Expression of tissue factor procoagulant activity: regulation by cytosolic calcium.

Intact bovine fibroblasts, pericytes, and kidney cells manifested significantly less tissue factor procoagulant activity than their disrupted counterparts. Addition of calcium ionophore A23187 rapidly and reversibly enhanced the cell-surface expression of tissue factor in intact cells up to the level achieved by disruption. Inhibitors of calmodulin blocked the ionophore-dependent enhancement of procoagulant activity. Similar kinetic parameters were obtained for factor X hydrolysis by tissue factor-factor VIIa on unperturbed pericytes and phosphatidylcholine vesicles. Increase in Vmax and decrease in apparent Km for this reaction were seen after either disruption or ionophore stimulation of the pericytes. Addition of phosphatidylserine to the reconstituted phospholipid vesicles also increased the Vmax and decreased the apparent Km for factor X hydrolysis. These data agree with the hypothesis that the expression of tissue factor procoagulant activity on cell surfaces is modulated by calcium-mediated changes in the asymmetric distribution of phosphatidylserine in plasma membrane.     

Evidence that plasma lipoproteins inhibit the factor VIIa-tissue factor complex by a different mechanism that extrinsic pathway inhibitor

Factor VIIa participates in blood clotting by activating factor X and/or factor IX by limited proteolysis. The proteolytic activity of factor VIIa is absolutely dependent on a lipoprotein cofactor designated tissue factor. We have examined the ability of purified preparations of human plasma high density, low density and very low density lipoproteins, as well as apolipoproteins A-I and A-II, to inhibit the factor VIIa-tissue factor mediated activation of either factor X or factor IX before and after treatment of the lipoprotein preparation with polyclonal antibody directed against partially- purified human plasma extrinsic pathway inhibitor (EPI). In the absence of anti-EPI IgG, HDL, LDL, VLDL, and apolipoprotein A-II noncompetitively inhibited factor X activation by factor VIIa-tissue factor with apparent Ki values of 3.39 mumol/L, 124 nmol/L, 33 nmol/L, and 10.5 mumol/L, respectively. Apolipoprotein A-I had no effect on this reaction. The inhibitory activity of HDL, LDL, VLDL, and apolipoprotein A-II in this reaction was unaffected by the presence of high levels of anti-EPI IgG. In the absence of exogenous factor Xa, none of the lipoproteins studied inhibited the activation of factor IX using the tritiated peptide release assay. In the presence of added factor Xa (1 nmol/L), LDL and VLDL, but not HDL and apolipoprotein A- II, inhibited the activation of factor IX by factor VIIa-tissue factor. This inhibition was completely blocked by prior incubation of the lipoprotein with anti-EPI IgG indicating association of EPI with these particles. Taken collectively, our data indicate that HDL, LDL, and VLDL, at or below their plasma concentration, each selectively inhibits the factor VIIa-tissue factor mediated activation of factor X by a mechanism that appears to be distinct from extrinsic pathway inhibitor. These lipoproteins may not only play a role in the regulation of extrinsic blood coagulation, but may also selectively promote the activation of factor IX by factor VIIa-tissue factor in vivo at low tissue factor concentrations.     

Cultured normal human hepatocytes do not synthesize lipoprotein-associated coagulation inhibitor: evidence that endothelium is the principal site of its synthesis.

Human plasma contains a factor Xa-dependent inhibitor of tissue factor/factor VIIa complex termed lipoprotein-associated coagulation inhibitor (LACI). The present study examines the site(s) of LACI synthesis. In this study, cultured hepatocytes isolated from normal human liver were found to be essentially negative in LACI mRNA as revealed by Northern blot analysis using a full-length LACI cDNA as probe. The conditioned media from these cultures were also essentially negative for LACI activity. Similarly, poly(A)+ RNA obtained from normal human liver did not contain detectable LACI mRNA. In contrast, cultured human umbilical vein endothelial cells and human lung tissue (rich in endothelium) both contained abundant amounts of LACI mRNA. Moreover, erythrocyte lysates and culture media from normal monocytes, lymphocytes, or neutrophils did not contain measurable LACI activity; these cells were also negative for LACI mRNA. Platelets, however, contained LACI activity. The likely source of platelet LACI is the megakaryocyte cell since a megakaryocyte cell line (MEG-01) was found to contain LACI mRNA and to secrete small amounts of LACI activity. Additionally, human vascular smooth muscle cells and lung fibroblasts were also found to synthesize only small amounts of LACI. From these observations, we conclude that normal liver does not synthesize LACI and that endothelium is the principal source of plasma LACI. The undegraded LACI synthesized by endothelial cells had a molecular weight of approximately 41,000.     

Apolipoprotein A-II,HDL metabolism and atherosclerosis

Apolipoprotein (Apo) A-I and apo A-II are the major apolipoproteins of HDL. It is clearly demonstrated that there are inverse relationships between HDL-cholesterol and apo A-I plasma levels and the risk of coronary heart disease (CHD) in the general population. On the other hand, it is still not clearly demonstrated whether apo A-II plasma levels are associated with CHD risk. A recent prospective epidemiological (PRIME) study suggests that Lp A-I (HDL containing apo A-I but not apo A-II) and Lp A-I:A-II (HDL containing apo A-I and apo A-II) were both reduced in survivors of myocardial infarction, suggesting that both particles are risk markers of CHD. Apo A-II and Lp A-I:A-II plasma levels should be rather related to apo A-II production rate than to apo A-II catabolism. Mice transgenic for both human apo A-I and apo A-II are less protected against atherosclerosis development than mice transgenic for human apo A-I only, but the results of the effects of trangenesis of human apo A-II (in the absence of a co-transgenesis of human apo A-I) are controversial. It is highly suggested that HDL reduce CHD risk by promoting the transfer of peripherical free cholesterol to the liver through the so-called 'reverse cholesterol transfer'. Apo A-II modulates different steps of HDL metabolism and therefore probably alters reverse cholesterol transport. Nevertheless, some effects of apo A-II on intermediate HDL metabolism might improve reverse cholesterol transport and might reduce atherosclerosis development while some other effects might be deleterious. In different in vitro models of cell cultures, Lp A-I:A-II induce either a lower or a similar cellular cholesterol efflux (the first step of reverse cholesterol transport) than Lp A-I. Results depend on numerous factors such as cultured cell types and experimental conditions. Furthermore, the effects of apo A-II on HDL metabolism, beyond cellular cholesterol efflux, are also complex and controversial: apo A-II may inhibit lecithin-cholesterol acyltransferase (LCAT) (potential deleterious effect) and cholesteryl-ester-transfer protein (CETP) (potential beneficial effect) activities, but may increase the hepatic lipase (HL) activity (potential beneficial effect). Apo A-II may also inhibit the hepatic cholesteryl uptake from HDL (potential deleterious effect) probably through the SR-BI depending pathway. Therefore, in terms of atherogenesis, apo A-II alters the intermediate HDL metabolism in opposing ways by increasing (LCAT, SR-BI) or decreasing (HL, CETP) the atherogenicity of lipid metabolism. Effects of apo A-II on atherogenesis are controversial in humans and in transgenic animals and probably depend on the complex effects of apo A-II on these different intermediate metabolic steps which are in weak equilibrium with each other and which can be modified by both endogenous and environmental factors. It can be suggested that apo A-II is not a strong determinant of lipid metabolism, but is rather a modulator of reverse cholesterol transport.     

Inhibition of the intrinsic factor X activating complex by protein S: evidence for a specific binding of protein S to factor VIII

Protein S is a vitamin K-dependent nonenzymatic anticoagulant protein that acts as a cofactor to activated protein C. Recently it was shown that protein S inhibits the prothrombinase reaction independent of activated protein C. In this study, we show that protein S can also inhibit the intrinsic factor X activation via a specific interaction with factor VIII. In the presence of endothelial cells, the intrinsic activation of factor X was inhibited by protein S with an IC50 value of 0.28 +/- 0.04 mumol/L corresponding to the plasma concentration of protein S. This inhibitory effect was even more pronounced when the intrinsic factor X activation was studied in the presence of activated platelets (IC50 = 0.15 +/- 0.02 mumol/L). When a nonlimiting concentration of phospholipid vesicles was used, the plasma concentration of protein S (300 nmol/L) inhibited the intrinsic factor X activation by 40%. Thrombin-cleaved protein S inhibited the endothelial cell-mediated factor X activation with an IC50 similar to that of native protein S (0.26 +/- 0.02 mumol/L). Protein S in complex with C4b-binding protein inhibited the endothelial cell-mediated factor X activation more potently than protein S alone (IC50 = 0.19 +/- 0.03 mumol/L). Using thrombin activated factor VIII, IC50 values of 0.53 +/- 0.09 mumol/L and 0.46 +/- 0.10 mumol/L were found for native protein S and thrombin-cleaved protein S, respectively. The possible interactions of protein S with factor IXa, phospholipids, and factor VIII were investigated. The enzymatic activity of factor IXa was not affected by protein S, and interaction of protein S with the phospholipid surface could not fully explain the inhibitory effect of protein S on the factor X activation. Using a solid-phase binding assay, we showed a specific, saturable, and reversible binding of protein S to factor VIII with a high affinity. The concentration of protein S where half-maximal binding was reached (B1/2max) was 0.41 +/- 0.06 mumol/L. A similar affinity was found for the interaction of thrombin-cleaved protein S with factor VIII (B1/2max = 0.40 +/- 0.04 mumol/L). The affinity of the complex protein S with C4B-binding protein appeared to be five times higher (B1/2max = 0.07 +/- 0.03 mumol/L). Because the affinities of the interaction of the different forms of protein S with factor VIII correspond to the IC50 values observed for the intrinsic factor X activating complex, the interaction of protein S with factor VIII may explain the inhibitory effect of protein S on the intrinsic factor X activating complex.(ABSTRACT TRUNCATED AT 400 WORDS)     

Utilization of a continuous flow reactor to study the lipoprotein-associated coagulation inhibitor (LACI) that inhibits tissue factor

A microperfusion system containing a glass capillary, the inner surface of which is coated with a phospholipid bilayer containing tissue factor, was used to explore the requirement for factors VIIa and Xa in the complex formed with the lipoprotein-associated coagulation inhibitor (LACI). Various combinations of factors VIIa, Xa, and LACI were perfused together or sequentially at a wall shear rate of 300 sec-1; a final perfusion of factors X and VIIa was performed to evaluate the residual tissue factor activity. Factor Xa concentration at the outlet of the tube was determined using a chromogenic substrate. In the presence of factors VIIa, Xa, and LACI, complete inhibition of tissue factor was observed on both phosphatidylcholine (neutral surfaces) and on phosphatidylserine/phosphatidylcholine (acidic) surfaces; omission of factors Xa or LACI resulted in no inhibition. The absence of factor VIIa in the initial perfusion steps resulted in no inhibition on neutral surfaces whereas about 90% inhibition was observed on acidic surfaces. Initial perfusion with factor Xa, but not LACI, followed by the remaining protein components, resulted in an inhibitory complex. Thus, it appears that a tissue factor:factor Xa:LACI complex can form in the absence of factor VIIa on acidic surfaces; moreover, our data imply a tissue factor binding site for factor Xa, but not for LACI.     

Studies on the biological activity of triiodothyronine sulfate.

Hepatic microsomes and isolated hepatocytes in short term culture desulfate T3 sulfate (T3SO4). We, therefore, wished to determine whether T3SO4 could mimic the action of thyroid hormone in vitro. T3SO4 had no thyromimetic effect on the activity of Ca(2+)-ATPase in human erythrocyte membranes at doses up to 10,000 times the maximally effective dose of T3 (10(-10) mol/L). In GH4C1 pituitary cells, T3SO4 failed to displace [125I]T3 from nuclear receptors in intact cells or soluble preparations. Thus, T3SO4 was not directly thyromimetic in either an isolated human membrane system or a pituitary cell system in which nuclear receptor occupancy correlates with GH synthesis. Thyroid hormones inhibit [3H]glycosaminoglycan synthesis by cultured human dermal fibroblasts, and T3SO4 displayed about 0.5% the activity of T3 at 72 h. Human fibroblasts contained roughly the same level of microsomal p-nitrophenyl sulfatase activity as that previously observed in hepatic microsomes. Propylthiouracil (50 mumol/L) did not affect the action of T3SO4, suggesting that deiodination was not important for this activity of T3SO4. Thus, it appears T3SO4 has no intrinsic biological activity, but, under certain circumstances, may be reactivated by desulfation.     

Isolation, purification, and reconstitution of the Na+ gradient-dependent Ca2+ transporter (Na+-Ca2+ exchanger) from brain synaptic plasma membranes.

A [Na+]-gradient-dependent Ca2+ transporter from brain synaptic plasma membranes has been isolated, purified, and reconstituted into brain phospholipid vesicles. The purification was achieved by sucrose-gradient centrifugation after solubilization of the synaptic membranes in cholate in the presence of a 30-fold excess (by weight) of added brain phospholipids and [Na+]-gradient-dependent Ca2+ loading of the reconstituted vesicles. A 128-fold increase in specific activity of [Na+]-gradient-dependent Ca2+ uptake per mg of protein has been obtained. The purified and reconstituted vesicles took up Ca2+ only in response to an outward-oriented [Na+] gradient. The Ca2+ uptake could be inhibited by dissipation of the [Na+] gradient with nigericin. Successful purification was based on the initial [Na+]-gradient dependency of the Ca2+-transport process, the magnitude of the [Na+]-gradient-dependent uptake, and the presence of purified brain phospholipids. Analysis of the sucrose-gradient-purified reconstituted vesicles on NaDodSO4/polyacrylamide gels showed that the activity coincided with enriched appearance of a 70,000-Da protein.     

Relationship between plasma phospholipid transfer protein activity and HDL subclasses among patients with low HDL and cardiovascular disease

Low levels of high density lipoproteins (HDL) are associated with an increased risk for premature cardiovascular disease. The plasma phospholipid transfer protein (PLTP) is believed to play a critical role in lipoprotein metabolism and reverse cholesterol transport by remodeling HDL and facilitating the transport of lipid to the liver. Plasma contains two major HDL subclasses, those containing both apolipoproteins (apo) A-I and A-II, Lp(A-I, A-II), and those containing apo A-I but not A-II, Lp(A-I). To examine the potential relationships between PLTP and lipoproteins, plasma PLTP activity, lipoprotein lipids, HDL subclasses and plasma apolipoproteins were measured in 52 patients with documented cardiovascular disease and low HDL levels. Among the patients, plasma PLTP activity was highly correlated with the percentage of plasma apo A-I in Lp(A-I) (r=0.514, p<0.001) and with the apo A-I, phospholipid and cholesterol concentration of Lp(A-I) (r=0.499, 0.478, 0.457, respectively, p≤0.001). Plasma PLTP activity was also significantly correlated with plasma apo A-I (r=0.413, p=0.002), HDL cholesterol (r=0.308, p=0.026), and HDL₂ and HDL₃ cholesterol (r=0.284 and 0.276, respectively, p<0.05), but no significant correlation was observed with Lp(A-I, A-II), plasma cholesterol, triglycerides, or apo B, very low density lipoprotein cholesterol or low density lipoprotein cholesterol. These associations support the hypothesis that PLTP modulates plasma levels of Lp(A-I) particles without significantly affecting the levels of Lp(A-I, A-II) particles.     

Short-term effects of prednisone on serum lipids and high density lipoprotein subfractions in normolipidemic healthy men

To study the effects of short term low dose prednisone administration on serum lipids and lipoproteins we measured the concentration and composition of serum lipoproteins; serum apoproteins (apo) A-I, A-II, and B; and plasma lipolytic enzymes before and during prednisone administration (30 mg/day for 7 days) in eight normal men. We also measured insulin binding to adipocytes. Serum high density lipoprotein (HDL) cholesterol increased significantly after 2 days of prednisone administration; the maximal increase was 27% (P less than 0.01 after 5 days). The rise of HDL cholesterol was accounted for by that of HDL2 cholesterol. There were marked changes in the distribution of HDL particles; HDL2 increased, whereas HDL3 decreased. These changes were also apparent after 2 days of prednisone administration and were maximal at 5 days [mean, 1.58 +/- 0.12 (+/- SE) vs. 2.00 +/- 0.14 g/L (P less than 0.001) for HDL2; 1.82 +/- 0.11 vs. 1.61 +/- 0.06 g/L (P less than 0.05) for HDL3], and they were due to opposing changes in cholesterol, phospholipids, and proteins in the HDL subfractions. The change in HDL2 protein correlated inversely with that in HDL3 protein (r = -0.73; P less than 0.05). Notably, prednisone did not change the apo A-I concentration, but that of apo A-II decreased (0.32 +/- 0.02 vs. 0.27 +/- 0.01 g/L; P less than 0.05). Consequently, the lipid to protein ratio of HDL increased. Prednisone induced no significant changes in very low density or low density (LDL) lipoproteins. Adipose tissue LPL activity did not increase until after 7 days of prednisone intake (1.10 +/- 0.28 vs. 3.43 +/- 1.02 mumol FFA/g.h; P less than 0.05), and the same was true for muscle LPL (0.49 +/- 0.14 vs. 0.82 +/- 0.11 mumol FFA/g.h; n = 4; P = 0.06). Specific insulin binding was normal, but both basal and maximal insulin-stimulated glucose transport decreased significantly. In summary, prednisone induces changes in serum HDL which are characterized by redistribution of particles within HDL density toward less dense particles and a quantitative rise of lipids in the HDL2 fraction.     

The lipoprotein-associated coagulation inhibitor that inhibits the factor VII-tissue factor complex also inhibits factor Xa: insight into its possible mechanism of action

Blood coagulation is initiated when plasma factor VII(a) binds to its essential cofactor tissue factor (TF) and proteolytically activates factors X and IX. Progressive inhibition of TF activity occurs upon its addition to plasma. This process is reversible and requires the presence of VII(a), catalytically active Xa, Ca2+, and another component that appears to be associated with the lipoproteins in plasma, a lipoprotein-associated coagulation inhibitor (LACI). A protein, LACI(HG2), possessing the same inhibitory properties as LACI, has recently been isolated from the conditioned media of cultured human liver cells (HepG2). Rabbit antisera raised against a synthetic peptide based on the N-terminal sequence of LACI(HG2) and purified IgG from a rabbit immunized with intact LACI(HG2) inhibit the LACI activity in human serum. In a reaction mixture containing VIIa, Xa, Ca2+, and purified LACI(HG2), the apparent half-life (t1/2) for TF activity was 20 seconds. The presence of heparin accelerated the initial rate of inhibition threefold. Antithrombin III alpha alone had no effect, but antithrombin III alpha with heparin abrogated the TF inhibition. LACI(HG2) also inhibited Xa with an apparent t1/2 of 50 seconds. Heparin enhanced the rate of Xa inhibition 2.5-fold, whereas phospholipids and Ca2+ slowed the reaction 2.5-fold. Xa inhibition was demonstrable with both chromogenic substrate (S-2222) and bioassays, but no complex between Xa and LACI(HG2) could be visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Nondenaturing PAGE, however, showed that LACI(HG2) bound to Xa but not to X or Xa inactivated by diisopropyl fluorophosphate. Thus, LACI(HG2) appears to bind to Xa at or near its active site. Bovine factor Xa lacking its gamma-carboxyglutamic acid-containing domain, BXa(-GD), through treatment with alpha-chymotrypsin, was used to further investigate the Xa requirement for VIIa/TF inhibition by LACI(HG2). LACI(HG2) bound to BXa(-GD) and inhibited its catalytic activity against a small molecular substrate (Spectrozyme Xa), though at a rate approximately sevenfold slower than native BXa. Preincubation of LACI(HG2) with saturating concentrations of BXa(-GD) markedly retarded the subsequent inhibition of BXa. The VII(a)/TF complex was not inhibited by LACI(HG2) in the presence of BXa(-GD), and further, preincubation of LACI(HG2) with BXa(-GD) slowed the inhibition of VIIa/TF after the addition of native Xa. The results are consistent with the hypothesis that inhibition of VII(a)/TF involves the formation of a VIIa-TF-XA-LACI complex that requires the GD of XA.(ABSTRACT TRUNCATED AT 400 WORDS)     

Flow and the inhibition of prothrombinase by antithrombin III and heparin

Inhibition of prothrombinase by antithrombin III (ATIII) and heparin was investigated in a continuous-flow system. Phospholipid-coated capillaries, containing phospholipid-bound factor Xa and factor Va, were perfused with 1.0 mumol/L prothrombin and 0.5 nmol/L factor Va. At 25 degrees C and a flow rate of 32 microL/min (shear rate 28 seconds-1) the steady-state rates of prothrombin conversion depended linearly on the surface concentration of prothrombinase up to 2 fmol/cm2. The rate of thrombin generation was 952 +/- 43 (SE) mol/min/mol prothrombinase. When ATIII was included in the perfusate for 10 minutes, the free thrombin concentration at the outlet of the capillary was markedly reduced: a 50% neutralization was obtained at 0.7 mumol/L ATIII. However, the prothrombinase activity was not inhibited, as could be established after a subsequent perfusion with prothrombin and factor Va. At an ATIII concentration typical of normal plasma (2 mumol/L) a slight neutralization of prothrombinase was observed: 10% neutralization following a 10-minute perfusion. During a perfusion with ATIII in the absence of prothrombin, or in its presence with hirudin (2 mumol/L) also included in the perfusate, a more pronounced neutralization of prothrombinase was observed: 40% residual activity was obtained after a 10-minute perfusion. From this observation the suggestion comes forward that thrombin, continuously produced at the surface, consumes ATIII in the boundary layer. In this case the true ATIII concentration in the vicinity of surface-bound prothrombinase will be but a small fraction of the initial ATIII concentration in the bulk fluid. Unfractionated heparin and an ultra-low molecular weight heparin (pentasaccharide) did enhance the ATIII-dependent neutralization of prothrombinase, but to a much lesser extent than observed with small unilaminar phospholipid vesicles as the catalytic sites for prothrombinase assembly. The findings reported here support the notion that regulation of prothrombinase by heparin under in vivo conditions occurs at the stage of its formation, ie, through inhibition of free factor Xa and/or the generation of factor Va, rather than by direct inhibition of the prothrombinase activity.     

The effect of lipoproteins on the synthesis of prostacyclin, von Willebrand factor and apoliproteins A-I and A-II in cultured human endothelial cells

Primary cultures of confluent human endothelial cells (ECM) were grown in media containing the major lipoproteins (LP) and lipoprotein deficient serum (LDS). The release of 6-keto-PGF₁, von Willebrand factor (VIII RAg) and apolipoproteins (apo) A-I and A-II were investigated by radioimmunoassay. The cell-associated VIII RAg, apo A-I and apo A-II were also confirmed by fluorescein antibodies, and the synthesis of the apolipoproteins was examined by incorporation of [^3H]leucine.

Apo A-I and apo A-II were located and synthesized in ECM, yet only apo A-I was released into the medium. Very low density (VLDL) and low density lipoproteins (LDL) in concentrations of 50–600 μg/ml stimulated release of apo A-I. Stimulation of ECM for 5 min with thrombin (T) or arachidonic acid (A) did not induce apo A-I release.

VIII RAg was always released into the media from ECM. The release was not affected by the lipoproteins. VIII RAg was also localized on the cell surface (VIII RAgC) and approximately 80% was released by trypsin. LDL stimulated the occurrence of factor VIII RAg on the cell surface.

6-Keto PGF₁ was always released into the medium and the production was stimulated by T and AA. The main lipoproteins (50–600 μg/ml) and apo A-I and A-II did not affect the release of 6-keto-PGF₁

This study shows that endothelial cells synthesize and release proteins important for thrombogenesis and atherosclerosis. The release of apolipoproteins A-I was stimulated by VLDL and LDL, and the concentration of cell-related factor VIII RAg was stimulated by LDL.     

Reconstitution in vitro of neurotoxin-responsive ion efflux by using membrane glycoproteins of neuroblastoma cells.

Glycoproteins were purified from a clonal cell line of mouse neuroblastoma, N-18, labeled metabolically with L-[3H]fucose. The purified radioactive glycoproteins were reconstituted into artificial phosphatidylcholine vesicles. When the vesicles were preloaded with cesium acetate and treated with neurotoxins to activate the Na+ channel, a shift in intravesicular density was observed to a less dense position after centrifugation on sucrose gradients. This shift was partially inhibited by tetrodotoxin, which prevents the activation of the Na+ channel. A similarly derived fraction of [14C]fucose-containing glycoproteins from a neuroblastoma cell line that does not possess excitable membranes, N1A-103, was reconstituted into phospholipid vesicles, and, after preloading with cesium ions, the fraction was combined with those of the 3H-labeled glycoproteins of the differentiated cells, N-18, which have excitable membranes. Only the 3H-labeled glycoprotein-containing vesicles were responsive to the neurotoxins, as shown by a shift in intravesicular density on sucrose gradients. These results are interpreted as a demonstration of the reconstitution of glycoproteins to form the activated Na+ channel. Comparison of the radioactive glycoprotein profiles after polyacrylamide gel electrophoresis showed that glycoproteins of Mr 200,000, Mr 165,000, and Mr 65,000 were common to the reconstituted fractions that were biologically active.     

Alkaline phosphatase activity of glutaraldehyde-treated bovine pericardium used in bioprosthetic cardiac valves

Bioprosthetic valves fail frequently because of pathological mineralization, a process that begins in cell remnants of the glutaraldehyde (GLUT) fixed tissue. Other pathological cardiovascular calcification and physiological mineralization in skeletal/dental tissues are both largely initiated in cell-derived membranous structures (often called "matrix vesicles"), and the enzyme alkaline phosphatase (AP) likely has an important function in the pathogenesis of mineral nucleation. This study tested the hypothesis that AP might also be present in and contribute to calcification of bioprosthetic valves. AP activity of fresh and GLUT-treated bovine pericardium was measured by the conversion of p-nitrophenyl phosphate to p-nitrophenol. Following 24 hours in 0.6% HEPES-buffered GLUT and storage for 2 weeks in 0.2% GLUT, considerable AP hydrolytic activity remained in GLUT-treated tissue relative to that of fresh tissue (Vmax, 24 vs. 45 mumol reaction product/min/mg tissue protein, respectively), although binding was somewhat reduced (Km, 1.9 X 10(3) vs. 1.4 X 10(3) microM substrate, respectively). Enzyme reaction product was demonstrated in both fixed and fresh tissue by light microscopic histochemical studies, confirming the biochemical results. Reaction product was noted along membranes of vascular endothelial cells and interstitial fibroblasts, the sites of early calcific deposits in bioprosthetic valves, by ultrastructural examination of GLUT-treated tissue. We conclude that GLUT-treated bovine pericardium retains much of the hydrolytic activity of AP, an enzyme associated with normal skeletal and pathological cardiovascular and noncardiovascular mineralization, and suggest that further examination of the mechanistic role of this enzyme may stimulate new approaches for slowing or preventing calcification of bioprosthetic tissue.     

Preliminary report: kinetic studies on the modulation of high-density lipoprotein, apolipoprotein, and subfraction metabolism by sex steroids in a postmenopausal woman

To investigate the effects of estrogens and androgens on the metabolism of high density lipoproteins (HDL) and low density lipoproteins (LDL), a normolipidemic postmenopausal woman was studied under the following conditions: (1) during supplementation with ethinyl estradiol (0.06 mg/d); (2) without sex steroid therapy; (3) during treatment with stanozolol, an androgenic, anabolic steroid (6 mg/d). During these manipulations HDL and LDL cholesterol levels fluctuated widely but reciprocally: during estrogen supplementation HDL increased while LDL decreased; during stanozolol HDL-C decreased while LDL-C increased. Simultaneous changes in post-heparin plasma hepatic triglyceride lipase activity paralleled those of LDL (and opposed those of HDL), decreasing with estrogen and increasing with stanozolol. During all three phases, autologous 125I-HDL turnover studies disclosed similarities between HDL2 and apolipoprotein A-I metabolism and between HDL3 and apolipoprotein A-II metabolism. In the untreated state the residence times of HDL2 and apo A-I were only half those of HDL3 and apo A-II. During estrogen treatment HDL2 and apo A-I, residence times were selectively prolonged, coming to resemble those of HDL3 and apo A-II, which remained unchanged. By contrast, during stanozolol treatment HDL3 and apo A-II residence times were selectively reduced, coming to resemble those of HDL2 and apo A-I, which remained unchanged. Apo A-I levels increased on estrogen and decreased on stanozolol, while apo A-II remained stable. Hence, estrogen increased HDL primarily by retarding the catabolism of the HDL2 subfraction rich in apo A-I, whereas stanozolol decreased HDL by accelerating the catabolism of HDL3, relatively rich in apo A-II.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relevance of different apolipoprotein content in binding of homocysteine to plasma lipoproteins

BACKGROUND AND AIMS: In plasma the atherogenic thiol homocysteine (Hcy) circulates either free or bound to proteins (Pb-Hcy). The present study sets out to evaluate the lipoprotein-Hcy (LP-Hcy) binding in vivo and the possible influence of different apolipoprotein content in this binding, being lipoprotein oxidation a possible mechanism of Hcy-induced damage. METHODS AND RESULTS: In 34 healthy subjects we assayed fasting plasma lipoprotein and correspondent apolipoprotein (apo A-I, apo A-II, apo C-II, apo C-III, apo B, apo(a) and apo E content, and Hcy bound to different plasma protein fractions; moreover ten subjects underwent an oral methionine load in order to evaluate possible "dynamic" modifications of Pb-Hcy and LP-Hcy after induction of hyperhomocysteinemia. Pb-Hcy (mean values 9.22 +/- 1.7 mumol/L) represented about 78% of total plasma Hcy (mean values 11.8 +/- 1.8 mumol/L). Pb-Hcy distribution between the different fractions was as follows (mumol/L): VLDL = 0.25 +/- 0.08 (2.7%); LDL = 0.88 +/- 0.22 (9.5%); HDL = 1.40 +/- 0.36 (15.2%); fractions with density greater than 1.21 g/mL (Lipoprotein-Free Protein Fraction, LPDS) = 6.7 +/- 1.2 (72.6%). Hcy/protein ratios (nmol/mg of protein) in each protein fraction were: VLDL = 0.32 +/- 0.19, LDL = 0.43 +/- 0.37, HDL = 0.26 +/- 0.18, LPDS < 0.1, thus suggesting a higher binding capacity for Hcy by VLDL and LDL. These data were confirmed by the higher increase in Hcy content in LDL and VLDL (76 and 90%, respectively vs 36% and 3.1% for HDL and LPDS fractions) after hyperhomocysteinemia. Lp-Hcy binding significantly correlated with the apo B content of VLDL and LDL and Apo A-I content of HDL. CONCLUSIONS: An important fraction of plasma Hcy circulates bound to LP (about 27% of Pb-Hcy); VLDL and LDL show the highest binding capacity for Hcy, probably due to their content in Apo B, a possible high capacity binding site for Hcy.