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S Niewiarowski

Publications and source records attributed to S Niewiarowski.

At least 145 records · Page 8Linked to original sources
PubMed1983

Purification of two heparin-binding proteins from porcine platelets and their homology with human secreted platelet proteins.

Two heparin-neutralizing proteins secreted by thrombin-stimulated platelets were purified to homogeneity by means of heparin-agarose affinity chromatography. These proteins, termed porcine platelet basic protein (PBP) and porcine platelet factor 4 (PF4), were eluted from a heparin-agarose column at 0.6-0.9 M NaCl and at 1-1.4 M NaCl, respectively. The molecular weight of porcine platelet basic protein was 7,000-7,700 daltons, as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and amino acid analysis. The isoelectric point of this protein was at pH 9.0. The amino acid composition of porcine platelet basic protein resembled that of human low affinity platelet factor 4 (LA-PF4), except that the porcine protein did not contain tyrosine. The molecular weight of porcine platelet factor 4 ranged from 10,000 (estimated from amino acid analysis) to 14,000 (estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis). The amino acid compositions of human platelet factor 4 and of porcine platelet factor 4 were similar. Monospecific antibodies against porcine platelet factor 4 and porcine platelet basic protein were raised in rabbits. Competitive radioimmunoassay demonstrated a low but significant immunologic cross-reactivity between human and porcine platelet factor 4, and between porcine platelet basic protein and a group of human secreted platelet proteins that bind to heparin with low affinity (beta-thromboglobulin [beta TG] and low affinity platelet factor 4). Experiments with direct immuno-precipitation of 125I-labeled antigens suggested that all four proteins investigated (human platelet factor 4, porcine platelet factor 4, human low affinity platelet factor 4 or human beta-thromboglobulin, and porcine platelet basic protein) share common antigenic determinants. However, there was a higher degree of immunologic cross-reactivity between heterologous antigens with similar heparin binding affinity (human platelet factor 4 and porcine platelet factor 4) than between heterologous antigens with different binding affinity (human platelet factor 4 and porcine platelet basic protein). In conclusion, our finding suggests a significant structural homology among the four proteins.

Animals↗
PubMed1983

Effect of heparin on the in vivo release and clearance of human platelet factor 4.

Intravenous injection of heparin (100 U/kg) into normal volunteers resulted in an increase of platelet factor 4 (PF4) level in platelet-poor plasma from a mean value of 18.1 +/- 6.6 ng/ml before the injection to 257.9 +/- 68.3 ng/ml at 5 min after injection. PF4 antigen isolated from "postheparin plasma" by adsorption on heparin-agarose and elution with 2.0 M NaCl and "authentic PF4" isolated from human platelets showed identical patterns of migration as determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Material released by washed human platelets was injected intravenously into rats. The clearance of PF4 followed a biphasic exponential pattern. The half-lives (T1/2) for the fast and slow components for control rats were 1.2 and 17.1 min. Heparin significantly extended the half-life of human PF4 in rat circulation. The clearance of PF4 injected together with heparin followed a single component model with a half-life of 27.6 min. Administration of heparin to rats that had been previously injected with human platelet releasate resulted in a 30-fold increase of plasma PF4 level in their circulation. The clearance of PF4 from the circulation of these rats (T1/2 = 45 min) fitted a single component model. We propose that PF4 is originally secreted by platelets into circulation and subsequently bound reversibly to vascular sites from which it can be released back into the circulation by heparin. The fast component of PF4 clearance that is abolished by heparin may reflect binding of this protein to the endothelial cells.

Animals↗
PubMed1982

Human platelet basic protein. Its relation to low affinity platelet factor 4 and beta-thromboglobulin.

Human beta-thromboglobulin, low affinity platelet factor 4 and platelet basic protein have been purified to homogeneity from the material released by thrombin-stimulated platelets. Purification steps included isoelectric focusing and heparin-agarose chromatography. Antibodies against each of these proteins have been raised in rabbits. Antigenic identity of the proteins has been demonstrated in radioimmunoassay using 125I-labelled platelet basic protein or 125I-labelled low affinity platelet factor 4 and a variety of antibodies. The molecular weight of platelet basic protein estimated by gel filtration in 6 M guanidine hydrochloride using Sepharose 6B corresponded to approx. 10 000 daltons, slightly higher than that of beta-thromboglobulin (8851 daltons) and low affinity platelet factor 4 (9278 daltons). These findings raise the possibility that the formation of low affinity platelet factor 4 beta-thromboglobulin may be a consequence of the action of proteolytic enzymes on platelet basic protein.

Amino Acid Sequence↗
PubMed1982

Platelet function during cardiac operation: comparison of membrane and bubble oxygenators.

The effects of cardiopulmonary bypass with bubble and membrane oxygenator systems on platelet function were studied in 26 patients who had elective coronary arterial bypass grafts. Fourteen patients were perfused with spiral coil membrane oxygenator systems, 12 with bubble oxygenator systems. During and after bypass, platelet counts decreased in both groups; however, when corrected for dilution, platelet counts did not change significantly in patients perfused with membrane oxygenators and increased slightly but significantly in those perfused with membrane oxygenators and increased slightly but significantly in those perfused with bubble oxygenator systems. During and 1 hour after bypass, the concentration of adenosine diphosphate (ADP) required to cause complete aggregation increased in both groups. Plasma low affinity platelet factor 4 (LA-PF4) increased significantly during and after bypass in both groups. However, the concentration of platelet adenine nucleotides and LA-PF4, measured only in patients perfused with membrane oxygenator systems, did not change. Bleeding times increased postoperatively in both groups and 18 hour blood losses were similar. Cardiopulmonary bypass with membrane and bubble oxygenator systems causes qualitatively similar losses in sensitivity to ADP and similar increases in bleeding times. The mechanism by which platelets are altered during cardiopulmonary bypass in obscure but is not due to partial depletion of granule contents in patients perfused with membrane oxygenators.

Adenosine Diphosphate↗
PubMed1981

Affinity labeling of a human platelet membrane protein with 5'-p-fluorosulfonylbenzoyl adenosine. Concomitant inhibition of ADP-induced platelet aggregation and fibrinogen receptor exposure.

Incubation of washed human blood platelets with 5'-p-fluorosulfonylbenzoyl [3H]adenosine (FSBA) covalently labels a single polypeptide of Mr = 100,000. Protection by ADP has suggested that an ADP receptor on the platelet surface membrane was modified. The modified cells, unlike native platelets, failed to aggregate in response to ADP (100 microM) and fibrinogen (1 mg/ml). The extent of binding of 125I-fibrinogen and aggregation was inhibited to a degree related to the incorporation of 5'-p-sulfonylbenzoyl adenosine (SBA) into platelets, indicating FSBA could inhibit the exposure of fibrinogen receptors by ADP necessary for aggregation. Incubation of SBA platelets with alpha-chymotrypsin cleaved the covalently labeled polypeptide and concomitantly reversed the inhibition of aggregation and fibrinogen binding. Platelets proteolytically digested by chymotrypsin prior to exposure to FSBA did not require ADP for aggregation and fibrinogen binding. Moreover, subsequent exposure to FSBA did not inhibit aggregation or fibrinogen binding. The affinity reagent FSBA can displace fibrinogen bound to platelets in the presence of ADP, as well as promote the rapid disaggregation of the platelets. The apparent initial pseudo-first order rate constant of dissociation of fibrinogen was linearly proportional to FSBA concentrations. These studies suggest that a single polypeptide can be altered either by ADP-induced conformational changes or proteolysis by chymotrypsin to reveal latent fibrinogen receptors and promote aggregation of platelets after fibrinogen binding.

Adenosine↗
PubMed1981

Role of kidney in the catabolic clearance of human platelet antiheparin proteins from rat circulation.

UNLABELLED: Stimulated platelets release at least two antiheparin proteins: platelet factor 4 (PF4) and low affinity platelet factor 4 (LA-PF4) from which beta-thromboglobulin (beta TG) is derived. We have found previously marked elevation of LA-PF4/beta TG antigen in platelet poor plasma of patients with chronic renal failure, whereas levels of PF4 remained normal. Therefore, we examined the role of the kidneys in the metabolic clearance of LA-PF4/beta TG and PF4. The supernates of aggregates of thrombin-stimulated human platelets were injected into sham operated control rats, nephrectomized rats, and into rats with acute ureteral ligation. The disappearance of human LA-PF4/beta TG antigen and PF4 in rat plasma determined by specific radioimmunoassays followed biphasic exponential curves. The half-lives (t1/2) for the fast and slow components of LA-PF4 in control rats were 6.4 and 68.4 min. Nephrectomy significantly increased these times to 9.7 and 144 min, while ureteral ligation resulted in no significant change. Comparison of the level of LA-PF4/beta TG antigen and of creatinine in aorta and in renal vein showed 25%-30% extraction of these compounds by the kidney. Less than 0.1% of the total LA-PF4 antigen injected was recovered in the urine of control rats. In contrast to these results, the clearance of PF4 was not affected by nephrectomy. IN CONCLUSION: (1) functional renal tissue is necessary for normal clearance of LA-PF4/beta TG, but renal excretion does not play a major role in its elimination suggesting that the protein is catabolized by the kidney; and (2) catabolic clearance of PF4 does not depend on functioning kidney tissue.

Animals↗
PubMed1981

Acquired granular pool defect in stored platelets.

Platelets stored as concentrates (PC) for 72 h at 22 degrees C develop a functional defect. Alterations in adenine nucleotides of platelets have been shown to affect platelet function. Adenine nucleotide content of platelets was measured before and after storage and a decrease of 27.1 /+- 1.7% (mean /+- SE) in ATP and 39.1 /+- 2.6% in ADP were found in 34 PC stored with final volume of 50 ml. In 11 PC with 30 ml volume. ATP and ADP decreased by 39.4 /+- 3.2% and 49.4 /+- 2.1%, respectively. The mean ATP to ADP ratio of stored platelets was significantly higher than of fresh platelets in both groups, suggesting a relatively greater decrease in granular than metabolic pool nucleotides. Levels of low affinity platelet factor 4 measured by radioimmunoassay in plasma from 0.86 /+- 0.08 microgram/ml in the fresh PC to 8.59 /+- 0.39 microgram/ml in stored PC, indicating a concomitant alpha-granular secretion. Labeling of metabolic pool with 14C-adenine revealed a mean decrease in the adenylate energy charge of 2.0 /+- 0.4% in 12 of 16 stored PC, with a lower ATP and higher hypoxanthine labeling in stored as compared to fresh platelets. These observations suggest that stored platelets develop an acquired defect in both dense and alpha granules and in their ability to maintain ATP homeostasis.

Adenosine Diphosphate↗
PubMed1980

Conversion of low-affinity platelet factor 4 to beta-thromboglobulin by plasmin and trypsin.

Low-affinity platelet factor 4 and beta-thromboglobulin are platelet-secreted proteins that bind with low affinity to heparin. They show extensive immunological cross-reactivity and appear to differ in amino acid sequence only by an amino-terminal peptide unique to low-affinity platelet factor 4. The possibility that beta-thromboglobulin is derived from low-affinity platelet factor 4 by proteolysis was investigated by exposing this protein to the action of plasmin, thrombin and trypsin. While thrombin had no effect, plasmin and trypsin converted low-affinity platelet factor 4 to a species with the same electrophoretic mobility and isoelectric point as beta-thromboglobulin. We conclude that beta-thromboglobulin is a breakdown product of low-affinity platelet factor 4.

Amino Acid Sequence↗
PubMed1980

Human platelet basic protein associated with antiheparin and mitogenic activities: purification and partial characterization.

Platelet basic protein (PBP) (isoelectric point, 10.0-10.5; apparent Mr, 11,000-15,000) has been purified to homogeneity from material secreted by fresh human platelets after stimulation by thrombin. The purification, using preparative isoelectric focusing and chromatography on heparin-Sepharose, yielded two additional peptides with antiheparin activity that were immunologically identical with PBP: low-affinity platelet factor 4 and beta-thromboglubulin. The purity of the peptides was confirmed by immuoelectrophoresis and by NH2-terminal amino acid analysis. Dansyl chloride-treated PBP yielded a single dansylated amino acid residue (glycine). By using a specific radioimmunoassay it was shown that 10(9) human platelets contain 2-3 microgram of PBP which can be released in response to specific stimulation. PBP is associated with mitogenic activity as assayed in Swiss 3T3 mouse cells cultured in low-serum (0.4-1.5%) medium at levels of about 1 ng/ml and saturating at 10-40 ng/ml. The biological activity of different PBP preparations was variable, presumably due to inhibition by the varying amounts of ampholytes that interfered with the mitogenic activity of the peptide. Mitogenic activity was eluted from NaDodSO4/polyacrylamide gels and shown to comigrate with immunoreactive material and with conventional marker proteins of 14,000-17,000 daltons or with histones of 11,000-15,000 daltons. Evidence is presented that PBP is different from cationic platelet-derived growth factor which has an apparent Mr of 30,000.

Amino Acid Sequence↗
PubMed1980

Identification and separation of secreted platelet proteins by isoelectric focusing. Evidence that low-affinity platelet factor 4 is converted to beta-thromboglobulin by limited proteolysis.

Low-affinity platelet factor 4 and beta-thromboglobulin are low molecular weight platelet secretory proteins that have common antigenic determinants. Four amino acids (Asn-Leu-Ala-Lys) at the amino terminus of beta-thromboglobulin are deleted, but the remaining sequences of the two peptides appear to be identical. Low-affinity platelet factor 4 and beta-thromboglobulin have respective isoelectric points at pH 8.0 and at pH 7.0. Identification, quantitation, and separation of both proteins was achieved by a method combining preparative isoelectric focusing and specific radioimmunoassay with anti-low-affinity platelet factor 4 antibody. It has been determined that the supernate processes immediately after platelet aggregation induced by ionophore A23187 or thrombin contains approximately 80% low-affinity platelet factor 4, 8% beta-thromboglobulin, and 12% highly cationic immunoreactive material (platelet basic protein). Experimental evidence suggests that low-affinity platelet factor 4 is originally secreted by platelets and then converted to beta-thromboglobulin by a platelet-derived, heat-labile protease that is inhibited by phenylmethylsulfonyl fluoride.

Amino Acid Sequence↗
PubMed1980

In vivo release and turnover of secreted platelet antiheparin proteins in rhesus monkey (Macaca mulatta).

Human and rhesus monkey platelets secrete at least two antiheparin proteins: platelet factor 4 (PF4) and low affinity platelet factor 4 (LA-PF4). Neither of these proteins showed species-related antigenic differences. As determined by radioimmunoassay, the levels of PF4 and LA-PF4 antigen per 10(9) monkey platelets amounted to 10.7 and 20.3 microgram, respectively. One milliliter of monkey plasma prepared from blood collected into an anticoagulant composed of EDTA, prostaglandin E1, and theophylline solution contained 22.4 ng LA-PF4 and 8.0 ng PF4. Concentrations of these two platelet-specific proteins in monkeys closely resembled levels found in human platelets and plasma. Infusion of prostacyclin (PGI2) (100 or 300 ng/kg/min) into monkeys for 15 min resulted in a significant decrease of plasma levels of LA-PF4 antigen and of PF4 by 40%--60% (p < 0.0001). This decrease was related to the inhibitory effect of PGI2 on the secretion of platelets stimulated by a catheter or by venipuncture. Longer infusion of PGI2 did not produce further significant change. The supernate obtained after aggregation of human platelets stimulated by thrombin was injected into monkeys receiving PGI2 infusion. The disappearance of LA-PF4 antigen in monkey plasma followed a biphasic exponential curve with half-lives for the fast and slow components of 8.4 and 63 min. PF4 disappeared faster but followed the same pattern (half-lives for the fast and slow component of 2.1 and 70 min). Analysis of the experimental data suggests that the low levels of secreted platelet proteins in monkey plasma are related to their minimal in vivo release and to their rapid clearance.

Animals↗
PubMed1980

Identification of two distinct adenosine diphosphate receptors in human platelets.

(1) FSBA, an inhibitor of platelet shape change and aggregation, inhibits and reverses the binding of fibrinogen to washed platelets induced by ADP in the presence of calcium ion; (2) FSBA does not inhibit either aggregation of or fibrinogen binding to platelets treated with chymotrypsin; (3) additional evidence is provided that FSBA does not inhibit aggregation by stimulating cyclic AMP accumulation; (4) FSBA does not inhibit PGE1-induced cyclic AMP accumulation and has little ability to inhibit the action of ADP on cyclic AMP or the binding of the ADP analog [beta 32P] 2-MeSADP; and (5) these results provide additional support for the view that the effects of ADP on aggregation and on cyclic AMP are mediated by distinct receptors, one of which, designated "A", is labeled by FSBA; the second receptor designated "C", has selective affinity for 2-MeSADP.

Adenosine↗