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S Niewiarowski

Publications and source records attributed to S Niewiarowski.

At least 91 records · Page 5Linked to original sources
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

Human platelet factor 4 and its C-terminal peptides: heparin binding and clearance from the circulation.

Human platelet factor 4 (PF4), a high affinity heparin binding protein, is released from stimulated platelets and stored at vascular sites, predominantly in liver, from where it can be brought back into circulation by heparin. We attempted to define structural requirements for PF4 binding to heparin and for the pattern of its clearance from the circulation. Intact PF4 bound strongly to heparin agarose and was eluted at 1.4 M NaCl, while reduced PF4 and PF4 C-terminal peptides PF4 (47-70) and PF4 (58-70) bound weakly and were eluted at 0.2-0.5 M NaCl. 125I-radiolabeled intact PF4, reduced PF4 and C-terminal PF4 peptides injected into rabbits were cleared from the circulation in a biphasic pattern with components having half-life time of 1-2 min and 20-140 min. Heparin eliminated the fast component of PF4 clearance, but it did not affect clearance of reduced PF4 or C-terminal PF4 peptides. In contrast to reduced PF4 and PF4 (47-70), intact PF4 that accumulated in the liver and spleen, was displaced by heparin into circulating blood. In conclusion, specific binding sites and native conformation of the molecule are critical for high affinity PF4 binding to insolubilized heparin and for a pattern of PF4 clearance from the circulation in the presence of heparin.

Animals↗
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

Inhibition of platelet adhesion to surfaces of extracorporeal circuits by disintegrins. RGD-containing peptides from viper venoms.

Previous studies indicate that exposure of fibrinogen receptors associated with glycoprotein IIb/IIIa complex contributes to platelet loss during cardiopulmonary bypass. Recently, we isolated a number of RGD (Arg-Gly-Asp)-containing, low molecular weight, cysteine-rich peptides from viper venoms. These peptides, which we propose to call "disintegrins," block platelet-fibrinogen interaction and platelet aggregation. We compared the effect of RGDS (Arg-Gly-Asp-Ser) and four disintegrins (echistatin, flavoridin, albolabrin, and bitistatin) on platelet behavior in a membrane oxygenator. During simulated extracorporeal circulation for 2 hours, platelet count decreased to about 30% of initial values. Addition of echistatin (60-200 nM), albolabrin (60-200 nM), bitistatin (60 nM), and flavoridin (45 nM) significantly inhibited platelet loss in the circuit. RGDS (33 microM) did not show any significant inhibitory effect. ADP-induced platelet aggregation was inhibited in samples of platelet-rich plasma taken from the circuits containing disintegrins. However, echistatin appeared to be a more potent inhibitor of platelet aggregation, whereas albolabrin and flavoridin interfered more selectively with platelet loss from the circuit. Echistatin prevented the accumulation of glycoprotein IIIa on the surface of the circuit. Echistatin (60-200 nM), flavoridin (45 nM), bitistatin (60 nM), and albolabrin (200 nM) significantly inhibited the loss of beta-thromboglobulin from platelets into circulating plasma. Electron microscopy studies demonstrated shape change but not degranulation in platelets circulating in the presence of 200 nM echistatin. On the other hand, this peptide (up to 1,000 nM) did not prevent loss of alpha granules and beta-thromboglobulin from thrombin-stimulated platelets, although it prevented their aggregation. In conclusion, disintegrins protect platelets in the circuit by preventing their adhesion to surfaces and, therefore, preventing fragmentation of adhered platelets under the shear stress of flowing blood. This study indicates that disintegrins may be potential candidates for platelet protection during cardiopulmonary bypass.

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↗
PubMed1989

Separation of different receptor-mediated effects of a prostaglandin H2 analogue (U46619) on human platelets by means of human granulocytic elastase and chymotrypsin.

Previous investigations indicated two classes of thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptors on human platelets and suggested that shape change and myosin light chain phosphorylation correlated with the occupancy of high affinity receptors while serotonin release was related to a putative low affinity binding component (Morinelli TA et al., Am J Physiol 253: H1035-H1043, 1987). The current study shows that chymotrypsin destroyed three receptor-mediated responses of platelets to U46619 (a TXA2/PGH2 agonist), i.e. shape change, myosin light chain phosphorylation and serotonin release. Human granulocyte elastase selectively inactivated platelet ability to release serotonin following stimulation with U46619, but it did not affect significantly shape change and myosin light chain phosphorylation. In conclusion, it is possible to separate different receptor-mediated effects of U46619 on human platelets by means of human granulocytic elastase and chymotrypsin.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1989

Structural and functional characterization of major platelet membrane components derived by limited proteolysis of glycoprotein IIIa.

The authors isolated a product of proteolytic degradation of glycoprotein IIIa (GPIIIa) which is formed on the surface of human platelets during incubation with chymotrypsin and which was previously described as the 66 kDa platelet membrane component. This component migrated with an apparent Mr 62,400 in a non-reduced system of sodium dodecyl sulfate polyacrylamide gel electrophoresis. In a reduced system it yielded two major subunits migrating with apparent Mr 14,000-17,000 and 65,000. The low-molecular weight component began with the NH2-terminal sequence of GPIIIa (GPNICTTR...) and the larger component with residue 348 of GPIIIa (GKIRSKKA...) as deduced from a cDNA clone of this glycoprotein. The two subunits appeared to be linked by one or more S-S bridges supporting the contention that GPIIIa is a highly folded molecule on the platelet membrane. In contrast to GPIIIa, the '66 kDa component' did not bind to GRGDSPK-agarose, to fibrinogen-agarose nor to insolubilized monoclonal antibody recognizing the GPIIb/IIIa complex. The exposure of fibrinogen receptors during the course of incubation of platelets with chymotrypsin preceded the formation of the '66 kDa component' characterized in this study. An intermediate product of GPIIIa proteolysis migrating with an apparent Mr 120,000 in a non-reduced system and Mr 80,000 in a reduced system was identified as a precursor of the '66 kDa component'. The '120 kDa component' was not retained on GRGDSPK-agarose or on fibrinogen-agarose but it was retained on insolubilized antibody recognizing the GPIIb/IIIa complex. Incubation of platelets with porcine pancreatic elastase or human granulocytic elastase resulted in the formation of similar proteolytic degradation fragments.

Amino Acid Sequence↗
PubMed1989

Trigramin: primary structure and its inhibition of von Willebrand factor binding to glycoprotein IIb/IIIa complex on human platelets.

Trigramin, a naturally occurring peptide purified from Trimeresurus gramineus (T. stejnegeri formosensis) snake venom, inhibits platelet aggregation and the binding of 125I-fibrinogen to ADP-stimulated platelets (Ki = 2 X 10(-8) M) without affecting the platelet-release reaction. 125I-trigramin binds to ADP-stimulated and to chymotrypsin-treated normal platelets but not to thrombasthenic platelets. 125I-trigramin binding to platelets is blocked by monoclonal antibodies directed against the glycoprotein IIb/IIIa complex and by Arg-Gly-Asp-Ser (RGDS) [Huang et al. (1987) J. Biol. Chem. 262, 161]. We determined the primary structure of trigramin, which is composed of a single polypeptide chain of 72 amino acid residues and six disulfide bridges. The molecular weight of trigramin calculated on the basis of amino acid sequence was 7500, and the average pI was 5.61. An RGD sequence appeared in the carboxy-terminal domain of trigramin. An amino-terminal fragment (7-33) of trigramin showed 39% homology with a region (1555-1581) of von Willebrand factor (vWF). Trigramin also showed 36% identity in a 42 amino acid overlap and 53% identity in a 15 amino acid overlap when compared with two adhesive proteins, collagen alpha 1 (I) and laminin B1, respectively. Trigramin blocked binding of human vWF to the glycoprotein IIb/IIIa complex in thrombin-activated platelets in a dose-dependent manner. Reduction of trigramin resulted in a marked decrease in its ability to block vWF binding to human platelets.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1989

High molecular weight kininogen inhibits fibrinogen binding to cytoadhesins of neutrophils and platelets.

Fibrinogen inhibited 125I-high molecular weight kininogen (HMWK) binding and displaced bound 125I-HMWK from neutrophils. Studies were performed to determine whether fibrinogen could bind to human neutrophils and to describe the HMWK-fibrinogen interaction on cellular surfaces. At 4 degrees C, the binding of 125I-fibrinogen to neutrophils reached a plateau by 30 min and did not decrease. At 23 and 37 degrees C, the amount of 125I-fibrinogen bound peaked by 4 min and then decreased over time because of proteolysis of fibrinogen by human neutrophil elastase (HNE). Zn++ (50 microM) was required for binding of 125I-fibrinogen to neutrophils at 4 degrees C and the addition of Ca++ (2 mM) increased the binding twofold. Excess unlabeled fibrinogen or HMWK completely inhibited binding of 125I-fibrinogen. Fibronectin degradation products (FNDP) partially inhibited binding, but prekallikrein and factor XII did not. The binding of 125I-fibrinogen at 4 degrees C was reversible with a 50-fold molar excess of fibrinogen or HMWK. Binding of 125I-fibrinogen, at a concentration range of 5-200 micrograms/ml of added radioligand, was saturable with an apparent Kd of 0.17 microM and 140,000 sites/cell. The binding of 125I-fibrinogen to neutrophils was not inhibited by the peptide RGDS derived from the alpha chain of fibrinogen or by the mAb 10E5 to the platelet glycoprotein IIb/IIIa heterodimer. Fibrinogen binding was inhibited by a gamma-chain peptide CYGHHLGGAKQAGDV and by mAb OKM1 but was not inhibited by OKM10, an mAb to a different domain of the adhesion glycoprotein Mac-1 (complement receptor type 3 [CR3]). HMWK binding to neutrophils was not inhibited by OKM1. These observations were consistent with a further finding that fibrinogen is a noncompetitive inhibitor of 125I-HMWK binding to neutrophils. Fibrinogen binding to ADP-stimulated platelets was increased twofold by Zn++ (50 microM) and was inhibited by HMWK. These studies indicate that fibrinogen specifically binds to the C3R receptor on the neutrophil surface through the carboxy terminal of the gamma-chain and that HMWK interferes with the binding of fibrinogen to integrins on both neutrophils and activated platelets.

Antibodies, Monoclonal↗
PubMed1989

Inhibition of platelet hemostatic plug formation by trigramin, a novel RGD-peptide.

Trigramin, a cysteine rich, RGD (Arg-Gly-Asp)-containing peptide from Trimeresurus gramineus snake venom (Mr 7,500) has been previously reported to inhibit fibrinogen binding to ADP-activated platelets and platelet aggregation (disassociation constant 10(-8) M). The present study demonstrates that the infusion of trigramin (17-212 micrograms/100 g body wt) significantly prolonged the bleeding time of severed mesenteric arteries in hamsters (anesthetized with 65 mg/kg pentobarbital), whereas the infusion of RGDS (Arg-Gly-Asp-Ser, 0.45-1.0 mg/100 g body wt) failed to increase the bleeding time in this model. The bleeding time immediately returned to normal after cessation of trigramin infusion. The pattern of the disappearance of 125I-labeled trigramin from the circulation fit a two-compartment model with the half-life for the fast component between 0.7 and 2.0 min and with the half-life for the slow component between 31 and 105 min. It appeared that the kidney and liver are major routes of elimination of trigramin from the circulation. The ability of trigramin to prolong bleeding time, as well as its rapid disappearance from the circulation, indicates that this peptide may be a useful compound to transiently prevent the ability of platelets to form thromboemboli without impairing their long-term hemostatic function.

Animals↗
PubMed1989

Inhibition of human megakaryocytopoiesis in vitro by platelet factor 4 (PF4) and a synthetic COOH-terminal PF4 peptide.

We report that highly purified human platelet factor 4 (PF4) inhibits human megakaryocytopoiesis in vitro. At greater than or equal to 25 micrograms/ml, PF4 inhibited megakaryocyte colony formation approximately 80% in unstimulated cultures, and approximately 58% in cultures containing recombinant human IL 3 and granulocyte-macrophage colony-stimulating factor. Because PF4 (25 micrograms/ml) had no effect on either myeloid or erythroid colony formation lineage specificity of this effect was suggested. A synthetic COOH-terminal PF4 peptide of 24, but not 13 residues, also inhibited megakaryocyte colony formation, whereas a synthetic 18-residue beta-thromboglobulin (beta-TG) peptide and native beta-TG had no such effect when assayed at similar concentrations. The mechanism of PF4-mediated inhibition was investigated. First, we enumerated total cell number, and examined cell maturation in control colonies (n = 200) and colonies (n = 100) that arose in PF4-containing cultures. Total cells per colony did not differ dramatically in the two groups (6.1 +/- 3.0 vs. 4.2 +/- 1.6, respectively), but the numbers of mature large cells per colony was significantly decreased in the presence of PF4 when compared with controls (1.6 +/- 1.5 vs. 3.9 +/- 2.3; P less than 0.001). Second, by using the human leukemia cell line HEL as a model for primitive megakaryocytic cells, we studied the effect of PF4 on cell doubling time, on the expression of both growth-regulated (H3, p53, c-myc,and c-myb), and non-growth-regulated (beta 2-microglobulin) genes. At high concentrations of native PF4 (50 micrograms/ml), no effect on cell doubling time, or H3 or p53 expression was discerned. In contrast, c-myc and c-myb were both upregulated. These results suggested the PF4 inhibited colony formation by impeding cell maturation, as opposed to cell proliferation, perhaps by inducing expression of c-myc and c-myb. The ability of PF4 to inhibit a normal cell maturation function was then tested. Megakaryocytes were incubated in synthetic PF4, or beta-TG peptides for 18 h and effect on Factor V steady-state mRNA levels was determined in 600 individual cells by in situ hybridization. beta-TG peptide had no effect on FV mRNA levels, whereas a approximately 60% decrease in expression of Factor V mRNA was found in megakaryocytes exposed to greater than or equal 100 ng/ml synthetic COOH-terminal PF4 peptide. Accordingly, PF4 modulates megakaryocyte maturation in vitro, and may function as a negative autocrine regulator of human megakaryocytopoiesis.

Amino Acid Sequence↗