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S Niewiarowski

Publications and source records attributed to S Niewiarowski.

At least 19 recordsLinked to original sources
PubMed1992

The disulfide bridge pattern of snake venom disintegrins, flavoridin and echistatin.

Flavoridin and echistatin, isolated from the venom of Trimeresurus flavoviridis and Echis carinatus, respectively, belong to the disintegrin family of integrin beta 1 and beta 3 inhibitors of low molecular weight RGD-containing, cysteine-rich peptides. Since disulfide bonds are critical for expression of biological activity, we sought to determine their location in these two proteins. In flavoridin, direct evidence for the existence of linkage between Cys4-Cys19 and between Cys45 and Cys64 was obtained by analysis of proteolytic products, and indirect evidence suggests links between Cys6-Cys14 and Cys13-Cys36. In echistatin, links between Cys8-Cys37 and Cys20-Cys39 were identified by direct chemical analysis.

Amino Acid Sequence↗
PubMed1992

Binding of glycoprotein IIIa-derived peptide 217-231 to fibrinogen and von Willebrand factors and its inhibition by platelet glycoprotein IIb/IIIa complex.

Based on previous reports in the literature and the high homology between platelet glycoprotein (GP) IIIa 217-231 and similar portions of other beta subunits of integrin receptors, we hypothesized that this region may participate in ligand binding. Using a polyclonal antibody against GPIIIa 217-231(YC), we tested the interaction of a synthetic peptide representing this region with fibrinogen (Fg), in the enzyme-linked immunosorbent assay (ELISA) system. Results show a calcium-independent, dose-related, direct interaction between GPIIIa 217-231(Y) and immobilized Fg. This peptide also bound to von Willebrand Factor (vWF) and fibronectin (Fn), but did not attach to a 50 kDa Fn fragment which is deficient in the cell attachment site. In addition, purified GPIIb/IIIa displaced GPIIIa 217-231(Y) from Fg and vWF. Binding of 125I-GPIIIa 217-231(Y) to Fg coated tubes was inhibited by soluble Fg and by the GPIIb/IIIa complex. We synthesized this peptide with several alterations; similar peptides with Pro-219 replaced with an Ala showed significantly reduced binding to Fg and vWF. The decreased binding of the peptides with Pro-219 substitutes suggests that the confirmation of GPIIIa 217-230 is important for its ability to bind to adhesive ligands. In conclusion, the amino acid residues between 217 and 231 of GPIIIa appear to be involved in ligand binding and Pro-219 probably plays a significant role in this interaction.

Amino Acid Sequence↗
PubMed1992

Platelet factor 4 efficiently reverses heparin anticoagulation in the rat without adverse effects of heparin-protamine complexes.

BACKGROUND: It has been observed that the reversal of heparin anticoagulation in humans by protamine sulfate (PS) results in various adverse reactions including leukopenia, thrombocytopenia, activation of complement, increased vascular permeability, systemic hypotension, pulmonary vasoconstriction, and pulmonary edema. The purpose of this study was to compare the efficacy and effects of native platelet factor 4 (PF4) and recombinant platelet factor 4 (rPF4) with those of PS in heparin neutralization in vivo, using a rat model. METHODS AND RESULTS: Sprague-Dawley rats were anesthetized with sodium pentobarbital, and the right femoral vein and carotid artery were cannulated. For determination of activated partial thromboplastin time, platelet count, white blood cell count, and complement titer, arterial blood samples were taken before and immediately after heparin (10 units/100 g) infusion and at several time points after the infusion of the neutralizing agent (PS, 0.1 mg/100 g; PF4, 0.5 mg/100 g). In separate groups of animals, mean arterial blood pressure was monitored throughout identical protocols and the lungs were prepared for histological examination. The anticoagulant activity of heparin was effectively reversed by all of the neutralizing agents (PS, PF4, and rPF4). Platelet count (48% of initial), white blood cell count (52% of initial), complement titer (60% of initial), and mean arterial pressure (20% decrease) decreased significantly in heparinized animals receiving PS but not in those receiving PF4 or rPF4. Lung interstitium appeared normal when heparin was followed by PF4; however, interstitial edema and hemorrhage were observed with heparin-PS. CONCLUSIONS: These results suggest that PF4 efficiently reverses heparin anticoagulation in the rat without the adverse effects of heparin-protamine complexes. Therefore, rPF4 may be an appropriate substitute for PS in patients undergoing cardiovascular surgery and other procedures that require heparin anticoagulation.

Animals↗
PubMed1992

Isolation, characterization, and immunological detection of neutrophil-activating peptide 2: a proteolytic degradation product of platelet basic protein.

Neutrophil-activating peptide 2 (NAP-2), corresponding to platelet basic protein fragment 25-94, was prepared by chymotryptic digestion of its precursors, low affinity platelet factor 4 or beta-thromboglobulin, followed by purification by high performance liquid chromatography. NAP-2 (0.1-1.5 microns) caused the release of human granulocyte elastase from cytochalasin B-treated neutrophils in a dose-dependent manner. In the same system, beta-thromboglobulin, human platelet factor 4, S-pyridylethyl NAP-2, and platelet basic protein C-terminal fragment (77-94) were inactive, whereas platelet basic protein fragment 22-89 had low, but significant, activity. Sensitive immunological identification of NAP-2 based on nonequilibrium isoelectric focusing and immunoblotting is described.

Amino Acid Sequence↗
PubMed1992

Protection of platelets during long-term extracorporeal membrane oxygenation in sheep with a single dose of a disintegrin.

BACKGROUND: Both short- and long-term extracorporeal membrane oxygenation (ECMO) causes platelet loss and dysfunction. Bitistatin is a reversible inhibitor of the platelet glycoprotein IIb/IIIa receptor. This study tests the hypothesis that inhibition of platelets by bitistatin during initial contact with the perfusion circuit preserves platelet number and function during long-term ECMO in sheep. METHODS AND RESULTS: Bitistatin, purified from crude snake venom, was tested for its effect on platelet count, responsiveness to ADP, release of platelet factor 4, and prevention of surface-adsorbed glycoprotein IIIa in vitro and during 24 hours of ECMO in nine splenectomized sheep. During simulated extracorporeal circulation, 0.5-1.0 microgram/ml bitistatin significantly prevented platelet adhesion, attenuated release of sheep platelet factor 4, and preserved platelet responsiveness to ADP. During ECMO at 1.8 l/min for 24 hours, a single dose of bitistatin (200 micrograms/kg) (n = 4) produced higher platelet counts (p = 0.0002) and suppressed release of platelet factor 4 (p = 0.035) for 16 hours compared with five control animals. This dose of bitistatin caused an immediate inhibition of platelet aggregation; however, between 4 and 24 hours of perfusion, platelets of bitistatin-treated animals were more responsive to ADP (p < 0.0001) compared with platelets in control animals. The amount of glycoprotein IIIa antigen extracted by Triton X-100 from the perfusion circuits was reduced in bitistatin-treated sheep. CONCLUSIONS: A single dose of bitistatin given before blood contact with the ECMO circuit briefly inhibits platelet adhesion and aggregation but thereafter preserves platelet numbers and function and suppresses alpha-granule release for 12-16 hours of ECMO.

Adenosine Diphosphate↗
PubMed1991

Ovine platelet factor 4: purification, amino acid sequence, radioimmunoassay and comparison with platelet factor 4 of other species.

A simple method of purification of ovine platelet factor 4 (PF4) is described. Material released by freezing and thawing suspension of washed sheep platelets was fractionated by heparin-agarose chromatography and reverse phase HPLC. Purified ovine PF4 contained 85 amino acids (Mr 9130) and showed 78% homology with bovine PF4, 76% with porcine PF4, 71% homology with human PF4, and 61% with rat PF4. The heparin binding site of ovine PF4 localized in the C-terminal region of the molecule was identified as LYKKIIKRLL. The content of PF4 determined by radioimmunoassay was 22.6 (+/- 1.6 S.D.) micrograms per 10(9) platelets and 46.8 (+/- 19.6 S.D.) ng per ml platelet poor plasma collected in acid citrate dextrose in the presence of prostaglandin E1. PF4 was rapidly released during stimulation of ovine platelets by collagen.

Amino Acid Sequence↗
PubMed1991

Identification of the disulfide bond pattern in albolabrin, an RGD-containing peptide from the venom of Trimeresurus albolabris: significance for the expression of platelet aggregation inhibitory activity.

Albolabrin is a 73 amino acid peptide isolated from the venom of Trimeresurus albolabris. It contains an RGD sequence and 12 cysteines and is a potent inhibitor of both platelet aggregation and fibrinogen binding to the GPIIb/IIIa complex. This protein shows a high degree of analogy (primarily due to the alignment of all cysteines and the RGD) with a number of other viper venom proteins which inhibit cell adhesion and platelet aggregation and are referred to as disintegrins: rhodostomin, trigramin, flavoridin, applagin, elegantin, and batroxostatin. In this study, we found that the reduction and vinylpyridylethylation of albolabrin and flavoridin decreased their platelet aggregation inhibitory activity approximately 40-50 times. It can be postulated that the higher potency of native and reduced flavoridin as compared to albolabrin depends on the substitution of the Asp of albolabrin with a Phe at the C-terminal end of the RGD in flavoridin. The activity of a synthetic C-terminal peptide derived from flavoridin (residues 35-65) containing four cysteines was about 75 times lower than that of the original flavoridin. The substitution of a pair of cysteine residues with alanines in this peptide resulted in further loss of activity. In order to identify the disulfide bonds in albolabrin, the molecule was digested consecutively by trypsin and porcine pancreatic elastase. Peptides resulting from this digestion were isolated by reverse-phase HPLC and identified by amino acid composition and mass spectrometry.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate↗
PubMed1991

Inhibition of murine melanoma cell-matrix adhesion and experimental metastasis by albolabrin, an RGD-containing peptide isolated from the venom of Trimeresurus albolabris.

Albolabrin, a 7.5-kDa cysteine-rich protein isolated from the venom of Trimeresurus albolabris, contains the arginine-glycine-aspartic acid (RGD) cell recognition sequence found in many cell adhesion-promoting extracellular matrix proteins, such as fibronectin and laminin. Albolabrin belongs to a family of RGD-containing peptides, termed disintegrins, recently isolated from the venom of various vipers and discovered to be potent inhibitors of both platelet aggregation and cell-substratum adhesion. Here we report that albolabrin inhibited the attachment of B16-F10 mouse melanoma cells to either fibronectin or laminin absorbed on plastic. When immobilized on plastic, albolabrin promoted B16-F10 melanoma cell attachment; this was inhibited by either RGD-serine (RGDS) or antibodies to integrins, suggesting that albolabrin binds via its RGD amino sequence to integrin receptors expressed on the melanoma cell surface. In an in vivo experimental metastasis system, albolabrin at a concentration of 300-600 nM inhibited C57BL/6 mouse lung colonization by tail vein-injected mouse melanoma cells and was at least 2000 times more active than RGDS in this assay. We propose that albolabrin inhibits tumor cell metastasis by inhibiting integrin-mediated attachment of melanoma cells to RGD-containing components of the extracellular matrix in the mouse lung.

Animals↗
PubMed1991

Cyclosporin A augments human platelet sensitivity to aggregating agents by increasing fibrinogen receptor availability.

Clinical use of cyclosporin A (CsA) has been associated with platelet hypersensitivity and an increased incidence of thrombotic and vasoactive events. The purpose of this study was (1) to confirm that CsA enhances platelet sensitivity to the soluble agonists, adenosine diphosphate (ADP) and epinephrine (EPI), and (2) to determine if this enhancement is mediated by alteration in the availability of platelet surface fibrinogen receptors, a final mediator of platelet activation. Mean log dose of ADP required to achieve complete second-wave platelet aggregation in vitro decreased from 1.90 to 1.49 microM (n = 19, paired t test, P less than 0.05) and 2.86 to 2.11 microM (n = 16, P less than 0.05) following a 15-min and 3-hr incubation in the absence (saline) and presence of CsA (1000 ng/ml), respectively. At the threshold dose of ADP, concurrent thromboxane B2 levels at 15 min were 245 +/- 44 ng/ml (n = 12, saline) and 265 +/- 54 ng/ml (n = 9, CsA; P greater than 0.05). At 3 hr respective levels were 333 +/- 57 and 442 +/- 81 ng/ml (P greater than 0.05). Similar results were obtained with EPI. The number of fibrinogen binding sites in response to 50 microM ADP was determined in washed platelets in the absence and presence of CsA by radioligand binding. In 6 of 7 volunteers, CsA increased fibrinogen receptors from 26,635 +/- 4841 to 35,925 +/- 7290 sites/platelet (means +/- SEM; P less than 0.05). No change in receptor affinity was noted. In conclusion, cyclosporine does augment platelet reactivity.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate↗
PubMed1991

Characterization of an antibody raised against reduced glycoprotein IIIa of human platelets.

A polyclonal antibody against reduced and vinylpyridylethylated human glycoprotein IIIa was raised in rabbits. Its reactivity with reduced GPIIIa was about 500 times higher than that of the antibody against native GPIIIa. The lowest amounts of purified reduced and native GPIIIa recognized by the antibody against reduced GPIIIa were 25 and 400 ng, respectively. The antibody did not recognize native GPIIIa (about 1-2 micrograms) in platelet extracts and chymotryptic degradation products of GPIIIa. It inhibited ADP-induced platelet aggregation but it did not inhibit fibrinogen binding to ADP-stimulated platelets. Our experiments suggest that the antigenicity of GPIIIa (beta 3 integrin) depends on the conformation of the molecule determined by numerous S-S bridges between cysteine residues.

Antibodies↗
PubMed1990

Platelet thromboxane A2/prostaglandin H2 receptors in human volunteers on low doses of aspirin.

Administration of aspirin (81 mg/day for 2-3 weeks) in nine healthy volunteers (out of an initial ten subjects, only nine qualified) resulted in a greater than 95% decrease of thromboxane B2 production by thrombin-stimulated platelets. At the same time, ligand binding studies with a thromboxane A2 antagonist, 125I-PTA-OH, measurements of shape change, and aggregation of platelets stimulated with U46619, a prostaglandin H2 analogue, indicated that administration of aspirin to normal human subjects does not result in the up-regulation of platelet thromboxane A2/prostaglandin H2 receptors.

Adult↗
PubMed1990

Batroxostatin, an Arg-Gly-Asp-containing peptide from Bothrops atrox, is a potent inhibitor of platelet aggregation and cell interaction with fibronectin.

A potent inhibitor of platelet aggregation and cell adhesion was isolated from the venom of Bothrops atrox. This peptide, referred to as batroxostatin, was composed of 71 amino acids and showed a high degree of homology with other snake venom peptides including trigramin, albolabrin, elegantin and applagin: all 12 cysteines and the RGD sequence (standard one-letter amino acid codes) aligned in the same position. Compared on a molar basis, the anti-platelet aggregation activity of batroxostatin was about 1000-times higher than that of RGDS. In addition, batroxostatin was about 400-times more potent than GRGDS at inhibiting melanoma cell adhesion to fibronectin. Batroxostatin covalently attached to plastic promoted adhesion of melanoma cells. The anti-GP140 antibody, recognizing beta 1 integrins, completely inhibited adhesion of mouse melanoma cells to batroxostatin. This observation, in addition to the inhibitory effect of batroxostatin on the adhesion of chick fibroblasts to fibronectin, suggests that batroxostatin interacts with integrins from both the beta 1 and beta 3 subfamilies.

Amino Acid Sequence↗
PubMed1990

Elegantin and albolabrin purified peptides from viper venoms: homologies with the RGDS domain of fibrinogen and von Willebrand factor.

The RGD-containing peptides isolated from the venoms of the Viperidae constitute a new class of small cysteine-rich peptides of variable amino acid composition and biological activity (Huang, T.-F., et al. (1987) J. Biol. Chem. 262, 16157-16163; Gan, Z.R., et al. (1988) J. Biol. Chem 263, 19827-19832; Huang, T.-F., et al. (1989) Biochemistry 28, 661-668), which it is proposed by Gould et al. (unpublished data) that we call 'disintegrins'. These peptides bind to the glycoprotein IIb-IIIa receptor on the platelet surface and inhibit aggregation induced by ADP, thrombin, platelet-activating factor and collagen. These peptides are also potent inhibitors of cell adhesion to fibrinogen (Knudsen, K.M., et al. (1988) Exp. Cell Res. 179, 42-49). We report the isolation of two further RGD-peptides from the venoms of Trimeserusus elegans and Trimeserusus albolabris, purified to homogeneity with high yield by a novel, rapid reverse-phase HPLC method. The primary structures of these two peptides were determined to be single polypeptide chains of 73 amino acids. Albolabrin differed from trigramin by eight residues whilst elegantin differed by 22 residues. The molecular mass of albolabrin calculated on the basis of amino acid sequence was 7574 Da and the pI similarly calculated was 4.27. The molecular mass of elegantin was calculated to be 7806 Da and the theoretical pI to be 4.69. RGD is maintained in the same position (51-53 AA) and all 12 cysteines are identical. Our data suggest that the presence of RGD, the conserved secondary and tertiary structure, are essential for the expression of biological activity by these peptides. Both peptides inhibited ADP-induced platelet aggregation. Extended homologies around the RGDS sequences in human von Willebrand Factor and bovine fibrinogen were found with both peptides.

Amino Acid Sequence↗
PubMed1990

Disintegrins: a family of integrin inhibitory proteins from viper venoms.

Disintegrins represent a new class of low molecular weight, RGD-containing, cysteine-rich peptides isolated from the venom of various snakes. They interact with the beta 1 and beta 3 families of integrins and their potency is at least 500-2000 times higher than short RGDX peptides. Analysis of the amino acid sequences of 14 different disintegrins suggests that the RGD sequence, in the spatial configuration determined by the appropriate pairing of the cysteine residues, functions as a cell recognition site. However, certain nonconserved amino acids appear to modify the activity of disintegrins, their specificity for various receptors, and their ability to compete specifically with various ligands.

Amino Acid Sequence↗
PubMed1979

Thrombocytin, a serine protease from Bothrops atrox venom. 1. Purification and characterization of the enzyme.

Thrombocytin, a platelet-activating enzyme from Bothrops atrox venom, has been purified to homogeneity by precipitation with sodium salicylate and chromatography on heparin--agarose. Thrombocytin is a single-chain glycoprotein with a molecular weight of 36 000 which contains 5.6% carbohydrate. It causes platelet aggregation, release of platelet serotonin, and activation of factor XIII. The most sensitive substrate for the amidolytic activity of thrombocytin was Tos-Gly-Pro-Arg-p-nitroanilide hydrochloride. The activity of thrombocytin on this substrate and on platelets was inhibited by diisopropyl fluorophosphate (DFP), soybean trypsin inhibitor, and several arginine chloromethyl ketones. Active site titration with nitrophenyl guanidinobenzoate demonstrated that approximately 86% of the preparation was in the active form. These experiments demonstrate the presence of serine and histidine in the active site of thrombocytin and suggest that thrombocytin is a classical serine protease with a platelet-activating activity similar to thrombin.

Amidohydrolases↗
PubMed1979

Thrombocytin, a serine protease from Bothrops atrox venom. 2. Interaction with platelets and plasma-clotting factors.

Thrombocytin, a serine protease from Bothrops atrox venom, caused platelet aggregation and release of platelet constituents at a concentration of 10(-7) M and clot retraction at a concentration of 2 x 10(-9) M. Thrombocytin was slightly more active when tested on platelets in plasma than on washed platelets suspended in Tyrode--albumin solution. Thrombin was 5 times more active than thrombocytin when tested on platelets in plasma and 50 times more active when tested on washed platelets. The patterns or release induced by thrombocytin and thrombin were similar. Prostaglandin E1 (10(-5) M) produced complete inhibition of platelet release induced by thrombocytin and thrombin. Indomethacin (10(-4) M) was without any effect. Antithrombin III, in the presence of heparin, inhibited the action of thrombocytin on platelets and on a synthetic peptide substrate (Tos-Gly-Pro-Arg-pNA.HCl). formation of an antithrombin III--thrombocytin complex was demonstrated on NaDodSO4--polyacrylamide gel electrophoresis. Hirudin and alpha 1-antitrypsin did not inactivate thrombocytin. Thrombocytin had a low fibrinogen-clotting activity (less than 0.06% that of thrombin). Thrombocytin also caused progressive degradation of the alpha chain of human fibrinogen, and it cleaved prothrombin, releasing products similar to intermediate 1 and fragment 1 produced by thrombin. Thrombocytin activated factor XIII by limited proteolysis and increased the procoagulant activity of factor VIII in a manner analogous to that of thrombin.

Animals↗