Affinity of binding of radiolabeled (125I) heparin and low molecular weight heparin fraction CY 222 to endothelium in culture

Binding of radiolabeled (125I) heparin and its low molecular weight fraction CY 222 (Choay) to human and porcine cultured endothelium was investigated. The binding was measured over a wide range of heparin or CY 222 concentration in culture medium, from less than 20-time up to more than 30-times of the therapeutic heparin level. A relatively small fraction (less than 1%) of tested products was bound to endothelium. The process of binding was temperature-independent. A comparable number of endothelial binding sites (approx. 10(12)/cm2) for both 125I-heparin and 125I-CY 222 was calculated. About 40% totally bound 125I-heparin and 30% of 125I-CY 222 was found in extracellular matrix of cultured endothelium. The endothelium exhibited a 2.4-times lower affinity for 125I-CY 222 (Kd = 5.59 +/- 1.77 microM) than that for 125I-heparin (Kd = 2.35 +/- 0.78 microM). A similar affinity of human (venous) and porcine (aortic) endothelium for 125I-heparin was demonstrated. Cultured endothelium exhibited the same affinity for unlabeled heparin as for 125I-heparin. The endothelium depleted of sialic acid residues bound 1.5-times more of 125I-CY 222 than the control endothelium in culture.     

Binding and endocytosis of heparin by human endothelial cells in culture

Binding of heparin and low molecular weight heparin fragments (CY 222, Mr range 1500-8000) to human vascular endothelial cells was studied. Primary culture of human umbilical vein endothelial cells and either 125I or 3H-labeled heparin or [125I]CY 222 were used. Slow, saturable and specific binding was found. No other tested glycosaminoglycan, excepting a highly sulfated heparan fraction, was able to compete for heparin binding. Two groups of binding sites for [3H]heparin could be distinguished: one with high affinity (Kd = 0.12 microM) and another with lower affinity (Kd = 1.37 microM) and a relative large capacity of binding (1.16 X 10(7) molecules/cell) was calculated. The Kd for unlabeled heparin, as calculated from competition experiments, was 0.23 microM. Much lower affinity was calculated for unlabeled low molecular weight heparin fragments CY 222 (Kd = 4.3 microM) from competition experiments with [125I]CY 222. The binding reversibility was only partial for unfractionated heparin. Even by chasing with unlabeled compound, a fraction of 25-30% was not dissociable from endothelial cells. This fraction was much lower if incubation was carried out at 4 degrees C. The addition of basic proteins (histones) to the incubation medium greatly enhanced the undissociable binding at 37 degrees C, but not at 4 degrees C. The undissociable fraction of heparin was not available to degradation by purified microbial heparinase. These results suggest that a fraction of bound heparin is internalized by the vascular endothelium.     

Inhibition of coagulation and platelet adhesion to extracellular matrix by unfractionated heparin and a low molecular weight heparin

In 7 healthy volunteers, the effect of a single i.v. injection of 52 mg (7,500 IU) of an unfractionated heparin (UFH) and of 52.5 mg (5,000 anti XaU) of a low molecular weight heparin (LMWH) on coagulation parameters and platelet function has been studied. Thrombin-induced platelet aggregation was inhibited after the injection of both heparins. There was no significant change of ADP or collagen-induced aggregation after LMWH of UFH. Platelet adhesion to bovine extracellular matrix was not inhibited by UFH but was significantly reduced after addition in vitro and ex vivo after administration of LMWH. Further investigation should establish the time course of LMWH effects on platelet adhesion. A long duration of this effect could be partially correlated with the antithrombotic effects of LMWH.     

Antithrombin III-dependent anti-prothrombinase activity of heparin and heparin fragments

Heparin and heparin fragments in the molecular mass range 1,700-20,000 Da were examined for their ability to accelerate the antithrombin III (AT III)-dependent inhibition of human factor Xa and the prothrombin converting complex (prothrombinase) during human prothrombin activation. The prothrombinase reaction was modeled by a 3-parameter 2-exponential equation to determine the initial rate of prothrombin activation and the pseudo-first order rate constants of inhibition of prothrombinase and in situ generated thrombin activity. The catalytic specific activities of the heparins increased with increasing molecular size for both the inhibition of prothrombinase and factor Xa. A 10-fold increase over the entire Mr range was found. In contrast to results obtained by others (Ellis, V., Scully, M. F., and Kakkar, V. V. (1986) Biochem. J. 233, 161-165; Barrowcliffe, T. W., Havercroft, S. J., Kemball-Cook, G., and Lindahl, U. (1987) Biochem. J. 243, 31-37), all the heparins showed a 5-fold higher rate of inhibition of factor Xa when compared with the inhibition of prothrombinase, indicating that the factor Va-mediated protection of factor Xa from inhibition by AT III/heparin is independent of the molecular size of the heparin. Our original approach has also revealed a hitherto unrecognized phenomenon, namely, in addition to the accelerating effect of the heparins on the rate of formation of the inactive AT III-factor Xa complex, heparins with Mr greater than 4,500 reduce the initial rate of thrombin generation in the presence of AT III in a concentration-dependent way. We hypothesize that the formation of the dissociable ternary AT III-heparin-factor Xa complex results in a (partial) loss of factor Xa activity towards its natural substrate prothrombin.     

Anti-thrombin activities of heparin. Effect of saccharide chain length on thrombin inhibition by heparin cofactor II and by antithrombin.

The interactions of two proteinase inhibitors, heparin cofactor II and antithrombin, with thrombin are potentiated by heparin. Using two methods, we have studied the potentiating effects of a series of heparin (poly)saccharides with high affinity for antithrombin and mean Mr ranging from approx. 1700 to 18,800. First, catalytic amounts of heparin (poly)saccharide were added to purified systems containing thrombin and either heparin cofactor II or antithrombin. Residual thrombin activity was determined with a chromogenic substrate. It was found that only the higher-Mr polysaccharides (Mr greater than 8000) efficiently catalysed thrombin inhibition by heparin cofactor II, there being a progressive catalytic effect with increasing Mr of the polysaccharide. Weak accelerating effects were noted with low-Mr saccharides (Mr less than 8000). This contrasted with the well-characterized interaction of heparin with antithrombin and thrombin, where heparin oligosaccharides of Mr less than 5400 had absolutely no ability to accelerate the reaction, while (poly)saccharides of Mr exceeding 5400 showed rapidly increasing catalytic activity with increasing Mr. Secondly, these and other heparin preparations were added in a wide concentration range to plasma with which 125I-labelled thrombin was then incubated for 30 s. Inhibited thrombin was determined from the distribution of labelled thrombin amongst inhibitor-thrombin complexes, predominantly antithrombin-thrombin and heparin cofactor II-thrombin complexes. In this situation, where the inhibitors competed for thrombin and for the (poly)saccharides, it was found that, provided the latter were of high affinity for antithrombin and exceeded a Mr of 5400, thrombin inhibition in plasma was mediated largely through antithrombin. Polysaccharides of Mr exceeding 8000 that were of low affinity for antithrombin accelerated thrombin inhibition in plasma through their interaction with heparin cofactor II. High concentrations of saccharides of Mr 1700-5400 exhibited a size-dependent acceleration of thrombin inhibition, not through their interaction with antithrombin, but through their interaction with heparin cofactor II.     

Biosyntheses of interstitial collagens and fibronectin by porcine aorta smooth muscle cells. Modulation by low-molecular-weight heparin fragments

The effect of low-molecular-weight heparin fragments (CY222) on the biosynthetic phenotype of porcine aortic smooth muscle cells (SMC) was investigated in vitro on overconfluent cell cultures. Addition of increasing concentrations of CY222 to the culture medium of early passage SMC resulted in a dose-dependent decrease of type III to type I collagen ratio without change in total collagen biosynthesis. In the same range of concentrations CY222 did not affect the biosynthesis of fibronectin. However, heparin fragments decreased the proportion of the freshly synthesized pericellular form of fibronectin with a concomitant increase of neosynthesized intracellular fibronectin, indicating an inhibitory effect of CY222 on fibronectin secretion. Our results demonstrate that the biosynthetic phenotype of SMC in vitro can be modulated by low-molecular-weight heparin fragments and confirm also that interactions between cells and extracellular matrix molecules can modify the biosynthetic pattern of mesenchymal cells.     

Inhibition of allergic airway responses by inhaled low-molecular-weight heparins: molecular-weight dependence

Martinez-Salas, José, Richard Mendelssohn, William M. Abraham, Bernard Hsiao, and Tahir Ahmed. Inhibition of allergic airway responses by inhaled low-molecular-weight heparins:molecular-weight dependence. J. Appl.Physiol. 84(1): 222-228, 1998.Inhaled heparin prevents antigen-induced bronchoconstriction and inhibitsanti-immunoglobulin E-mediated mast cell degranulation. We hypothesizedthat the antiallergic action of heparin may be molecular weightdependent. Therefore, we studied the effects of three differentlow-molecular-weight fractions of heparin [medium-, low-, andultralow-molecular-weight heparin (MMWH, LMWH, ULMWH,respectively)] on the antigen-induced acute bronchoconstrictorresponse (ABR) and airway hyperresponsiveness (AHR) in allergic sheep.Specific lung resistance was measured in 22 sheep before and afterairway challenge with Ascarissuum antigen, without and afterpretreatment with inhaled fractionated heparins at doses of0.31-5.0 mg/kg. Airway responsiveness was estimated before and 2 hpostantigen as the cumulative provocating dose of carbachol in breathunits that increased specific lung resistance by 400%. Allfractionated heparins caused a dose-dependent inhibition of ABR andAHR. ULMWH was the most effective fraction, with the inhibitory dosecausing 50% protection (ID₅₀)against ABR of 0.5 mg/kg, whereasID₅₀ values of LMWH and MMWH were1.25 and 1.8 mg/kg, respectively. ULMWH was also the most effective fraction in attenuating AHR; theID₅₀ values for ULMWH, LMWH, andMMWH were 0.5, 2.5, and 4.7 mg/kg, respectively. These data suggestthat 1) fractionatedlow-molecular-weight heparins attenuate antigen-induced ABR and AHR;2) there is an inverse relationship between the antiallergic activity of heparin fractions and molecular weight; and 3) ULMWH is the mosteffective fraction preventing allergic bronchoconstriction and airwayhyperresponsiveness.

     

Analysis of heparin binding to human leukocytes using a fluorescein-5-isothiocyanate labeled heparin fragment

The binding of LMWH-tyr-FITC to granulocytes, monocytes, and lymphocytes was analyzed by flow cytometry using a low-molecular-weight heparin (LMWH) labeled with fluorescein-5-isothiocyanate (FITC). FITC was covalently bound to tyramine, which was synthesized to LMWH by endpoint-attachment (Malsch et al.: Anal Biochem 217:255-264, 1994). The binding was rapid, specific, dose-dependent, saturable, and reversible. To investigate the molecular weight dependence of heparins, heparin-derived di- to dodecasaccharides were used. With decreasing molecular weight, the amount of oligosaccharides increased; these were bound to granulocytes, monocytes, and lymphocytes (r = -0.77). The degree of sulfation of non-heparin glycosaminoglycans influenced the binding to leukocytes. Decreasing the degree of sulfation decreased the binding. The pentasaccharide did not bind as strongly as the other heparin-derived oligosaccharides, indicating an AT III-independent mechanism. Two classes of heparin binding sites were identified on granulocytes and one class of binding sites on monocytes and lymphocytes. The lowest amount of LMWH-tyr-FITC detected was 1 ng on granulocytes, 0.18 ng on monocytes and 0.01 ng on lymphocytes. The data suggest that heparin and other sulfated polysaccharides may play a role in the physiology of thrombosis, arteriosclerosis, and inflammation by binding to granulocytes, monocytes, and lymphocytes.     

Effects of heparin and derivatives on podocytes: An in vitro functional and morphological evaluation

Unfractionated heparin (UFH) and low molecular heparin derivatives (LMWH) display numerous biological properties in addition to their anticoagulant effects. However, due to the physicochemical heterogeneity of these drugs, a better understanding concerning their effects on human cells is clearly needed. Considering that heparins are mainly excreted by the kidney, we focused our attention on the effect of UFH and LMWH on human podocytes by functional and morphological/phenotypic in vitro analyses. We demonstrated that these products differentially modulate the permeability of podocyte monolayer to albumin. The functional perturbations observed were correlated to significant cellular morphological and cytoskeletal changes, as well as a decrease in the expression of proteins involved in podocyte adherence to the extracellular matrix or intercellular interactions. This point confirms that UFH and the different LMWHs exert specific effects on podocyte permeability and underlines the need of in vitro tests to evaluate new biological nonanticoagulant properties of LMWH.     

Small-angle x-ray scattering of high- and low-affinity heparin

Solution characterization of heparin with high affinity (HA) and low affinity (LA) for antithrombin III was performed using the methods of small-angle x-ray scattering (SAXS), viscometry, and aqueous gel permeation chromatography (GPC). SAXS provided various topological parameters including the radius of gyration ([S2]1/2), radius of gyration of cross-section ([S2]q1/2), persistence length (a*), contour length (L), and mass parameters, e.g., overall molecular mass (Mr), and mass per unit length (Mq). The molecular weights of HA and LA pig mucosal heparins were found to be 14,900 and 11,500 and the respective radii of gyration were 40.1 and 33.6 A. The persistence lengths of HA and LA were 21.3 and 20.3 A, respectively. These parameters were compared to SAXS data of heparin [S. S. Stivala, M. Herbst, O. Kratky, and I. Pilz (1968) Arch. Biochem. Biophys. 127, 795-802] fractionated according to molecular weight only. It was found that the various experimental values of this heparin lie somewhere in between those of HA and LA heparins. It appears that there are no appreciable differences in the physico-chemical properties, including conformation, among the heparins in H2O at 25 degrees C.     

Energetics of hydrogen bond switch, residue burial and cavity analysis reveals molecular basis of improved heparin binding to antithrombin

Antithrombin III (ATIII) is the main inhibitor of the coagulation proteases like factor Xa and thrombin. Anticoagulant activity of ATIII is increased by several thousand folds when activated by vascular wall heparan sulfate proteoglycans (HSPGs) and pharmaceutical heparins. ATIII isoforms in human plasma, alpha-ATIII and beta-ATIII differ in the amount of glycosylation which is the basis of differences in their heparin binding affinity and function. Crystal structures and site directed mutagenesis studies have mapped the heparin binding site in ATIII, however the hydrogen bond switch and energetics of interaction during the course of heparin dependent conformational change remains largely unclear. An analysis of heparin bound conformational states of ATIII using PEARLS software showed that in heparin bound intermediate state, Arg 47 and Arg 13 residues make hydrogen bonds with heparin but in the activated conformation Lys 11 and Lys 114 have more hydrogen bond interactions. In the protease bound-antithrombin-pentasaccharide complex Lys 114, Pro 12 and Lys 125 form important hydrogen bonding interactions. The results showed that A-helix and N-terminal end residues are more important in the initial interactions but D-helix is more important during the latter stage of conformational activation and during the process of protease inhibition. We carried out the residue wise Accessible Surface Area (ASA) analysis of alpha and beta ATIII native states and the results indicated major differences in burial of residues from Ser 112 to Ser 116 towards the N-terminal end. This region is involved in the P-helix formation on account of heparin binding. A cavity analysis showed a progressively larger cavity formation during activation in the region just adjacent to the heparin binding site towards the C-terminal end. We hypothesize that during the process of conformational change after heparin binding beta form of antithrombin has low energy barrier to form D-helix extension toward N and C-terminal end as compared to alpha isoform.     

Endothelial binding sites for heparin. Specificity and role in heparin neutralization.

The specificity of endothelial binding sites for heparin was investigated with heparin fractions and fragments differing in their Mr, charge density and affinity for antithrombin III, as well as with heparinoids and other anionic polyelectrolytes (polystyrene sulphonates). The affinity for endothelial cells was estimated by determining I50 values in competition experiments with 125I-heparin. We found that affinity for endothelial cells increases as a function of Mr and charge density (degree of sulphation). Binding sites are not specific receptors for heparin. Other anionic polyelectrolytes, such as pentosan polysulphates and polystyrene sulphonates, competed with heparin for binding to endothelial cells. Fractions of standard heparin with high affinity for antithrombin III also had greater affinity for endothelium. However, these two properties of heparin (affinity for antithrombin III and affinity for endothelial cells) could be dissociated. Oversulphated heparins and oversulphated low-Mr heparin fragments had lower anticoagulant activity and higher affinity for endothelial cells than did their parent compounds. Synthetic pentasaccharides, bearing the minimal sequence for binding to antithrombin III, did not bind to endothelial cells. Binding to endothelial cells involved partial neutralization of heparin. Bound heparin exhibited only 5% and 7% of antifactor IIa and antifactor Xa specific activity, respectively. In the presence of 200 nM-antithrombin III, and in the absence of free heparin, a limited fraction (approx. 30%) of bound heparin was displaced from endothelial cells during a 1 h incubation period. These data suggested that a fraction of surface-bound heparin could represent a pool of anticoagulant.     

The relative molecular mass dependence of the anti-factor Xa properties of heparin.

The effect of heparin fractions of various Mr, with high affinity for antithrombin III, on the kinetics of the reaction between factor Xa and antithrombin III have been studied using purified human proteins. Each of the heparin fractions, which varied between pentasaccharide and Mr 32,000, accelerated the inhibition of factor Xa although an increasing rate of inhibition was observed with increasing Mr. The chemically synthesized pentasaccharide preparation (Mr 1714) gave a maximum inhibition rate constant of 1.2 X 10(7) M-1 X min-1, compared with 6.3 X 10(4) M-1 X min-1 in the absence of heparin, and this rose progressively to 4.2 X 10(8) M-1 X min-1 with the two fractions of highest Mr (22,500 and 32,000). The 35-fold difference in inhibition rates observed with the high-affinity fractions was virtually abolished by the presence of 0.3 M-NaCl. The disparity in these rates of inhibition was shown to be due to a change in the Km for factor Xa when a two-substrate model of heparin catalysis was used. The Km for factor Xa rose from 28 nM for the fraction of Mr 32,000 to 770 nM for the pentasaccharide, whilst 0.3 M-NaCl also caused an increase in Km with the high-Mr fraction. These data suggest that the increased rates of inhibition observed with heparins of higher Mr may be due to an involvement of heparin binding to factor Xa as well as to antithrombin III.     

A selective protein sensor for heparin detection

No clinical assays for the direct detection of heparin in blood exist. To create a heparin sensor, the hyaluronan (HA)-binding domain (HABD) of a protein that binds heparin and HA was engineered. GST fusion proteins containing one to three HABD modules were cloned, expressed, and purified. The affinities of each construct for heparin and for HA were determined by a competitive enzyme-linked immunosorbent assay using immobilized HA or heparin. Each of the constructs showed modest affinity for immobilized HA. However, heparin was 100-fold more potent than HA as a competing ligand. With immobilized heparin, affinity increased as the HABD copy number increased. The three-copy construct, GST-HB3, detected unfractionated free heparin (UFH) as low as 39ng/ml (equivalent to approximately 0.1U/ml) with a signal-to-noise ratio of 5.6. GST-HB3 also showed 100-fold selectivity for heparin in preference to other glycosaminoglycans. The plot of logKd vs log [Na+] showed 2.5 ionic interactions per heparin-HB3 interaction. GST-HB3 showed a linear detection of both UFH (15kDa) and low-molecular-weight heparin (LMWH; 6kDa) added to human plasma. For UFH, the range examined was 78 to over 2000ng/ml (equivalent to 0.2 to 5.0U/ml). For LMWH, the useful range was 312 to over 2000ng/ml. The coefficient of variance for the assay was < 9% for six serial heparin dilutions and <12% for three plasma samples. In clinical use, GST-HB3 could accurately measure therapeutic heparin levels in plasma (0.2 to 2U/ml).     

Multiple complexes of thrombin and heparin

Fluorescence polarization has been used to study the interaction of thrombin and heparin, and the catalysis by heparin of the combination of thrombin and antithrombin. At low ionic strength (20 mM Tris, pH 7.4), the addition of heparins of known molecular weights to thrombin led to the formation of large complexes (defined as 'complex 1'). Further addition of heparin led to a rearrangement of these large complexes to form smaller complexes (defined as 'complex 2'). The molar ratio of thrombin to heparin in complex 1 increased with increasing heparin molecular weight, and corresponded to one thrombin molecule for every heparin segment of Mr 3000. The stoichiometry of complex 2 was 1 heparin to 1 thrombin, irrespective of the heparin molecular weight. At higher ionic strength (150 mM NaCl) some complex 1 was still formed. However, by reversing the titration and adding thrombin to fluorescein-heparin the dissociation constant for complex 2 was estimated to be 1-3 microM and independent of the heparin molecular weight. The complex formed between thrombin and heparin, to which antithrombin was attached, has a dissociation constant of 1-2 microM, again irrespective of the heparin molecular weight. In the heparin-catalysed thrombin-antithrombin reaction, an increase in the size of heparin leads to a lowering of the observed Km for thrombin. A possible explanation is that thrombin, after initial binding to the heparin, moves rapidly to the site where it combines with antithrombin.     

Catalytic and molecular properties of highly purified phosvitin/casein kinase type II from human epithelial cells in culture (HeLa) and relation to ecto protein kinase

Phosvitin/casein type II kinase was purified from HeLa cell extracts to homogeneity and characterized. The kinase prefers phosvitin over casein (Vmax phosvitin greater than Vmax casein; apparent Km 0.5 microM phosvitin and 3.3 microM casein) and utilizes as cosubstrate ATP (apparent Km 3-4 microM), GTP (apparent Km 4-5 microM) and other purine nucleoside triphosphates, including dATP and dGTP but not pyrimidine nucleoside triphosphates. Enzyme reaction is optimal at pH 6-8 and at 10-25 mM Mg2+.Mg2+ cannot be replaced by, but is antagonized by other divalent metal ions. The kinase is stimulated by polycations (spermine) and monovalent cations (Na+,K+), and is inhibited by fluoride, 2,3-diphosphoglycerate, and low levels of heparin (50% inhibition at 0.1 microgram/ml). The HeLa enzyme is composed of three subunits with Mr of approximately 43,000 (alpha), 38,000 (alpha'), and 28,000 (beta) forming alpha alpha'beta 2 and alpha'2 beta 2 structures with obvious sequence homology of alpha with alpha' but not with beta. Photoaffinity labeling with [alpha-32P]- and [gamma-32P]8-azido-ATP revealed high affinity binding sites on subunits alpha and alpha' but not on subunit beta. The kinase autophosphorylates subunit beta and, much weaker, subunits alpha and alpha'. Ecto protein kinase, detectable only by its enzyme activity but not yet as a protein (J. Biol. Chem. 257, 322-329), was characterized in cell-bound form and in released form, and the released form both with and without prior separation from phosvitin which was employed to induce the kinase release from intact HeLa cells (Proc. Natl. Acad. Sci. U.S.A. 80, 4021-4025). Ratios of phosvitin/casein phosphorylation (greater than 2) and of ATP/GTP utilization (1.5-2.1), inhibition by heparin (50% inhibition at 0.1 microgram/ml), and amino-acid side chains phosphorylated in phosvitin and casein (serine, threonine) are comparable for cell-bound and released form. These properties resemble those of type II kinase as does Mr of released ecto kinase (120-150,000). Consistently, a protein with Mr 125,000 in calf serum and a protein (possibly two) with Mr greater than 300,000 in calf plasma which are selectively phosphorylated by the ecto kinase are also substrates of the type II kinase. Thus, nearly all properties examined of the ecto kinase are characteristic for a type II kinase.     

Reactive site peptide structural similarity between heparin cofactor II and antithrombin III

Heparin cofactor II (Mr = 65,600) was purified 1800-fold from human plasma to further characterize the structural and functional properties of the protein as they compare to antithrombin III (Mr = 56,600). Heparin cofactor II and antithrombin III are functionally similar in that both proteins have been shown to inhibit thrombin at accelerated rates in the presence of heparin. There was little evidence for structural homology between heparin cofactor II and antithrombin III when high performance liquid chromatography-tryptic peptide maps and NH2-terminal sequences were compared. A partially degraded form of heparin cofactor II was also obtained in which a significant portion (Mr = 8,000) of the NH2 terminus was missing. The rates of thrombin inhibition (+/- heparin) by native and partially degraded-heparin cofactor II were not significantly different, suggesting that the NH2-terminal region of the protein is not essential either for heparin binding or for thrombin inhibition. A significant degree of similarity was found in the COOH-terminal regions of the proteins when the primary structures of the reactive site peptides, i.e. the peptides which are COOH-terminal to the reactive site peptide bonds cleaved by thrombin, were compared. Of the 36 residues identified, 19 residues in the reactive site peptide sequence of heparin cofactor II could be aligned with residues in the reactive site peptide from antithrombin III. While the similarities in primary structure suggest that heparin cofactor II may be an additional member of the superfamily of proteins consisting of antithrombin III, alpha 1-antitrypsin, alpha 1-antichymotrypsin and ovalbumin, the differences in structure could account for differences in protease specificity and reactivity toward thrombin. In particular, a disulfide bond which links the COOH-terminal (reactive site) region of antithrombin III to the remainder of the molecule and is important for the heparin-induced conformational change in the protein and high affinity binding of heparin does not appear to exist in heparin cofactor II. This observation provides an initial indication that while the reported kinetic mechanisms of action of heparin in accelerating the heparin cofactor II/thrombin and antithrombin III/thrombin reactions are similar, the mechanisms and effects of heparin binding to the two inhibitors may be different.     

Baculovirus-directed expression of the human insulin receptor and an insulin-binding ectodomain

In this report we describe the use of the baculovirus expression system to overproduce the human insulin holoreceptor (HIR) and a truncated, secretory version of the HIR cDNA (HIRsec) consisting of the alpha subunit and the extracellular portion of the beta subunit (beta'). Sf9 cells infected with the full-length HIR viruses synthesize recombinant HIR (rHIR) with an insulin-binding alpha subunit of apparent Mr = 110,000 and a beta subunit of apparent Mr = 80,000. Uncleaved alpha beta proreceptor accumulates in infected cells. Both of these forms assemble into higher order disulfide-linked dimers or heterotetramers of apparent Mr greater than 350,000. Insulin-binding activity in cells infected with rHIR viruses is present predominantly on the extracellular aspect of the plasma membrane (greater than 80%). Insulin binding to the full-length rHIR occurs with typical complex kinetics with Kd1 = 0.5-1 x 10(-9) M and Kd2 = 10(-7) M and receptors are present in large amounts in infected cells (1 x 10(6) receptors/cell; 1-2 mg HIR/10(9) cells). The full-length rHIR undergoes insulin-dependent autophosphorylation; half-maximal activation of beta subunit autophosphorylation occurs at 1-2 x 10(-8) M. The alpha beta proreceptor also becomes phosphorylated in vitro. Analysis of tryptic phosphopeptides derived from in vitro autophosphorylated beta subunit and alpha beta proreceptor reveals a pattern of phosphorylation that is indistinguishable from that of authentic placental HIR. Sf9 cells infected with rHIRsec viruses synthesize and secrete an (alpha beta')2 heterotetrameric complex having an insulin-binding alpha subunit of apparent Mr = 110,000 and a truncated beta' subunit of apparent Mr = 45,000 that lacks kinase activity. The rHIRsec complex purified from the conditioned medium of infected cells binds insulin with high affinity (Kd = 10(-9) M).     

Dependence on heparin chain-length of the interaction of heparin with human plasma low density lipoproteins

Successive rechromatography of commercial bovine lung heparin on human plasma low density lipoproteins (LDL) immobilized to AffiGel-10 yielded four high reactive heparin (HRH-I to IV) fractions and an unreactive fraction (URH). HRH-I was the most sulphated HRH fraction whereas URH had the least sulphation. In the presence of 10 mM Ca2+, LDL were precipitated by these heparins in the following order: HRH-II greater than HRH-III greater than HRH-IV greater than HRH-I greater than URH. The average molecular weight of HRH-I to IV was 8600, 11400, 10,100, and 10,000, respectively. A plot of log molecular weight versus the concentration of HRH required to give half-maximal precipitation of LDL showed a negative correlation (r = -0.880). These results indicate that heparin chain length is an important determinant of heparin binding to LDL in solution and may have relevance to the binding and precipitation of LDL in the arterial wall.     

Isolation and characterization of an antithrombin III variant with reduced carbohydrate content and enhanced heparin binding

Two distinct forms of antithrombin III were isolated by chromatography of normal human plasma on heparin-Sepharose. The predominant antithrombin species present (AT-III alpha), which eluted from the affinity column in 1 M NaCl, was identified as the antithrombin III form which has been previously characterized. Ionic strength of the buffer was increased to elute a variant form of antithrombin III, designated as AT-III beta. The molecular weight of AT-III beta is less than that of AT-III alpha, but physicochemical studies do not indicate measureable differences in the polypeptide portion of the proteins. Carbohydrate determination revealed the sole detectable structural difference in the two antithrombins: levels of hexosamine, neutral sugars, and sialic acid in AT-III beta were all 25-30% less than in AT-III alpha. Kinetic studies of thrombin inactivation by both antithrombins, in the presence of nonsaturating amounts of heparin, indicated that AT-III beta inhibited thrombin more rapidly. AT-III beta is also distinguishable from AT-III alpha on the basis of heparin-binding affinity estimated from titration of protein fluorescence with heparin. Thus, antithrombin III exists as two molecular entities in human plasma which differ both structurally, in carbohydrate content, and functionally, in their heparin-binding behavior.