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Biomedical subjects

P Ganguly

Publications and source records attributed to P Ganguly.

At least 37 records · Page 2Linked to original sources
PubMed1986

Thrombin-reactive polypeptides of platelets may regulate inhibition of thrombin by antithrombin.

The central enzyme involved in blood coagulation and activation of platelets is the serine proteinase thrombin. The principal inhibitor of this proteinase in plasma is antithrombin. The mechanism of regulation of the thrombin-antithrombin reaction remains unknown. Two polypeptides of 74 and 55 kDa present on the platelet surface and in plasma are known to specifically enhance the activity of thrombin on different substrates. This study was undertaken to assess the effects of these platelet proteins on thrombin-antithrombin interaction. Direct measurements of residual thrombin activity in mixtures of thrombin and antithrombin, in the presence or absence of the platelet proteins, were made utilizing a specific chromogenic substrate. Under these conditions, when 60% of thrombin activity was inhibited by antithrombin in controls, 100% of enzyme activity was retained in the presence of the platelet proteins. When heparin was used in these assays, the rate of inhibition of thrombin by antithrombin was much more rapid and 62% of thrombin activity remained after 1 min. Under these conditions, the platelet proteins continued to protect thrombin from inactivation with 98% activity remaining at 1 min and 85% activity at 5 min. In contrast, the inhibition of trypsin by antithrombin was not affected by the platelet proteins. Additional studies in platelet aggregation showed that the platelet polypeptides have two effects on thrombin: (i) protection of the enzyme inhibition by antithrombin and (ii) stabilization of thrombin from loss of activity due to aging. The results suggest a novel role for the platelet proteins in hemostasis - regulation of the inhibition of thrombin by antithrombin.

Antithrombins↗
PubMed1985

Protein phosphorylation and activation of platelets by wheat germ agglutinin.

The incorporation of 32Pi into the 47 and 20 kDa polypeptides in platelets activated by wheat germ agglutinin (WGA) was studied. The pattern of enhanced phosphorylation produced by the lectin was comparable to that by thrombin. The 47 kDa polypeptide was phosphorylated at both serine and threonine while the 20 kDa protein was mainly labeled at serine residues. However, the ratio of phosphoserine to phosphothreonine in the 47 kDa polypeptide in WGA-activated platelets was higher than thrombin-stimulated platelets. Addition of N-acetylglucosamine at different times blocked platelet activation by WGA. There was a concomitant modification in the phosphorylation of the 47 kDa protein. These data suggest that the phosphorylation of the 47 kDa polypeptide may modulate the WGA-receptor mediated activation of platelets. Our studies also demonstrate that activation of platelets by different stimuli may lead to differential phosphorylation of different amino acid residues in the same protein.

Acetylglucosamine↗
PubMed1985

Thrombin-reactive polypeptides of human blood. Some biochemical and immunological properties.

Two polypeptides of 74 kDa and 55 kDa have been isolated from human platelets by immunoaffinity and lectin affinity chromatography and their effects on thrombin reactivity have been examined. These proteins in combination enhanced the aggregation of platelets by thrombin while aggregation induced by trypsin, collagen and adenosine diphosphate was not significantly affected. An enhancement in the action of thrombin on fibrinogen, N-benzoylarginine ethyl ester and H-D-phenylalanyl-L-pipecolyl-L-arginine-p-nitroanilide dihydrochloride was also observed in the presence of the platelet proteins. Under similar conditions, the proteins did not influence the esterolytic activity of trypsin or plasmin. Studies at different thrombin and protein concentrations showed maximum enhancement of enzyme reactivity when the ratio between the peptides and thrombin was optimal. In the presence of these proteins, the affinity of thrombin for N-benzoylarginine ethyl ester was about twofold higher than in the control. Two polypeptides with properties similar to those described above have also been isolated from human plasma. Antibodies to the above proteins isolated from either platelets or plasma were raised in rabbits. Intact platelets solubilized in Triton X-100 or plasma showed two precipitin lines in immunoelectrophoresis against both of the above antisera and a similar pattern was observed with the isolated polypeptides. The polypeptides did not interact in immunoelectrophoresis with antisera to whole serum, antithrombin, C4 binding protein or protein S. These 74-kDa and 55-kDa polypeptides contained radioactivity when radioiodinated platelets were used suggesting that they are located on the cell surface. Fresh plasma was analyzed by gel electrophoresis under nondenaturing and denaturing conditions and the proteins were transferred to nitrocellulose sheets. Staining with antibody to these thrombin-reactive proteins and 125I-protein A showed several reactive plasma proteins under nondenaturing conditions with the major band migrating in the albumin area. In plasma treated with sodium dodecyl sulfate, the 74-kDa and 55-kDa components were observed. A prominent 74-kDa band and a fainter 55-kDa component were again observed when platelets solubilized in sodium dodecyl sulfate were analysed by the above procedure. It is proposed that human platelets and plasma contain polypeptides which may directly modulate thrombin reactivity.

Animals↗
PubMed1984

Estimation of the amount of 5-methylcytosine in Drosophila melanogaster DNA by amplified ELISA and photoacoustic spectroscopy.

We have previously reported a sensitive immunochemical method for detecting 5-methylcytosine in DNA which involves spotting DNA samples on nitrocellulose paper and detection of 5-methylcytosine, if any, by a combination of the double antibody method and a staining reaction brought about by biotin-avidin and peroxidase. We report here a linear relationship between the concentration of 5-methylcytosine in DNA and staining intensity, as recorded by photoacoustic spectroscopy. It appears possible to obtain, by this method, reliable quantitative estimates of 5-methylcytosine in nanogram quantities of intact DNA. When Drosophila melanogaster DNA was assayed for the presence of 5-methylcytosine by this method, a faint but clearly positive reaction was obtained. When the photoacoustic intensity of this stained spot is compared with a calibration plot derived from phi X174 DNA whose 5-methylcytosine content is known, we obtain, for D. melanogaster DNA, one 5-methylcytosine residue in approximately 12 500 bases or 0.008 mol% methylation.

5-Methylcytosine↗
PubMed1984

The role of sialic acid in the activation of platelets by wheat germ agglutinin.

Sialic acid is believed to play a critical role in the survival of blood platelets in circulation. Wheat germ agglutinin, which shows specificity for sialic acid, N-acetylglucosamine, and N-acetylgalactosamine, strongly activates platelets. The role of sialic acid in platelet activation by this lectin was studied utilizing neuraminidase-treated platelets and the succinylated lectin that has been reported not to recognize sialic acid. The succinylated lectin had a dimeric structure similar to the native lectin, but migrated more slowly in gel electrophoresis. The modified lectin bound to about 2.8 X 10(5) sites/cell, with an apparent dissociation constant of 2 microM compared to 5 X 10(5) sites/cell and a dissociation constant of 0.4 microM for the native lectin. The succinylated lectin neither aggregated nor agglutinated platelets, but agglutination of red cells in microtiter plates was normal. Aggregation of platelets by either wheat germ agglutinin or ristocetin was not affected by the succinylated lectin. Since the native wheat germ agglutinin is a strong activator of platelets and the succinylated derivative was devoid of all activity, it appears that a sialoprotein acts as the biologic receptor of wheat germ agglutinin in human platelets. This suggestion was strengthened by the observation that platelets treated with different concentrations of neuraminidase had a decreased capacity to bind this lectin. These platelets also showed reduced aggregation and serotonin secretion when activated with the native lectin. Since sialic acid has been implicated in the removal of platelets from circulation, wheat germ agglutinin may prove to be a useful tool to explore those clinical conditions in which platelet survival is shortened.

Agglutination Tests↗
PubMed1983

A novel thrombin enhancement factor in human plasma.

A protein has been isolated from human plasma by gel filtration followed by affinity chromatography with a derivative of wheat germ agglutinin and ion exchange chromatography. This protein showed one peak in high performance liquid chromatography but in gel electrophoresis, in the presence of sodium dodecyl sulfate and beta-mercaptoethanol, revealed two major components of 74 kDa and 55 kDa. These results indicate that the protein probably exists as a complex of the two polypeptides. This protein complex enhanced platelet aggregation by thrombin while aggregation induced by ADP was not significantly affected. Similarly, the rate of thrombin action on fibrinogen and N-benzoylarginine ethyl ester as measured in a spectrophotometer was increased in the presence of this plasma protein. These results suggest the presence of a protein complex in human plasma which can directly interact with thrombin and enhance its reactivity.

Blood Coagulation↗
PubMed1983

Preparation and some properties of a derivative of wheat germ agglutinin with altered biological activity.

An inactive derivative of wheat germ agglutinin, which is a strong activator of blood platelets, was prepared by selective chemical modification of the lectin with cyanogen bromide at acid pH. The derivative was then used as a probe to learn about the initial events in platelet stimulation by physiological agents. Amino acid analysis of the modified lectin confirmed specific cleavage of a methionine residue. Gel filtration studies indicated a molecular weight for the lectin derivative similar to the unmodified lectin. In gel electrophoresis in the presence of sodium dodecyl sulfate, reduced samples of the derivative showed two bands and the main component migrated slightly faster than the native lectin. The derivative retained the capacity to precipitate an antibody to the lectin although at least one of the antigenic sites was lost due to chemical modification. The derivative did not compete with the unmodified lectin for binding to platelets. Unlike the parent lectin, the derivative did not aggregate platelets even at a ten fold higher concentration. Under similar conditions, there were about 1.0 X 10(5) binding sites/platelet for the lectin derivative with an apparent dissociation constant of 1.7 microM compared to 5 X 10(5) sites/cell and a dissociation constant of 0.4 microM for the native lectin. Overnight incubation of platelets or red cells with the derivative in microtiter plates showed about 2-5% agglutinating activity for the derivative compared to the unmodified lectin. Incubation of platelets with the lectin derivative inhibited platelet aggregation by thrombin while aggregation induced by a number of other agents was not significantly affected. This inhibitory effect of the lectin derivative on thrombin-induced platelet aggregation could be readily reversed with GlcNAc. The lectin derivative may be a useful tool to explore the structure-function relationship of cell surface components.

Amino Acids↗
PubMed1983

Inhibition of thrombin binding and serotonin secretion from platelets by a 74,000 dalton protein.

A 74,000 dalton protein has been isolated from solubilized human platelet membranes by affinity chromatography with a nonagglutinating derivative of wheat germ agglutinin. The protein as well as the lectin derivative blocked platelet aggregation by thrombin while aggregation induced by several other agents was unchanged. This protein inhibited the binding of thrombin to platelets in a competitive manner. The secretion of serotonin from platelets by thrombin was also blocked by the protein. These effects of the protein were observed when platelets were suspended in either plasma or buffer. This protein may have functional significance in the activation of platelets by thrombin.

Binding Sites↗
PubMed1983

A novel thrombin-reactive protein complex in human platelets.

Attempts were made to isolate a 74,000-dalton protein which specifically and competitively blocked platelet aggregation by thrombin (Ganguly, P., and Fossett, N. G. (1981) Blood 57, 343-352) by two independent affinity chromatography methods. The protein isolates showed a main band migrating slightly faster than albumin in gel electrophoresis under nondenaturing conditions. In the presence of sodium dodecyl sulfate, electrophoresis of protein preparations consistently revealed the presence of four polypeptides of 74,000, 55,000, 27,000, and 20,000 daltons. The results suggest that these polypeptides are probably present as a multiprotein complex in platelets. The 55,000-dalton protein, like the 74,000-dalton protein, inhibited thrombin-induced platelet aggregation, while aggregation induced by adenosine diphosphate or trypsin was not significantly affected. However, incubation of the 55,000-dalton protein with the 74,000-dalton protein prior to the addition of thrombin did not inhibit platelet aggregation by thrombin, while the individual components under the same conditions did. In fact, the two proteins in combination could significantly enhance platelet aggregation by thrombin. These results suggest the existence of a multiprotein complex in human platelets, the components of which may modulate the action of thrombin on platelet aggregation.

Blood Platelets↗
PubMed1982

Inhibition of platelet aggregation by primary amines. Evidence for a possible role of membrane-associated calcium.

The aggregation of human blood platelets by thrombin, adenosine diphosphate, wheat germ agglutinin or ristocetin was inhibited by primary amines. In general, thrombin-induced platelet aggregation was strongly affected by the amines while the effect was weak on cell aggregation by ristocetin. Usually, the diamines were stronger inhibitors of aggregation than the monoamines with cadaverine as the strongest and ethylamine as the weakest inhibitor. At concentration where platelet aggregation was inhibited, the amines neither displaced serotonin from serotonin-loaded platelets nor caused lysis of human red cells. The lectin activity of wheat germ agglutinin on human red cells was not affected by the amines indicating that the amines probably acted on platelets and not on the agglutinin. The clotting activity of thrombin on fibrinogen was partially inhibited by the amines while its esterolytic activity remained unaltered. The inhibitory action of the amines on platelet aggregation could be overcome with small amounts of calcium while other divalent cations tested had little effect. It is suggested that the amines affect platelet aggregation by interfering with the actions of membrane-associated calcium.

Adenosine Diphosphate↗