp and fragments with Mw 48,000 from the amino-terminus. The carboxy1-terminal fragment with Mw 160,000 was found to be composed of approximately seven polypeptides with Mw 25,000, which were linked by disulfide bonds.The experiments using these fragments and the monoclonal antibodies showed that: (1) Protein S bound not only to the intact C4bp, but also to the fragment with Mw 160,000. (2) The fragment with Mw 160,000 inhibited the binding of protein S to C4bp, but the fragment with Mw 48,000 did not. (3) One of the seventeen monoclonal antibodies blocked the inhibition of C4bp on the cofactor activity of protein S. (4) This antibody inhibited C4bp binding to protein S. (5) The antibody bound to the fragment with Mw 160,000. Based on these results, protein S was suggested to lose its cofactor activity for activated protein C by binding to the carboxyl-terminal core of C4bp where seven subunits are linked by disulfide bonds.

REDUCTION OF THE ANTICOAGULANT ACTIVITY OF PROTEIN C AND PROTEIN S DURING THE POSTOPERATIVE PERIOD

1987 ◽  
Author(s):  
S Vigano D'Angelo ◽  
F Gilardoni ◽  
M P Seveso ◽  
A Marassi ◽  
G Mari ◽  
...  
Keyword(s):  
Postoperative Period ◽  
Protein C ◽  
Comprehensive Evaluation ◽  
Binding Protein ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Protein S ◽  
Major Surgery ◽  
Thrombotic Risk ◽  
Free Protein

Protein S circulates in plasma as free protein S and in complex with C4b-binding protein, an inhibitor of complement activation. Only free protein S functions as the cofactor for the anticoagulant and profibrinolytic effects of activated protein C. Since isolated reductions of protein C and protein S result in increased thrombotic risk, only measurement of both proteins permits comprehensive evaluation of the antithrombotic potential of the protein C system. No information is available on protein C and protein S functional levels during the postoperative period, an established prothrombotic condition. The plasma changes of protein C, protein S and C4b-binding protein were followed in 40 patients with no malignancy undergoing abdominal surgery. No significant change of protein C and protein S activi ty was observed following minor operations. After major surgery, protein C anticoagulant activity dropped to 80% of preoperative levels during the first postoperative week (p<0.00l). Significant increase of both total protein S antigen (110%, p< 0.01) and C4b-binding protein (130%, p<0.001) were observed after major surgery resulting in reduction of free protein S antigen to 86% of pre operative values (p<0.001). Protein S anticoagulant activity matched the changes of free protein S antigen.Albeit transient and moderate, the observed reductions of both protein C and protein S may act synergistically to cause significant impairment of the antithrombotic potential during the postoperative period. The effect of heparin prophylaxis on protein C and protein S postoperative levels is currently under investigation.

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.

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