DETECTION AND EFFECTS OF THROMBOMODULIN ACTIVITY IN CRUDE THROMBOPLASTIN PREPARATIONS FROM PLACENTA AND LUNG

1987 ◽  
Author(s):  
M L Wiesel ◽  
R Spaethe ◽  
J-M Freyssinet ◽  
T Tran ◽  
H-J Kolde ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Vitamin K ◽  
Human Placenta ◽  
Protein C ◽  
Anticoagulant Activity ◽  
Protein S ◽  
Coagulation System ◽  
Factor X ◽  
Coagulant Activity ◽  
The Stability

The activation of protein C (PC) by thrombin requires the presence of an endothelial membrane cofactor, thrombomodulin (TM). Activated PC (APC) exerts its anticoagulant activity by degrading factors (F) Va and Villa in the presence of phospholipids and of a vitamin K-dependent cofactor, protein S. Tissue factor (TF) is the essential cofactor of factor Vll/VIIain the activation of factor X. TF is synthetized by several cell lines including endothelial cells. Using a specific TM assay, up to 0.85 units of TM activity could be detected in commercial thromboplastin (TP) preparations from human placenta or rabbit or porcine lung, when the amount of TP was adjusted to contain 1 unit of TF activity. Preparations from brain contained very low amounts, if any, of this activity (< 0.02 TM units). In order to evaluate the effects of the presence of TM activity in some TP preparations, the stability of F V and VIII activities was examined after activation of the coagulation system by these TP in various plasmas. PC deficient plasmas, plasmas lacking F V, VIII or IX and immunoadsorbed PC deficient plasma supplemented with purified human PC (5 Ug/ml) were used. After activation with placenta or lung TP, F V and VIII activities were markedly reduced ( ∼ 90 % reduction) in normal and hemophiliac plasmas, whereas they remained high after activation with brain TP. F V and VIII activities were preserved in protein C deficient plasma after activation by all TP preparations. The same decrease of F V and VIII activities was observed after activation of immunoadsorbed PC deficient plasma supplemented with purified PC with placenta or lung TP only. Preincubation of TP from human placenta with antibodies to human TM raised in laying hens abolished the capacity of this preparation to destroy F V activity of PC containing plasmas. These results establish the presence of TM activity in crude thromboplastin preparations from placenta or from lung. Surprisingly, this anti-coagulant activity seems to be absent from brain. TM from placenta or lung extracts is responsible for the degradation of F V and VIII.

Platelet Factor 4 Mediates Activated Protein C Resistance by Impairment of Protein S Cofactor Enhancement

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 20-20
Author(s):  
Roger JS Preston ◽  
Jennifer A Johnson ◽  
Fionnuala Ni Ainle ◽  
Shona Harmon ◽  
Owen P. Smith ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Protein C ◽  
Anticoagulant Activity ◽  
Protein S ◽  
Endothelial Barrier ◽  
Platelet Factor ◽  
Barrier Permeability ◽  
Gla Domain ◽  
Anti Inflammatory

Abstract Platelet factor 4 (PF4) is an abundant platelet α-granule chemokine released following platelet activation. PF4 interacts with thrombomodulin and the γ-carboxyglutamic acid (Gla) domain of protein C to significantly enhance activated protein C (APC) generation by the thrombin-thrombomodulin complex on the surface of endothelial cells. However, the protein C Gla domain not only mediates protein C activation in vivo, but also plays a critical role in modulating the diverse functional properties of APC once generated. The functional consequences of the interaction between the APC Gla domain and PF4 in relation to APC anticoagulant, anti-inflammatory and anti-apoptotic functions have not previously been fully defined. In a tissue factor-initiated thrombin generation assay, APC impaired thrombin generation as previously described. However PF4 inhibited APC anticoagulant activity in a concentration-dependent manner (IC50 for PF4 inhibition of APC anticoagulant function, 11μg/ml). In contrast, addition of two other cationic polypeptides protamine and polybrene, both significantly enhanced APC anticoagulant activity in plasma. To elucidate the mechanism through which PF4 inhibits APC anticoagulant activity, we utilized a phospholipid-dependent FVa proteolysis time course assay. In the absence of protein S, PF4 had no effect upon FVa proteolysis by APC, indicating that PF4 does not influence the ability of APC to interact with either anionic phospholipids or FVa. However, in the presence of protein S, PF4 significantly inhibited APC-mediated FVa proteolysis (3–5 fold). Collectively, these findings demonstrate that in addition to enhancing APC generation, PF4 also significantly attenuates APC anticoagulant activity in plasma by impairing critical protein S cofactor enhancement of FVa proteolysis, and suggest that PF4 contributes to the poorly-understood APC resistance phenotype associated with activated platelets. APC bound to the endothelial cell protein C receptor (EPCR) via its Gla domain can activate PAR-1 on endothelial cells, triggering complex intracellular signaling that result in anti-inflammatory and anti-apoptotic cellular responses. To ascertain whether PF4 interaction with the protein C/APC Gla domain might impair APC-EPCR-PAR-1 cytoprotective signaling, APC protection against thrombin-induced endothelial barrier permeability and staurosporine-induced apoptosis in the presence of PF4 was determined. APC significantly attenuated thrombin-induced endothelial cell barrier permeability, as expected. PF4 alone (up to 1μM) had no independent effect upon endothelial barrier permeability, and did not protect against thrombin-mediated increased permeability. In contrast to its inhibition of APC anticoagulant activity, PF4 did not significantly inhibit the endothelial barrier protective properties of APC. To determine whether PF4 might interfere with APC-mediated cytoprotection, staurosporine-induced apoptosis in EAhy926 cells was assessed by RT-PCR quantification of pro-apoptotic (Bax) to anti-apoptotic (Bcl-2) gene expression. Pre-treatment of EAhy926 cells with APC decreased the Bax/Bcl-2 ratio close to that determined for untreated EAhy926 cells. PF4 alone, or in combination with APC, had no effect upon apoptosis-related gene expression as determined by alteration of Bax/Bcl-2 expression ratios in response to staurosporine. In summary, PF4 inhibits APC anticoagulant function via inhibition of essential protein S cofactor enhancement in plasma, whilst retaining EPCR/PAR-1 mediated cytoprotective signalling on endothelial cells. This provides a rationale for how PF4 can exert prothrombotic effects in vivo, but also mediate enhanced APC generation on the surface of endothelial cells to induce both anti-inflammatory and anti-apoptotic events. Based on these observations, we propose that PF4 acts as a critical regulator of APC generation in vivo, but also targets APC towards cytoprotective, rather than anticoagulant functions at sites of vascular injury with concurrent platelet activation.

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.

HUMAN PLACENTAL ANTICOAGULANT PROTEIN

1987 ◽  
Author(s):  
K Fujikawa ◽  
T Funakoshi ◽  
R L Heimark ◽  
J F Tait
Keyword(s):  
Molecular Weight ◽  
Endothelial Cells ◽  
Anticoagulant Activity ◽  
Gel Filtration ◽  
Factor Xa ◽  
Protein S ◽  
Factor X ◽  
Aortic Endothelial Cells ◽  
Bovine Aortic Endothelial Cells

Endothelium is important to maintain blood fluidity preventing coagulation. Glycosaminoglycan in the endothelial cell plasma membrane has been thought to prevent activation of blood coagulation. Heparin-like compound, which is a potent anticoagulant activity, has been localized on the surface of the cultured endothelial cells. Anticoagulant action associated with thrombomodulin, which is present in endothelial cells, is another mechanism to provide hemostatic nature of endothelial cells.We wondered whether any other intracellular protein(s) is involved in coagulation. We looked for such a protein(s) in cultured bovine aortic endothelial cells. We soon found an anticoagulant activity in the soluble fraction of endothelial cells and it was partially purified. This activity was adsorbed to DEAE-Sepharose and eluted from a gel filtration column in a molecular weight range of 30,000-40,000. However, limited amounts of the cells made it difficult to purify this activity. We then chose human placenta as a substitute source of this protein and have continued the purification of this anticoagulant activity.In this communication, we describe the isolation and characterization of a placental anticoagulant protein, called "PAP", which is silmilar or possible same as the endothelial anticoaguant protein. PAP was purified from the soluble fraction of human placenta by ammonium sulfate precipitation and column chromatography on DEAE-Sepharose, Sephadex G-75, and mono S (Pharmacia). Approximately 20 mg of the protein was purified from one placenta. The purified protein gave a single band by SDS polyacrylamide gel electrophoresis with a molecular weight of 36,500. This protein inhibited both kaolin- and thromboplastin-induced partial thromboplastin times of normal human plasma. It also inhibited the clotting time of platelet-rich plasma induced by factor Xa, but did not affect the thrombin activity of fibrinogen-fibrin conversion. The purified protein completely inhibited the prothrombin activation by reconstituted prothrombinase. The protein neither inhibited the amidolytic activity of factor Xa nor bound factor Xa. This protein specifically bound to phospholipid vesicles (20% phosphatidylserine and 80% phosphatidylcholine) in the presence of calcium ions. These results indicate that PAP inhibits coagulation through the binding to phospholipid vesicles. The study on the amino acid sequence of PAP is in progress in our laboratory. Surprisingly, the sequence analysis of the cyanogen bromide fragments revealed that PAP is a new member of the lipocortin or calpactin family. The sequences of several cyanogen bromide fragments of PAP aligns with the sequences of lipocortin I and II with over 50% identity.Since PAP interacts directly with phospholipid rather than factor Xa, other activation steps in the coagulation cascade, in which phospholipid is involved, are pro^|bly affected by PAP. These reactions are the activation of factor X by a complex of factor IXa-factor VIIIa-phospholipid-Ca++ and the activations of factor X and factor IX by a tissue factor-factor VIIa-Ca++ complex.Reutelingsperger et. al,, have reported the isolation of a novel inhibitor from arteries of human umbilical cord. This protein inhibited the prothrombin activation by prothrombinase. The authors proposed that the inhibition mechanism of this inhibitor was a competition with factor Xa for binding to phospholipid. This protein is very similar to PAP as to the mode of inhibition. The molecular weight of this inhibitor is 32,000, which is slightly smaller than PAP. With the limited chemical characterization of this protein, presently it is difficult to identify this inhibitor with PAP.At the present time, the physiological role and origin of PAP is not known. PAP may originate from the endothelium of placenta, because we have detected a PAP-like anticoagulant activity in bovine aortic endothelial cells. This activity and PAP were quite alike in the purification up to the gel filtration step. If PAP antibody recognizes the antigen in the endothelial cells, it is interesting to see whether PAP localizes on the surface or inside the cells. Nevertheless, if PAP is present in the endothelial cells, it may play an important role to maintain the hemostatic nature of endothelium. PAP may bind phospholipid components at injured sites, before coagulation factors come in contact with lipid components and initiate thrombolytic events.

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.

INHIBITION OF HUMAN PROTEIN C ACTIVATION BY VITAMIN K-DEPENDENT PROTEINS, INVOLVEMENT OF THE γ-CARBOXIGLUTAMIC ACID DOMAIN IN DISTINCT INTERACTIONS WITH THE HUMAN THROMBIN-THROMBOMODULIN COMPLEX AND PHOSPHOLIPIDS

1987 ◽  
Author(s):  
J M Freyssinet ◽  
A Beretz ◽  
C Klein-Soyer ◽  
J Gauchy ◽  
J Gauchy ◽  
...  
Keyword(s):  
Vitamin K ◽  
Protein C ◽  
Competitive Inhibition ◽  
Protein S ◽  
Factor X ◽  
Amidolytic Activity ◽  
Carboxyglutamic Acid ◽  
Human Thrombin ◽  
Gla Domain ◽  
Acid Domain

Protein C (PC) activation by thrombin (T) occurs at the endothelial cell (EC) surface in the presence of thrombomodulin (TM). Reconstitution of purified TM into phospholipid (PL) vesicles results in an increased activation of PC but not of Gla-domainless-PC (GD-PC), a chymotryptic derivative of PC lacking the γ-carboxyglutamic acid domain (Gla-domain). We show that several human vitamin K-dependent proteins can interfere in the activation of human PC by the human T-TM complex either in the presence or in the absence of PL. Prothrombin fragment 1 (F1), peptide 1 -4.1, the N-terminal chymotryptic Gla-domain of prothrombin (F II), FI I and factor X (FX) were able to inhibit PC activation. They had no effect on the amidolytic activity of activated PC. Non-competitive inhibition was observed in the presence of 10 yM F1 when PC, at various concentrations, was activated by the 8 μM T-TM complex, at 2 mM Ca2+, with or without PL-reconstituted TM. In any case the apparent Km remained unchanged at 2 μM. In the presence of optimal PL concentrations and in the absence of F1, the Vmax could be enhanced up to 9-fold. When F1 was present, the extents of inhibition with and without PL were comparable and resulted in a 3fold decrease of the Vmax. These effects were independent of Ca2+ between 1 and 5 mM and of T between 10 and 50 nM. At 0.6 μM PC, half maximal inhibition occurred at 8μM F1 and 1 μM peptide 1-41 in the presence or in the absence of PL. Protein S and factors VII and IX had only minimal effect. The inhibition due to F1 and FX was also noticed when PC was activated by T in the presence of cultured human vascular EC. A Ki of 4 μM could be determined for F1 with EC-bound TM. The non-competitive character was confirmed by the observation that F1 could also inhibit the activation of GD-PC by the T-TM complex. Incomplete heat-decarboxylation of F1 and FII, partially abolished their capacity to inhibit PC activation. These results suggest that the Gla domain of PC is involved in two distinct types of interactions. This vitamin K-dependent functional entity is necessary to allow the enhancement of PC activation by anionic PL and also interacts with the T-TM complex.

The Gla26 Residue of Protein C Is Required for the Binding of Protein C to Thrombomodulin and Endothelial Cell Protein C Receptor, but not to Protein S and Factor Va

1996 ◽  
Vol 75 (02) ◽  
pp. 275-282 ◽  
Author(s):  
Junji Nishioka ◽  
Masaru Ido ◽  
Tatsuya Hayashi ◽  
Koji Suzuki
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Protein C ◽  
Anticoagulant Activity ◽  
Protein S ◽  
Cell Protein ◽  
Human Endothelial Cells ◽  
Factor Va ◽  
Activation Rate ◽  
Carboxyglutamic Acid

SummaryA functionally defective protein C (PC)-Mie, detected in the plasma of a patient with hereditary thrombophilia, has Lys substituted for γ-carboxyglutamic acid (Gla)26 residue. The activation rate of PC-Mie by Protac or thrombin in the absence of Ca2+ and that by thrombin with native thrombomodulin (TM), recombinant soluble truncated TM or on cultured endothelial cells in the presence of Ca2+ were all apparently lower than that of normal PC. The anticoagulant activity of Protac-activated PC (APC)-Mie on the plasma clotting time and the rate of inactivation of factor Va by APC-Mie in the presence of phospholipids were lower than those seen with normal APC. APC-Mie and normal APC bound equally to protein S and to biotinyl-factor Va. However, neither PC-Mie nor APC-Mie bound to phospholipids and to cultured human endothelial cells. It was similar to that observed with Gla-domainless PC/APC, but different from that seen with normal PC/APC. These results suggest that Gla26-dependent conformation is required for the binding of PC/APC to phospholipids, TM and the surface of endothelial cell PC/APC receptor, but not to protein S and factor Va.

Covalent antithrombin-heparin effect on thrombin-thrombomodulin and activated protein C reaction with factor V/Va

2010 ◽  
Vol 103 (05) ◽  
pp. 910-919 ◽  
Author(s):  
Maria Christina Van Walderveen ◽  
Leslie Roy Berry ◽  
Helen Mary Atkinson ◽  
Anthony Kam Chuen Chan
Keyword(s):  
Endothelial Cells ◽  
Chondroitin Sulfate ◽  
Protein C ◽  
Umbilical Vein ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Protein S ◽  
Factor V ◽  
Western Blots ◽  
Umbilical Vein Endothelial Cells

SummaryThrombomodulin (TM), which variably contains a chondroitin sulfate (±CS), forms an anticoagulant complex with thrombin (IIa). IIaTM(±CS) converts protein C (PC) into activated PC (APC), which then inactivates activated factors V (FVa) and VIII (FVIIIa). This reduces prothrombinase and tenase complexes that generate IIa. Heparin (H) increases the rate of IIa-TM inhibition by antithrombin (AT) and enhances FV cleavage by APC. Our novel covalent AT-H (ATH) product, has superior anticoagulant activity compared to AT + unfractionated H (UFH). We studied mechanisms by which ATH versus AT + UFH inhibits IIaTM(±CS), and ATH influences on APC cleavage of FV/FVa compared to UFH. Findings would determine how these reactions moderate ATH’s overall effects as an anticoagulant. Discontinuous second order rate inhibition assays of IIa-TM(±CS) inhibition by AT + UFH or ATH were performed in presence or absence of human umbilical vein endothelial cells (HUVECs). FV/FVa cleavage by APC in the presence of UFH or ATH was analysed by Western blots. ATH increased IIa-TM(±CS) inhibition to a greater degree than AT + UFH, both on plastic and HUVEC surfaces. Unlike UFH, ATH did not accelerate FV cleavage by APC, but ATH did enhance FVa cleavage relative to UFH. Increased IIa-TM inhibition by ATH downregulates PC activation. However, ATH does accelerate downstream inactivation of FVa, which increases its potency for IIa generation inhibition compared to UFH. This trend holds true in the presence of APC’s cofactor, protein S. Overall, ATH may have a balanced function towards inhibiting or accelerating PC pathway activities.

Severe Protein S Deficiency in a Newborn

PEDIATRICS ◽  
1992 ◽  
Vol 89 (4) ◽  
pp. 674-676
Author(s):  
CHARLES H. PEGELOW ◽  
MARUIES LEDFORD ◽  
JONELL YOUNG ◽  
GASTON ZILLERUELO
Keyword(s):  
Vitamin K ◽  
Protein C ◽  
Anticoagulant Activity ◽  
Purpura Fulminans ◽  
Protein S ◽  
Protein S Deficiency ◽  
Protein C Deficiency ◽  
Newborn Period ◽  
Thrombotic Disease ◽  
S Deficiency

Protein S is a vitamin K-dependent glycoprotein which acts as a cofactor for the anticoagulant activity of protein C.1,2 With production under autosomal control, heterozygotes produce half-normal levels and thrombotic disease may develop.3-6 Although thromboses occur primarily in adults, there are isolated reports of their occurrence in affected children.7-13 Severe protein C deficiency results in a syndrome in which affected children develop multiple thromboses in the newborn period.14 A recent report described a child with homozygous protein S deficiency who presented with neonatal purpura fulminans and other thromboses similar to those found in protein C deficiency.15,16 In this report, we

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