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S Niewiarowski

Publications and source records attributed to S Niewiarowski.

At least 55 records · Page 3Linked to original sources
PubMed1995

Effects of the disintegrin eristostatin on individual steps of hematogenous metastasis.

Adhesion molecules, including integrins, are important for interactions of cancer cells with vessel walls, a step leading to cancer metastasis. Disintegrins block the action of integrins by binding to them. We tested the hypothesis that the disintegrin eristostatin would block metastasis by interfering with cancer cell adhesion to vessel walls, thus preventing extravasation. Experimental metastasis assays, in which B16F1 melanoma cells (controls vs eristostatin-treated, 25 micrograms/ml) were injected via mesenteric veins of anesthetized C57BL/6 mice, showed that eristostatin reduced (P < 0.05) the mean number of liver metastases from 14.4 to 0.6 at 11 days postinjection (p.i.). We examined three different steps in metastasis at which eristostatin could have exerted its effect, namely, cell arrest, extravasation, and migration. Control and eristostatin-treated B16F1 cells were fluorescently labeled and examined by videomicroscopy in liver microcirculation in vivo at various times up to 14 days p.i. Measurements of vessel size in which cell arrest occurred and length/width ratio of arrested cells showed only small differences between control and eristostatin-treated cells. Eristostatin treatment did not prevent extravasation, and the timing and process of extravasation were similar for both treated and control cells; by 3-4 days p.i. more than 90% of the cells had extravasated or were in the process. Eristostatin also did not affect the ability of extravasated cells to migrate through the extracellular matrix to the subcapsular region where tumors later form. Therefore, we conclude that eristostatin exerted its primary effect by regulating the number of individual cancer cells that grow after extravasation.

Animals↗
PubMed1995

Iloprost and echistatin protect platelets during simulated extracorporeal circulation.

Temporary, reversible inhibition of platelets during cardiopulmonary bypass is an attractive strategy to protect platelets and normalize postoperative bleeding times. Iloprost, an analogue of prostacyclin, and the disintegrins reversibly inhibit platelets by different mechanisms. We tested the hypothesis that reduced doses of iloprost and either echistatin, a natural disintegrin, or RO43-5054, a peptidomimetic, in combination provide better platelet protection than any drug alone during simulated extracorporeal circulation. Thirty-five recirculation studies using fresh, heparinized human blood in an extracorporeal perfusion circuit that contained a 0.45-m2 spiral coil membrane oxygenator were performed. Iloprost, but neither echistatin nor RO43-5054, increased platelet cyclic adenosine monophosphate. Combinations of iloprost and either fibrinogen receptor antagonist at reduced doses submaximally increased platelet cyclic adenosine monophosphate. Platelet adhesion and release of beta-thromboglobulin antigen was completely inhibited by combinations of the two classes of drugs, but only partially inhibited by each drug alone. Combinations of drugs also completely inhibited platelet aggregation to adenosine diphosphate; these platelets retained full sensitivity to adenosine diphosphate after 90 minutes of recirculation when drugs were removed by gel filtration. We conclude that combinations of iloprost and a fibrinogen receptor antagonist at doses that are unlikely to produce clinical side effects completely inhibit platelet activation and preserve platelet function during in vitro extracorporeal circulation.

Adenosine Diphosphate↗
PubMed1995

Reversal of heparin anticoagulation by recombinant platelet factor 4 and protamine sulfate in baboons during cardiopulmonary bypass.

The ability of recombinant platelet factor 4 and protamine to neutralize heparin after cardiopulmonary bypass was compared in anesthestized baboons. Clotting titration curves of heparinized baboon blood demonstrate an anticoagulant effect of protamine that is not seen with recombinant platelet factor 4. Neither drug caused meaningful changes in central pressures or cardiac output within 30 minutes after injection. After 30 minutes of cardiopulmonary bypass, recombinant platelet factor 4 normalized thrombin times and activated partial thromboplastin times within minutes of injection, but protamine did not. Neither drug altered bleeding times. Recombinant platelet factor 4 caused a species-specific leukopenia in baboons and significantly increased activated complement protein 3 (C3a) more than protamine. However, the increase in plasma C3a was small and neither drug caused a significant increase in plasma neutrophil elastase-alpha 1 proteinase inhibitor complex. We conclude that recombinant platelet factor 4 is effective and safe in baboons, does not have an anticoagulant effect with excess concentration, and reverses in vivo heparin more rapidly than protamine. The data support progression to a clinical trial.

Animals↗
PubMed1995

Effect of four disintegrins on the adhesive and metastatic properties of B16F10 melanoma cells in a murine model.

Four disintegrins, eristostatin, albolabrin, barbourin and echistatin, injected IV into C57BL/6 mice in combination with B16F10 murine melanoma cells, inhibited formation of experimental lung metastases with ID50s of 0.05, 1.0, 0.9, and 3.7 mumoles per mouse, respectively. When injected 1 h after tumor cells, albolabrin, echistatin and barbourin had the same antimetastatic activity, while eristostatin was not active. Eristostatin (IC50 7-8 nM) was more potent than echistatin (IC50 74-75 nM), barbourin (IC50 46-60 nM), and albolabrin (IC50 130-165 nM) as an inhibitor of murine platelet aggregation induced by ADP or tumor cells. Fibronectin was the best substrate for melanoma cell adhesion (95%), followed by laminin (47%) and vitronectin (24%). Albolabrin was the strongest and eristostatin the weakest inhibitor of cell adhesion to all substrata. Adhesion of melanoma cells to albolabrin, echistatin, and barbourin was partially inhibited by monoclonal antibody against mouse alpha v subunit. This antibody bound to B16F10 melanoma cells in suspension and inhibited binding of fluorescein isothiocyanate (FITC)-labeled disintegrins to these cells, being the most effective with FITC-labeled albolabrin. Our study suggests that a major contribution of eristostatin to inhibition of lung colonization is via preferential binding to platelet alpha IIb beta 3 integrin and blocking tumor cells interaction with platelets. A major contribution of albolabrin, barbourin and echistatin appears to be by interference with other integrin receptors on the tumor cell surface. Albolabrin appeared to inhibit RGD-dependent integrins containing alpha v subunit, such as alpha v beta 3 and alpha v beta 1.

Amino Acid Sequence↗
PubMed1995

Interaction of echicetin with a high affinity thrombin binding site on platelet glycoprotein GPIb.

Echicetin, a protein isolated from Echis carinatus snake venom, inhibited platelet aggregation and secretion induced by low concentrations of thrombin ( < 0.2 U/ml), by binding to platelet glycoprotein Ib (GPIb). The inhibition was not observed when the platelets were stimulated with higher concentrations of thrombin ( > 0.2 U/ml). Echicetin competed with thrombin for binding to the high affinity site on GPIb. Thrombin also inhibited 50% of the binding of 125I-echicetin to the platelets.

Amino Acid Sequence↗
PubMed1995

A comparison of the effect of decorsin and two disintegrins, albolabrin and eristostatin, on platelet function.

Naturally-occurring fibrinogen receptor antagonists and platelet aggregation inhibitors that are found in snake venom (disintegrins) and leeches share many common features, including an RGD sequence, high cysteine content, and low molecular weight. There are, however, significant selectivity and potency differences. We compared the effect of three proteins on platelet function: albolabrin, a 7.5 kDa disintegrin, eristostatin, a 5.4 kDa disintegrin in which part of the disintegrin domain is deleted, and decorsin, a 4.5 kDa non-disintegrin derived from the leech Macrobdella decora, which has very little sequence similarity with either disintegrin. Decorsin was about two times less potent than albolabrin and six times less potent than eristostatin in inhibiting ADP-induced human platelet aggregation. It had a different pattern of interaction with glycoprotein IIb/IIIa as compared to the two disintegrins. Decorsin bound with a low affinity to resting platelets (409 nM) and to ADP-activated platelets (270 nM), and with high affinity to thrombin activated platelets (74 nM). At concentrations up to 685 nM, it did not cause expression of a ligand-induced binding site epitope on the beta 3 subunit of the GPIIb/IIIa complex. It did not significantly inhibit isolated GPIIb/IIIa binding to immobilized von Willebrand Factor. At low doses (1.5-3.0 micrograms/mouse), decorsin protected mice against death from pulmonary thromboembolism, showing an effect similar to eristostatin. This suggested that decorsin is a much more potent inhibitor of platelet aggregation in vivo than in vitro, and it may have potential as an antiplatelet drug.

Amino Acid Sequence↗
PubMed1995

Methylcellulose protects the ability of anchorage-dependent cells to adhere following isolation and holding in suspension.

Preparation of suspension of anchorage-dependent cells growing in tissue cultures requires removal of the cells from their substrata by means of trypsin and/or EDTA. The purpose of the experiments was to investigate the effect of methylcellulose on the adhesive ability of cells removed from substrata by EDTA and trypsin. Human umbilical cord vein endothelial cells (HUVEC), after detachment from substrata, adhere well to fibronectin at 37 degrees C but not at 0 degrees C. During a 60-min incubation at 37 degrees C, these cells lose about 70% of their ability to adhere to fibronectin. The ability of cells to adhere was restored in the presence of 0.2% methylcellulose. Methylcellulose also prevented human skin fibroblasts, human melanoma cells and mouse lung fibroblasts from losing adhesive properties. By contrast, it did not affect the adhesive ability of B16F10 melanoma cells incubated at 37 degrees C. In conclusion, our study suggests that methylcellulose can be a useful reagent for preservation of cell function in suspension; it may also simplify some experimental procedures including radioiodination of cell surface components and cross-linking of radiolabeled ligands to the cell surface.

Animals↗
PubMed1994

Isolation, characterization and amino acid sequence of echicetin beta subunit, a specific inhibitor of von Willebrand factor and thrombin interaction with glycoprotein Ib.

Echicetin is a dimeric protein isolated from the venom of Echis carinatus that is a potent inhibitor of von Willebrand Factor and thrombin binding to glycoprotein Ib. Here, we report isolation and amino acid sequence of the beta subunit of echicetin that contains 123 amino acids, including 7 cysteines, and shows similarity with amino acid sequences of botrocetin and Factor IXa/Xa binding protein. We provide evidence that biological activity of echicetin which resides in this beta subunit is relatively resistant to reduction of the molecule.

Amino Acid Sequence↗
PubMed1994

Structural requirements of platelet chemokines for neutrophil activation.

Using recombinantly expressed proteins and synthetic peptides, we examined the structural/functional features of the platelet chemokines, neutrophil-activating peptide-2 (NAP-2) and platelet factor 4 (PF4); that were important in their activation of neutrophils. Previous studies with the chemokine interleukin-8 (IL-8) had shown that the N-terminal region preceding the first cysteine residue was critical in defining neutrophil-activating properties. We examined whether NAP-2 and PF4 had similar structural requirements. In the Ale-glu-leu-arg (AELR) N-terminus of NAP-2, substitution of E or R abolished Ca2+ mobilization and elastase secretion. Unlike the parent molecule PF4, AELR/PF4, the hybrid formed by replacing the N-terminal sequence of PF4 before the first cysteine residue with the homologous sequence of NAP-2, stimulated Ca2+ mobilization and elastase secretion. Furthermore, the effect of amino acid substitutions in the ELR motif differed from those seen with NAP-2 in that conserved substitutions of E or R in NAP-2 abolished activity, but only reduced neutrophil activation in the hybrid. These studies show that just as with IL-8, the N-termini of NAP-2 and PF4 are critical for high-level neutrophil-activating function. Desensitization studies provided information on receptor binding. NAP-2, which binds almost exclusively to the type 2 IL-8 receptor (IL-8R), did not desensitize neutrophils to activation by IL-8 because IL-8 could bind to and activate via both type 1 and 2 IL-8R. AELR/PF4 appears to bind to both types of receptors because it desensitized neutrophils to NAP-2 activation; but was not desensitized by NAP-2, and because it desensitized to and was desensitized by IL-8. Thus, although NAP-2 and AELR/PF4 share approximately 60% amino acid homology, they have different receptor affinities. Studies were performed to define the role of the C-termini of these platelet chemokines in receptor binding. Heparin and a monoclonal antibody specific for the heparin-binding domain of PF4 both inhibited Ca2+ mobilization and elastase release, further suggesting that the C-terminus of these chemokines is important in receptor binding. Synthetic NAP-2(51-70) failed to mobilize Ca2+, whereas PF4(47-70) and PF4(58-70) induced Ca2+ mobilization and secretion of elastase at high concentrations. Pertussis toxin inhibited neutrophil activation by 40% to 50%, establishing a role for G-protein-coupled receptors such as the IL-8Rs in activation by the PF4 C-terminal peptides.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1994

Characterization of the cross-linking site of disintegrins albolabrin, bitistatin, echistatin, and eristostatin on isolated human platelet integrin GPIIb/IIIa.

Disintegrins, a family of low molecular weight, RGD-containing peptides found in snake venoms prevent the binding of adhesive ligands to a number of integrin receptors. Albolabrin, bitistatin, echistatin, and eristostatin bind to the platelet fibrinogen receptor (GPIIb/IIIa) acting thus as potent inhibitors of platelet aggregation. Here, we have determined the cross-linking of these disintegrins on isolated GPIIb/IIIa. The cross-linking site of all of them was within GPIIIa 217-302, a domain that has been implicated in a number of receptor functions including heterodimer association, activation-dependent conformational changes, and fibrinogen binding.

Amino Acid Sequence↗
PubMed1994

Interaction of disintegrins with the alpha IIb beta 3 receptor on resting and activated human platelets.

Viper venom disintegrins contain the RGD/KGD motif. They inhibit platelet aggregation and cell adhesion, but show structural and functional heterogeneity. We investigated the interaction of four prototypic disintegrins with alpha IIb beta 3 expressed on the surface of resting and activated platelets. The binding affinity (Kd) of 125I-albolabrin, 125I-echistatin, 125I-bitistatin and 125I-eristostatin toward resting platelets was 294, 153, 48 and 18 nM respectively. The Kd value for albolabrin decreased 3-fold and 6-fold after ADP- or thrombin-induced activation. The Kd values for bitistatin and echistatin also decreased with ADP, but there was no further decrease with thrombin. In contrast, eristostatin bound with the same high affinity to resting and activated platelets. The pattern of fluorescein isothiocyanate (FITC)-eristostatin and FITC-albolabrin binding to resting and activated platelets was consistent with observations using radiolabelled material. Eristostatin showed faster and more irreversible binding to platelets, and greater potency compared with albolabrin in inducing conformational neo-epitopes in beta 3. The anti-alpha IIb beta 3 monoclonal antibody OP-G2 that is RGD-dependent inhibited disintegrin binding to activated platelets more strongly than binding to resting platelets and it inhibited the binding to platelets of albolabrin more strongly than eristostatin. The specificity of disintegrin interaction with alpha IIb beta 3 was confirmed by demonstrating cross-linking of these peptides to alpha IIb beta 3 on normal platelets, but not to thrombasthenic platelets deficient in alpha IIb beta 3.

Adenosine Diphosphate↗
PubMed1994

Comparison of disintegrins with limited variation in the RGD loop in their binding to purified integrins alpha IIb beta 3, alpha V beta 3 and alpha 5 beta 1 and in cell adhesion inhibition.

The inhibitory capacities of six different disintegrins and one related neurotoxin analogue for the binding of RGD-dependent integrins to either fibrinogen, vitronectin or fibronectin were compared in solid phase assays. Echistatin and flavoridin were the most active inhibitors for alpha V beta 3 and alpha 5 beta 1 integrins and moderately exceeded the activity of the natural protein ligands. The same disintegrins together with eristostatin, bitistatin and barbourin were also very potent inhibitors of fibrinogen binding to alpha IIb beta 3 integrin. For all three integrins, albolabrin showed the lowest affinity, but it still clearly exceeded that of synthetic GRGDS. However, assay conditions may determine these relative affinities, as shown for the alpha IIb beta 3 and alpha V beta 3 integrins when used either in immobilized or soluble form. For alpha IIb beta 3, however, a close correlation was found between KD values determined in platelet binding assays and the concentrations required for half maximal inhibition of three disintegrins. The inhibiting capacity of disintegrins in assays with purified integrins also correlated reasonably well with their inhibition of cell attachment to RGD-dependent protein substrates. However, sequence differences in the RGD loops of the various disintegrins may not fully account for the 20-100-fold difference in their binding capacities. This was particularly evident for echistatin and albolabrin, which differ in this region only by two conservative substitutions but have considerably different inhibitory activities. More remote regions of the disintegrins and alignment of disulfide bridges are therefore likely to contribute to their affinity and selectivity.

Amino Acid Sequence↗
PubMed1994

Alpha 1-antitrypsin Pittsburgh (Met358-->Arg) inhibits the contact pathway of intrinsic coagulation and alters the release of human neutrophil elastase during simulated extracorporeal circulation.

Cardiopulmonary bypass prolongs bleeding time, increases postoperative blood loss, and triggers activation of plasma proteolytic enzyme systems and blood cells referred to as the "whole body inflammatory response". Contact of blood with synthetic surfaces leads to qualitative and quantitative alterations in platelets, neutrophils, contact and complement systems. Contact and complement pathway proteins both induce neutrophil activation. alpha 1-antitrypsin Pittsburgh (Met358-->Arg), a mutant of alpha 1-antitrypsin, is a potent inhibitor of plasma kallikrein and thrombin. We investigated whether this recombinant mutant protein inhibited platelet activation, as well as contact and/or complement-induced neutrophil activation during simulated extracorporeal circulation. Arg358 alpha 1-antitrypsin did not prevent the 34% drop in platelet count at 5 min of recirculation, did not block the 50% decrease in ADP-induced platelet aggregation at 120 min of recirculation, nor inhibit the release of 6.06 +/- 1.07 micrograms/ml beta-thromboglobulin at 120 min of recirculation suggesting that the inhibitor had little effect on platelet activation. However, Arg358 alpha 1-antitrypsin totally blocked kallikrein-C1-inhibitor complex formation but not C1-C1-inhibitor complex formation. Most importantly, Arg358 alpha 1-antitrypsin decreased the release of 1.11 +/- 0.16 micrograms/ml human neutrophil elastase by 43%. The attenuation of neutrophil activation in the absence of an effect on complement activation via the classical pathway, supports the concept that kallikrein is a major mediator of neutrophil degranulation during cardiopulmonary bypass.

Arginine↗
PubMed1994

Disintegrins and other naturally occurring antagonists of platelet fibrinogen receptors.

Disintegrins represent a family of low-molecular weight, cysteine-rich, RGD-containing peptides that inhibit fibrinogen binding to glycoprotein IIb/IIIa complex as well as binding of other ligands to RGD-dependent integrins on the surface of other cells. Disintegrins occur in the venom of various vipers. Disintegrin domains have been identified in viper venom hemorrhagins and in sperm proteins involved in the sperm-egg fusion. Amino acid sequences of 25 disintegrins, alignment of S-S bridges in four disintegrins, and stereo models of five disintegrins are presented. Primary structures of disintegrins differ significantly from other fibrinogen receptor antagonists occurring in the venoms of Elapidae, leeches, and ticks. Several aspects of structure-function relationship of disintegrins, their biological activities, and possible clinical applications are discussed.

Amino Acid Sequence↗