Sheep Platelets Research Articles

Purification and characterization of sheep platelet cyclo-oxygenase. Acetylation by aspirin prevents haemin binding to the enzyme.

Arachidonate cyclo-oxygenase (prostaglandin synthetase; prostaglandin endoperoxide synthetase; EC 1.14.99.1) was purified from sheep platelets. The purification procedure involved hydrophobic column chromatography using either Ibuprofen-Sepharose, phenyl-Sepharose or arachidic acid-Sepharose as the first step followed by metal-chelate Sepharose and haemin-Sepharose affinity chromatography. The purified enzyme (Mr approximately 65,000) was homogeneous as observed by SDS/polyacrylamide-gel electrophoresis and silver staining. The enzyme was a glycoprotein with mannose as the neutral sugar. Haemin or haemoglobin was essential for activity. The purified enzyme could bind haemin exhibiting a characteristic absorption maximum at 410 nm. The enzyme after metal-chelate column chromatography could undergo acetylation by [acetyl-3H]aspirin. The labelled acetylated enzyme could not bind to haemin-Sepharose, presumably due to acetylation of a serine residue involved in the binding to haemin. The acetylated enzyme also failed to show its characteristic absorption maximum at 410 nm when allowed to bind haemin.

Identification and purification of sheep platelet phospholipase A2 isoforms. Activation by physiologic concentrations of calcium ion.

Two families of platelet phospholipase A2 activity, were chromatographically resolved by anion exchange chromatography and were functionally distinguishable by their differential phospholipid subclass substrate specificity and calcium ion requirements. The major phospholipase A2 activity was present in the cytosolic compartment, eluted from DEAE-cellulose at 230 mM NaCl (hereafter referred to as phospholipase A2(beta)), and demonstrated a 100-fold selectivity in catalyzing the hydrolysis of 1-(O)-(Z)-hexadecenyl-2-oleoyl-sn-glycero-3-phosphocholine (plasmenylcholine) in comparisons with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (phosphatidylcholine). Phospholipase A2(beta) was purified to homogeneity by sequential gel filtration and Mono Q column chromatographies. Phospholipase A2(beta) eluted with an apparent molecular mass of 58 kDa during gel filtration chromatography and migrated as a single band with an apparent molecular mass of 30 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that its native quaternary structure is dimeric. Fast protein liquid chromatography demonstrated that the polypeptides catalyzing this activity were comprised of multiple isoforms which possessed different specific activities. Each isoform required Ca2+ ion for activity and was completely activated over the range through which Ca2+ ion concentration is augmented in stimulated platelets (i.e. 300-800 nM).

Lipid composition of subcellular particles from sheep platelets. Location of phosphatidylethanolamine and phosphatidylserine in plasma membranes and platelet liposomes

The lipid composition of whole sheep platelets and their subcellular fractions was determined. The basic lipids show similar distributions in granules, microsomes, plasma membranes and whole platelets. Phospholipid (about 70% of total lipids) and cholesterol (25% of total lipids) are the principal lipid components. Free cholesterol represents about 98% of the total, whereas cholesteryl ester is a minor component. The phospholipid composition found in intact platelets and their subcellular particles is about: 35% phosphatidylethanolamine (PE), 30% phosphatidylcholine (PC), 20% sphingomyclin and 15% phosphatidylserine (PS). We also investigated aminophospholipid topology in intact platelet plasma membranes and platelet liposomes by using the nonpenetrating chemical probe trinitrobenzenesulfonic acid (TNBS), because they are the major components of total lipids. In intact platelets, PS is not accessible to TNBS during the initial 15 min of incubation, whereas 18% PE is labelled after 15 min. In contrast, in phospholipid extracted from platelets 80% PE and 67% PS react with TNBS within 5 min, while 27 and 25% PE and 15 and 19% PS from liposomes and isolated plasma membranes, respectively, were modified after 15 min of incubation. In view of this chemical modification, it is concluded that 22% of PE and less than 1% of PS are located on the external surface of intact platelet plasma membranes. The asymmetric orientation of aminophospholipids is similar between liposomes and isolated plasma membrane. PS (23 and 28%) and PE (34 and 31%) are scarcely represented outside the bilayer. The data found are consistent with the nonrandom phospholipid distribution of blood cell surface membranes.

Angiotensin II and eicosanoids in the control of glomerular size in the rat and human.

We examined the possibility that glomerular prostaglandin E2 (PGE2) regulates the action of angiotensin II (ANG II) on mesangial contraction and filtration surface area. Using isolated rat glomeruli we indirectly assessed mesangial contraction and filtration surface area through measurements of glomerular planar surface area (GPSA) by image-analysis microscopy. ANG II reduced GPSA by approximately 20% in human and rat glomeruli, with threshold concentrations of 1 X 10(-13) M and maximum effect at 5 X 10(-11) M ANG II. Inhibition of glomerular PG synthesis with indomethacin or meclofenamate potentiated the threshold response of ANG II to reduce GPSA. Arachidonic acid (5 micrograms/ml) blunted both the threshold and the maximum responses of GPSA to ANG II. PGE2, 10(-8) and 10(-9) M, also decreased glomerular contraction to ANG II. Endoperoxide (EP) analogues decreased GPSA and EP 045, a thromboxane A2 (TXA2) receptor blocker, eliminated the contractile responses of glomeruli to the EP analogues. Authentic TXA2, synthesized from sheep platelet microsomes, also reduced GPSA. We conclude that glomerular products of arachidonate cyclooxygenation may either relax or contract the mesangium, thereby preserving or reducing filtration surface area. PGE2 exerts protective actions to reduce the mesangial contraction of ANG II, primarily through postreceptor effects. TXA2 may decrease glomerular filtration rate in certain diseases through direct actions on the mesangium.

Lack of production of thromboxane A 2 by the sheep heart following coronary occlusion

1. Blood pressure, heart rate and ECG were monitored continuously and coronary venous blood samples assayed for thromboxane B 2. Arterio-coronary venous lactic acid differences were estimated. 2. In anaesthetized sheep, 60 sec occlusions of the left circumflex coronary artery caused hypotension with little change in heart rate. Five minute occlusions produced similar changes accompanied by cardiac arrhythmias and a larger fall in blood pressure. 3. In conscious sheep heart rate rose and blood pressure was maintained. 4. ECG and lactic acid changes indicated severe myocardial ischaemia but no change in thromboxane release was detected. 5. The apparent lack of involvement of thromboxane may reflect a relative inability of sheep platelets to produce this substance or a failure to provide an adequate stimulus for synthesis.

The solubilization of platelet membrane-bound acetylcholinesterase and aryl acylamidase by exogenous or endogenous phosphatidylinositol specific phospholipase C

Phosphatidylinositol specific phospholipase C from Staphylococcus aureus could solubilize acetylcholinesterase up to 55% from sheep platelets in the presence of ethylenediaminetetra acetic acid (EDTA). The endogenous phosphatidylinositol specific phospholipase C of platelets activated by deoxycholate (at 3–5 mM) could also solubilize the enzyme to a similar extent. The solubilized enzyme could be further purified to apparent homogeneity by affinity chromatography without the use of any detergents. It is suggested that phosphatidylinositol specific phospholipase C will be a useful tool in the solubilization of acetylcholinesterase from mammalian sources and its purification free of detergents. The present study also demonstrates the parallel behaviour of acetylcholinesterase and aryl acylamidase in platelets confirming their identity.

A comparative study of collagen induced thromboxane release from platelets of different species: Implications for human athero sclerosis models

The role of thromboxane in human, rabbit, dog, pig and sheep platelet aggregation has been studied. Each of these species showed a concentration-dependent relationship between collagen concentrations and the thromboxane B 2 (TXB 2) released from aggregating platelets, however sheep platelets produced only 17.5% of the amount of TXB 2 released from human platelets under the same collagen stimulus. Indomethacin did not inhibit sheep platelet aggregation in the concentration range 0.114 mM – 0.114 μM, yet proved to be a potent inhibitor of human platelet aggregation. This prostaglandin endoperoxide analogue, U.46619, induced human platelet aggregation at a dose of 100 ng, however the analouge, at doses as high as 500 μg was much less active on sheep platelets when compared to human platelets. Thus we have demonstrated that there are differences in the extent of thromboxane involvement in platelet aggregation between different species, which must be considered with other criteria, before choosing an animal model for studying human atherosclerosis.

Serotonin-sensitive aryl acylamidase activity of platelet acetylcholinesterase

Serotonin-sensitive aryl acylamidase (AAA, EC 3.5.1.13) was purified to apparent homogeneity from sheep platelets by affinity chromatography and it was shown to be associated with the platelet acetylcholinesterase (AChE, EC 3.1.1.7). The basis for the association of the two enzymes was the following. Both enzyme activities co-eluted from the affinity columns with constant ratios of specific activities and percentage recoveries. Both enzymes co-migrated on gel electrophoresis. Both enzymes co-eluted during Sepharose 6B gel filtration. Potent inhibitors of AChE such as bis(4-allyldimethyl ammoniumphenyl) pentan-3-one dibromide (BW 284C51), neostigmine and eserine also inhibited AAA potently. Both enzymes lost significant activity on treatment with deoxycholate or taurodeoxycholate and the loss could be partly restored by a mixture of phospholipids. The platelet AAA was specifically inhibited by serotonin and to a lesser extent by tryptamine but not by several other amines. It was also inhibited by acetylcholine and several of its analogues and homologues. It is suggested that in the platelets the two enzymes (AAA and AChE) are probably identical.

Isolation, characterization and chemical composition of the membrane from sheep platelets

A procedure of the isolation of platelets from the blood of adult sheep ( Ovis aries L. var domestica) is reported. This procedure is based on differential centrifugation and a specific lysis for elimination of erythrocytes. We have obtained platelets with a purity at least 99% and a relative high yield (2.3 ± 0.4 g wet/1 whole blood = 235 ± 40 mg platelet proteins/1 whole blood). After disruption, homogenisation and ultracentrifugation onto a discontinuous sucrose gradient (1.6 M, 1.1 M, 1.0 M and 0.6 M sucrose), four fractions were obtained. We have separated, for the first time, a particulate preparation enriched in the whole sheep plasma membrane. This fraction was characterized by: (i) the typical membrane morphology as shown by electron micrographs; (ii) the highest activities in membrane marker enzymes such as bis( p-nitrophenyl)phosphate phosphodiesterase (EC 3.1.16.1) and 5′-dTMP- p-nitrophenyl ester phosphodiesterase (EC 3.1.3.35), and the relatively low activity for marker enzymes associated to other subcellular fractions; (iii) the highest sialic acid; cholesterol and phospholipid concentrations. The chemical composition of the platelet membrane isolated is: total proteins, 49%; lipids, 47%; carbohydrates, ≅3.4% (the content of hexoses is twice as high as that of hexosamines and sialic acid). The similarities and differences of this preparation with others from several sources are discussed.

Thrombocytolysis in gel.

Cytolysis test in agarose gel was applied to study surface antigens of platelets. By means of rabbit antisera to nucleated cells and/or erythrocytes, species-specific antigens were demonstrated on platelets of various species. In studying reactions of human platelets with rabbit antisera, it was found that species antigens shared by nucleated cells and erythrocytes play a major role in thrombocytolysis. Evidence was also presented that human platelets contain surface antigens specific for platelets. Forssman antigen was detected on platelets of Forssman-positive species guinea pig, hamster and chicken. Heterophile antigen (Paul-Bunnell antigen) was demonstrated on bovine and sheep platelets. Lytic titers of most infectious mononucleosis sera were considerably higher against bovine than against sheep platelets. Results obtained in preliminary studies indicated that thrombocytolysis in gel may be used for detection of human alloantigens on platelets.

Comparative aspects of sugar penetration into blood platelets

1. 1. The relative rates of penetration of pentoses and hexoses into blood platelets of dog ( Canis familiaris) and sheep ( Ovis aries) were studied with a photoelectric densimeter. 2. 2. In both species of platelets D-ribose and L-sorbose penetrated more rapidly than D-xylose. No penetration of D-glucose into these cells was detected by the methods employed. 3. 3. It was shown also that dog platelets are more permeable to some sugars than sheep platelets.

Platelet Aggregation by Staphylococcal Toxins

SummaryAggregation by some staphylococcal toxins resulted in the following observations. Staphylocoagulase in lower concentrations causes aggregation in a similar way as thrombin, whereas in higher concentrations, clotting occurs which is always preceded by quick platelet aggregation. Alpha-hemolysin aggregates human and rabbit platelets, whereas sheep platelets are not affected. Preincubation of susceptible platelets with this toxin, results in their lysis. Betahemolysin causes also aggregation. Leukocidin is inactive. All these phenomena are well visible visually and in the phase contrast microscope. Staphylocoagulase and alpha-hemolysin, when added to the platelet-rich plasma simultaneously with the adenosine diphosphate, interfere and the platelet aggregation is partly inhibited. Pathological aggregation of platelets by bacterial products and its possible significance is discussed.