Membrane Glycoproteins of Human and Rabbit Platelets: Studies In Vitro and after Circulation in Vivo

1975 ◽  
Author(s):  
J. N. George ◽  
P. C. Lewis ◽  
D. A. Sears
Keyword(s):  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Age Distribution ◽  
Human Platelets ◽  
Membrane Glycoproteins ◽  
Rabbit Platelets ◽  
Trypsin Hydrolysis ◽  
Initial Recovery

The initial events of hemostasis and thrombosis involve platelet contact interactions and may be mediated by surface glycoproteins. Human and rabbit platelets were labeled with 125I-diazotized diiodosulfanilic acid (I), which reacts covalently with proteins, and proteins were separated by SDS-polyacrylamide gel electrophoresis. Only exposed membrane proteins were labeled because: 1) protein specific activity of membranes was 4-7 times that of whole platelets, 2) different proteins were labeled when I was reacted with isolated membranes, and 3) trypsin-hydrolysis of labeled intact platelets altered the radioactive peaks. Like Phillips (Biochem. 11, 4582, 72) and Nachman et al. (JBC 248, 2928, 73) we found that lactoperoxidase iodinated the 93,000 dalton glycoprotein (GP) of human platelets. In contrast, I labeled both the 93,000 and 118,000 dalton membrane GP of human platelets, and all 3 membrane GP of rabbit platelets.Rabbit platelets labeled simultaneously with I and 51Cr had identical density and therefore age distribution of the 2 labels. After infusion into rabbits, initial recovery of I was 23% of the Cr recovery. After 3 hrs, I disappearance was exponential and more rapid (T/2 = 17 hrs) than the linear Cr disappearance (T/2 = 30 hrs, p < .01). This was due to in vivo removal of I from circulating platelets since 1 did not elute more rapidly from platelets harvested after 3 hrs circulation and incubated in plasma at 37° (T/2 of I elution = 43 hrs, Cr = 33 hrs). Platelets harvested after 14-20 hrs circulation had the same distribution of I on the membrane GP as before circulation. We postulate that this symmetrical label loss indicates uniform loss of membrane GP, suggesting that platelets lose pieces of their plasma membrane during circulation. This could occur during contact interaction in the process of hemostasis.

scholarly journals Assessment of Acid Phosphatase Production by In vitro Cultures of Atropa acuminata

2016 ◽  
Vol 26 (1) ◽  
pp. 15-23
Author(s):  
Saima Khan ◽  
Meenu Katoch ◽  
Sharada Mallubhotla ◽  
Suphla Gupta ◽  
Manju Sambyal ◽  
...  
Keyword(s):  
Acid Phosphatase ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Growth Period ◽  
Callus Cultures ◽  
Shoot Cultures ◽  
Crude Enzyme Extract ◽  
Atropa Acuminata

The potential of various culture lines of Atropa acuminata were investigated for resourcing acid phosphatase (ACP) (3.1.3.2). Crude enzyme extract comprised of a mixture of four isoforms, distinguishable by polyacrylamide gel electrophoresis (PAGE) with molecular weight ranging from 39 to 215 kDa. In vitro regenerated proliferative shoots, callus and roots showed higher specific activity (2.49, 3.41, 2.91 U/mg protein, respectively) as compared to in vivo grown plants (0.71 U/mg protein). ACP activity in root cultures increased progressively up to 4.6 U/mg during the entire growth period (2 ? 24 weeks), whereas in case of shoot cultures, the specific activity escalated to 2.49 U/mg at 8 weeks, which then declined subsequently (1.95 U/mg). Similarly, callus cultures initially showed a higher phosphohydrolytic activity (3.41 U/mg protein) until 8 weeks by which period, it decreased with the passage of growth period. The present studies reveal an alternate system for resourcing of ACP from Atropa acuminata.Plant Tissue Cult. & Biotech. 26(1): 15-23, 2016 (June)

scholarly journals Comparative Studies of Platelet Survival by Different Methods in the Rabbit

Blood ◽  
1965 ◽  
Vol 25 (4) ◽  
pp. 548-566 ◽  
Author(s):  
SHIRLEY EBBE ◽  
MARIO BALDINI ◽  
JANET DONOVAN
Keyword(s):  
Survival Time ◽  
Whole Blood ◽  
Comparative Studies ◽  
Significant Degree ◽  
Human Platelets ◽  
Sodium Chromate ◽  
Platelet Concentrates ◽  
Rabbit Platelets

Abstract Four methods for measuring the survival of homologous platelets in rabbits were studied: (1) transfusion of nonradioactive platelet concentrates to thrombocytopenic recipients, (2) transfusion of concentrates of platelets labeled in vitro with Cr51-sodium chromate, (3) transfusion of concentrates of platelets labeled in vivo with P32-orthophosphate and (4) transfusion of whole blood labeled in vivo with P32-orthophosphate. The survival time of platelets in normal rabbits was 3-4 days. From comparison of the 3 methods using platelet concentrates, the following conclusions were drawn. (1) All the platelets in a platelet concentrate were capable of recirculating after transfusion. (2) Labeling with P32 or Cr51 did not damage platelets. (3) About one-third of the Cr51 was immediately eluted from viable platelets after they were transfused. (4) Further exchange of the label in vivo did not occur to a significant degree with either Cr51 or P32. (5) Cr51 did not elute from platelets during storage of the platelets. (6) Studies of rabbit platelets had applicability in predicting the behavior of human platelets.

Interaction of Bacterial Endotoxin and Liquoid with Blood Platelets: Aggregation and Decrease in the Electrokinetic Charge

1969 ◽  
Vol 22 (01) ◽  
pp. 192-202 ◽  
Author(s):  
K. A Gröttum ◽  
P. F Hjort ◽  
M Jeremic
Keyword(s):  
Platelet Aggregation ◽  
Electrophoretic Mobility ◽  
Blood Platelets ◽  
Human Platelets ◽  
Dextran Sulphate ◽  
Rabbit Platelets ◽  
High Concentrations ◽  
Very High

SummaryThe effects of Endotoxin and Liquoid on the electrophoretic mobility of human platelets and erythrocytes in vitro and on rabbit platelets and erythrocytes in vivo and in vitro have been investigated.Liquoid reduced the electrophoretic mobility of human platelets to 74% of normal and rabbit platelets to 59% in vitro and to 68% of normal in vivo, while the erythrocytes were unchanged. Liquoid induced massive aggregation of both human and rabbit platelets. In very high concentrations, Liquoid increased the electrophoretic mobility of human platelets and did not induce aggregation.Endotoxin reduced the electrophoretic mobility of rabbit platelets to 83% of normal and aggregated the platelets, but had none of these effects on human platelets.The effects of Endotoxin and Liquoid were inhibited by EDTA, but not by ADPase, suggesting that aggregation was not mediated through ADP.We conclude that Liquoid has the same pattern of effects on the electrokinetic charge of platelets and platelet aggregation as the acid polymeric agents dextran sulphate and heparin. There was good correlation between reduction in the electrokinetic charge of the platelets and platelet aggregation. There were striking similarities between the effects of these agents and Endotoxin.

Comparison Of The Major Membrane Glycoproteins Of Human, Rabbit And Rat Platelets

1981 ◽  
Author(s):  
B Toor ◽  
J L McGregor ◽  
K J Clemetson ◽  
L McGregor ◽  
M Dechavanne ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Surface Composition ◽  
Sodium Dodecyl ◽  
Human Platelets ◽  
Membrane Glycoproteins ◽  
Neuraminidase Treatment ◽  
Platelet Surface ◽  
Rabbit Platelets ◽  
Rat Platelets

Rabbit and rat platelets have been extensively investigated under in vitro or in vivo conditions to try to understand the pathology of thrombosis in man. Here, surface-labelling techniques have been used to find out if the platelet surface has a similar composition in these two animals and in man or not. Human, rabbit and rat platelets were isolated, washed and surface-labelled by techniques specific for protein or for sugars (sialic acid or penultimate galactose/N-acetyl galactosamine residues). Labelled platelets were solubilized in sodium dodecyl sulphate and separated under reducing conditions on 7.5 % Laemmli polyacrylamide gels. Dried gels were exposed to film by fluorography or indirect autoradiography. Terminal Gal/Gal NAc residues (no neuraminidase treatment) were strongly labelled with rat and rabbit platelets compared to human platelets which labelled very poorly. Terminal sialic acid labelling with rat and rabbit platelets showed a weak labelling of a glycoprotein (GP) with the same M.Wt. as GPIb which is the most intensely labelled GP in man. However two GP (with rabbits) and one GP (in rats) were intensely labelled at a M.Wt. similar to that of GPIa in man. These GP had a different M.Wt. with terminal Gal/Gal NAc labelling. Bands with a similar M.Wt. to GPIIb and IIIa in man were strongly iodinated with rabbit platelets but with rat platelets only a single band at the position of GPIIb was strongly iodinated. These results strongly indicate that there are considerable differences in surface composition between rabbit, rat and human platelets.

scholarly journals Radioactive Diisopropyl Fluorophosphate as a Platelet Label: An Evaluation of in Vitro and in Vivo Technics

Blood ◽  
1967 ◽  
Vol 29 (3) ◽  
pp. 354-372 ◽  
Author(s):  
T. C. BITHELL ◽  
J. W. ATHENS ◽  
G. E. CARTWRIGHT ◽  
M. M. WINTROBE
Keyword(s):  
Specific Activity ◽  
Human Subjects ◽  
Survival Curve ◽  
Human Platelets ◽  
Detectable Effect ◽  
Survival Curves ◽  
Diisopropyl Fluorophosphate ◽  
Platelet Survival

Abstract A technic for the in vitro labeling of human platelets with DFP32 is presented, critically evaluated, and compared to in vivo methods employing DFP32 and to in vitro methods using Cr51. The initial recovery of platelets labeled in vitro with DFP32 averaged 79 per cent, but the survival curve was characterized by an irreversible initial loss of platelet radioactivity. Experiments in which platelets were simultaneously labeled in vitro with both DFP32 and Cr51 suggest that this is not due to elution of DFP32. The survival curve of platelets labeled in vivo with DFP32 shows an initial transient reduction in platelet radioactivity. It is suggested that both of these aberrations in initial survival are the result of platelet injury by DFP32. Significant "tailing" was observed in the survival curves obtained with DFP32, and possible explanations of this phenomenon are discussed. DFP32-labeled platelets circulating after 5 hours apparently survive normally and disappear from the circulation as a rectilinear function over the next 6-8 days. Although both in vitro and in vivo labeling methods employing DFP32 provide a meaningful approximation of platelet lifespan, the initial and terminal aberrations of the survival curves greatly complicate further interpretation. Dextran had no detectable effect on platelet survival, and epinephrine, Mecholyl, and cutaneous vasodilatation did not alter the platelet count or the specific activity of circulating labeling platelets in human subjects. The problem of initial platelet survival and the question of an extravascular or marginal platelet pool is discussed in the light of these data.

Investigation of In Vivo Factors that May Influence Platelet Survival and Density

1979 ◽  
Author(s):  
M.A. Packham ◽  
J.F. Mustard ◽  
M.A. Guccione ◽  
P. D. Winocour ◽  
H.M. Groves ◽  
...  
Keyword(s):  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Platelet Membrane ◽  
Membrane Glycoproteins ◽  
Aortic Endothelium ◽  
Platelet Survival ◽  
Rabbit Platelets

Platelet survival CPS) is shortened in a number of conditions but the mechanisms responsible are unclear. In rabbits, removal of the aortic endothelium or injury of the neointima does not shorten PS. However, induction of thrombi in rabbit aortae with Indwelling cannulae (IDC) shortens FS (IDC 37.0 hr, control 79.6 hr), and Increases the proportion of platelets in the lightest fraction upon straetan density gradient centrifugation. Therefore we examined the effect of agents to which platelets may be exposed during thromboembolism (ADP, thrombin, plasmin) on PS and platelet density. ADP treatnent of washed rabbit platelets did not alter their survival but did increase the proportion in the lightest fraction. Treatment of platelets with thrombin did not shorten PS but increased the proportion in the lightest fraction. Treatment with plasmin in vitro shortened PS (plasmin, 57.6 ± 6.0 hr, control 80.2 ± 4,2 hr) and increased the proportion in the lightest fraction. Thus changes in platelet density are not necessarily associated with changes in PS. Of the factors investigated that are known to be involved in thromboembolism, only plasmin shortened PS. This may be due to its ability to alter major platelet membrane glycoproteins(principally glycoproteins I and II of rabbit platelets).

scholarly journals Purification and properties of heparin-releasable lipoprotein- associated coagulation inhibitor

Blood ◽  
1991 ◽  
Vol 78 (2) ◽  
pp. 394-400 ◽  
Author(s):  
WF Novotny ◽  
M Palmier ◽  
TC Wun ◽  
GJ Jr Broze ◽  
JP Miletich
Keyword(s):  
Vascular Endothelium ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Size Exclusion ◽  
Plasma Lipoproteins ◽  
Major Band ◽  
Coagulation Inhibitor

The lipoprotein-associated coagulation inhibitor (LACI) is present in vivo in at least three different pools: sequestered in platelets, associated with plasma lipoproteins, and released into plasma by intravenous heparin, possibly from vascular endothelium. In this study we have purified the heparin-relesable form of LACI from post-heparin plasma and show that it is structurally different from lipoprotein LACI. The purification scheme uses heparin-agarose chromatography, immunoaffinity chromatography, and size-exclusion chromatography and results in a 185,000-fold purification with a 33% yield. Heparin- releasable LACI (HRL), as analyzed by sodium dodecyl sulfate- polyacrylamide gel electrophoresis, under reducing conditions, appears as a major band at 40 Kd and a minor band at 36 Kd. Immunoblot analysis suggests that the 36-Kd band arises from carboxyl-terminus proteolysis that occurs during the purification. HRL has a specific activity similar to that of HepG2 or lipoprotein LACI. HRL and lipoprotein LACI combine with lipoproteins in vitro while purified HepG2 LACI does not. I125-labeled HRL, injected into a rabbit, is cleared more slowly than I125-labeled HepG2 LACI, which may be due to attachment to lipoproteins in vivo. Preliminary evidence suggests that HRL is associated with vascular endothelium, possibly by attachment to glycosaminoglycans.

Loss Of Platelet Membrane Glycoproteins Or Terminal Sialic Acid Detected By Fitc-Lectins

1981 ◽  
Author(s):  
L McGregor ◽  
J L McGregor ◽  
K J Clemetson ◽  
M Dechavanne ◽  
E F Lüscher
Keyword(s):  
Sialic Acid ◽  
Ricinus Communis ◽  
Lectin Binding ◽  
Human Platelets ◽  
Membrane Glycoproteins ◽  
Binding Studies ◽  
Platelet Surface ◽  
Terminal Sialic Acid

Pre-thrombic conditions in certain individuals resulting from enhanced platelet-vessel wall or platelet-platelet interactions are perhaps characterized by a reduction in certain membrane glycoproteins or loss of terminal sialic acid. In order to investigate if such changes are detectable, the binding of FITC-lectins to human platelets treated under in vitro conditions with certain proteases to mimic possible in vivo changes occuring on the platelet surface, has been examined. Human platelets were isolated, washed and either treated with neuraminidase (10 U) or plasmin (1 CU) before fixing with formaldehyde. Binding studies were performed by the method of Monsigny et al. using FITC labelled wheat germ agglutinin (WGA), Lens culinaris lectin (LCL), Ricinus communis agglutinin (RCA) and concanavalin A (ConA). The number of lectin-binding sites (n) and the dissociation constant (Kd) were obtained by Steck and Wallach reciprocal plots. After neuraminidase or plasmin treatment n was reduced but Kd remained approximately the same with WGA. FITC-RCA-60 gave a slight fluorescence with untreated and very strong fluorescence with neuraminidase treated platelets. Platelet glycoproteins separated by 2-dimensional gel electrophoresis were identified by binding of fluorescent lectins. Plasmin decreased the intensity of GP Ib and IIb and removed Ia completely. Neuraminidase decreased the labelling of Ib by WGA. These techniques show promise as methods of detecting pre-thrombotic conditions.

Studies of Heparin Affinity to Antithrombin III and Other Proteins in Vitro and in Vivo

1977 ◽  
Vol 38 (03) ◽  
pp. 0685-0695 ◽  
Author(s):  
Y Takeda ◽  
N Kobayashi
Keyword(s):  
Gel Electrophoresis ◽  
Antithrombin Iii ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Heparin Binding ◽  
Life Values ◽  
Canine Plasma ◽  
Heparin Affinity

SummaryProperties of human, canine, and porcine heparin and S-35-heparin were first studied. Their electrophoretic mobility through 10 g % polyacrylamide gels, specific activity and their filtration patterns through Sephadex G-200 columns were closely similar. Then, S-35-heparin (0.5 mg) and cold heparin (27 mg) were simultaneously injected intravenously into 5 dogs and their plasma behavior was compared. The plasma half-lives of S-35-heparin averaged 1.18 ± 0. 13 (SD) hr and was identical with that of cold heparin, but the half-lives were much shorter and averaged 0.46 ± 0.08 (SD) hr in 5 dogs when S-35-heparin alone was injected. Studies were next made of heparin affinity to proteins including canine antithrombin III (AT) by the use of Sephadex G-200 chromatography, polyacrylamide gel electrophoresis, and S-35-heparin as a tracer. It was found that S-35-heparin-binding to alpha1, beta, and gammaglobulins and fibrinogen was readily separable upon addition of 5 mg cold heparin, but that the binding to AT was inseparable by addition of cold heparin or by electrophoresis. However, in canine plasma, both S-35-heparin and cold heparin were mostly bound to proteins other than AT, suggesting that this might be the case in vivo. To further substantiate this, studies were made of the comparative behavior of I-125-labeled AT (I-125-AT) and S-35-heparin in dogs with the idea that the plasma half-lives of both should be equal if they were irreversibly bound to each other. The plasma half-lives of I-125-AT averaged 2.10 ± 0.05 (SD) days in 5 male dogs and 1.99 ± 0.04 (SD) days in 5 female dogs, and were much different from the half-life values of S-35-heparin as given above. These results indicate that heparin in vitro is more tightly bound to AT than to other proteins, that heparin in vivo is not irreversibly bound to AT and suggest that it is mostly bound to proteins other than AT in vivo.

scholarly journals Purification and properties of heparin-releasable lipoprotein- associated coagulation inhibitor

Blood ◽  
1991 ◽  
Vol 78 (2) ◽  
pp. 394-400 ◽  
Author(s):  
WF Novotny ◽  
M Palmier ◽  
TC Wun ◽  
GJ Jr Broze ◽  
JP Miletich
Keyword(s):  
Vascular Endothelium ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Size Exclusion ◽  
Plasma Lipoproteins ◽  
Major Band ◽  
Coagulation Inhibitor

Abstract The lipoprotein-associated coagulation inhibitor (LACI) is present in vivo in at least three different pools: sequestered in platelets, associated with plasma lipoproteins, and released into plasma by intravenous heparin, possibly from vascular endothelium. In this study we have purified the heparin-relesable form of LACI from post-heparin plasma and show that it is structurally different from lipoprotein LACI. The purification scheme uses heparin-agarose chromatography, immunoaffinity chromatography, and size-exclusion chromatography and results in a 185,000-fold purification with a 33% yield. Heparin- releasable LACI (HRL), as analyzed by sodium dodecyl sulfate- polyacrylamide gel electrophoresis, under reducing conditions, appears as a major band at 40 Kd and a minor band at 36 Kd. Immunoblot analysis suggests that the 36-Kd band arises from carboxyl-terminus proteolysis that occurs during the purification. HRL has a specific activity similar to that of HepG2 or lipoprotein LACI. HRL and lipoprotein LACI combine with lipoproteins in vitro while purified HepG2 LACI does not. I125-labeled HRL, injected into a rabbit, is cleared more slowly than I125-labeled HepG2 LACI, which may be due to attachment to lipoproteins in vivo. Preliminary evidence suggests that HRL is associated with vascular endothelium, possibly by attachment to glycosaminoglycans.

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