Isolation and sequence of the cDNA for human protein S, a regulator of blood coagulation.

Lundwall, Åke; Dackowski, William; Cohen, Edward H.; Shaffer, Marilee; Mahr, Anna; Dahlbäck, Björn; Stenflo, Johan; Wydro, Robert

doi:10.1073/pnas.83.18.6716

Cited by 195 publications

(144 citation statements)

References 34 publications

Supporting

Mentioning

135

Contrasting

Unclassified

Order By: Relevance

“…The slightly higher molecular mass of rPS observed on SDSjPAGE was explainable by differences in Lhe carbohydrate side chains ; after deglycosylation the difference disappeared. The human protein S molecule contains three potential N-glycosylation sites (Asn-458, 468 and 489), but it has not yet been determined which is glycosylated [22]. Treatment of rPS and pPS with endoglycosidase F resulted in a reduction in the molecular mass of both rPS and pPS, and the difference in apparent molecular mass disappeared.…”

Section: Discussionmentioning

confidence: 99%

“…Construction of vector and expression qf recombinant protein S A cDNA clone, MI 17S, for human protein S [22], coding for amino acids Leu-15 of the leader sequence to the termination codon after Ser-635, was used for expression of rPS. Since the human protein S cDNA did not encode the complete leader peptide, the missing part of the leader sequence of bovine protein S cDNA was spliced onto the human protein S cDNA using state-of-the-art techniques [38,39].…”

Section: Experimental Procedures Proteinsmentioning

confidence: 99%

“…Ala (73) Ala (58) Ala Asn (11) Asn (1 2) Asn Ser (22) Ser (10) Ser Leu (19) Leu (28) Leu Leu (26) Leu (34) Leu -Gla…”

unclassified

See 2 more Smart Citations

Expression of completely γ‐carboxylated and β‐hydroxylated recombinant human vitamin‐K‐dependent protein S with full biological activity

Malm¹,

Cohen²,

Dackowski³

et al. 1990

European Journal of Biochemistry

View full text Add to dashboard Cite

Human anticoagulant vitamin-K-dependent protein S was expressed in mouse C127 cells using a bovine papilloma virus vector system. A full-length cDNA construct was introduced into the vector in the 5' untranslated region of the mouse metallothionein-I gene. Transfected cells expressed approximately 10 pg/ml of the recombinant protein which was purified by ion-exchange chromatography followed by affinity chromatography using Ca2'-dependent monoclonal antibodies against the region of protein S containing 4-carboxyglutamic acid. Recombinant protein S was structurally and functionally similar to protein S purified from plasma. On SDS/polyacrylamidegel electrophoresis recombinant protein S had a slightly higher molecular mass than plasma protein S. After treatment with endoglycosidase F, the proteins comigrated suggesting the observed molecular mass difference to be due to alterations in the N-linked carbohydrate side chains. Recombinant and plasma protein S demonstrated identical amino-terminal sequences, similar amino acid composition and number of 4-carboxyglutamyl and 3-hydroxyaspartyl/asparaginyl residues. Recombinant protein S had the same affinity for Ca2+ as protein S from plasma and the two proteins had the same activated protein C cofactor activity in a functional assay. In addition, both forms of protein S formed complexes with C4b-binding protein with the same apparent Kd. Protein S is the most extensively post-translationally modified vitamin-K-dependent protein, and all the modifications were carried out in the recombinant DNA system yielding a recombinant protein S with full biological activity.Protein S is an anticoagulant vitamin-K-dependent glycoprotein ( M , 75000) functioning as a cofxtor to activated protein C in the degradation of the activated forms of coagulation factors V and VIII [I -81. In plasma, approximately 60% of protein S is non-covalently complexed with C4b-binding protein [9 -1 I], a regulatory protein in the classical complement pathway. Only the free form of protein S functions as an anticoagulant [12-141. The physiological importance of the anticoagulant activity of protein S is demonstrated by the high incidence of thromboembolic diseases in patients with hereditary heterozygous deficiency of protein S [12, cDNA clones for bovine and human protein S have recently been isolated [22 -251, and the gene(s) for human protein S localized to chromosome 3 [25, 261. Human protein S contains 635 amino acid residues in a single chain (Fig. 1). The amino acid sequence demonstrates that protein S is not a serine protease. The N-terminal region contains 11 vitamin-K-dependent 4-carboxyglutamyl residues (Gla). This part of protein S contains several Gla-dependent calcium-binding sites [27]; the protein also has two or three Gla-independent 15-21].Correspondence to J. Malm, Department of Clinical Chemistry, University o f Lund, Malmo General Hospital, S-214 01 Malmo, SwedenAhhreviutions. rPS, recombinant protein S; pPS, plasma protein S; Gla, 4-carboxyglutamic acid; EGF, epidermal growth factor;...

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Experimental Procedures Proteinsmentioning

confidence: 99%

See 1 more Smart Citation

Expression of completely γ‐carboxylated and β‐hydroxylated recombinant human vitamin‐K‐dependent protein S with full biological activity

Malm¹,

Cohen²,

Dackowski³

et al. 1990

European Journal of Biochemistry

View full text Add to dashboard Cite

show abstract

“…[3][4][5] Another determinant is FV, which acts in synergy with PS in FVIIIa inactivation by APC in purified systems. 6 PS also demonstrates APC-independent anticoagulant activity by inhibiting prothrombinase and tenase activities, 7,8 and this may contribute to the overall anticoagulant activity of PS in plasma.PS is a single-chain, 635-amino acid glycoprotein composed of an N-terminal domain rich in ␥-carboxyglutamic acid (Gla domain; amino acids 1-45), a thrombin-sensitive region (TSR; amino acids 46-74), 4 epidermal growth factor (EGF)-like domains (amino acids 75-242), 9 and a large C-terminal sex hormone-binding globulin (SHBG)-like region (amino acids 243-635). 10 The SHBGlike domain of PS binds tightly to C4b-binding protein (C4BP), a regulatory component of the complement system.…”

mentioning

confidence: 99%

The γ-carboxyglutamic acid domain of anticoagulant protein S is involved in activated protein C cofactor activity, independently of phospholipid binding

Saller

Villoutreix

Amelot

et al. 2005

Blood

View full text Add to dashboard Cite

We expressed 2 chimeras between human protein S (PS) and human prothrombin (FII) in which the prothrombin ␥-carboxyglutamic acid (Gla) domain replaced the PS Gla domain in native PS (Gla FII -PS) or in PS deleted of the thrombinsensitive region (TSR) (Gla FII -⌬TSR-PS). Neither PS/FII chimera had activated protein C (APC) cofactor activity in plasma clotting assays or purified systems, but both bound efficiently to phospholipids. This pointed to a direct involvement of the PS Gla domain in APC cofactor activity through molecular interaction with APC. Using computational methods, we identified 2 opposite faces of solventexposed residues on the PS Gla domain (designated faces 1 and 2) as potentially involved in this interaction. Their importance was supported by functional characterization of a PS mutant in which the face 1 and face 2 PS residues were reintroduced into Gla FII -PS, leading to significant APC cofactor activity, likely through restored interaction with APC. Furthermore, by characterizing PS mutants in which PS face 1 and PS face 2 were individually replaced by the corresponding prothrombin faces, we found that face 1 was necessary for efficient phospholipid binding but that face 2 residues were not strictly required for phospholipid binding and were involved in the interaction with APC. IntroductionProtein S (PS) is a vitamin K-dependent protein involved in regulating the anticoagulant activity of activated protein C (APC). In purified systems, PS functions as a nonenzymatic cofactor for APC in proteolytic inactivation of activated factor V (FVa) and activated factor VIII (FVIIIa), 1 reflecting the ability of PS to interact with APC. However, these effects are relatively weak and are inconsistent with the major physiologic role of PS. Indeed, the importance of PS as a natural anticoagulant is clearly demonstrated by the occurrence of severe thrombotic complications in infants with homozygous hereditary PS deficiency and by a mild thrombotic tendency in heterozygous subjects. 2 This suggests that additional components may determine the expression of the APC cofactor activity of PS in plasma. Thus, activated factor X (FXa) and activated factor IX (FIXa) have been shown to protect FVa and FVIIIa from inactivation by APC, and, in this system, PS is able to abolish these protective effects. [3][4][5] Another determinant is FV, which acts in synergy with PS in FVIIIa inactivation by APC in purified systems. 6 PS also demonstrates APC-independent anticoagulant activity by inhibiting prothrombinase and tenase activities, 7,8 and this may contribute to the overall anticoagulant activity of PS in plasma.PS is a single-chain, 635-amino acid glycoprotein composed of an N-terminal domain rich in ␥-carboxyglutamic acid (Gla domain; amino acids 1-45), a thrombin-sensitive region (TSR; amino acids 46-74), 4 epidermal growth factor (EGF)-like domains (amino acids 75-242), 9 and a large C-terminal sex hormone-binding globulin (SHBG)-like region (amino acids 243-635). 10 The SHBGlike domain of PS binds tightly to C4b...

show abstract

“…9 It comprises an N-terminal g-carboxylated glutamic acid (Gla) domain, a thrombin-sensitive region (TSR), 4 epidermal growth factor-like (EGF) domains and a C-terminal sex hormone-binding globulin (SHBG)-like domain. 18 The SHBG-like domain is composed of 2 laminin G-type subunits, LG1 and LG2. 19,20 Protein S circulates in plasma at concentrations of 20 to 25 mg/mL, with approximately 60% bound to C4b-binding protein (C4BP).…”

Section: Introductionmentioning

confidence: 99%

TFPI cofactor function of protein S: essential role of the protein S SHBG-like domain

Reglińska-Matveyev

Andersson

Rezende

et al. 2014

Blood

View full text Add to dashboard Cite

Key Points• The protein S SHBG-like domain and, more specifically, its LG1 subunit are important for binding and enhancement of TFPI.• TFPI binding to the protein S SHBG-like domain likely positions TFPI Kunitz domain 2 for optimal interaction with the active site of FXa.Protein S is a cofactor for tissue factor pathway inhibitor (TFPI), accelerating the inhibition of activated factor X (FXa). TFPI Kunitz domain 3 residue Glu226 is essential for enhancement of TFPI by protein S. To investigate the complementary functional interaction site on protein S, we screened 44 protein S point, composite or domain swap variants spanning the whole protein S molecule for their TFPI cofactor function using a thrombin generation assay. Of these variants, two protein S/growth arrestspecific 6 chimeras, with either the whole sex hormone-binding globulin (SHBG)-like domain (Val243-Ser635; chimera III) or the SHBG laminin G-type 1 subunit (Ser283-Val459; chimera I), respectively, substituted by the corresponding domain in growth arrest-specific 6, were unable to enhance TFPI. The importance of the protein S SHBG-like domain (and its laminin G-type 1 subunit) for binding and enhancement of TFPI was confirmed in FXa inhibition assays and using surface plasmon resonance. In addition, protein S bound to C4b binding protein showed greatly reduced enhancement of TFPI-mediated inhibition of FXa compared with free protein S. We show that binding of TFPI to the protein S SHBG-like domain enables TFPI to interact optimally with FXa on a phospholipid membrane. (Blood. 2014;123(25):3979-3987) IntroductionTissue factor pathway inhibitor (TFPI) is a ;41 kDa Kunitz-type protease inhibitor that consists of an acidic amino-terminal polypeptide, followed by 3 tandem Kunitz-type domains (Kunitz domains 1, 2, and 3) and a basic carboxy (C)-terminal tail. 1 It circulates in plasma at a concentration of ;1.6 nM. 2,3 The majority (;80%) of plasma TFPI is truncated and bound to lipoproteins, ;5% is localized in storage granules within platelets, ;5% circulates as free truncated variants, and only around 10% is considered to be free full-length TFPI, 4 with this having the greatest anticoagulant activity. [5][6][7] TFPI and its cofactor protein S downregulate tissue factor (TF)-induced thrombin generation, and a deficiency of either protein has been linked to an increased risk of venous thrombosis. [8][9][10] TFPI specifically inhibits the initiation of coagulation through direct binding and inhibition of factor Xa (FXa) and, in a FXa-dependent manner, inhibition of the TF/factor VIIa (FVIIa) complex by forming a quarternary TF/FVIIa/FXa/TFPI complex. 11,12 The P1 residue in the Kunitz domain 2 of TFPI is required for binding to FXa, whereas the P1 residue in Kunitz domain 1 is required for binding and inhibition of TF/FVIIa. 13 The kinetic mechanism of FXa inhibition by TFPI is described as a 2-step process where TFPI first forms an immediate encounter complex with FXa (FXa/TFPI), followed by a slow isomerization into a final tight complex (FXa/TFPI*...

show abstract

Isolation and sequence of the cDNA for human protein S, a regulator of blood coagulation.

Cited by 195 publications

References 34 publications

Expression of completely γ‐carboxylated and β‐hydroxylated recombinant human vitamin‐K‐dependent protein S with full biological activity

Expression of completely γ‐carboxylated and β‐hydroxylated recombinant human vitamin‐K‐dependent protein S with full biological activity

The γ-carboxyglutamic acid domain of anticoagulant protein S is involved in activated protein C cofactor activity, independently of phospholipid binding

TFPI cofactor function of protein S: essential role of the protein S SHBG-like domain

Contact Info

Product

Resources

About