The association of bovine prothrombin fragment 1 with phospholipid. Quantitative characterization of the Ca2+ ion-mediated binding of prothrombin fragment 1 to phospholipid vesicles and a molecular model for its association with phospholipids.

Dombrose, Frederick A.; Gitel, Sanford N.; Zawalich, Kathleen C.; Jackson, CM

doi:10.1016/s0021-9258(18)50556-1

Cited by 90 publications

(71 citation statements)

References 59 publications

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“…lb and 2). It has been generally recognized that Ca2 + mediates the interaction of vitamin Kdependent proteins with membranes that contain negatively charged phospholipids (Stenflo & Suttie, 1977;Nelsestuen et al, 1978;Dombrose et al, 1979). In the preceding paper (Mertens et al, 1984), we have determined the binding parameters for the Ca2 +-mediated interaction of human Factor IXa and Factor X with PS/PC menmbranes.…”

Section: Discussionmentioning

confidence: 99%

The contribution of Ca2+ and phospholipids to the activation of human blood-coagulation Factor X by activated Factor IX

Mertens¹,

Bertina²

1984

Biochemical Journal

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The role of the cofactors Ca2+ and phospholipid in the activation of human Factor X by Factor IXa was investigated. By use of a sensitive spectrophotometric Factor Xa assay, it was demonstrated that human Factor IXa can activate Factor X in the absence of cofactors. The presence of Ca2+ as the only cofactor resulted in a 7-fold stimulation of the Factor Xa formation. Kinetic analysis of the Ca2+-stimulated reaction showed that the apparent Km of Factor X was 4.6 microM, whereas the apparent Vmax. for Factor Xa formation was 0.0088 mol of Xa/min per mol of IXa. The presence of phospholipid as the only cofactor had no effect on the rate of Factor Xa formation. However, a several-hundred-fold stimulation was observed when Ca2+ and phospholipid were present in combination. The activation of Factor X in the presence of Ca2+ and phospholipid was found to be kinetically heterogeneous, involving both phospholipid-bound and free reactants. Quantitative data concerning the phospholipid binding of Factors IXa and X were used to study the relation between the rate of Factor Xa formation and the binding of enzyme and substrate to the phospholipid membrane. The results support the hypothesis that phospholipid-bound Factor X is the substrate in the phospholipid-stimulated reaction; however, phospholipid-bound and free Factor IXa seem to be equally efficient in catalysing the activation of phospholipid-bound Factor X.

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Section: Discussionmentioning

confidence: 99%

The contribution of Ca2+ and phospholipids to the activation of human blood-coagulation Factor X by activated Factor IX

Mertens¹,

Bertina²

1984

Biochemical Journal

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“…Thus, we determined from densitometry that MzIIa des F1 constituted about 30% of the total protein in our meizothrombin preparation (see Results). Since the labeled MzIIa des F1 no longer contained the membrane-binding domain (Dombrose et al, 1979), we assumed that it did not contribute to the FRET that accompanies binding of protein to membranes. Thus, we subtracted 30% of the total fluorescence from both Q D and Q DA (after correcting Q DA for nonspecific FRET) to obtain data sets to analyze in terms of our binding analysis.…”

Section: Methodsmentioning

confidence: 99%

Fluorescence Resonance Energy Transfer Study of Shape Changes in Membrane-Bound Bovine Prothrombin and Meizothrombin

Qing

Lentz

1997

Biochemistry

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Prothrombin activation to thrombin is a key control reaction in blood coagulation. During the process, prothrombin is sequentially cleaved at two peptide bonds (Arg323-Ile and Arg274-Thr) by factor X(a) to generate meizothrombin and then thrombin. Phosphatidylserine (PS)-containing membranes from platelets are believed to facilitate this two-step process. Using fluorescence energy transfer (FRET), we determined the distances of closest approach between a specifically located C-terminal fluorescein of a double mutant bovine prothrombin (P(S528A, G581C)-FM) or meizothrombin (M(S528A, G581C)-FM) and phosphatidylethanolamine-N-rhodamine B (PE-Rh; 0-8.7 mol %) contained in membranes composed of PS (25 mol %) and phosphatidylcholine (66.3-75 mol %). Plots of the energy transfer efficiency as a function of membrane concentration, at six PE-Rh surface densities, were analyzed globally to obtain dissociation constants and binding stoichiometries as global parameters and saturating energy transfer efficiencies characteristic of each surface density. From the global analysis, the dissociation constants were estimated to be 0.32 +/- 0.10 and 0.28 +/- 0.12 microM with stoichiometries of 42 +/- 12 and 44 +/- 9 lipid/protein for prothrombin and meizothrombin, respectively. The distance of closest approach was obtained from the dependence of the saturating energy transfer efficiency on the acceptor (PE-Rh) surface density. With the assumptions of kappa2 = 2/3 and n = 1.4, the distances were 94 +/- 3 A for prothrombin and 114 +/- 2 A for meizothrombin. Since both prothrombin and meizothrombin behave in solution as oblate ellipsoids of revolution with a long axis of 120 A, our FRET measurements suggest that binding to PS-containing membranes induced tighter folding of the prothrombin molecule but not of the meizothrombin intermediate. This observation is consistent with our hypothesis that membrane binding plays an essential role in the sequential alignment of the bond Arg323-Ile in prothrombin and Arg274-Thr in meizothrombin with the active site of the membrane-bound prothrombinase in the two-step thrombin-generating process.

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“…While a recent study examined both rate constants directly, k dissn was measured by displacement by another protein (Kung et al, 1994), a technique that does not result in variation of protein density on the membrane. Slight heterogeneity of equilibrium binding to SUV has been reported as prothrombin reached high packing density on the membrane surface (Wei et al, 1982;Dombrose et al, 1979). Other proteins show extensive heterogeneity as a function of protein packing density on the membrane (Abbott & Nelsestuen, 1987;Bazzi & Nelsestuen, 1991b).…”

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confidence: 99%

Dynamic Features of Prothrombin Interaction with Phospholipid Vesicles of Different Size and Composition: Implications for Protein−Membrane Contact

Nelsestuen

1996

Biochemistry

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The dynamics of prothrombin interaction with membrane vesicles of different size and composition was investigated to ascertain the impact of membrane surface characteristics and particle size on this interaction. Dissociation rates were highly sensitive to membrane composition and varied from about 20/s for membranes of 10% PS to 0.1/s for membranes of 50% PS. Overall affinity also varied by more than two orders of magnitude. Very small differences between prothrombin binding to SUV versus LUV were found. Association with large unilamellar vesicles (LUV of 115 nm diameter) was about 4-fold slower, when expressed on the basis of binding sites, than association with small unilamellar vesicles (SUV, 30 nm diameter) of the same composition. Both reactions proceeded at less than 25% of the collisional limit so that the differences were largely due to intrinsic binding properties. Vesicles of 325 nm diameter showed even slower association velocities. Dissociation rates from LUV were about 2-fold slower than from SUV. Again, these differences arose primarily from intrinsic binding properties. Dissociation conformed to a single first order reaction over a wide range of protein occupancy on the membrane. At very high packing density, the dissociation rate increased by about 2-fold. At equilibrium, prothrombin preferred binding to SUV over LUV by about 2-fold. This very small difference, despite substantial differences in phospholipid headgroup packing and hydrocarbon exposure, appeared inconsistent with an important role for protein insertion into the hydrocarbon region of the membrane. However, prothrombin-membrane interaction may arise from a series of interaction forces that have compensating features at equilibrium. The small differences in prothrombin binding to SUV versus LUV, together with differences in the number of protein binding sites per vesicle, were important to identify mechanisms of substrate delivery to the active site of the prothrombinase enzyme [Lu, Y., & Nelsestuen, G. L. (1996) Biochemistry 35, 8201-8209].

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The association of bovine prothrombin fragment 1 with phospholipid. Quantitative characterization of the Ca2+ ion-mediated binding of prothrombin fragment 1 to phospholipid vesicles and a molecular model for its association with phospholipids.

Cited by 90 publications

References 59 publications

The contribution of Ca2+ and phospholipids to the activation of human blood-coagulation Factor X by activated Factor IX

The contribution of Ca2+ and phospholipids to the activation of human blood-coagulation Factor X by activated Factor IX

Fluorescence Resonance Energy Transfer Study of Shape Changes in Membrane-Bound Bovine Prothrombin and Meizothrombin

Dynamic Features of Prothrombin Interaction with Phospholipid Vesicles of Different Size and Composition: Implications for Protein−Membrane Contact

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