Cooperative calcium binding by the phospholipid binding region of bovine prothrombin: A requirement for intact disulfide bridges

Henriksen, Ruth Ann; Jackson, Craig M.

doi:10.1016/0003-9861(75)90106-x

Cited by 97 publications

(24 citation statements)

References 36 publications

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“…43,44 The modification of glutamic acid residues endows these proteins with the property of binding to negatively charged phospholipid membranes at physiological calcium concentration. 45 The calcium binding is characterized by an initial positive cooperativity 46 and induces two conformational transitions in the molecule. 47 The first of these is cation nonselective, whereas the second transition, necessary for membrane binding, is specifically dependent on calcium ions.…”

Section: Discussionmentioning

confidence: 99%

Association of Vitamin K–Dependent Coagulation Proteins and C4b Binding Protein With Triglyceride-Rich Lipoproteins of Human Plasma

Dahlbäck

Öhlin

et al. 1998

ATVB

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Abstract-The triglyceride (TG) concentration in plasma is an independent risk factor for coronary heart disease. There is evidence that TG-rich lipoprotein (TGRLP), ie, chylomicrons (CMs), chylomicron remnants (CMRs), and VLDLs associate with factor VII and prothrombin and that the association enhances a platelet factor Xa-mediated prothrombin activation when the CM-prothrombin complex is exposed to platelets. In this study, we examined the association of the vitamin K-dependent coagulation factors VII, IX, X, and prothrombin, as well as the anticoagulation protein C and its cofactor protein S, in plasma lipoproteins obtained from human fasting and postprandial plasma. We also analyzed some other proteins that are related to the coagulation system but not to vitamin K-dependent proteins, including factor V, serum amyloid P component (SAP), C4b binding protein (C4BP), and thrombomodulin (TM), and as a control, Ig G. Human TGRLP (dϽ1.006 kg/L), LDL (dϭ1.006 to 1.063 kg/L), and HDL (dϭ1.063 to 1.210 kg/L) were separated from normal subjects both in fasting and 2 to 3 hours after the ingestion of a meal containing 100 g fat. The different coagulation proteins, SAP, C4BP, TM, and Ig G were determined by SDS-polyacrylamide gel electrophoresis combined with Western blotting, using specific polyclonal or monoclonal antibodies, and were visualized by peroxidase staining. All the vitamin K-dependent proteins associate with TGRLP in both fasting and postprandial plasma, but not with LDL or HDL. Factor V, SAP, TM, and Ig G were not found in any lipoprotein classes. C4BP, which is a regulatory protein of the classic pathway of the complement system and which binds protein S in vivo to regulate blood coagulation, was present in TGRLP, especially postprandial, but not in LDL or HDL. The amounts of prothrombin, protein S, and C4BP in postprandial TGRLP were larger than those in fasting TGRLP. Vitamin K-dependent procoagulation and anticoagulation proteins, as well as C4BP, could be associated with TGRLP in vivo.

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Section: Discussionmentioning

confidence: 99%

Association of Vitamin K–Dependent Coagulation Proteins and C4b Binding Protein With Triglyceride-Rich Lipoproteins of Human Plasma

Dahlbäck

Öhlin

et al. 1998

ATVB

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“…Ca 2ϩ binding is also required for PS regulation of factor X a proteolytic activity (1). Ca 2ϩ binds mainly to the Gla module (13), but there also appears to be a high affinity Ca 2ϩ binding site (k d ϳ 160 M) in the catalytic domain (9, 14 -16) and a lower affinity Ca 2ϩ binding site (k d ϳ 0.7-1.2 m M) on the isolated first EGF-like module (4,12,16,17). A Ca 2ϩ -dependent interaction between the EGF-like and Gla modules appears to enhance the affinity of the site on the EGF-like module to the point that it is tighter (17, 18) (k d ϳ 120 M) than the catalytic domain site.…”

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confidence: 99%

Localization of Phosphatidylserine Binding Sites to Structural Domains of Factor Xa

Srivastava¹,

Wang²,

Majumder³

et al. 2002

Journal of Biological Chemistry

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Binding of short chain phosphatidylserine (C6PS) enhances the proteolytic activity of factor X a by 60-fold (Koppaka, V., Wang, J., Banerjee, M., and Lentz, B. R. (1996) Biochemistry 35, 7482-7491). In the present study, we locate three C6PS binding sites to different domains of factor X a using a combination of activity, circular dichroism, fluorescence, and equilibrium dialysis measurements on proteolytic and biosynthetic fragments of factor X a . Our results demonstrate that the structural responses of human and bovine factor X a to C6PS binding are somewhat different. Despite this difference, data obtained with fragments from both human and bovine factor X a are consistent with a common hypothesis for the location of C6PS binding sites to different structural domains. First, the ␥-carboxyglutamic acid (Gla) domain binds C6PS only in the absence of Ca 2؉ (k d ϳ 1 mM), although this PS site does not influence the functional response of factor X a . Second, a Ca 2؉ -dependent binding site is in the epidermal growth factor domains (EGF NC ) that are linked by Ca 2؉ and C6PS binding to the Gla domain. This site appears to be the lipid regulatory site of factor X a . Third, a Ca 2؉ -requiring site seems to be in the EGF C -catalytic domain. This site appears not to be a lipid regulatory site but rather to share residues with the substrate recognition site. Finally, the full functional response to C6PS requires linkage of the Gla, EGF NC , and catalytic domains in the presence of Ca 2؉ , meaning that PS regulation of factor X a involves linkage between widely separated parts of the protein.The substantial effects of soluble phosphatidylserine (C6PS 1 ) on the kinetics of prothrombin activation by factor X a(1) and on the structure of factor X a , as documented here, indicate that phosphatidylserine (PS) may act as an allosteric regulator of prothrombin activation. PS located on the cytoplasmic face of resting platelet plasma membranes is exposed on the surface of activated platelet vesicles (2, 3). The implication of this PS exposure and of the effect of PS on factor X a and on its ability to catalyze activation of prothrombin is that PS may act as a second messenger in regulating thrombin formation. Because of the crucial role of thrombin in hemostasis, the exposure of PS may be a crucial regulatory step in blood coagulation. To better define this regulatory process, it is important to know the locations of the PS binding sites on factor X a . The organization of factor X into structural domains is illustrated below in Fig. 1. Factor X consists of two peptides. The light chain consists of an N terminus ␥-carboxyglutamic acidrich region (Gla module) and two Cys-rich cassette modules. The heavy chain consists of the serine protease catalytic domain. The two cassette modules of the light chain show strong sequence and structural homology to epidermal growth factor (EGF) (4) and are thus referred to as EGF N and EGF C , where N and C indicate the domain nearer to the N and C termini, respectively. Crystal structures o...

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“…Nelsestuen (1976 and Prendergast & Mann (1977) have observed a relationship, at neutral pH, between Ca2+-induced fluorescence quenching in prothrombin fragment 1 and the ability of fragment 1 to associate with phospholipid vesicles. Perhaps related to the lipidbinding processes discussed above and co-operativity noted for Ca2+ binding to fragment 1 (Henriksen & Jackson, 1975;Bajaj et al, 1975) is the observed intrinsic and Ca2+-mediated association offragment-I molecules (Prendergast & Mann, 1977;Agarwal et al, 1977). Thus, whether it is itself sufficient to initiate phospholipid binding or not, a conformational change in the protein, monitored by fluorescence quenching in fragment 1, has been implicated in Ca2+-mediated phospholipid binding in this system.…”

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confidence: 99%

“…The presence of Ca2+ and phospholipid, along with Factor Va, are necessary for the optimal rate of conversion of prothrombin into thrombin by Factor Xa in the blood-coagulation process (Papahadjopoulos & Hanahan, 1964;Cole et al, 1965;Gitel et al, 1973;Henriksen & Jackson, 1975;Nelsestuen, 1976). At neutral pH, the formation of a functional prothrombin-membrane complex depends on the presence of y-carboxyglutamic acid residues near the prothrombin N-terminus, and to some extent on intact protein tertiary structure.…”

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The effects of calcium ions and pH on bovine prothrombin fragment 1. Intrinsic fluroescence studies

Scott¹,

Koehler²,

Hiskey³

1979

Biochemical Journal

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The effects of pH and Ca2+ on the intrinsic fluorescence of bovine prothrombin fragment 1 were investigated to deduce the nature of protein functional groups involved in Ca2+ binding to fragment 1. From pH values of 9 to 3, increasing the H30+ concentration results in quenching of the fluorescence of fragment 1. Reversible pH-titration curves are obtained which appear to consist of two regions. From pH4 to pH6.5 a broad titration curve is obtained, whereas from pH6.5 to 9 a more pronounced titration behaviour is evidenced by a group or groups on fragment 1 with an apparent pKa of approx. 7.5. In contrast, the apparent association constant for Ca2+ and fragment 1 shows a sharp pHdependence in the region between pH7 and 8 with tighter Ca2+ binding at higher pH values. A pKa of approx. 7.5 can be estimated for the group or groups on fragment 1 linked to the tight binding of Ca2+. Both H30+ and Ca2+ result in blue-shifts in the wavelengths of fragment-I emission. These results are interpreted in terms of H+-and Ca2+-induced changes in the conformation of fragment 1 as a result of surface-charge neutralization.

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Cooperative calcium binding by the phospholipid binding region of bovine prothrombin: A requirement for intact disulfide bridges

Cited by 97 publications

References 36 publications

Association of Vitamin K–Dependent Coagulation Proteins and C4b Binding Protein With Triglyceride-Rich Lipoproteins of Human Plasma

Association of Vitamin K–Dependent Coagulation Proteins and C4b Binding Protein With Triglyceride-Rich Lipoproteins of Human Plasma

Localization of Phosphatidylserine Binding Sites to Structural Domains of Factor Xa

The effects of calcium ions and pH on bovine prothrombin fragment 1. Intrinsic fluroescence studies

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