MOLECULAR REPRODUCTION AND DEVELOPMENT 30:207-213 (1991)
Studies on Murine Embryo-Derived Platelet-Activating Factor (EPAF) L.M. ADAMSON, B. PODSIADLY, Y.C. SMART, J.D. STANGER, AND T.K. ROBERTS Department of Biological Sciences and Discipline of Surgical Science, University of Newcastle and Lingard Fertility Centre, Lingard Hospital, Newcastle, New South Wales, Australia Studies were carried out using the ABSTRACT splenectomized mouse bioassay (SMB)to investigate the nature of embryo-derived platelet-activating factor (EPAF) and its relationship to synthetic platelet activating factor (PAF). While both C16-PAFand embryo conditioned media (ECM) induced a significant platelet decline in the SMB at 15 min postinjection, C18-PAF induced a similar effect at 30 min postinjection. The degree of EPAF activity in ECM was not altered with increasing embryo number from 2 to 40/ml of media. In contrast, PAF (C16/C18 mixture) induced a linear increase in activity with increasing concentration,leading to lethal effects at high concentrations. While EPAF activity was not significantly altered when ECM was diluted 1/1,000, PAF activity was abolished at 1/10 dilution. EPAF in ECM was not inactivated by mouse plasma; however, lipid extracted ECM, like PAF, underwent rapid inactivation in the presence of plasma. Aggregometer studies using horse platelets showed that ECM and lipid-extracted ECM were unable to induce platelet aggregation, while thin-layer chromatography (TLC) purified ECM (Rf 0.23) successfully aggregated horse platelets in vitro. Results suggested that EPAF and PAF are not homologous. EPAF might consist of PAF bound to a regulatory carrier molecule and appears to be associated with EPAF-inhibitor substance(s)in ECM. Key Words: Embryo, Platelet activating factor (PAF), Pregnancy, Platelets, Mouse, Embryo conditioned medium
INTRODUCTION One preimplantation embryo-released factor thought to play a role in the establishment of pregnancy is embryo-derived platelet-activating factor (EPAF). EPAF release has been reported for mouse (O’Neill, 1985; Roberts et al., 1987) and human (O’Neill and Saunders, 1984) embryos grown from the one-cell stage to the blastocyst stage in vitro. EPAF activity is also detectable in the maternal peripheral circulation following in vitro fertilization (IVF) and embryo transfer (ET) treatment for infertility (Roberts et al., 1987). The detection of EPAF in IVF-treated patients is possible within 1 day of embryo transfer, implicating EPAF as a potential marker of embryonic viability and treatment success. Currently, no information concerning the molecular 0 1991 WILEY-LISS, INC.
structure of EPAF is available. Some evidence implies a similarity between EPAF and the phospholipid mediator of platelet activation, platelet-activating factor (PAF) (O’Neill, 1985; Adamson et al., 1987). More recently, Angle et al. (1988) and Amiel e t al. (1989) have reported their failure to detect PAF in embryo conditioned medium (ECM), suggesting that EPAF and PAF might not be homologous. Perhaps these conflicting results are in part due to the fact th a t PAF consists of a t least 16 molecular species containing saturated or unsaturated alkyl or acyl residues (Ludwig and Pinckard, 1987). The two major molecular species of PAF have either a hexadecyl (C16) or a n octadecyl (C18) alkyl moiety, and these are compared with EPAF in terms of biological activity in the present study. In view of the potential role of EPAF in mammalian early pregnancy, it is important to understand the biological nature of this molecule and its relationship to PAF. This study was undertaken to address these areas.
MATERIALS AND METHODS Materials A semisynthetic mixture of C16 and C18 PAF (L-(Y phosphatidylcholine, P-acetyl, y-alkyl) was obtained as 2 mg/ml solution in chloroform (Sigma Chemical Co., St. Louis, MO). Except where stated, this PAF mixture preparation (C16/C18) was used in all SMB experiments. Synthetic C16 PAF and C18 PAF were also purchased a s separate preparations (Boehringer Mannheim, Sydney, Australia). A stock solution of 5 (*g/ml was made for each PAF preparation in modified Tyrode’s (T6) medium (Stanger and Quinn, 19821, containing 0.4% bovine serum albumin (BSA; Fraction V; Sigma. The stock solution was diluted to the required concentration with T6 + 0.4% BSA, stored a t 4”C, and used within 48 hr. T6 containing 0.4% BSA and chloroform a t a volume equivalent to that in the PAF solution was used a s the control medium (CM). Unless otherwise stated, PAF was used at a concentration that elicted 30% peripheral platelet decline in the splenectomized mice within 15 min of intraperitoneal injecReceived January 18, 1991; accepted June 3, 1991. Address reprint requests to Dr. T.K. Roberts, Department of Biological Sciences, University of Newcastle, Newcastle, New South Wales, 2308 Australia.
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tion. This PAF concentration was termed “platelet decline 30% (PD,,)” and was reestablished whenever a new batch of PAF was used. The 30% level was chosen to mirror the 30% platelet reduction found in the maternal murine system during early pregnancy (Roberts et al., 1987). ECM, which was used as the EPAF source, was collected from the 5 day (i.e., one cell to blastocyst) in vitro growth of embryos collected from 3-4-week-old F1 (C57BLi6 x CBA) mice, as described previously (Roberts et al., 1987). Embryos were cultured in groups of 20 in 1 ml T6 medium containing 0.4% BSA, unless otherwise stated. Following expanded blastocyst development, the embryos were removed, and the ECM was stored at -20°C until used. The T6 medium + 0.4% BSA was used as the CM.
Assay for PAF and EPAF Quackenbush strain (QS) mice (4-6 weeks old) were splenectomized as previously described (Roberts et al., 1987) and allowed to recover for 7-10 days before use. The test samples were injected intraperitoneally in 400 pl volumes. Mice were bled via the retroorbital plexus at 0 min and 15 min after injection, and manual platelet counts were performed on the blood samples, as previously described (Roberts et al., 1987). Results were presented as the percentage change in platelet counts between 0 and 15 min.
the original volume of T6 BSA, and tested in the aggregometer.
SPECIFIC EXPERIMENTS Activity of ECM, C16-PAF, and C18-PAF in the Splenectomized Mouse Bioassay (SMB) C16 PAF, C18 PAF (PD,, concentration), and ECM were each tested in four splenectomized mice. To compare the activity of these samples, platelet counts were performed on sequential blood samples collected a t 5, 15, 30, 60, and 90 min postinjection. Effect of Embryo Number on EPAF Activity Embryo cultures were set up with varying numbers of embryos (between 1and 40) per milliliter of medium. All cultures were of 5 days duration (i.e., one cell to blastocyst stage). ECM samples were collected, and each was tested undiluted in six splenectomized mice. In a n attempt to compare these EPAF results with the PAF dose response, varying PAF (C16/C18 mixture) concentrations (5-1.25 pgiml) and CM were each tested in five splenectomized mice.
Dilution Effect of EPAF, PAF, and Lipid-Extracted Material 1. ECM and PAF (PD,, concentration) were diluted repeatedly tenfold with control medium. The diluted and undiluted samples and control medium were each tested in a t least four splenectomized mice. In Vitro Platelet Aggregation 2. The total lipids were extracted from ECM (20 Horse blood was collected in acid citrate dextrose and embryosiml) and PAF (PD,, concentration) using the centrifuged (2OOg, 10 min) to obtain platelet-rich Bligh-Dyer protocol (Bligh and Dyer, 1959). The explasma (PRP). Platelets were isolated using ficoll- tracted lipids were resuspended in T6 medium + BSA. hypaque gradient centrifugation and resuspended in The lipid-extracted ECM was diluted repeatedly tencalcium-free Tyrode’s medium prior to use. Platelet- fold, and each dilution was tested in at least four poor plasma (PPP)was obtained by centrifuging (9OOg, splenectomized mice. Similarly, both lipid-extracted 10 min) a n aliquot of PRP. Both PRP and PPP were and untreated PAF were diluted repeatedly twofold used to calibrate a Payton dual-channel aggregometer (because a tenfold dilution had caused complete activ(Houston Instruments, Australia). Immediately before ity loss), and each dilution was tested in a t least four use, horse platelets were diluted in calcium chloride splenectomized mice. (5:1), and platelet samples of 475 p1 were placed in Effect of Mouse Plasma on PAF and siliconized glass cuvettes to which 25 pl ECM or PAF EPAF Activity was added. Aggregation was detected a s a change in the percent light transmission with time, which increased Since it is well known that plasma contains a n as platelets aggregated. acetylhydrolase capable of inactivating PAF (Pinckard et al., 1979), the effect of mouse plasma on PAF and Thin-Layer Chromatography (TLC) EPAF activity was investigated. Whole blood was The lipid-extracted ECM samples were dissolved in a collected via the retroorbital plexus of nonpregnant QS minimal amount (50 p1) of ch1oroform:methanol (2:l mice and placed immediately into tubes containing viv), spotted (25 p1) onto silica gel TLC plates (250 pm EDTA (5% wtivol solution) anticoagulant. Blood was hard surface analytical layer; J.T. Baker, Australian centrifuged a t 9OOg for 1 5 min, and the plasma was Chromatography Company, Sydney, Australia), and collected. PAF (PD,, concentration), lipid-extracted allowed to run a t least 150 mm using a chloroform: (1ilO dilution), and untreated (1/1,000 dilution) ECM methano1:water (65:35:4) solvent. Sections (10 mm x 8 and CM were added to separate tubes containing a n mm) were scraped from the plate and suspended in equal volume of plasma. The tubes were incubated a t ch1oroform:methanol (2:l viv) to elute the active frac- 37”C, and aliquots were removed from each tube at 0, tions. Silica was removed by centrifugation. Eluted 30, and 60 min. Each sample was tested immediately substances were dried under nitrogen, resuspended in for activity in six splenectomized mice.
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TABLE 1. Mean (kSE) Percentage Change in Platelet Counts of Splenectomized Mice (N = 4) at Various Time Intervals Following Injection of C16 PAF. C18 PAF. and ECM Time (min) C16 PAF C18 PAF 0 0.0 0.0 5 -15.0 (k6.8) -4.86 (k0.3) 15 -25.8 (+2.7)* -12.9 (k3.4) 30 -16.7 (+2.4)* -30.6 (+2.4)* 60 -12.8 (f2.4) -13.7 (k6.2) 90 -7.7 (k4.8) -13.3 (k2.3) *Platelet counts significantly (P < 0.05) different from time 0 count.
TABLE 2. Mean (kSE) Percentage Change in Platelet Counts of Splenectomized Mice (N = 6) 15 rnin Following Injection of ECM From Cultures Containing Varying Embryo Number per 1 ml Medium Percent change in Embryo number/ml medium platelet counts 0 -1.7 (k3.3) 1 0.5 (f5.0) 2 5 10 20 30 40
-17.9 -30.8 -24.2 -25.4 -23.3 -23.5
(k5.0)* (+2.9)* (+2.5)* (k2.1)* (f4.6)* (k3.8)*
ECM 0.0 -12.9 (k6.4) -28.6 (k2.2)* -17.3 (+2.7)* -10.4 (k6.2) -11.0 (k8.0)
TABLE 3. Mean (&SE)Percentage Change in Platelet Counts of Splenectomized Mice (N = 5) 15 rnin Following Injection of Various PAF Concentrations Percent change PAF concentration in platelet counts (/.&mu 0 0.5 (f3.3) 1.25 2.50 3.75 5.00
-10.4 -23.8 -25.1 -46.4
(k4.0) (+3.3)* (k3.3)* (k3.2)*
*Platelet counts significantly (P< 0.05) different between 0 and 15 min.
*Platelet counts significantly (P< 0.05) different between 0 and 15 min.
Statistical Analysis The changes in platelet counts of splenectomized mice between 0 and 15 min were analyzed for statistical significance using paired Student’s t tests. A significant (P < 0.05) response is indicated in the tables by a n asterisk.
Effect of Embryo Number on EPAF Activity Table 2 illustrates the effect of ECM in the SMB. Neither CM nor ECM from one embryo/ml medium induced a significant decline in platelet counts in splenectomized mice. ECM samples collected from the growth of from two to 40 embryodm1 medium induced significant reductions in platelet counts within 15 min of injection. The degree of platelet reduction induced by these samples was consistently on the order of 20-30% and was not significantly altered by increasing embryo number from two to 40. In no case was ECM found to be lethal in the SMB. In contrast, increasing the concentration of PAF in the SMB induced a linear increase in activity (Table 3). The highest PAF concentration indicated in Table 3 resulted in a response of between 40% and 50% platelet reduction, a level of activity found to be lethal in about 20% of the cases. The approximate LD,o concentration of PAF was found to correspond to that causing 55-65% reduction in platelet counts (data not shown).
RESULTS EPAF Activity in the SMB: Comparison With C16 and C18 PAF A significant platelet decline was found in the SMB a t 15 min postinjection of both ECM and C16 PAF.A similar platelet decline was detected a t 30 min postinjection of C18 PAF (Table 1).
Dilution Effect of EPAF, PAF, and Lipid-Extracted Material in SMB 1. ECM induced a significant decline in platelet counts of splenectomized mice over the range of undiluted to 1/1,000 dilution. A linear decline in activity was found over the range of 1/1,000 to 1/100,000 dilution, the latter dilution showing negligible activity (Table 4).Dilution of PAF (PDS0)resulted in the loss of
In Vitro Platelet Aggregation Induced by PAF (C16 and C18) and ECM (Untreated, Lipid-Extracted, and TLC Separated) In vitro aggregation of horse platelets was used to assess the activity of C16 PAF (20 pg), C18 PAF (20 pg), ECM, and CM. These samples were tested either untreated, after lipid extraction, or after TLC separation (as described above). Twenty-five microliters of each sample was tested for activity in the platelet aggregation module.
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TABLE 4. Mean (fSE)Percentage Change in Platelet Counts of Splenectomized Mice 15 rnin Following Injection of Diluted ECM (N = 6) and Diluted PAF (N = 4) Dilution ECM PAF
Neat 1/10
moo
1/ 1,000 1/10,000 1/100,000
-30.4 (+2.9)* -25.8 (51.7*) -24.5 (+2.9)* -24.2 (40.6)* -11.3 (52.9) -3.8 (k2.l) 0.0 (f1.5)
-28.1 (+3.2)* 2.9 (f3.2) -7.2 (f2.8) 0.9 (H.3)
NT NT CM -2.2 (f2.6) *Platelet counts significantly (P< 0.05) different between 0 and 15 min. NT, not tested.
TABLE 5. Mean (kSE)Percentage Change in Platelet Counts of Splenectomized Mice 15 min Following Injection of Untreated (N = 6) and Extracted (N = 4) ECM Dilutions
Dilution ECM Extracted ECM Neat -28.9 (+0.6)* -31.3 (+0.8)* 1/10 -26.0 (+1.2)* -24.2 (k1.3)* 1/100 -24.6 (+2.5)* -10.6 (f3.3) 1/1,000 -24.8 (+0.6)* -7.9 (f1.7) 1/10,000 -10.8 (f2.5) -7.5 (k2.5) 1/100,000 -3.9 (*2.l) -2.9 (f0.8) CM -1.2 (f2.5) 0.4 (k0.8) *Platelet counts significantly (P
Studies on Murine Embryo-Derived Platelet-Activating Factor (EPAF) L.M. ADAMSON, B. PODSIADLY, Y.C. SMART, J.D. STANGER, AND T.K. ROBERTS Department of Biological Sciences and Discipline of Surgical Science, University of Newcastle and Lingard Fertility Centre, Lingard Hospital, Newcastle, New South Wales, Australia Studies were carried out using the ABSTRACT splenectomized mouse bioassay (SMB)to investigate the nature of embryo-derived platelet-activating factor (EPAF) and its relationship to synthetic platelet activating factor (PAF). While both C16-PAFand embryo conditioned media (ECM) induced a significant platelet decline in the SMB at 15 min postinjection, C18-PAF induced a similar effect at 30 min postinjection. The degree of EPAF activity in ECM was not altered with increasing embryo number from 2 to 40/ml of media. In contrast, PAF (C16/C18 mixture) induced a linear increase in activity with increasing concentration,leading to lethal effects at high concentrations. While EPAF activity was not significantly altered when ECM was diluted 1/1,000, PAF activity was abolished at 1/10 dilution. EPAF in ECM was not inactivated by mouse plasma; however, lipid extracted ECM, like PAF, underwent rapid inactivation in the presence of plasma. Aggregometer studies using horse platelets showed that ECM and lipid-extracted ECM were unable to induce platelet aggregation, while thin-layer chromatography (TLC) purified ECM (Rf 0.23) successfully aggregated horse platelets in vitro. Results suggested that EPAF and PAF are not homologous. EPAF might consist of PAF bound to a regulatory carrier molecule and appears to be associated with EPAF-inhibitor substance(s)in ECM. Key Words: Embryo, Platelet activating factor (PAF), Pregnancy, Platelets, Mouse, Embryo conditioned medium
INTRODUCTION One preimplantation embryo-released factor thought to play a role in the establishment of pregnancy is embryo-derived platelet-activating factor (EPAF). EPAF release has been reported for mouse (O’Neill, 1985; Roberts et al., 1987) and human (O’Neill and Saunders, 1984) embryos grown from the one-cell stage to the blastocyst stage in vitro. EPAF activity is also detectable in the maternal peripheral circulation following in vitro fertilization (IVF) and embryo transfer (ET) treatment for infertility (Roberts et al., 1987). The detection of EPAF in IVF-treated patients is possible within 1 day of embryo transfer, implicating EPAF as a potential marker of embryonic viability and treatment success. Currently, no information concerning the molecular 0 1991 WILEY-LISS, INC.
structure of EPAF is available. Some evidence implies a similarity between EPAF and the phospholipid mediator of platelet activation, platelet-activating factor (PAF) (O’Neill, 1985; Adamson et al., 1987). More recently, Angle et al. (1988) and Amiel e t al. (1989) have reported their failure to detect PAF in embryo conditioned medium (ECM), suggesting that EPAF and PAF might not be homologous. Perhaps these conflicting results are in part due to the fact th a t PAF consists of a t least 16 molecular species containing saturated or unsaturated alkyl or acyl residues (Ludwig and Pinckard, 1987). The two major molecular species of PAF have either a hexadecyl (C16) or a n octadecyl (C18) alkyl moiety, and these are compared with EPAF in terms of biological activity in the present study. In view of the potential role of EPAF in mammalian early pregnancy, it is important to understand the biological nature of this molecule and its relationship to PAF. This study was undertaken to address these areas.
MATERIALS AND METHODS Materials A semisynthetic mixture of C16 and C18 PAF (L-(Y phosphatidylcholine, P-acetyl, y-alkyl) was obtained as 2 mg/ml solution in chloroform (Sigma Chemical Co., St. Louis, MO). Except where stated, this PAF mixture preparation (C16/C18) was used in all SMB experiments. Synthetic C16 PAF and C18 PAF were also purchased a s separate preparations (Boehringer Mannheim, Sydney, Australia). A stock solution of 5 (*g/ml was made for each PAF preparation in modified Tyrode’s (T6) medium (Stanger and Quinn, 19821, containing 0.4% bovine serum albumin (BSA; Fraction V; Sigma. The stock solution was diluted to the required concentration with T6 + 0.4% BSA, stored a t 4”C, and used within 48 hr. T6 containing 0.4% BSA and chloroform a t a volume equivalent to that in the PAF solution was used a s the control medium (CM). Unless otherwise stated, PAF was used at a concentration that elicted 30% peripheral platelet decline in the splenectomized mice within 15 min of intraperitoneal injecReceived January 18, 1991; accepted June 3, 1991. Address reprint requests to Dr. T.K. Roberts, Department of Biological Sciences, University of Newcastle, Newcastle, New South Wales, 2308 Australia.
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tion. This PAF concentration was termed “platelet decline 30% (PD,,)” and was reestablished whenever a new batch of PAF was used. The 30% level was chosen to mirror the 30% platelet reduction found in the maternal murine system during early pregnancy (Roberts et al., 1987). ECM, which was used as the EPAF source, was collected from the 5 day (i.e., one cell to blastocyst) in vitro growth of embryos collected from 3-4-week-old F1 (C57BLi6 x CBA) mice, as described previously (Roberts et al., 1987). Embryos were cultured in groups of 20 in 1 ml T6 medium containing 0.4% BSA, unless otherwise stated. Following expanded blastocyst development, the embryos were removed, and the ECM was stored at -20°C until used. The T6 medium + 0.4% BSA was used as the CM.
Assay for PAF and EPAF Quackenbush strain (QS) mice (4-6 weeks old) were splenectomized as previously described (Roberts et al., 1987) and allowed to recover for 7-10 days before use. The test samples were injected intraperitoneally in 400 pl volumes. Mice were bled via the retroorbital plexus at 0 min and 15 min after injection, and manual platelet counts were performed on the blood samples, as previously described (Roberts et al., 1987). Results were presented as the percentage change in platelet counts between 0 and 15 min.
the original volume of T6 BSA, and tested in the aggregometer.
SPECIFIC EXPERIMENTS Activity of ECM, C16-PAF, and C18-PAF in the Splenectomized Mouse Bioassay (SMB) C16 PAF, C18 PAF (PD,, concentration), and ECM were each tested in four splenectomized mice. To compare the activity of these samples, platelet counts were performed on sequential blood samples collected a t 5, 15, 30, 60, and 90 min postinjection. Effect of Embryo Number on EPAF Activity Embryo cultures were set up with varying numbers of embryos (between 1and 40) per milliliter of medium. All cultures were of 5 days duration (i.e., one cell to blastocyst stage). ECM samples were collected, and each was tested undiluted in six splenectomized mice. In a n attempt to compare these EPAF results with the PAF dose response, varying PAF (C16/C18 mixture) concentrations (5-1.25 pgiml) and CM were each tested in five splenectomized mice.
Dilution Effect of EPAF, PAF, and Lipid-Extracted Material 1. ECM and PAF (PD,, concentration) were diluted repeatedly tenfold with control medium. The diluted and undiluted samples and control medium were each tested in a t least four splenectomized mice. In Vitro Platelet Aggregation 2. The total lipids were extracted from ECM (20 Horse blood was collected in acid citrate dextrose and embryosiml) and PAF (PD,, concentration) using the centrifuged (2OOg, 10 min) to obtain platelet-rich Bligh-Dyer protocol (Bligh and Dyer, 1959). The explasma (PRP). Platelets were isolated using ficoll- tracted lipids were resuspended in T6 medium + BSA. hypaque gradient centrifugation and resuspended in The lipid-extracted ECM was diluted repeatedly tencalcium-free Tyrode’s medium prior to use. Platelet- fold, and each dilution was tested in at least four poor plasma (PPP)was obtained by centrifuging (9OOg, splenectomized mice. Similarly, both lipid-extracted 10 min) a n aliquot of PRP. Both PRP and PPP were and untreated PAF were diluted repeatedly twofold used to calibrate a Payton dual-channel aggregometer (because a tenfold dilution had caused complete activ(Houston Instruments, Australia). Immediately before ity loss), and each dilution was tested in a t least four use, horse platelets were diluted in calcium chloride splenectomized mice. (5:1), and platelet samples of 475 p1 were placed in Effect of Mouse Plasma on PAF and siliconized glass cuvettes to which 25 pl ECM or PAF EPAF Activity was added. Aggregation was detected a s a change in the percent light transmission with time, which increased Since it is well known that plasma contains a n as platelets aggregated. acetylhydrolase capable of inactivating PAF (Pinckard et al., 1979), the effect of mouse plasma on PAF and Thin-Layer Chromatography (TLC) EPAF activity was investigated. Whole blood was The lipid-extracted ECM samples were dissolved in a collected via the retroorbital plexus of nonpregnant QS minimal amount (50 p1) of ch1oroform:methanol (2:l mice and placed immediately into tubes containing viv), spotted (25 p1) onto silica gel TLC plates (250 pm EDTA (5% wtivol solution) anticoagulant. Blood was hard surface analytical layer; J.T. Baker, Australian centrifuged a t 9OOg for 1 5 min, and the plasma was Chromatography Company, Sydney, Australia), and collected. PAF (PD,, concentration), lipid-extracted allowed to run a t least 150 mm using a chloroform: (1ilO dilution), and untreated (1/1,000 dilution) ECM methano1:water (65:35:4) solvent. Sections (10 mm x 8 and CM were added to separate tubes containing a n mm) were scraped from the plate and suspended in equal volume of plasma. The tubes were incubated a t ch1oroform:methanol (2:l viv) to elute the active frac- 37”C, and aliquots were removed from each tube at 0, tions. Silica was removed by centrifugation. Eluted 30, and 60 min. Each sample was tested immediately substances were dried under nitrogen, resuspended in for activity in six splenectomized mice.
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TABLE 1. Mean (kSE) Percentage Change in Platelet Counts of Splenectomized Mice (N = 4) at Various Time Intervals Following Injection of C16 PAF. C18 PAF. and ECM Time (min) C16 PAF C18 PAF 0 0.0 0.0 5 -15.0 (k6.8) -4.86 (k0.3) 15 -25.8 (+2.7)* -12.9 (k3.4) 30 -16.7 (+2.4)* -30.6 (+2.4)* 60 -12.8 (f2.4) -13.7 (k6.2) 90 -7.7 (k4.8) -13.3 (k2.3) *Platelet counts significantly (P < 0.05) different from time 0 count.
TABLE 2. Mean (kSE) Percentage Change in Platelet Counts of Splenectomized Mice (N = 6) 15 rnin Following Injection of ECM From Cultures Containing Varying Embryo Number per 1 ml Medium Percent change in Embryo number/ml medium platelet counts 0 -1.7 (k3.3) 1 0.5 (f5.0) 2 5 10 20 30 40
-17.9 -30.8 -24.2 -25.4 -23.3 -23.5
(k5.0)* (+2.9)* (+2.5)* (k2.1)* (f4.6)* (k3.8)*
ECM 0.0 -12.9 (k6.4) -28.6 (k2.2)* -17.3 (+2.7)* -10.4 (k6.2) -11.0 (k8.0)
TABLE 3. Mean (&SE)Percentage Change in Platelet Counts of Splenectomized Mice (N = 5) 15 rnin Following Injection of Various PAF Concentrations Percent change PAF concentration in platelet counts (/.&mu 0 0.5 (f3.3) 1.25 2.50 3.75 5.00
-10.4 -23.8 -25.1 -46.4
(k4.0) (+3.3)* (k3.3)* (k3.2)*
*Platelet counts significantly (P< 0.05) different between 0 and 15 min.
*Platelet counts significantly (P< 0.05) different between 0 and 15 min.
Statistical Analysis The changes in platelet counts of splenectomized mice between 0 and 15 min were analyzed for statistical significance using paired Student’s t tests. A significant (P < 0.05) response is indicated in the tables by a n asterisk.
Effect of Embryo Number on EPAF Activity Table 2 illustrates the effect of ECM in the SMB. Neither CM nor ECM from one embryo/ml medium induced a significant decline in platelet counts in splenectomized mice. ECM samples collected from the growth of from two to 40 embryodm1 medium induced significant reductions in platelet counts within 15 min of injection. The degree of platelet reduction induced by these samples was consistently on the order of 20-30% and was not significantly altered by increasing embryo number from two to 40. In no case was ECM found to be lethal in the SMB. In contrast, increasing the concentration of PAF in the SMB induced a linear increase in activity (Table 3). The highest PAF concentration indicated in Table 3 resulted in a response of between 40% and 50% platelet reduction, a level of activity found to be lethal in about 20% of the cases. The approximate LD,o concentration of PAF was found to correspond to that causing 55-65% reduction in platelet counts (data not shown).
RESULTS EPAF Activity in the SMB: Comparison With C16 and C18 PAF A significant platelet decline was found in the SMB a t 15 min postinjection of both ECM and C16 PAF.A similar platelet decline was detected a t 30 min postinjection of C18 PAF (Table 1).
Dilution Effect of EPAF, PAF, and Lipid-Extracted Material in SMB 1. ECM induced a significant decline in platelet counts of splenectomized mice over the range of undiluted to 1/1,000 dilution. A linear decline in activity was found over the range of 1/1,000 to 1/100,000 dilution, the latter dilution showing negligible activity (Table 4).Dilution of PAF (PDS0)resulted in the loss of
In Vitro Platelet Aggregation Induced by PAF (C16 and C18) and ECM (Untreated, Lipid-Extracted, and TLC Separated) In vitro aggregation of horse platelets was used to assess the activity of C16 PAF (20 pg), C18 PAF (20 pg), ECM, and CM. These samples were tested either untreated, after lipid extraction, or after TLC separation (as described above). Twenty-five microliters of each sample was tested for activity in the platelet aggregation module.
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TABLE 4. Mean (fSE)Percentage Change in Platelet Counts of Splenectomized Mice 15 rnin Following Injection of Diluted ECM (N = 6) and Diluted PAF (N = 4) Dilution ECM PAF
Neat 1/10
moo
1/ 1,000 1/10,000 1/100,000
-30.4 (+2.9)* -25.8 (51.7*) -24.5 (+2.9)* -24.2 (40.6)* -11.3 (52.9) -3.8 (k2.l) 0.0 (f1.5)
-28.1 (+3.2)* 2.9 (f3.2) -7.2 (f2.8) 0.9 (H.3)
NT NT CM -2.2 (f2.6) *Platelet counts significantly (P< 0.05) different between 0 and 15 min. NT, not tested.
TABLE 5. Mean (kSE)Percentage Change in Platelet Counts of Splenectomized Mice 15 min Following Injection of Untreated (N = 6) and Extracted (N = 4) ECM Dilutions
Dilution ECM Extracted ECM Neat -28.9 (+0.6)* -31.3 (+0.8)* 1/10 -26.0 (+1.2)* -24.2 (k1.3)* 1/100 -24.6 (+2.5)* -10.6 (f3.3) 1/1,000 -24.8 (+0.6)* -7.9 (f1.7) 1/10,000 -10.8 (f2.5) -7.5 (k2.5) 1/100,000 -3.9 (*2.l) -2.9 (f0.8) CM -1.2 (f2.5) 0.4 (k0.8) *Platelet counts significantly (P
