Sheep Platelets Research Articles

Differences in human and sheep platelet adherence, aggregation and activation induced by glass beads in a modified chandler loop-system.

In human cardiovascular research, sheep in particular are used as a large animal model in addition to pigs. In these animals, medical products, developed and tested for human medical purposes, are almost exclusively used in interventional studies. Therefore, the extent to which platelets from human and ovine blood differ in terms of adherence, aggregation and activation after a 4- or 8-minutes exposure to glass was investigated. Testing was performed with platelet-rich plasma (PRP) and a modified chandler loop-system, with 4- and 8-minute blood-material exposure times corresponding to 20 and 40 test cycles, respectively, through the entire silicone tube loop of the test system.In sheep and human PRP, contact with the silicone tubing resulted in a decrease in platelet count after 4 minutes and 20 test cycles, respectively. Four more minutes (20 additional test cycles) caused a further decrease of the platelet count only in sheep PRP. When the silicon tube was partly filled with glass beads, these effects were more pronounced and stronger in sheep then in human PRP.The mean platelet volume, which was used as parameter for platelet aggregation, did not change over time in human PRP without glass exposure. With glass exposure in human and sheep PRP the mean platelet volume increased within 40 test cycles, but this increase was stronger in sheep than in human PRP.Regarding activation behavior, the activation markers CD62P and CD63 were detectable only in < 30% (sheep) and < 45% (human) of platelets, whereas after 8 min of glass exposure, the proportion of CD62P+ and CD63+ cells was more increased than before only in sheep. These results indicate that ovine platelets adhere more strongly to glass and show stronger aggregation behavior after glass contact than human platelets, but that ovine and human platelets differ only slightly in activability by glass.

Comparative Analysis of the Hemostasiological Profile in Sheep and Patients with Cardiovascular Pathology as the Basis for Predicting Thrombotic Risks During Preclinical Tests of Vascular Prostheses.

The aim of the investigation was to study the details of hemostasiological profile in sheep and patients with coronary heart disease (CHD) and to find the possibility of predicting thrombotic risks during preclinical tests of vascular prostheses on a large laboratory animal model.Materials and Methods.The functional activity of platelets was measured in platelet-rich plasma with inductors: ADP, epinephrine, collagen. Prothrombin activity, international normalized ratio, activated partial thromboplastin time (APTT), thrombin time, fibrinogen concentration, antithrombin III and protein C activity, fibrinolysis were determined in blood plasma. Changes in clot formation and viscoelastic properties of clots were assessed using thromboelastography.Results.Significant differences were found in the hemostasiological profile of sheep and CHD patients. Sheep platelets had increased response to ADP induction and practically no response to epinephrine induction; collagen-induced aggregation was comparable in the study groups. Coagulation hemostasis of sheep was characterized by increased activity of the prothrombin complex, shortened thrombin time, while APTT and fibrinogen values remained comparable. At the same time, sheep exhibited a significant decrease in the activity of anticoagulant and fibrinolytic systems as compared to CHD patients. When assessing dynamic changes in clot formation, it was observed that initiation phase was faster in animals, while clot density exceeded that in patients.Conclusion.The hemostasiological profile of sheep is characterized by the increased speed of thrombus formation, greater strength of the formed clot, and lower lysis ability as compared to CHD patients. The revealed details of the hemostasiological profile of sheep can be potential targets for therapy with antithrombotic drugs that minimize thrombotic risks in preclinical testing of vascular prostheses.

Vaccination prepartum enhances the beneficial effects of melatonin on the immune response and reduces platelet responsiveness in sheep

BackgroundMelatonin regulates several physiological processes and its powerful action as antioxidant has been widely reported. Melatonin acts modulating the immune system, showing a protective effect on the cardiovascular system and improving vaccine administration as an adjuvant-like agent. Here, we have investigated the role of melatonin as an adjuvant of the Clostridium perfringens vaccine in prepartum sheep and whether melatonin modulates platelet physiology during peripartum.ResultsThe experiments were carried out in peripartum sheep from a farm located in an area of Mediterranean-type ecosystem. Plasma melatonin levels were determined by ELISA and sheep platelet aggregation was monitored using an aggregometer. Here we demonstrated for the first time that plasma melatonin concentration were higher in pregnant (125 pg/mL) than in non-pregnant sheep (15 pg/mL; P < 0.05). Administration of melatonin prepartum did not significantly modify platelet function but significantly improved the immune response to vaccination against C. perfringens.ConclusionAdministration of melatonin as an adjuvant provides a significant improvement in the immune response to vaccine administration prepartum against C. perfringens.

Melatonin, as an adjuvant‐like agent, enhances platelet responsiveness

Melatonin exerts immunomodulatory actions that enhance the magnitude and quality of immune responses specific for certain antigens; this has raised the possibility of using melatonin to design novel vaccine adjuvant systems. The present study investigated the effect of subcutaneous slow-release melatonin implants and subcutaneous melatonin injections on the responsiveness of circulating platelets in sheep after vaccination against Dichelobacter nodosus (A1 and C serotypes), the bacterium that causes ovine footrot, a major cause of lameness in sheep. The experiments were carried out in sheep from a farm located in an area of Mediterranean-type ecosystem. Plasma melatonin levels were determined by radioimmunoassay, sheep platelet aggregation was monitored using an aggregometer and Ca2+ mobilization was determined by spectrofluorimetry using fura-2. Administration of melatonin either by implants or subcutaneous injections increased plasma melatonin concentrations, an effect that was found to be greater and more sustained when melatonin was administered via implants. Vaccination per se, as well as melatonin, increased the percentage and rate of platelet aggregation and reduced the lag-time in response to the physiological agonist thrombin, an effect that was found to be significantly greater when melatonin was administered to vaccinated animals. Melatonin enhanced thrombin-evoked Ca2+ release and entry and further increased Ca2+ mobilization observed in platelets from vaccinated sheep. These observations suggest that the use of melatonin, as a novel adjuvant, induces beneficial effects on platelet function and haemostasis, and opens new perspectives for therapeutic manipulation of immune responses to vaccination.

Mechanical Stress Activates Platelets at a Subhemolysis Level: An In Vitro Study

A feasibility study is performed to quantify sheep platelets (PLTs) and to identify the relationship between PLT count and hemolysis as a consequence of mechanical stress. Six adult, healthy Dorset sheep have been used for in vitro blood sampling test procedures in a hemoresistometer device (HRM). In each experiment, blood of the same animal was exposed to six different shear rates. Free hemoglobin levels and PLT count for each shear rate were detected. In all animals (A-F), hemolysis increased significantly between the shear rates of 2325 and 3100/s (P < 0.05) and the mean PLT count dropped immediately (contact, low shear) 40% in the beginning, between the shear rates of 0 and 775/s (P < 0.05). PLT count increased slightly as soon as hemolysis started. At higher shear rates, hemolysis increased and PLTs reduced further. Precise counting of PLTs indicates that PLTs are consumed dramatically at very low shear (by contact) and further by applied mechanical stress when hemolysis is obvious. A repetition of these tests with human blood could indicate species differences.

Anticoagulation of Juvenile Sheep and Goats With Heparin, Warfarin, and Clopidogrel

Little data exist on anticoagulation of young sheep and goats. We tested the effect of heparin, warfarin, and clopidogrel in two sheep and two goats weighing 17-35 kg. Each animal received heparin boluses of 80, 100, and 200 units/kg; goats also received 300, 350, and 400 units/kg. All animals received continuous heparin 40, 60, and 80 units/kg/hour; oral warfarin 0.3, 0.6, and 0.9 mg/kg/day; and oral clopidogrel 75 and 150 mg/day (2.8-3.4 and 5.6-6.9 mg/kg/day). Results were in the form of complete blood counts, activated clotting times (ACT), partial thromboplastin times, prothrombin times, thromboelastograms, and whole-blood lumiaggregometry. After heparin boluses of 200 units/kg, sheep and goats reached mean peak ACTs over 400 seconds. After continuous infusions of 40, 60 and 80 units/kg/hour, sheep and goats exceeded our therapeutic range for ACTs (195-215 seconds for sheep, 155-175 seconds for goats). For warfarin therapy, both sheep and goats required treatment with >0.6 mg/kg/day to achieve INRs over 2.5. Clopidogrel treatment, after 14-17 days of 75-150 mg/day, inhibited sheep platelets by 25-36% and goat platelets by 35-46%. We conclude that young sheep and goats can be safely and effectively anticoagulated with heparin and warfarin, and can also show a modest antiplatelet response to clopidogrel. Doses for each drug were generally higher than those used for humans, and warfarin therapy in sheep may be unpredictable. These results should be useful for developing anticoagulation protocols to test pediatric mechanical circulatory support devices.

Adhesive Properties of Platelets from Different Animal Species

The use of large animals (e.g., pig and sheep) in human medicine, and the need to develop new therapeutic strategies for domestic animal diseases related to platelet disorders, require better characterization of the physiology of animal platelets. In this study, the ability of platelets from buffaloes, horses, pigs and sheep to adhere to immobilized autologous fibrinogen was compared with that of human platelets. Blood samples were collected from the jugular vein of six healthy subjects of each species and platelet-rich plasma (PRP) was obtained by centrifugation. Platelets, isolated by further centrifugation of PRP, were washed by gel-filtration on Sepharose-2B, counted and added to the wells of 96-well plates pre-coated with autologous fibrinogen. After different times of incubation, non-adherent platelets were removed, and the number of adherent platelets was assessed by measuring endogenous acid phosphatase activity. Horse platelets showed the strongest ability to adhere to autologous immobilized fibrinogen, being 1·7-, 3·1- and 2·3-fold more active than human, buffalo and porcine platelets, respectively. Sheep platelets were unable to adhere to autologous immobilized fibrinogen. Platelet activation by adenosine 5-diphosphate (ADP) increased both human and animal platelet adhesive response. ADP-stimulated sheep platelets were able to adhere to autologous immobilized fibrinogen, albeit to a lesser extent than platelets from the other animal species. The observed interspecies variability in adhesive properties of platelets may reflect structural differences, or differences in the availability of the fibrinogen receptor (glycoprotein IIb/IIIa) on the platelet surface.

Species Variability in Platelet Aggregation Response to Different Agonists

Conflicting data on platelet function in animal species are reported in the literature. In this study, the response of buffalo, horse, pig and sheep platelets to different agonists was assessed. Blood samples were collected from the jugular vein of six healthy subjects of each species and platelet-rich plasma was obtained by centrifugation. Platelet aggregation responses to increasing doses of adenosine 5′-diphosphate (ADP), arachidonic acid, collagen, platelet activating factor (PAF) and ristocetin were measured by a turbidimetric method. Horse platelets were the most responsive to ADP, collagen and PAF, whereas sheep platelets were the most responsive to ristocetin. The response to arachidonic acid varied least between species. PAF was the most effective agonist, inducing a maximum aggregation response at a concentration of 1 μM for platelets of each species. Conversely, concentrations of ristocetin higher than 1 mg/ml induced a maximum aggregation response only with sheep and horse platelets. The different responses of platelets from the four animal species to various agonists may reflect either (1) structural differences (including composition of the platelet membrane and presence of specific agonist receptors), or (2) activation of distinct signalling pathways by the agonist.

Comparison between in vitro lipid peroxidation in fresh sheep platelets and peroxidative processes during sheep platelet ageing under storage at 4°C

Incubation of sheep platelet crude membranes with xanthine oxidase (XO)/hypoxanthine/Fe 2+-ADP revealed: (i) a fast peroxidative response – with a maximal linear rate of 14 nmol malondialdehyde (MDA) equivalents/mg protein, as evidenced by the thiobarbituric acid test – and a decrease in the polyunsaturated fatty acid (PUFA) content of the platelet crude membranes; (ii) a decrease in the lipid fluidity in the deep lipid core of the membranes but not at the membrane surface; (iii) a dramatic inhibitory effect on glucose 6-phosphatase (Glc-6-Pase) but not on acetylcholinesterase activity. Platelets were also aged by storage at 4°C in their own plasma or in Seto additive solution. In these media, platelet aggregates were visible and the effects on platelet phospholipids, PUFA, lipid extract fluorescence, crude membrane fluidity and membrane-bound enzyme activities were assessed for comparison with those observed in in vitro lipid peroxidation. The sensitivity of membranes from stored platelets to lipid peroxidation was also assessed. Storage of platelets in plasma for 5 days was associated with different changes in their crude membranes such as decreases in arachidonic acid contents, the decrease not being avoided by the presence of phospholipase A 2 inhibitors, increases in MDA equivalents, conjugated dienes and lipid extract fluorescence, decreases in the amounts of MDA equivalents formed by platelet crude membranes treated with the oxidizing agents, changes in membrane fluidity and inhibition of Glc-6-Pase. All these alterations were less pronounced or even abolished after platelet storage in Seto. These findings suggest that platelet lipid peroxidation due to XO/hypoxanthine/Fe 2+-ADP and platelet membrane alterations observed after platelet ageing under storage at 4°C share common features. Also, as regards the prevention of peroxidative processes, Seto solution permits better storage of sheep platelets than plasma.

Amphiphilic and hydrophilic nature of sheep and human platelet phosphotyrosine phosphatase forms

To date, although at least 75 different PTPases (protein-tyrosine-phosphate-phosphohydrolase, EC 3.1.3.48) have been identified, those detected in platelets are rather scarce. Based on previous results from our laboratory, we investigated the existence of new PTPases in platelets. Triton X-114 phase partitioning of Triton X-100-solubilized human and sheep platelet membranes allowed PTPase to be recovered in the detergent-rich (40–35%, respectively) and -poor phases (60–65%, respectively). Sedimentation analyses of both phases from the sheep species revealed hydrophilic 6S and 3.7S, and amphiphilic 7.5S and 10.3S PTPase forms. Sedimentation analyses of human platelet membrane-associated or cytosolic PTPase revealed hydrophilic 6.7S and 4.3S, and amphiphilic 5.5S and 10.8S forms, or hydrophilic 4S, 5.9S and 6.9S forms, respectively. Western blot analysis using monoclonal antibodies (MoAb) against human PTP1B, PTP1C, PTP1D and RPTPα (mouse anti-human PTPase MoAbs) showed that RPTPα was not present in platelets and that the PTP1C type and PTP1D type (but probably not the PTP1B type) were expressed in sheep species. Immunoblots also revealed that all PTPases detected were mainly membrane-associated, with similar percentages of cellular distribution in both species. All PTPases were mainly recovered in the detergent-poor phases from the Triton X-114 phase partitioning, although PTP1D from human species was also significantly present (30%) in the detergent-rich phase. Additionally, all PTPases sedimented within the same PTPase peak in sucrose gradients (sedimentation coefficients around 4S). These findings indicate that amphiphilic and hydrophilic PTPases different from PTP1B, PTP1C, PTP1D or RPTPα, with higher sedimentation coefficients and with higher activity when O-phosphotyrosine or a synthetic peptide phosphorylated on tyrosine were used as substrates, are present in platelets.

Sheep, pig, and human platelet–material interactions with model cardiovascular biomaterials

Sheep, pig, and human platelet–material interactions with model cardiovascular biomaterials

Oxidative inactivation of human and sheep platelet membrane-associated phosphotyrosine phosphatase activity

Incubation of human or sheep platelet crude membranes with xanthine oxidase/hypoxanthine in the presence of Fe 2+/ADP inactivated phosphotyrosine phosphatase (PTPase, protein-tyrosine-phosphate-phosphohydrolase, EC 3.1.3.48) activity in a time-dependent manner, this inhibition being significant within 5 min of treatment. The dynamics of protein thiols differed depending on the platelet species, but in any case decreases in protein thiols were only visible 20–45 min after the start of the treatment. The inhibition of PTPase activity in general showed good a correlation with the production of thiobarbituric acid-reactive substances (TBARS). The results with several antioxidants suggest that the inhibition of PTPase activity is related to the generation of alkoxyl and/or peroxyl radicals. Furthermore, the formation of fluorescent products and changes in amino groups were observed only after long incubation times with the oxidizing agents, these fluorescent products and the residual enzyme activity remaining in the membrane fraction. Treatment of platelet membranes with trans-2-nonenal and n-heptaldehyde, but not with acetaldehyde, also inhibited membrane-associated PTPase activity. However, the amount of protein thiols was reduced only by treatment with trans-2-nonenal. Fluorescence product formation was always higher with trans-2-nonenal, these products being mainly located in the protein fraction. The results with aldehydes suggest that secondary degraded products of lipid hydroperoxides affect PTPase activity. Kinetic studies of PTPase activity indicated that with all treatments enzyme inhibition is mainly due to a decrease in the V max value. The results of fluorescence anisotropy measurements of labeled platelet membranes did not support the notion of a contribution of the lipid organization to peroxidation-mediated PTPase inhibition. All the above results indicate that platelet membrane-associated PTPase inhibition due to treatment with xanthine oxidase/hypoxanthine in the presence of Fe 2+/ADP is a very complex, time-dependent process, and that it is probably related, at least after long periods of peroxidation, to changes in protein thiols and amino groups. We predict that the sensitivity of PTPase to lipid peroxidation must be physiologically relevant because of the increasing importance of tyrosine phosphorylation in signal transduction, in general, and in platelet activation and aggregation in particular.

Sheep, pig, and human platelet-material interactions with model cardiovascular biomaterials.

The relationship between cardiovascular device performance in animals and humans is not straightforward. As the principal formed element in a thrombus, platelets play a major role in determining the hemocompatibility of mechanical heart valves and other high-shear-rate cardiovascular devices. Since larger animals are required to test many such devices, sheep and porcine platelet responses were compared to humans. Adhesion, spreading, and the formation of thrombilike structures were examined in vitro on pyrolytic carbon mechanical heart valve leaflets, National Institutes of Health-reference polyethylene and silicone rubber, and Formvar. Principal findings were that platelet responses are strongly dependent upon the biomaterial and the species: Porcine and human platelets spread extensively on pyrolytic carbon, formed thrombuslike structures on Formvar, and were least active on silicone rubber. Human and porcine platelets responded differently to polyethylene: Human platelets spread extensively, while porcine platelets remained pseudopodial. In contrast, sheep platelets attached much less, never reached fully spread shapes, and were far less active overall. Since porcine responses were generally similar to humans, pigs may be a useful predictor of in vivo platelet-biomaterial interaction in humans. Conversely, as ovine platelets were much less active, this must be accounted for in the evaluation of cardiovascular devices tested in sheep.

Amphiphilic and hydrophilic forms of acetylcholinesterase from sheep platelets

Acetylcholinesterase (AChE, EC 3.1.1.7) was extracted from sheep platelets by successive homogenizations, yielding low-salt soluble (LSS), high-salt soluble (HSS) and detergent-soluble (DS) fractions. These accounted, respectively, for about 30%, 7% and 60% of total AChE activity. Applications of hydrophobic chromatography on phenyl-agarose to three solubilized fractions revealed that hydrophilic forms were almost exclusively located in the LSS fraction (≈27% of total AChE), whereas most amphiphilic forms were present in DS extracts (≈59% of total AChE), the remaining forms being distributed among aqueous soluble fractions. Enzyme molecular forms in the solubilized extracts were identified by centrifugation in 5–20% sucrose gradients containing Triton X-100 or Brij 97 to differentiate between hydrophilic or amphiphilic species. A predominance of hydrophilic dimeric forms (≈22%), with small amounts of hydrophilic monomers (5%) and amphiphilic dimers and monomers (3%), was found in soluble AChE (LSS fraction). Amphiphilic AChE forms extracted in the HSS and DS fractions had a single peak in the sedimentation profiles with sedimentation coefficients of about 6S in gradients with Triton X-100; these were slightly shifted in the presence of Brij 97. After treatment with dithiothreitol, this molecular form solubilized in DS was converted to another molecular form with a lower sedimentation coefficient. Our results show that amphiphilic globular dimers are the dominant molecular form in sheep platelet AChE, suggesting a partial conversion of this membrane-bound form into soluble dimers and monomers, mainly with a hydrophilic character, through the action of either endogenous proteases and phospholipases or residual endogenous reducing agents.

Characterization of Phosphotyrosine Phosphatase Activity in Sheep Platelets: Amphiphilic and Hydrophilic Forms

Using O-phosphotyrosine as a substrate, we characterized the phosphotyrosine phosphatase (PTPase; protein-tyrosine-phosphate-phosphohydrolase, EC 3.1.3.48) activity from sheep platelets. PTPase was found to be located in three particulate subcellular fractions and in the cytosol, with K m values in the millimolar range. PTPase was strongly inhibited by vanadate, molybdate and HgCl 2 and only weakly inhibited by Zn 2+. Other divalent cations and NaF had no significant effect on the activity associated with the membrane fraction but were slightly stimulatory as regards cytosolic activity. Heparin inhibited cytosolic activity 2-fold more than membrane-bound activity and dithiothreitol only inhibited cytosolic PTPase. Polycationic compounds were seen to be weak stimulators of all the PTPase activity. Solubilization of the PTPase from membranes always required a detergent. When subjected to Triton X-114 phase partitioning, PTPase was recovered in the detergent-rich (35%) and in the detergent-poor (65%) phases. Sedimentation analysis of the cytosolic PTPase showed a peak of 3.2S that remained unmodified when Triton X-100 or Brij 97 sucrose gradients were used. Sedimentation analysis of the membrane-associated PTPase showed 6S and 3.7S peaks unchanged in Triton X-100 or Brij 97 gradients together with 7.5S and 10.3S shoulders that shifted to smaller sedimentation coefficients in Brij 97 sucrose gradients. These results support the view that sheep platelets contain amphiphilic and hydrophilic forms of PTPase.

Decrease in platelet, erythrocyte and lymphocyte acetylcholinesterase activities due to the presence of protease inhibitors in the storage buffer.

The effect of the presence of protease inhibitors in the storage buffer on acetylcholinesterase (AChE) activity was studied in crude membrane preparations from sheep platelets before and after solubilization of the membranes with Triton X-100. Although sensitive to the action of trypsin, the biological activity of AChE remained unchanged for as long as 6 days at 4 degrees C in a protease-inhibitor-free medium. At 10(-5) M final concentration PMSF reduced AChE activity to 50% after 24 hours of storage. This reduction was abolished in mixtures in which PMSF was present together with 3 mM EGTA, 5 mM EDTA and 0.1 mg/mL trypsin inhibitor. Nevertheless, under these conditions, storage periods longer than 24 hours still drastically reduced AChE activity. A mixture of EDTA, EGTA and trypsin inhibitor also produced a decrease in AChE activity after 24 hours. A more complex cocktail of inhibitors including several commonly used peptides decreased AChE activity only if PMSF or EDTA were present in the mixtures. Similar results were obtained with sheep erythrocytes or lymphocytes, and bovine erythrocytes.

Cross-reactivity of human molecular markers for detection of prethrombotic states in various animal species

The aim of the present study was to investigate the reactivity of immunoreagents developed for clinical applications in humans in different animal species (hen, mouse, rat, rabbit, guinea-pig, dog, pig, sheep, baboon). Prothrombin fragment 1 + 2, thrombin-antithrombin III complex and fibrinopeptide A were tested for coagulation, platelet factor 4 and beta-thromboglobulin for platelet activation, glycoprotein IIb-IIIa, glycoprotein Ib and P-selectin for platelet membrane glycoproteins, D-dimers for fibrinolysis, thrombomodulin for activation of endothelial cells and thrombospondin and von Willebrand factor for adhesive proteins. Prothrombin fragment 1 + 2, platelet factor 4, beta-thromboglobulin and D-dimers were revealed only in baboons. Fibrinopeptide A was well detected in baboons but weakly in mice, dogs, pigs and sheep. Whereas glycoprotein IIb-IIIa was revealed on guinea-pig, dog and sheep platelets and glycoprotein Ib on rabbit and dog platelets, P-selectin and thrombomodulin were never detected. Thrombospondin was revealed in hens, mice, rats, guinea-pigs, pigs, sheep and baboons and von Willebrand factor in mice, rats, guinea-pigs, dogs, pigs, sheep and baboons. Interestingly, thrombin-antithrombin III complex (TAT) was detected in all species tested except the hen. A time- and dose-dependent increase in TAT was observed when rats, dogs or pigs were infused with thromboplastin (4.5-450 microliters/kg/h), while administration of hirudin (1 mg/kg) abolished this TAT generation. Thus, the TAT immunoassay could provide a tool for the screening of antithrombotic drugs in a number of animal species. However, the possibility of using a wider panel of human immunoreagents would appear to be restricted to baboons which display good species cross-reactivity.

Biochemical characterization of sheep platelet acetylcholinesterase after detergent solubilization

The biochemical characterization of detergent-solubilized acetylcholinesterase (AChE) from subcellular particles of sheep platelets and the effects of different effectors on AChE activity from solubilized platelet crude membranes have been undertaken and studied. Solubilization of AChE with detergent increased the thermal stability of the enzyme from all particulate fractions. Solubilized AChE from the mitochondria-granule fraction was the most thermostable at 55°C. The K m values against acetylthiocholine chloride and the Arrhenius plot obtained were very similar for the AChE from all the solubilized fractions. There were no differences in the ability of solubilized AChE from different subcellular fractions to bind concanavalin A (Con A). In solubilized platelet crude membranes, benzyl alcohol was a potent AChE inhibitor at a concentration of 10 −2 M, whereas ethanol was not. Mg 2+ cations and, to a lesser extent, Ca 2+ and Mn 2+ cations, activated AChE at concentrations higher than 1 mM. Serine hydrolase inhibitors and cholinesterase-specific inhibitors were very effective in the inactivation of AChE, whereas EDTA and EGTA had no effect. Of all the monosaccharides tested, only N-acetylneuraminic acid exerted an inhibitory effect on AChE activity. Immobilized-lectin binding studies demonstrated the interaction of solubilized crude membrane-bound AChE with Con A, lentil lectin and wheat germ agglutinin. Taken together, these data suggest the presence of a unique form of the membrane-bound AChE which has at least a-mannose and N-acetylglucosamine residues in the glycan chain.

The purification of megapoietin: a physiological regulator of megakaryocyte growth and platelet production.

The circulating blood platelet is produced by the bone marrow megakaryocyte. In response to a decrease in the platelet count, megakaryocytes increase in number and ploidy. Although this feedback loop has long been thought to be mediated by a circulating hematopoietic factor, no such factor has been purified. Using a model of thrombocytopenia in sheep, we have identified an active substance called megapoietin, which stimulated an increase in the number and ploidy of megakaryocytes in bone marrow culture. Circulating levels of this factor could be quantified with this assay and were found to be inversely proportional to the platelet count of the sheep. Levels increased from < 0.26 pM in normal sheep to 25-40 pM in thrombocytopenic sheep. From large amounts of thrombocytopenic sheep plasma we have purified a 31,200-Da protein and found that it retained the ability to stimulate both megakaryocyte number and ploidy in vitro. Injection of partially purified megapoietin into rats stimulated a 24% increase in megakaryocyte number and a 60% increase in mean ploidy as well as a 77% increase in the platelet count. Sheep platelets bound megapoietin and the amount of platelets required to eliminate half the activity in vitro was close to the amount associated with this same level of activity in vivo. We believe that megapoietin is the physiologically relevant mediator of megakaryocyte growth and platelet production. Moreover, our data suggest that the level of megapoietin is directly determined by the ability of platelets to remove megapoietin from the circulation.

Aspirin fails to inhibit platelet aggregation in sheep

This is the first report in which aspirin (ASA) has failed to inhibit aggregation of mammalian platelets. Preincubation of citrated sheep platelet rich plasma with a final concentration of 500 μM ASA for 3 minutes at 37°C did not inhibit aggregation induced by either arachidonic acid (AA; 1.6 mM), ADP (2.5 μM), collagen (5.6 μg/ml) or thrombin (0.04U). Instead, ASA potentiated the aggregation response produced by these agents except AA. Platelet aggregation that was reversible with ADP became irreversible after adding ASA. The inhibitory properties of ASA was confirmed with human platelets, challenged with AA, ADP, adrenalin and collagen. These findings suggest that sheep platelets have an ASA resistant cyclo oxygenase and may be able to aggregate by a pathway that is independent of arachidonic acid.