Britishjournal ofHaernatology, 1978.40, 31 1-33
I
Different Requirements for Intrinsic Factor-Xa Forming Activity and Platelet Factor 3 Activity and their Relationship to Platelet Aggregation and Secretion WITH
PETERN. WALSH THE TECHNICAL ASSISTANCE OF EILEEN CAMPA N D DONNA DENDE
Specialized Center on Thrombosis Research and Department ofMedicine, Temple University School of Medicine, Philadelphia, Pennsylvania SUMMARY. Platelets provide coagulant activity in part by promoting two essential reactions of intrinsic coagulation: ( I ) factor-)< activation by a complex of factor IXa, factor VIII, calcium and platelets (intrinsic factor-Xa forming activity or XaFA); and (2) prothrombin activation by a complex of factor Xa, factor V, calcium and platelets (platelet factor 3 activity or PF3A). W e found that a chloroform extract of acetone-dried brain had about half the XaFA of a petroleum ether extract of brain and over twice the PF3A of the petroleum ether extract; and that when purified phospholipids other than phosphatidylserine (PS) were added to PS, the resultant activity, compared with PS alone, was reduced in the assay for XaFA and enhanced in the assay for PF3A. In experiments with washed platelets stirred with collagen XaFA developed rapidly (100% activity at 3 0 s), well before maximum [14C]gHT release (2-4 min) and decayed to 1 5 % a t 10min. In contrast, PF3A developed slowly (100% activity after 20 min) well after maximal aggregation and release. Maximal XaFA developed in unstirred platelet suspensions incubated with collagen, whereas PF3A did not become available unless platelet suspensions were stirred and underwent secretion. Platelets stirred with ADP released [ 14C]gHT and developed PF3A but not XaFA. Both [14C]gHT release and PF3A by collagen were inhibited by p-chloromercuriphenylsulphonate, indomethacin and a combination of prostaglandin El and RA 233, but XaFA was only minimally affected by these inhibitors. In contrast concanavalin A inhibited collagen-induced PF3 A and XaFA in dose-dependent fashion but had no effect on [14C]5HTrelease. Both platelet coagulant activities and [14C]5HT release were inhibited by aspirin, EDTA and a combination of antimycin A and 2-deoxy-~-glucose. These results indicate that the biochemical determinants of XaFA and PF3A are different. PF3A developed only when aggregation and release occurred whereas XaFA was independent of aggregation and release. Correspondence: Dr Peter N. Walsh, Room 42 1-oms, Specialized Center on Thrombosis Research, Temple University School of Medicine, 3400 N. Broad Street, Philadelphia, Pennsylvania 19140, U.S.A. 0007-1048/78/1000-03 I I$OZ.OO 01978 Blackwell Scientific Publications 311
3 12
Peter N. W a l s h
Crude lipid extracts or mixtures of purified phospholipids have been shown to serve essential functions in two fundamental reactions of intrinsic coagulation (Milstone, 1964; Lundblad & Davie, 1964). It has also been demonstrated that platelets (Walsh & Biggs, 1972) participate in these two reactions, possibly by providing a phospholipoprotein surface which sequesters coagulation factors and calcium to form complexes with serine protease activity (Barton, 1969). In the first of these reactions, either phospholipids or platelets form a complex with calcium and factors IXa and VIII which activates factor X (Lundblad& Davie, 1964),a capacity of platelets referred to as intrinsic factor-Xa forming activity (Walsh & Biggs, 1972). The second reaction in which platelets or phospholipids are required is prothrombin activation by a complex consisting of factor-Xa, factor V, calcium and platelet phospholipids, generally referred to as platelet factor 3 activity (Papahadjopoulos & Hanahan, 1964; Barton et al, 1967; Hemker et al, 1967; Hemker & Kahn, 1967;Jobin & Esnouf, 1967). The coagulant activity of platelets is apparently present in ‘latent’ form, unavailable to participate in coagulation reactions until the platelet membrane is perturbed by various stimuli such as exposure to aggregating agents (Fantl & Ward, 1958). Although phospholipids can substitute for platelets in the two reactions under consideration, it is not known what biochemical components of platelets become exposed to promote these two reactions and whether the biochemical determinants are the same or different (Biggs & Bidwell, 1957; Marcus & Spaet, 1958). We have previously suggested that the requirements for intrinsic factor -Xa forming activity are different from those for platelet factor 3 activity (Walsh & Biggs, 1972). To examine this question further we have, in the study reported here, used two fundamentally different experimental approaches. The first is to study the functional activities of various crude lipid extracts and purified phospholipids in test systems designed to measure either intrinsic factor-Xa forming activity or platelet factor 3 activity. The second is to examine the coagulant activities, aggregation and secretion of washed platelets under various experimental conditions and after treatment with a variety of agents known to inhibit certain platelet functions. The results indicate that the biochemical determinants of intrinsic factor-Xa forming activity and platelet factor 3 activity are different and that whereas platelet factor 3 activity becomes available well after secretion has occurred, intrinsic factor-Xa forming activity develops and decays rapidly and is independent of aggregation and secretion. METHODS
Phospholipids and crude lipid extracts. Sections of the frontal lobes of human brains were macerated, extracted with acetone, dried and crude lipid extracts were made either by chloroform extraction (Bell & Alton, 1954; Biggs, 1972) or by petroleum ether extraction (Folch, 1942; Biggs, 1972). All lipid extracts and purified lipids were always handled and stored in an atmosphere of nitrogen. These total lipid extracts and purified phospholipids (obtained either from Applied Science Laboratories, State College, Pa., or from Supelco, Inc., Bellefonte, Pa.) were analysed by thin-layer chromatography (Rouser et al, 1966; Schick et al, 1976) on silica gel (Applied Science) in a solvent system of chloroform/methanol/concentrated N H 4 0 H , 140/60/10 (by vol). The plates were sprayed with 50% H 2 S 0 4 , charred to detect lipids and the spots were scraped (Rouser et al, 1966). After perchloric acid combustion (Bottcher et al, 1961), lipid phosphorus was determined (Bartlett, 1959), using K H 2 P 0 4 as a
Platelet Coagulant Activity
313
standard. Phospholipid content was calculated from the following formula (Bosmann et al, 196b), assuming 770 daltons as the average molecular weight of phospholipid: phospholipid content (mg) =inorganic phosphorus (pmol) x 0.77. Phospholipid analyses were also carried out on total lipid extracts which had been extracted three times with chloroform-methanol (2:1) at room temperature for 24 hand partitioned against NaCl (Folch et al, 1957).Phospholipid recovery after thin-layer chromatography and lipid phosphorus analysis showed the following composition of the petroleum ether extracted brain: phosphatidylserine (56Y0), phosphatidylinositol (8 %), sphingomyelin (7%), phosphatidylcholine (12%), phosphatidylethanolamine (9%), other (8 %). The chloroform extract of acetone-dried brain contained the following phospholipids: phosphatidylserine (19%), phosphatidylinositol(4%),sphingomyephosphatidylethanolamine (33 YO), other (8%). Protein lin (13 %), phosphatidylcholine (23 YO), determinations (Lowry et al, 1951) on crude lipid extract showed 5.48 p g protein/mg phospholipid in the petroleum ether extract and 45.73 pg proteinlmg phospholipid in the chloroform extract ofbrain. Purified phospholipids and crude lipid extracts were dried under a stream of nitrogen and aqueous lipid dispersions were made in 50 mM isotonic imidazole buffer, pH 7.3, by sonication on ice for 3 0 s, with an ultrasonic disintegrator (Biosonik IV, Bronwill Corp., Rochester, N.Y.). Preparation ofplatelet suspensions. Nine volumes of blood were collected by clean venipuncture with a 16 gauge needle and catheter directly into I volume of 3.8% trisodium citrate using plastic containers and equipment throughout. Platelet rich plasma was preincubated with 0.5 ~ L M [3-14C]~-hydr~xytryptamine (Amersham/Searle Company, Arlington Heights, Ill.) for 30 min at 37°C. Platelets were washed by albumin density gradient separation in calcium-free Tyrode’s solution by a modification (Walsh et al, 1977) of a previously described method (Walsh, 1972a). Apyrase was not used during the washing procedure. Platelets were counted by phase contrast microscopy (Brecher & Cronkite, 1950) and electronically using a Model ZBI Particle Counter (Coulter Electronics, Hialeah, Fla.) and the results were averaged. Experiments with washedplatelets. Imipramine (Ciba-Geigy Corp., Summit, N.J.) was added to platelet suspensions at a final concentration of 5 PM to prevent re-uptake of released [14C]gHT (Walsh & Gagnatelli, 1974). In experiments with ADP as a release inducer, human fibrinogen (grade ‘L’, 95% clottable, from A. B. Kabi, Stockholm) was added to platelet suspensionsin a concentration of 0.6 mg/ml. Platelet suspensions (I ml) were stirred at 37°C in a soliconized aggregometer cuvette (Payton, Scarborough, Ont.). Aggregating agents ( 100pl) were added and aggregation continuously recorded. Aliquots were subsampled at intervals for T as previously described (Walsh & Gagnatelli, 1974)and for determination of [ 1 4 C ] ~ H release assay of platelet factor 3 and intrinsic factor-Xa forming activity as described below. ADP (Sigma Chemical Co., St Louis, Mo.) was stored a t -60°C at I mM in 50 mM isotonic imidazole buffer, pH 7.3, and diluted appropriately before using. Acid soluble collagen was bovine achilles tendon in powdered form (Sigma), prepared as described by Holmsen et al (1973) to a concentration of approximately I mg/ml and used at a final dilution of I in ‘50 (approximately 20 pg/ml). Prostaglandin El (Upjohn Co., Kalamazoo, Mich.) was stored as a 2 mM solution in ethanol and RA 233 ([2,6-bis-diethylamin0]-4-piperidino pyrimido [ 5 ,qd] pyrimidine, Boehringer-Ingleheim, Ltd, Isleworth, Middlesex, England) was stored at -60°C as a 20 mM solution in acidified water. In experiments using a combination of PGEl and RA 233 as inhibitors of platelet aggregation a working solution of 5 mM RA 233 and 50 /.LM PGEl
314
Peter N. Walsh
was made up and added (40 pl) to I ml of a washed platelet suspension which was incubated for I min at 37°C before testing. Indomethacin (Sigma) was dissolved in ethanol at 10mM and added to washed platelets at a final concentration of 20 p ~ The . monosodium salt of p-chloromercuriphenyl-sulphonic acid (Sigma) was dissolved in 0.85 % NaC1, added a t a final concentration of I mM and incubated for I rnin before testing. Aspirin (Sigma) was stored as a stock solution of 20 mM in 0.12 M N a H C 0 3 and added to PRP to a final concentration of 0.5 mM for a 10min incubation at 37"C, after which the platelets were washed to remove excess aspirin. Antimycin A (Calbiochem, La Jolla, Cal.) was stored at -60°C at 2 mg/ml in ethanol and added (2 111) to I ml of washed platelets along with a-deoxy-~-glucose (Sigma) which was stored a t -60°C as a 600 mM solution in 5 0 mM isotonic imidazole buffer, pH 7.3. Antimycin A (final concentration 4 pg/ml) and a-deoxy-~-glucose (final concentration 6 mM) were incubated with washed platelets for 3 0 min a t 37°C before testing. Concanavahn A (Calbiochem) was added to washed platelets a t appropriate (as indicated in Results) final concentration from a stock solution of 5 mg/ml in water. Disodium ethylenediaminetetraacetate (EDTA, Fisher Scientific Co., Fairlawn, N.J.) was preincubated with washed platelets a t a final concentration of 5 mM for I min at 37°C before testing. Assaysfor platelet coagulant activities. Intrinsic factor-Xa forming activity (Walsh & Biggs, 1972) was assayed by a modification ofa previously reported method (Walsh, I972b). A IOO pl aliquot of a washed platelet suspension, treated as described above with an appropriate aggregating agent or control solution, was added to a 12 x 75 mm polypropylene tube (No. 2063, Becton, Dickinson & Co., Rutherford, N.J.) containing 300 pl of a solution prepared as follows: 8 ml of a solution containing I part 3.8% trisodium citrate and 5 parts of 50 mM isotonic imidazole buffer, pH 7.3; 2 ml of bovine plasma barium sulphate eluate (Sigma), 0.5 mg/ml in 50 mM isotonic imidazole buffer, pH 7.3, containing the activities of factors IX (ca 0.1-0.2 u/ml) and X (ca 0.1-0.2 u/ml) and no detectable prothrombin; and 2 ml of bovine factor VIII (Speywood Laboratories Ltd, Nottingham, England), z mg/ml in imidazole buffer containing ca 0.1-0.2 u/ml of factor VIII coagulant activity. To this mixture was added I 50 pl of a mixture containing the following reagents: 2 ml of 0. I M CaCl,; 2 ml of bovine thrombin (Parke, Davis & Co., Detroit, Mich.) 0.05 u/ml (to activate the factor VIII), and 2 ml of a celite eluate of human plasma (Nossel, 1964) containing the activity of factor-Xa (ca 0.01--0.02 u/ml). The contents of the tube were mixed and incubated at 37°C for exactly 5 min during which factor-Xa activity appears. At the end of the incubation an aliquot (100pl) of the incubation mixture was subsampled into a I .o x 75 mm soft soda glass clotting tube (No. 7810, Becton Dickinson) containing IOO pl of 0.025 M CaC12.A IOO pl quantity of bovine factor-X deficient substrate plasma (charcoal filtered bovine plasma, Diagnostic Reagents Ltd, Thame, Oxon, England) was then added and the clotting time determined as a measure of the amount of factor Xa formed. Frozen and thawed platelet suspensions tested a t a wide range of dilutions in the assay served as a standard curve for calculating the results of the assays in percentage terms, in which the shortest clotting time achieved by control platelets was assigned a value of 100%. Platelet factor 3 was assayed by a modification of a previously reported method (Walsh, 1972b) in which a 100 pl aliquot of the washed platelet suspension was subsampled into a 10x 75 mm glass tube containing 100 pl of pooled normal human plasma. To the mixture was added IOO pl of Russell's viper venom (StypvenB, Burroughs Wellcome & Co., Research Triangle Park, N.C.) 0.01 mg/ml in imidazole buffer diluted immediately before use. To the
Platelet Coagulant Activity
315
mixture 100 pl of 0.025 M CaClz was added and the clotting time determined. The results were calculated by reference to a dilution curve of frozen and thawed washed platelets as described for the assay for intrinsic factor-Xa forming activity.
RESULTS Experiments with Crude Lipid Extracts of Brain and Purijied Phospholipids Various crude lipid extracts of human brain and mixtures of purified phospholipids were tested for activity in the assays for intrinsic factor-Xa forming activity and platelet factor 3 activity (Table I). Serial dilutions of each of these preparations were tested a t similar total lipid concentrations in the two test systems described (see Methods) and the logarithm of clotting time was plotted against the logarithm of lipid concentration to permit comparisons with the activity of the petroleum ether extract of brain (Folch, 1942) arbitrarily assigned a value of 100%. The chloroform extract of acetone-dried brain (Bell & Alton, 1954) had about half the activity of the Folch lipid extract in the assay for intrinsic factor-Xa forming activity and over twice the activity of the Folch lipid extract in the assay for platelet factor 3 activity. Purified phospholipids were less active in both assays than crude lipid extracts of brain. Phosphatidylserine, which was more active by itself than any other single phospholipid, had less than half TABLE I. Coagulant activities of crude lipid extract of brain and purified phospholipids
Reagent tested Petroleum ether extract of brain Chloroform extract of acetone-dried brain Phosphatidylserine (PS) Phosphatidylinositol (PI) Phosphatidylethanolamine (PE) Phosphatidylcholine (PC) PS-PI PS-PE PS-PC PS-PI-PE PS-PE-PC PS-PI-PE-PC PI-PE PI-PC P E-P C PI-PE-PC
Intrinsicfactor forming activity 100
I00
52 ( + s . i ) *
46 ( k 9 . 4 ) 4 (ko.9) < I
27
(-e 4.6)
21
(k2.3)
21
(k3.9) (k7.0)
23 (k8.9) 31
(k22.9) 43 (k12.2) 17 ( k 4 . 9 ) 29 (k10.4)
222
I
Different Requirements for Intrinsic Factor-Xa Forming Activity and Platelet Factor 3 Activity and their Relationship to Platelet Aggregation and Secretion WITH
PETERN. WALSH THE TECHNICAL ASSISTANCE OF EILEEN CAMPA N D DONNA DENDE
Specialized Center on Thrombosis Research and Department ofMedicine, Temple University School of Medicine, Philadelphia, Pennsylvania SUMMARY. Platelets provide coagulant activity in part by promoting two essential reactions of intrinsic coagulation: ( I ) factor-)< activation by a complex of factor IXa, factor VIII, calcium and platelets (intrinsic factor-Xa forming activity or XaFA); and (2) prothrombin activation by a complex of factor Xa, factor V, calcium and platelets (platelet factor 3 activity or PF3A). W e found that a chloroform extract of acetone-dried brain had about half the XaFA of a petroleum ether extract of brain and over twice the PF3A of the petroleum ether extract; and that when purified phospholipids other than phosphatidylserine (PS) were added to PS, the resultant activity, compared with PS alone, was reduced in the assay for XaFA and enhanced in the assay for PF3A. In experiments with washed platelets stirred with collagen XaFA developed rapidly (100% activity at 3 0 s), well before maximum [14C]gHT release (2-4 min) and decayed to 1 5 % a t 10min. In contrast, PF3A developed slowly (100% activity after 20 min) well after maximal aggregation and release. Maximal XaFA developed in unstirred platelet suspensions incubated with collagen, whereas PF3A did not become available unless platelet suspensions were stirred and underwent secretion. Platelets stirred with ADP released [ 14C]gHT and developed PF3A but not XaFA. Both [14C]gHT release and PF3A by collagen were inhibited by p-chloromercuriphenylsulphonate, indomethacin and a combination of prostaglandin El and RA 233, but XaFA was only minimally affected by these inhibitors. In contrast concanavalin A inhibited collagen-induced PF3 A and XaFA in dose-dependent fashion but had no effect on [14C]5HTrelease. Both platelet coagulant activities and [14C]5HT release were inhibited by aspirin, EDTA and a combination of antimycin A and 2-deoxy-~-glucose. These results indicate that the biochemical determinants of XaFA and PF3A are different. PF3A developed only when aggregation and release occurred whereas XaFA was independent of aggregation and release. Correspondence: Dr Peter N. Walsh, Room 42 1-oms, Specialized Center on Thrombosis Research, Temple University School of Medicine, 3400 N. Broad Street, Philadelphia, Pennsylvania 19140, U.S.A. 0007-1048/78/1000-03 I I$OZ.OO 01978 Blackwell Scientific Publications 311
3 12
Peter N. W a l s h
Crude lipid extracts or mixtures of purified phospholipids have been shown to serve essential functions in two fundamental reactions of intrinsic coagulation (Milstone, 1964; Lundblad & Davie, 1964). It has also been demonstrated that platelets (Walsh & Biggs, 1972) participate in these two reactions, possibly by providing a phospholipoprotein surface which sequesters coagulation factors and calcium to form complexes with serine protease activity (Barton, 1969). In the first of these reactions, either phospholipids or platelets form a complex with calcium and factors IXa and VIII which activates factor X (Lundblad& Davie, 1964),a capacity of platelets referred to as intrinsic factor-Xa forming activity (Walsh & Biggs, 1972). The second reaction in which platelets or phospholipids are required is prothrombin activation by a complex consisting of factor-Xa, factor V, calcium and platelet phospholipids, generally referred to as platelet factor 3 activity (Papahadjopoulos & Hanahan, 1964; Barton et al, 1967; Hemker et al, 1967; Hemker & Kahn, 1967;Jobin & Esnouf, 1967). The coagulant activity of platelets is apparently present in ‘latent’ form, unavailable to participate in coagulation reactions until the platelet membrane is perturbed by various stimuli such as exposure to aggregating agents (Fantl & Ward, 1958). Although phospholipids can substitute for platelets in the two reactions under consideration, it is not known what biochemical components of platelets become exposed to promote these two reactions and whether the biochemical determinants are the same or different (Biggs & Bidwell, 1957; Marcus & Spaet, 1958). We have previously suggested that the requirements for intrinsic factor -Xa forming activity are different from those for platelet factor 3 activity (Walsh & Biggs, 1972). To examine this question further we have, in the study reported here, used two fundamentally different experimental approaches. The first is to study the functional activities of various crude lipid extracts and purified phospholipids in test systems designed to measure either intrinsic factor-Xa forming activity or platelet factor 3 activity. The second is to examine the coagulant activities, aggregation and secretion of washed platelets under various experimental conditions and after treatment with a variety of agents known to inhibit certain platelet functions. The results indicate that the biochemical determinants of intrinsic factor-Xa forming activity and platelet factor 3 activity are different and that whereas platelet factor 3 activity becomes available well after secretion has occurred, intrinsic factor-Xa forming activity develops and decays rapidly and is independent of aggregation and secretion. METHODS
Phospholipids and crude lipid extracts. Sections of the frontal lobes of human brains were macerated, extracted with acetone, dried and crude lipid extracts were made either by chloroform extraction (Bell & Alton, 1954; Biggs, 1972) or by petroleum ether extraction (Folch, 1942; Biggs, 1972). All lipid extracts and purified lipids were always handled and stored in an atmosphere of nitrogen. These total lipid extracts and purified phospholipids (obtained either from Applied Science Laboratories, State College, Pa., or from Supelco, Inc., Bellefonte, Pa.) were analysed by thin-layer chromatography (Rouser et al, 1966; Schick et al, 1976) on silica gel (Applied Science) in a solvent system of chloroform/methanol/concentrated N H 4 0 H , 140/60/10 (by vol). The plates were sprayed with 50% H 2 S 0 4 , charred to detect lipids and the spots were scraped (Rouser et al, 1966). After perchloric acid combustion (Bottcher et al, 1961), lipid phosphorus was determined (Bartlett, 1959), using K H 2 P 0 4 as a
Platelet Coagulant Activity
313
standard. Phospholipid content was calculated from the following formula (Bosmann et al, 196b), assuming 770 daltons as the average molecular weight of phospholipid: phospholipid content (mg) =inorganic phosphorus (pmol) x 0.77. Phospholipid analyses were also carried out on total lipid extracts which had been extracted three times with chloroform-methanol (2:1) at room temperature for 24 hand partitioned against NaCl (Folch et al, 1957).Phospholipid recovery after thin-layer chromatography and lipid phosphorus analysis showed the following composition of the petroleum ether extracted brain: phosphatidylserine (56Y0), phosphatidylinositol (8 %), sphingomyelin (7%), phosphatidylcholine (12%), phosphatidylethanolamine (9%), other (8 %). The chloroform extract of acetone-dried brain contained the following phospholipids: phosphatidylserine (19%), phosphatidylinositol(4%),sphingomyephosphatidylethanolamine (33 YO), other (8%). Protein lin (13 %), phosphatidylcholine (23 YO), determinations (Lowry et al, 1951) on crude lipid extract showed 5.48 p g protein/mg phospholipid in the petroleum ether extract and 45.73 pg proteinlmg phospholipid in the chloroform extract ofbrain. Purified phospholipids and crude lipid extracts were dried under a stream of nitrogen and aqueous lipid dispersions were made in 50 mM isotonic imidazole buffer, pH 7.3, by sonication on ice for 3 0 s, with an ultrasonic disintegrator (Biosonik IV, Bronwill Corp., Rochester, N.Y.). Preparation ofplatelet suspensions. Nine volumes of blood were collected by clean venipuncture with a 16 gauge needle and catheter directly into I volume of 3.8% trisodium citrate using plastic containers and equipment throughout. Platelet rich plasma was preincubated with 0.5 ~ L M [3-14C]~-hydr~xytryptamine (Amersham/Searle Company, Arlington Heights, Ill.) for 30 min at 37°C. Platelets were washed by albumin density gradient separation in calcium-free Tyrode’s solution by a modification (Walsh et al, 1977) of a previously described method (Walsh, 1972a). Apyrase was not used during the washing procedure. Platelets were counted by phase contrast microscopy (Brecher & Cronkite, 1950) and electronically using a Model ZBI Particle Counter (Coulter Electronics, Hialeah, Fla.) and the results were averaged. Experiments with washedplatelets. Imipramine (Ciba-Geigy Corp., Summit, N.J.) was added to platelet suspensions at a final concentration of 5 PM to prevent re-uptake of released [14C]gHT (Walsh & Gagnatelli, 1974). In experiments with ADP as a release inducer, human fibrinogen (grade ‘L’, 95% clottable, from A. B. Kabi, Stockholm) was added to platelet suspensionsin a concentration of 0.6 mg/ml. Platelet suspensions (I ml) were stirred at 37°C in a soliconized aggregometer cuvette (Payton, Scarborough, Ont.). Aggregating agents ( 100pl) were added and aggregation continuously recorded. Aliquots were subsampled at intervals for T as previously described (Walsh & Gagnatelli, 1974)and for determination of [ 1 4 C ] ~ H release assay of platelet factor 3 and intrinsic factor-Xa forming activity as described below. ADP (Sigma Chemical Co., St Louis, Mo.) was stored a t -60°C at I mM in 50 mM isotonic imidazole buffer, pH 7.3, and diluted appropriately before using. Acid soluble collagen was bovine achilles tendon in powdered form (Sigma), prepared as described by Holmsen et al (1973) to a concentration of approximately I mg/ml and used at a final dilution of I in ‘50 (approximately 20 pg/ml). Prostaglandin El (Upjohn Co., Kalamazoo, Mich.) was stored as a 2 mM solution in ethanol and RA 233 ([2,6-bis-diethylamin0]-4-piperidino pyrimido [ 5 ,qd] pyrimidine, Boehringer-Ingleheim, Ltd, Isleworth, Middlesex, England) was stored at -60°C as a 20 mM solution in acidified water. In experiments using a combination of PGEl and RA 233 as inhibitors of platelet aggregation a working solution of 5 mM RA 233 and 50 /.LM PGEl
314
Peter N. Walsh
was made up and added (40 pl) to I ml of a washed platelet suspension which was incubated for I min at 37°C before testing. Indomethacin (Sigma) was dissolved in ethanol at 10mM and added to washed platelets at a final concentration of 20 p ~ The . monosodium salt of p-chloromercuriphenyl-sulphonic acid (Sigma) was dissolved in 0.85 % NaC1, added a t a final concentration of I mM and incubated for I rnin before testing. Aspirin (Sigma) was stored as a stock solution of 20 mM in 0.12 M N a H C 0 3 and added to PRP to a final concentration of 0.5 mM for a 10min incubation at 37"C, after which the platelets were washed to remove excess aspirin. Antimycin A (Calbiochem, La Jolla, Cal.) was stored at -60°C at 2 mg/ml in ethanol and added (2 111) to I ml of washed platelets along with a-deoxy-~-glucose (Sigma) which was stored a t -60°C as a 600 mM solution in 5 0 mM isotonic imidazole buffer, pH 7.3. Antimycin A (final concentration 4 pg/ml) and a-deoxy-~-glucose (final concentration 6 mM) were incubated with washed platelets for 3 0 min a t 37°C before testing. Concanavahn A (Calbiochem) was added to washed platelets a t appropriate (as indicated in Results) final concentration from a stock solution of 5 mg/ml in water. Disodium ethylenediaminetetraacetate (EDTA, Fisher Scientific Co., Fairlawn, N.J.) was preincubated with washed platelets a t a final concentration of 5 mM for I min at 37°C before testing. Assaysfor platelet coagulant activities. Intrinsic factor-Xa forming activity (Walsh & Biggs, 1972) was assayed by a modification ofa previously reported method (Walsh, I972b). A IOO pl aliquot of a washed platelet suspension, treated as described above with an appropriate aggregating agent or control solution, was added to a 12 x 75 mm polypropylene tube (No. 2063, Becton, Dickinson & Co., Rutherford, N.J.) containing 300 pl of a solution prepared as follows: 8 ml of a solution containing I part 3.8% trisodium citrate and 5 parts of 50 mM isotonic imidazole buffer, pH 7.3; 2 ml of bovine plasma barium sulphate eluate (Sigma), 0.5 mg/ml in 50 mM isotonic imidazole buffer, pH 7.3, containing the activities of factors IX (ca 0.1-0.2 u/ml) and X (ca 0.1-0.2 u/ml) and no detectable prothrombin; and 2 ml of bovine factor VIII (Speywood Laboratories Ltd, Nottingham, England), z mg/ml in imidazole buffer containing ca 0.1-0.2 u/ml of factor VIII coagulant activity. To this mixture was added I 50 pl of a mixture containing the following reagents: 2 ml of 0. I M CaCl,; 2 ml of bovine thrombin (Parke, Davis & Co., Detroit, Mich.) 0.05 u/ml (to activate the factor VIII), and 2 ml of a celite eluate of human plasma (Nossel, 1964) containing the activity of factor-Xa (ca 0.01--0.02 u/ml). The contents of the tube were mixed and incubated at 37°C for exactly 5 min during which factor-Xa activity appears. At the end of the incubation an aliquot (100pl) of the incubation mixture was subsampled into a I .o x 75 mm soft soda glass clotting tube (No. 7810, Becton Dickinson) containing IOO pl of 0.025 M CaC12.A IOO pl quantity of bovine factor-X deficient substrate plasma (charcoal filtered bovine plasma, Diagnostic Reagents Ltd, Thame, Oxon, England) was then added and the clotting time determined as a measure of the amount of factor Xa formed. Frozen and thawed platelet suspensions tested a t a wide range of dilutions in the assay served as a standard curve for calculating the results of the assays in percentage terms, in which the shortest clotting time achieved by control platelets was assigned a value of 100%. Platelet factor 3 was assayed by a modification of a previously reported method (Walsh, 1972b) in which a 100 pl aliquot of the washed platelet suspension was subsampled into a 10x 75 mm glass tube containing 100 pl of pooled normal human plasma. To the mixture was added IOO pl of Russell's viper venom (StypvenB, Burroughs Wellcome & Co., Research Triangle Park, N.C.) 0.01 mg/ml in imidazole buffer diluted immediately before use. To the
Platelet Coagulant Activity
315
mixture 100 pl of 0.025 M CaClz was added and the clotting time determined. The results were calculated by reference to a dilution curve of frozen and thawed washed platelets as described for the assay for intrinsic factor-Xa forming activity.
RESULTS Experiments with Crude Lipid Extracts of Brain and Purijied Phospholipids Various crude lipid extracts of human brain and mixtures of purified phospholipids were tested for activity in the assays for intrinsic factor-Xa forming activity and platelet factor 3 activity (Table I). Serial dilutions of each of these preparations were tested a t similar total lipid concentrations in the two test systems described (see Methods) and the logarithm of clotting time was plotted against the logarithm of lipid concentration to permit comparisons with the activity of the petroleum ether extract of brain (Folch, 1942) arbitrarily assigned a value of 100%. The chloroform extract of acetone-dried brain (Bell & Alton, 1954) had about half the activity of the Folch lipid extract in the assay for intrinsic factor-Xa forming activity and over twice the activity of the Folch lipid extract in the assay for platelet factor 3 activity. Purified phospholipids were less active in both assays than crude lipid extracts of brain. Phosphatidylserine, which was more active by itself than any other single phospholipid, had less than half TABLE I. Coagulant activities of crude lipid extract of brain and purified phospholipids
Reagent tested Petroleum ether extract of brain Chloroform extract of acetone-dried brain Phosphatidylserine (PS) Phosphatidylinositol (PI) Phosphatidylethanolamine (PE) Phosphatidylcholine (PC) PS-PI PS-PE PS-PC PS-PI-PE PS-PE-PC PS-PI-PE-PC PI-PE PI-PC P E-P C PI-PE-PC
Intrinsicfactor forming activity 100
I00
52 ( + s . i ) *
46 ( k 9 . 4 ) 4 (ko.9) < I
27
(-e 4.6)
21
(k2.3)
21
(k3.9) (k7.0)
23 (k8.9) 31
(k22.9) 43 (k12.2) 17 ( k 4 . 9 ) 29 (k10.4)
222
