Comparison of the abilities of synthetic and platelet-derived membranes to enhance thrombin formation

Jones, Marcie E.; Lentz, Barry R.; Dombrose, Frederick A.; Sandberg, Helena

doi:10.1016/0049-3848(85)90255-5

Cited by 50 publications

(42 citation statements)

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“…It is known that the concentration of negatively charged phosphate groups on a surface will influence the degree of protein binding and the reaction rate. 50 Because lipoprotein particles consist of Ϸ5% negatively charged PLs, 51 whereas PCPS vesicles as we prepared them are Ϸ25% negatively charged PLs, this is a possible explanation for the difference between the rates on PCPS vesicles and the rates on lipoproteins. However, because the VLDL-dependent rates could be dramatically increased to approximately the same values as the PCPS-dependent rates by extracting the lipids, this suggests that rate differences cannot be explained simply by the different percentages of negatively charged PLs and that lipoprotein conformation has a major influence on prothrombinase rates.…”

Section: Discussionmentioning

confidence: 99%

Plasma Lipoproteins Support Prothrombinase and Other Procoagulant Enzymatic Complexes

Moyer

Tracy

et al. 1998

ATVB

105

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Abstract-The prothrombinase complex (factor [F]Xa, FVa, calcium ions, and lipid membrane) converts prothrombin to thrombin (FIIa). To determine whether plasma lipoproteins could provide a physiologically relevant surface, we determined the rates of FIIa production by using purified human coagulation factors, and isolated fasting plasma lipoproteins from healthy donors. In the presence of 5 nmol/L FVa, 5 nmol/L FXa, and 1.4 mol/L prothrombin, physiological levels of very low density lipoprotein (VLDL) (0.45 to 0.9 mmol/L triglyceride, or 100 to 200 mol/L phospholipid) yielded rates of 2 to 8 nmol FIIa ⅐ L Ϫ1 ⅐ s Ϫ1 in a donor-dependent manner. Low density lipoprotein (LDL) and high density lipoprotein (HDL) also supported prothrombinase but at much lower rates (Յ1.0 nmol FIIa ⅐ L Ϫ1 ⅐ s Ϫ1 ). For comparison, VLDL at 2 mmol/L triglyceride yielded Ϸ50% the activity of 2ϫ10 8 thrombin-activated platelets per milliliter. Although the FIIa production rate was slower on VLDL than on synthetic phosphatidylcholine/phosphatidylserine vesicles (Ϸ50 nmol FIIa ⅐ L Ϫ1 ⅐ s Ϫ1 ), the prothrombin K m values were similar, 0.8 and 0.5 mol/L, respectively. Extracted VLDL lipids supported rates approaching those of phosphatidylcholine/phosphatidylserine vesicles, indicating the importance of the intact VLDL conformation. However, the presence of VLDL-associated, factor-specific inhibitors was ruled out by titration experiments, suggesting a key role for lipid organization. VLDL also supported FIIa generation in an assay system comprising 0.1 nmol/L FVIIa; 0.55 nmol/L tissue factor; physiological levels of FV, FVIII, FIX, and FX; and prothrombin (3 These results indicate that isolated human VLDL can support all the components of the extrinsic coagulation pathway, yielding physiologically relevant rates of thrombin generation in a donor-dependent manner. This support is dependent on the intact lipoprotein structure and does not appear to be regulated by specific VLDL-associated inhibitors. Further studies are needed to determine the extent of this activity in vivo.

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Section: Discussionmentioning

confidence: 99%

Plasma Lipoproteins Support Prothrombinase and Other Procoagulant Enzymatic Complexes

Moyer

Tracy

et al. 1998

ATVB

105

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“…A natural variant of human factor IX with mutation of Phe 8 to a similar hydrophobic residue Leu (F8L) has been shown to have a small effect on the binding of factor IX to endothelial cells (Mayhew et ai., 1994). The electrostatic contributions from the interactions between the positively charged groups in the Gla domain of vitamin K-dependent proteins and the negatively charged groups on the PL membrane have been demonstrated (Higgins et al, 1985;Jones et al, 1985;Gerads et al, 1990). The recent discovery (Manfioletti et al, 1993) that the protein product (Gad) of the growth-arrest-specific gene gas6 is vitamin K-dependent and is homologous to protein S led us to compare its N-terminus to that of other Gla domain proteins (Fig.…”

Section: By Defining Two Centroids For Each Of the Side Chains Of Phementioning

confidence: 99%

Homology modeling and molecular dynamics simulation of human prothrombin fragment 1

Darden

Foley

et al. 1995

Protein Science

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The crystallographic structure of bovine prothrombin fragment 1 bound with calcium ions was used to construct the corresponding human prothrombin structure (hfl/Ca). The model structure was refined by molecular dynamics to estimate the average solution structure. Accommodation of long-range ionic forces was essential to reach a stable solution structure. The y-carboxyglutamic acid (Gla) domain and the kringle domain of hfl/Ca independently equilibrated. Likewise, the hydrogen bond network and the calcium ion coordinations were well preserved. A discussion of the phospholipid binding of the vitamin K-dependent coagulation proteins in the context of the structure and mutational data of the Gla domain is presented.Keywords: Gla domain; kringle domain; phospholipid binding; prothrombin; vitamin K-dependent proteins Prothrombin is a vitamin K-dependent coagulation protein that consists of a y-carboxyglutamic acid (G1a)-rich domain, two kringle domains, and a catalytic serine protease domain. Upon activation by prothrombinase complex, a membrane-bound complex of factor Xa and factor Va converts prothrombin to the active serine protease a-thrombin. The prothrombin structure can be divided into three components designated fragments 1 and 2 and prethrombin 2 . Fragment 1 is defined as the Gla domain (1-33, human numbering), the connecting region (34-a), and the first kringle domain (65-144) of prothrombin. Like other vitamin K-dependent proteins, the Gla domain of prothrombin is rich in y-carboxyglutamic acids (10 Gla residues). It is widely accepted that these y-carboxyglutamic acids are essential in calcium-mediated phospholipid binding, in maintaining the integrity of the Gla domain, and in calcium-dependent activities of these proteins (Borowski et al., 1985;Astermark et al., 1992;Kotkow et al., 1993;Colpitts & Castellino, 1994). Using site-specific mutagenesis techniques, it was proposed that while some y-carboxyglutamic acids are important for maintaining the basic structure of the Gla domain, others are involved in membrane binding and interdomain interaction (Ratcliffe et al., 1993). A similar conclusion was reached in a mutagenesis study of the Gla domain of human protein C . Because the Gla domain may be involved in several different interactions, the specific role(s) of the individual Gla residues remain unclear. A reliable model for the solution structure would clearly be useful.The crystal structure of bovine prothrombin fragment 1 bound with calcium ions (bfl/Ca) has been determined to 2.2 A resolution (Soriano-Garcia et al., 1992). The human and bovine proteins differ distinctly in the number of calcium ions bound (Deerfield et al., 1987) and in details of their Ca2+-induced fluorescence quench (Prendergast & Mann, 1977;Marsh et al., 1979). The crystal structure of bfl/Ca was used by our lab to develop a simulation model for the solution structure. The special challenge to model the highly ionic Gla domain was solved by establishing a careful simulation preparation protocol and by accommodating lo...

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“…Although the fatty acyl chains of membrane lipids have long been regarded as having little or no effect on the catalytic activity of coagulation factor complexes (9), more recent studies have reported that unsaturated acyl chains do increase the intrinsic k cat for PTase, compared with saturated acyl chains (10). This difference was evident even after accounting for changes in substrate transport due to altered lipid "fluidity" and was confirmed using the soluble substrate, prethrombin-1.…”

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confidence: 99%

Prothrombinase Acceleration by Oxidatively Damaged Phospholipids

Weinstein¹,

Li²,

Lawson³

et al. 2000

Journal of Biological Chemistry

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The optimally efficient production of thrombin by the prothrombinase complex relies on suitable positioning of its component factors and substrate on phosphatidylserine-containing lipid membranes. The presence of oxidatively damaged phospholipids in a membrane disrupts the normal architecture of a lipid bilayer and might therefore be expected to interfere with prothrombinase activity. To investigate this possibility, we prepared phosphatidylserine-containing lipid vesicles containing oxidized arachidonoyl lipids, and we examined their ability to accelerate thrombin production by prothrombinase. Oxidized arachidonoyl chains caused dosedependent increases in prothrombinase activity up to 6-fold greater than control values. These increases were completely attenuated by the presence of ␣-tocopherol, ␥-tocopherol, or ascorbate. Over the course of a 300-min oxidation, the ability of arachidonoyl lipids to accelerate prothrombinase peaked at 60 min and then declined to base-line levels. These results suggest that instead of being impeded by oxidative membrane damage, prothrombinase activity is enhanced by one or more products of nonenzymatic lipid oxidation.Thrombin production is controlled in vivo by a complex system of cascade and feedback mechanisms. By controlling thrombin production, these mechanisms regulate the various physiological activities of thrombin, including the maintenance of hemostasis, the signaling of smooth muscle and other cells, and the activation of platelets (1). Platelet plasma membranes are an especially important control point for thrombin production because upon activation they display anionic phosphatidylserine (PS) 1 lipids on their outer surface. PS-containing membranes accelerate thrombin production by facilitating the assembly of coagulation factors Va and Xa to form the prothrombinase complex (PTase), and the delivery of prothrombin to this complex for conversion into thrombin (2). To a large extent, PS lipids facilitate the assembly of PTase and the delivery of substrate by confining these factors to the membrane surface and reducing three-dimensional diffusional processes to two-dimensional processes. However, PS head groups can also accelerate PTase activity by functioning as a regulatory cofactor (3).There are compelling reasons to consider the possible effects of oxidative lipid damage on thrombin production. For instance, oxidation profoundly alters the architecture and chemical properties of phospholipid bilayers (4, 5), and the function of PTase is clearly sensitive to the physical state of the membrane (6). Many common cellular processes such as platelet activation release highly reactive oxygen species that can oxidize lipoproteins (7). Oxidation, in turn, dramatically increases the ability of lipoproteins to accelerate the activity of the PTase complex and produce more thrombin (8).The principal sites of both enzymatic and non-enzymatic oxidation in a membrane are the olefinic groups of unsaturated fatty acyl chains. Although the fatty acyl chains of membrane lipids have lon...

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Comparison of the abilities of synthetic and platelet-derived membranes to enhance thrombin formation

Cited by 50 publications

References 28 publications

Plasma Lipoproteins Support Prothrombinase and Other Procoagulant Enzymatic Complexes

Plasma Lipoproteins Support Prothrombinase and Other Procoagulant Enzymatic Complexes

Homology modeling and molecular dynamics simulation of human prothrombin fragment 1

Prothrombinase Acceleration by Oxidatively Damaged Phospholipids

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