Hypersulfated low molecular weight heparin with reduced affinity for antithrombin acts as an anticoagulant by inhibiting intrinsic tenase and prothrombinase.

In buffer systems, heparin and low molecular weight heparin (LMWH) directly inhibit the intrinsic factor X-activating complex (intrinsic tenase) but have no effect on the prothrombin-activating complex (prothrombinase). Although chemical modification of LMWH, to lower its affinity for antithrombin (LA-LMWH) has no effect on its ability to inhibit intrinsic tenase, N-desulfation of LMWH reduces its activity 12-fold. To further explore the role of sulfation, hypersulfated LA-LMWH was synthesized (sLA-LMWH). sLA-LMWH is not only a 32-fold more potent inhibitor of intrinsic tenase than LA-LMWH; it also acquires prothrombinase inhibitory activity. A direct correlation between the extent of sulfation of LA-LMWH and its inhibitory activity against intrinsic tenase and prothrombinase is observed. In plasma-based assays of tenase and prothrombinase, sLA-LMWH produces similar prolongation of clotting times in plasma depleted of antithrombin and/or heparin cofactor II as it does in control plasma. In contrast, heparin has no effect in antithrombin-depleted plasma. When the effect of sLA-LMWH on various components of tenase and prothrombinase was examined, its inhibitory activity was found to be cofactor-dependent (factors Va and VIIIa) and phospholipid-independent. These studies reveal that sLA-LMWH acts as a potent antithrombin-independent inhibitor of coagulation by attenuating intrinsic tenase and prothrombinase.

Comparison of heparin- and dermatan sulfate-mediated catalysis of thrombin inactivation by heparin cofactor II.

Heparin and dermatan sulfate activate heparin cofactor II (HCII) comparably, presumably by liberating the amino terminus of HCII to bind to exosite I of thrombin. To explore this model of activation, we systematically substituted basic residues in the glycosaminoglycan-binding domain of HCII with neutral amino acids and measured the rates of thrombin inactivation by the mutants. Mutant D, with changes at Arg(184), Lys(185), Arg(189), Arg(192), Arg(193), demonstrated a approximately 130-fold increased rate of thrombin inactivation that was unaffected by the presence of glycosaminoglycans. The increased rate reflects displacement of the amino terminus of mutant D because (a) mutant D inactivates gamma-thrombin at a 65-fold slower rate than alpha-thrombin, (b) hirudin-(54-65) decreases the rate of thrombin inactivation, and (c) deletion of the amino terminus of mutant D reduces the rate of thrombin inactivation approximately 100-fold. We also examined the contribution of glycosaminoglycan-mediated bridging of thrombin to HCII to the inhibitory process. Whereas activation of HCII by heparin was chain-length dependent, stimulation by dermatan sulfate was not, suggesting that dermatan sulfate does not utilize a template mechanism to accelerate the inhibitory process. Fluorescence spectroscopy revealed that dermatan sulfate evokes greater conformational changes in HCII than heparin, suggesting that dermatan sulfate stimulates HCII by producing more effective displacement of the amino terminus.

Biologically potent analogues of salmon calcitonin which do not contain an N-terminal disulfide-bridged ring structure.

The disulfide bridge formed between the cysteine residues at positions 1 and 7 of salmon calcitonin (sCT) is not required for biological activity. The analogues [Ala1,7]sCT,[AcmCys1,7]sCT and [AmcCys1,Ala7]sCT (AcmC = S-acetamido-methylcysteine) are linear sequences which retain full hypocalcemic activity in the intact rat and ability to activate adenylate cyclase of rat renal membranes. The secondary structure of these peptides in aqueous solution in the presence or absence of lipid is not greatly perturbed by the opening of the disulfide ring. In contrast with salmon calcitonin, substitution of Cys by AcmCys in human calcitonin results in greatly reduced hypocalcemic activity but no loss in the ability of the peptide to activate renal adenylate cyclase. Thus in vitro activation of adenylate cyclase by human calcitonin analogues is not always correlated with in vivo hypocalcemic potency.

Cellular binding and/or uptake of nickel(II) ions.

L-Histidine (L-His) and human serum albumin (HSA) at physiological concentrations, like the exogenous ligands D-penicillamine (D-PEN) and EDTA, are shown to inhibit the uptake of physiological levels of Ni2+ by B-lymphoblasts of human origin, human erythrocytes and rabbit alveolar macrophages. Evidence is also presented that illustrates the ability of these ligands to sequester Ni2+ from cells preloaded with this ion. The experimental observations are interpreted to indicate that serum concentrations of HSA and amino acids (especially L-His) exert a controlling influence on the cellular accumulation of Ni2+ in vitro, and further suggest the necessity to standardize the concentrations of these and related constituents in cell-culture media for meaningful comparisons of cellular uptake and toxicity of Ni2+ and other metal ions. Cells were lysed and the fractional distribution of 63Ni2+ in the lysate and residual pellet were assessed. About 60% of the radiolabel occurred in the pellet and 40% in the lysate for B-lymphoblasts, compared to 70 and 30% respectively for the alveolar macrophages. Diethyldithiocarbamate (DDC), unlike the other complexing agents, enhanced the cellular uptake of Ni2+ and prevented its removal from loaded cells. DDC also induced a transfer of the 63Ni2+ from the lysate to the residual pellet, suggesting that it promotes the deposition of Ni2+ in the lipid-rich components of cells (tissues). It is concluded that cellular association of Ni2+ is favoured by ligands forming lipophilic complexes, and extracellular localization by those giving hydrophilic complex compounds. The relevance of these contrasting roles in nickel detoxification by endogenous ligands and chelating drugs is discussed.

Chemical and biological reactivity of insoluble nickel compounds and the bioinorganic chemistry of nickel.

The basic concepts describing the surface properties of binary metal compounds at solid-solution interfaces are reviewed. For hydrated surfaces, the development of surface charge is dependent on pH and the extent of specific adsorption of cations and anions. It is concluded from data on haemolysis of human erythrocytes, and surface adsorption of proteins and cations that surface passivity (external smoothness/crystallinity, low surface charge) combined with low to moderate body fluid solubility correlate with published carcinogenicities. In a review of the coordination chemistry of Ni2+, the following chemical properties are identified as determinants of biological reactivity: size, stereochemistry, binding preferences, complex stability, kinetic lability, and redox properties. New developments in the association of Ni2+ with proteins suggest: a role for the Ni3+/Ni2+ redox couple in bacterial proteins; the involvement of the primary copper/nickel binding site of human serum albumin in antigen recognition by antibodies with nickel-related specificity; and that Ni2+ can act as an antagonist of essential metal ions. It is concluded that there is considerable scope for work elucidating the surface properties of binary nickel compounds, and that the in vivo displacement of Mg2+, Ca2+, Zn2+ and perhaps other ions by Ni2+ constitutes a useful mechanistic concept in nickel toxicology.