The Platelet Cytoskeleton Contains Elements of the Prothrombinase Complex*

Transcription

1 THE JOURNAL OF BIOLOGICAL CHEMISTRY by The American Society of Biological Chemists, Inc. Vol. 259, No. 11, Issue of June 10, pp ,1964 Printed in U. S. A. The Platelet Cytoskeleton Contains Elements of the Prothrombinase Complex* (Received for publication, September 7, 1983) George P. TuszynskiS, Gerard P. MaucoQ, Alice Koshy, Paul K. Schick, and Peter N. Walsh From the Thrombosis Research Center, Temple University School of Medicine, Philadelphia, Pennsylvania Triton-insoluble cytoskeletons prepared from Immunofluorescence studies and electron microscopic studies thrombin-activated platelets were found to potentiate have shown that concanavalin A receptors (2), histocompatithe activation of prothrombin (prothrombinase activ- bility antigens (3), Na+,K+-activated ATPase (41, and fibroity). Cytoskeletons prepared from red cells or lympho- nectin (5, 6) are associated with the internal cytoskeletal blasts contained no prothrombinase activity. The actomyosin network. Similar studies have shown that 01- platelet prothrombinase activity was dependent on cy- actinin (7) or related protein appear in the areas of cytoskeltoskeletal-associated Factor Va, and exogenously eton and surface membrane contact. (b) Myosin affinity chroadded Factor Xa and prothrombin. Cytoskeletons con- matography has revealed the association of H-2 antigen with tained 38% of the total platelet prothrombinase activ- the cytoskeleton of immune mastocytoma cells (8) and imity. Both platelets and cytoskeletons displayed half- munoglobulin with the cytoskeleton of activated lymphocytes maximal activities at similar prothrombin concentra- (9). (c) The association of glycoproteins GPIIIb and GPIII, tions. The role of lipids in the cytoskeletal prothromsurface proteins thought to mediate platelet aggregation (lo), binase activity was investigated. Cytoskeletons were found to contain 3.8% of the total platelet phosphowith the cytoskeleton of thrombin-activated platelets was lipids, consisting of the following lipids expressed as recently demonstrated by means of surface labeling studies percentage of total present in platelets: 6.0% sphingo- and SDS gels (10-12). Other studies have also revealed the myelin, 3.8% phosphatidylcholine, 2.9% phosphatidyl- association of a 130,000-dalton protein with the adhesion ethanolamine, 4.4% phosphatidylinositol, and 2.2% plaques of fibroblasts, suggesting that this protein may bind phosphatidylserine. The cytoskeletal prothrombinase the cell cytoskeleton to surface receptors (13). (d) Binding activity and the lipid phosphorus content of cytoskel- studies with purified proteins have revealed that protein 2.1, etons decreased after treatment of cytoskeletons with termed ankyrin, binds spectrin, the major protein comprising various doses of phospholipase C. Incubation of cyto- the red cell cytoskeleton, and band 3, a major red cell surface skeletons with the highest concentrations tested (10 protein (14). pg/ml) resulted in a 72% loss of phosphatidylserine and Our recent studies showing that coagulation Factor Va is 84% loss of cytoskeletal prothrombinase activity. Cy- associated with the platelet cytoskeleton of thrombin-actitoskeletal prothrombinase activity destroyed by phos- vated platelets (15) are consistent with the studies outlined pholipase C treatment could be restored to control lev- above and indicate that transmembrane associations of sur- els by treatment of hydrolyzed cytoskeletons with total face receptors and the internal platelet cytoskeleton may be cytoskeletal lipid or mixtures of phosphatidylserine/ important in mechanisms of platelet-coagulant protein interphosphatidylcholine (25:75% by weight). These results action. More specifically, our previous studies suggested that suggest that the cytoskeletal prothrombinase complex association of coagulation Factor Va with the platelet cytoin addition to containing Factor Va, as has been previously shown (15), contains a lipid cofactor activity skeleton has an important role in the mechanism of plateletconsisting in part of phosphatidylserine. Transmembrane associations of surface membrane proteins with the internal cell cytoskeleton have been postulated for many different cells (1). The evidence for these associations is varied and can be arranged into the following groups. (a) *This research was supported by Health and Human Services Grants HL 28149, HL 25455, HL 25661, and HL and by Grant CTR 1389 from the Council for Tobacco Research, Inc. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore he hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $To whom correspondence should be addressed at, Thrombosis Research Center, Temple University Health Sciences Center, 3400 North Broad Street, Philadelphia, PA Supported in part by Fondation pour la Recherche Medicale Francaise. On leave from the Facult6 de Medecine Purpan, Universite P. Sabatier, Toulouse, France. Present address, Institute National de la Santi! et de la Recherche Medicale U 101, Biochimie des Lipides, Hopital Purpan, Toulouse, Cedex, France mediated prothrombin conversion. We suggested that the surface prothrombinase complex is retained on the platelet cytoskeleton after solubilization of most of the membrane with Triton X-100. The present study expands on these previous observations. We present kinetic data that cytoskel- etons contain the important functional units of the prothrombinase complex present on the surface of activated platelets. We also characterize the lipid cofactor activity of the platelet cytoskeleton important in prothrombin activation. These studies may help to clarify how platelets act to potentiate prothrombin activation. EXPERIMENTAL PROCEDURES Materials-Proteolytic enzyme inhibitors, buffers, Sepharose 2B, reagents for clotting assays, chromatographic resins, and lipid standards were purchased from Sigma. Phospholipase C from Bacillus The abbreviations used are: SDS, sodium dodecyl sulfate; Hepes, N-2-hydroxyethylpiperazine-N -2-ethanesulfonic acid; PC, phosphatidylcholine; PS, phosphatidylserine; S-2222, N-benzoyl-L-isoleucyl- L-glutamylglycl-L-arginine-p-nitroanilide hydrochloride; $3-2238, H- D-phenylalanyl-L-pipecolyl-L-arginine-p-nitroanilide dihydrochloride.

2 6948 Platelet Cytoskeleton Contains Prothrombinase Complex Elements cereus was purchased from Boehringer Mannheim. Reagents for detectable levels of Factors 11, VIII, IX, X, or XI in our Factor V sodium dodecyl sulfate-gel electrophoresis were purchased from Bio- preparations. Rad. The chromogenic substrates N-benzoyl-L-isoleucyl-glutamylgly- Antibody Studies-Monoclonal antibody to Factor V was a genercyl-l-arginine-p-nitroanilide hydrochloride, H-D-phenylalanyl-L-pi- ous gift from Dr. Helen Glueck, University of Cincinnati Medical pecolyl-l-arginine-p-nitroanilide dihydrochloride, and A'-benzoyl-L- Center. Ten microliters of a cytoskeletal suspension, containing 1 phenylalanyl-l-valyl-l-arginine-p-nitroanilide hydrochloride for Fat- unitlml of Factor Va activity, were incubated in the presence and tor Xa, thrombin, and serine proteases, respectively, were purchased absence of a 1:lOOO dilution of antibody or control serum. Prothromfrom Ortho Diagnostics, Raritan, NJ. Highly purified human throm- bin generation rates were measured as a function of time of incubation bin was a gift from Dr. J. W. Fenton 11, New York State Department (4 "C). of Health. Phospholipase C Studies-Cytoskeletons from 6.0 X 10' platelets Isolation of Platelets-Platelet-rich plasma was prepared from titrated whole blood (0.38% citrate final concentration) by centrifuging the blood at 140 x g for 15 min. The platelet-rich plasma was gel filtered through Sepharose 2B (16) equilibrated in &pes-buffered ca2+-free Tyrode's solution (17) containing 138 mm NaC1,2.7 mm KCl, 1.0 mm MgC12.6H20, 3.3 mm NaH2P04. H,O, 3.5 mm Hepes, 5.5 mm dextrose, and 1.0 g/liter of bovine serum albumin, ph 7.4. Five ml of platelet-rich plasma were gel-filtered on a column con- structed from a 50-ml plastic syringe fitted with a porous polyethylene plug. Peak fractions were pooled (approximately 5 ml total), and final platelet counts were in the range of 200, ,000 platelets/pl. Preparation of Cytoskeletons-Gel-filtered platelets were treated at 37 "C with 1 unit/ml of thrombin for 3 min, and the reaction was stopped by the addition of 2.5 units/ml of hirudin and 1 mm diisopropyl fluorophosphate. The platelet suspensions were then treated with 0.5% Triton X-100, mixed, and centrifuged for 2 min at 10,000 x g. The pellet was washed twice in Hepes-buffered Tyrode's solution, ph 7.3, containing 0.5% Triton and once in Hepes-buffered Tyrode's solution without added Triton, resuspended in 100 p1 of Hepesbuffered Tyrode's solution, and sonicated briefly, and a portion was assayed for Factor Va clotting activity and prothrombin activating ability. Red cell cytoskeletons and cytoskeletons from other cell types were prepared in a manner similar to that described for platelets. Assays-Protein assays were performed according to the procedure of Lowry et al. (18). Factor V clotting assays were performed according to the procedure of Lewis and Ware (19). One unit of Factor V activity is defined as the amount present in 1 ml of normal citrated plasma. The ability of cytoskeletons to catalyze the conversion of prothrombin to thrombin was tested in an assay system that contained 78 pg/ ml of prothrombin, Factor Xa in the range of ng/ml, and 2.5 mm CaC1,. At various times, an aliquot was removed and added to a 50 mm Tris-C1 buffer, ph 8.3, solution containing 5 mm EDTA and mg/ml of S-2238, and the absorbance at 405 nm was measured. All incubations were carried out at 37 "C in Hepes-buffered Tyrode's solution, ph 7.3, containing 1 g/liter of bovine serum albumin. Units of thrombin generated were calculated from rates of S-2238 cleavage produced by solutions of thrombin of known concentration. Factor Xa activity in cytoskeletal preparations was measured spectrophotometrically at 405 nm using S-2222 in an assay system similar to that using the thrombin substrate S Phospholipase C was tested for proteolytic enzyme activity with New England Nuclear's general proteinase assay using radioactive a- casein (NEC-735 and Technical Report 3). Preparations showed activity equivalent to 10 ng/ml of trypsin assayed under the same conditions. Addition of 0.5 mm diisopropyl fluorophosphate to our phospholipase C solutions completely abolished the proteolytic activ- ity. All experiments were performed with diisopropyl fluorophosphate-treated phospholipase C. In addition, treated phospholipase C had no effect on the protein composition of cytoskeletons as revealed by SDS gels (data not shown) or on the Factor Va associated with the cytoskeleton as revealed by our reconstitution studies (see "Results'' and Table IV). Gel Electrophoresis-SDS-polyacrylamide gel electrophoresis was performed according to the procedure of Laemmli (20). Purification of Factor X, Prothrombin, and Factor V-Factor X and prothrombin were purified as previously reported (21,22). Both proteins were at least 95% pure as judged by SDS-gel electrophoresis. Factor X gave a specific activity of 125 clotting units/mg, a value that has been previously reported in the literature (22). Factor Xa was prepared as previously described (15). Factor V was partially purified as previously reported (23). Factor V preparations were fold purified as judged by the specific activity of the final preparations. Treatment of the purified Factor V preparations with thrombin increased their activity 20-fold, indicating our preparations were predominantly in the unactivated state. Clotting assays revealed no were suspended in 1 ml of Hepes-buffered Tyrode's solution containing 1.0 mm CaC12 and treated either with 1 p1 of buffer or phospholipase c (final concentration 1-10 pglml). Incubations were carried out for 10 min on ice, and the cytoskeletons were washed three times in Hepes-buffered Tyrode's solution, ph 7.3. Cytoskeletons were then assayed for their ability to activate prothrombin, analyzed on SDS gels, and analyzed for the presence of phospholipid. Lipid Analysis-Cytoskeletons and platelets were assayed for phospholipid content by the following procedure. Briefly, platelets (2 X 10') and cytoskeletons (3.6 X 10'") obtained from the same donor were extracted by a modified Bligh and Dyer procedure (24) in the presence of 20 mm EDTA. Partition of the aqueous phase was obtained by the addition of 1 volume of CHC13 followed by 1 volume of2.4 N HCl at 4 "C (25, 26). The individual phospholipids were separated by silica gel thin layer chromatography (Merck, Darmstadt, RFA) using CHC13/CH30H/CH3COOH/H20 (81:10:45:1, v/v) as the developing solvent (27). The chromatogram was revealed by UV fluorescence after spraying the plate with 0.1% primulin in methanol or by exposure to iodine vapor and staining with ninhydrin. To determine PS content, fatty acid analysis was performed. All the solvents contained 0.5% of butylated hydroxytoluene (w/v), and the methyl esters were obtained by reacting the silica scrapings with 14%BF3 in methanol at 100 "C for 20 min. After partition into pentane and concentration, these fatty acids methyl esters were dissolved in trimethyl-2,2,4-pentane, and 1 pl of the solution was injected into a glass column (6 X 0.25 inches) packed with 10% SP on l00/200 Chromosorb W AW (Supelco, Inc., Bellefonte, PA) fitted in a Hewlett-Packard 5730A gas chromotograph with flame ionization detector. After 2 min at 150 "C, the temperature was increased (4 'C/min) to 200 "C. Quantitation was obtained by adding heptadecanoic acid as an internal standard. Phosphorus was determined colorimetrically (28) after digesting the silica scrapings in 11.6 N HClO, at 180 "c for 90 min. Phospholipids used for the reconstitution studies were prepared by evaporating a chloroform solution of phospholipid under nitrogen and then resuspending the dried lipid in Hepes-buffered Tyrode's solution, ph 7.3, by brief sonication. Lipid samples were also sonicated just prior to use. The lipid concentrations given were calculated from the amount of lipid phosphorus (see above). RESULTS Association of Factor Va with the Platelet Cytoskeleton- We have previously shown that the Factor Va coagulant activity of cytoskeletons is inhibited in a time-dependent manner by a monoclonal antibody to Factor V (15). We now show that the ability of cytoskeletons to potentiate the activation of prothrombin as measured by our chromogenic assay system utilizing purified Factor Xa and prothrombin (see "Experimental Procedures") is also inhibited in a time-dependent manner by a monoclonal antibody to Factor V (Fig. 1). These results indicate that Factor Va is present on our washed cytoskeletons and is required for the ability of cytoskeletons to potentiate the Factor Xa-catalyzed activation of prothrombin. Cytoskeletons contained no intrinsic Factor Xa activity since they did not hydrolyze S-2222, the chromogenic substrate specific for Factor Xa. Comparison of Platelet and Cytoskeletal Prothrombinase Actiuities-The Factor Xa-catalyzed rates of prothrombin activation potentiated by platelets and cytoskeletons as a function of prothrombin concentration were compared in incubation mixtures containing 2-3 X 10' platelets or cytoskeletons/ml. The results showed that platelets gave 2.7-fold greater maximal rates than did cytoskeletons (Table I and

3 Platelet Cytoskeleton Contains Prothrombinase Complex Elements 6949 anti-v Time (min) I -I IO FIG. 1. Inhibition of cytoskeletal prothrombinase activity by anti-factor V antibody. Cytoskeletons were assayed for their ability to activate prothrombin as described under Experimental Procedures in the presence (0) of a 1:lOOO dilution of anti-factor V antibody and in the presence (0) of a 1:lOOO dilution of control serum. Incubations were carried out at 5 C. Fig. 2). In addition, the concentrations of prothrombin giving half-maximal rates of thrombin generation were similar (about 25 pg/ml) in the presence of platelets or cytoskeletons. The rates generated at low prothrombin concentration for both platelets and cytoskeletons were always consistently lower than would be predicted by simple Michaelis-Menten kinetics, and therefore the results given in Table I were estimated from direct plots of the data (Fig. 2) rather than from double reciprocal plots which consistently gave higher values for half-saturation and maximal rates. The reasons for this behavior at low substrate concentration is unknown although one explanation might be positive cooperativity. A more detailed analysis of the kinetics of prothrombin activation is beyond the scope of this study. In conclusion, these results indicate that platelets and cytoskeletons have similar prothrombinase activities, suggesting that the major elements of the platelet prothrombinase complex are retained on the cytoskeleton. Specificity of Associution of the Prothrombinase Complex with the Platelet Cytoskeleton-We have previously shown that, Factor Va is specifically associated with the platelet cytoskeleton (15). Additionally, we have now tested other cytoskeletal preparations isolated identically to the platelet cytoskeletons for activity in our assay system (Table 11). These cytoskeletons were prepared from 10 red cells or lymphoblast cells and possessed over 220-fold less activity than platelet cytoskeletons from 10 cells (Table 11). Addition of Factor V to the red cells or lymphoblasts either before preparation or after preparation of cytoskeletons increased the prothrombinase activity 4-8-fold, still far below that of the cytoskeletons. These results indicate that platelet cytoskeletons possess significant prothrombinase activity and that this activity is specific to platelets. Role of Cytoskeletal Lipid in Prothrombin Actiuation-Since lipid is thought to be an important component of the platelet surface prothrombinase complex (29), we investigated the role of lipid in the potentiation of prothrombin activation by cytoskeletons using the chromogenic assay system. Cytoskeletons were found to contain 3.8% of the total platelet phospholipids (Table 111). These lipids consisted of such procoagulant lipids as PS and phosphatidylethanolamine (29). When cytoskeletons were treated with increasing concentrations of phospholipase C, a decrease in cytoskeletal prothrombinase activity and a loss of cytoskeletal associated lipid phosphorus occurred (Fig. 3). At low phospholipase C concentrations, a significant amount of lipid phosphorus was removed (50%) with no effect on the rates, whereas at higher phospholipase C concentrations, a small change in the cytoskeletal lipid concentration produced a large decrease in the rate. At 10 fig/ ml of phospholipase C, 72.1 & 4.1% (mean k S.E. of the mean) of the total PS associated with the cytoskeleton was removed as measured by gas-liquid chromatography. When phospholipase C-treated cytoskeletons were incubated with aqueous suspensions of cytoskeletal lipid, 100% of the cytoskeletal associated prothrombinase activity could be restored (Table IV). When treated cytoskeletons were incubated with levels of PS roughly equivalent to the amount removed by phospholipase C treatment or a 10-fold excess, 100% of the activity was restored (Table IV). Therefore, it is possible that PS is a major lipid component of the cytoskeletal prothrombinase activity. We were unable to reconstitute the cytoskeletal activities to levels equaling the platelet activities. Apparently, some loss of prothrombinase activity occurs during isolation of the cytoskeleton despite the fact equal amounts of Factor TABLE I Rates of prothrombin activation obtained from thrombin-activated platelets and cytoskeletons All assays were performed at 37 C in the presence of 10 ng/ml of Factor Xa, 2.5 mm CaC12, pg/ml of prothrombin. The Factor Va concentration in all the preparations tested was approximately 1.0 unit/ml as determined by coagulation assays (see Experimental Procedures ). ~ _ Maximal Half-maximal Samples thrombin generated units/l@ platelets or eytoskeietons/min prothrombin concentrationb Factor Va coagulant activity unit/lp platelets or cytoskeietons Activated 4.62 * 0.90 (n = 5) 24.8 * 6.64 (n = 5) & (n = 3) platelets Cytoskeletonsd 1.74 k 0.78 (n = 5) 22.2 f 4.86 (n = 5) f (n = 6) The values given are the mean followed by the standard deviation with the number of determinations in parentheses. Each determination was performed on different donor platelets, determined at saturating prothrombin concentration pg/ml) and expressed per 10 platelets or cytoskeletons. The concentration of prothrombin that gives half-maximal rates of thrombin generation. The values given are the mean followed by the standard deviation with the number of determinations in parenthesis. Each determination was performed on different donor platelets. e 2-3 X 10 platelets/ml were activated with 1 unit/ml of thrombin for 1 min, and the thrombin was inactivated with 1 unit/ml of hirudin before assay. Cytoskeletons were prepared from 2-3 X 10 platelets/ml as described under Experimental Procedures, suspended in 1 ml, and assayed. -

4 ~ 6950 Platelet Cytoskeleton Contains Prothrombinase Complex Elements PROTHROMBIN ADDED(pg/ml) FIG. 2. Dependence of cytoskeletal and platelet potentiated rates of prothrombin activation on prothrombin concentration. Cytoskeletons were assayed for their ability to activate prothrombin as described under Experimental Procedures. 2-3 X lo8 platelets/ml were activated with 1 unit/ml of thrombin for 1 min, and the thrombin was inactivated with 1 unit/ml of hirudin before assay. 0, cytoskeletons; 0, thrombin-activated platelets. Cytoskeletons and platelets contained approximately equal Factor Va coagulant activities (see Table I and Ref. 15). TABLE I1 Specificity of the platelet cytoskeletal prothrombin activating ability -. Thrombin Cytoskeleton generated unitsl10 cytoskeletonslnin Platelets 1.63 Red cells Lymphoblasts Platelets 1.67 Vab Red cells Vab Lymphoblasts + Vab Cytoskeletons were assayed as described in the legend of Table I except that Factor Xa = 56 ng/ml and prothrombin = 78 pg/ml. Cell suspensions were 1o8/ml. * The final Factor Va concentration added was 1 unit/ml. Similar results were obtained when Factor Va was added to the cells prior to isolation of the cytoskeletons. TABLE I11 Phospholipid content of cytoskeletons and whole platelet The results given are the mean of five separate determinations on different donors followed by plus or minus the standard error of the mean. Platelets Cytoskeletons ~~~~~$~ nm11109 platelets Sphingomyelins 131 f 7 Phosphatidylcholine 169 f 11 Phosphatidylethanolamine 89 f 7 Phosphatidylinositol 41 f 3 Phosphatidylserine 83 f 9 Total phospholipids f 47 nn % cytoskeletons 7.3 f f f k k f V are retained (15). These results suggest that cytoskeletons possess a lipid cofactor activity in prothrombin activation and that PS is most likely a cofactor in the reaction. The nature of these essential catalytic components present on the cytoskeleton was investigated. One component is Factor V (15), which we now demonstrate in our purified assay PHOSPHOLIPASE C ADDED ()rg/ml) FIG. 3. Dependence of the rates of prothrombin activation and lipid phosphorus content of washed cytoskeletons on phospholipase C concentration. Values at each point were determined from cytoskeletons prepared from 6.0 X lo9 platelets as described under Experimental Procedures. The values presented for lipid phosphorus are expressed as nanomoles/108 cytoskeletons. TABLE IV The effect of removal or addition of phospholipid on cytoskeletons treated with Dhosoholiaase c Samples Untreated cytoskeleton Treated cytoskeleton Treated cytoskeleton + PL Treated cytoskeleton + PS-PC Treated cytoskeleton + PS-PCb Treated cytoskeleton + PC Treated cytoskeleton + SM Treated cytoskeleton + PC-SM Thrombin generated unitsl10 cytoskeletonslmin 1.51 f (n = 12) f 0.09 (n = 7) 1.66 f 0.39 (n = 2) 1.33 f (n = 2) Samples were assayed as described in the legendof Table I. Cytoskeletons from 3.6 X 1O O platelets were treated with 10 g/ml of phospholipase C or buffer as described under Experimental Procedures. Treated cytoskeletons were assayed in the presence of either 15 pg/ml of lipid extracted from cytoskeletons (designated PL) or 2 pg/ml of a 7525% mixture of PS and PC (designated PS-PC). The values given are the mean plus or minus the standard deviation followed by the number of determinations each performed on different donors in parentheses. * Treated cytoskeletons were assayed in the presence of 0.2 pg/ml of a 25:75% mixture of PS and PC (designated PS-PC). The amount of PS (0.15 pg) added back is roughly equivalent to the amount of PS (0.093 pg) removed by phospholipase treatment. Lipid = 5 pg/ml and the ratio of sphingomyelin (SM) to PC was 2 to 1. system to be necessary for optimum functioning of the cytoskeletal associated prothrombinase complex (Fig. 1). Another appears to be phospholipid. Both we (30) and others (31) have shown that cytoskeletons contain phospholipid. In the present study, we used an acid/chloroform/methanol extraction procedure to remove cytoskeletal associated lipid and demonstrated that 2.2% of the total platelet phosphatidylserine is present on the cytoskeletons. In general, the acid/chloroform/ methanol extraction procedure gives slightly different lipid compositions of platelets and cytoskeletons than previously shown (30, 31), since it allows for better recovery of acidic lipids. Upon treatment of cytoskeletons with phospholipase C, both PS and other phospholipids were hydrolyzed, resulting in an 84% loss of cytoskeletal associated prothrombinase activity. This activity could only be restored by adding back cytoskeletal lipid or phospholipid mixtures containing PS. Other phospholipids such as sphingomyelin, PC, or mixtures thereof did not restore the activity (Table IV). Therefore, we

5 Platelet Cytoskeleton Contains Prothrombinase Complex Elements 6951 conclude from these experiments that (a) cytoskeletons possess a lipid cofactor activity in prothrombin activation and (b) this lipid cofactor activity is most likely composed in part of PS. We feel the most striking observation of this study is that although cytoskeletons contain only 3.8% of the total platelet phospholipid they can potentiate prothrombin activation 38% as well as activated platelets. DISCUSSION REFERENCES We have shown that cytoskeletons prepared from throm- 1. Nicolson, G. L. (1976) Biochim. Biophys. Acta 457, Ash, J. F., and Singer, S. J. (1976) Proc. Natl. Acad. Sci. U. S. A. bin-activated platelets can potentiate the Factor Xa-catalyzed 73, activation of prothrombin. Cytoskeletons potentiate pro- 3. Bourguignon, L. Y. W., and Singer, S. J (1977) Proc. Natl. Acad. thrombin activation 2.7-fold less well when compared to the Sci. U. S. A. 74, parent platelets under conditions of saturating prothrombin 4. Ash. J. F., Louvard, D., and Singer, S. J. (1977) Proc. Natl. Acad. concentration and limiting Factor Xa concentration (Table SCi. U. S. A. 74, I). The concentration of prothrombin (approximately 25 pg/ 5. Hvnes. R. 0.. and Destree. A. T. (1978) Cell Scnger; I. I. (1979) Cell 16; ml) required to promote a half-maximal rate of thrombin 7. Lazarides, E., and Burridge, K. (1975) Cell 6, generation in the presence of either platelets or their corre- 8. Koch, G. L. E., and Smith, M. J. (1978) Nature (Lord.) 273, sponding cytoskeletons is nearly identical. Both the rates of the Factor Xa-catalyzed prothrombin activation potentiated 9. Flanagan, J., and Koch, G. L. E. (1978) Nature (Lond.) 273, by either cytoskeletons or platelets show a similar dependence on prothrombin concentration, displaying non-michaelis- 10. Phillips, D. R., Jennings, L. K., and Edwards, H. H. (1980) J. Cell Biol. 86, Menten saturation kinetics. The reason for this is unknown 11. Rotman, A., Heldman, J., and Linder, S. (1982) Biochemistry 21, * G. P. Tuszynski and G. J. Stewart, unpublished observations. close association with the prothrombinase complex. Our results are not inconsistent with the observations of Bevers et al. (33) who have shown that PS is exposed on the surface of platelets activated with thrombin and collagen and that this PS contributes to the platelet prothrombinase activity. Acknowledgments-We would like to thank Dr. Earl Henderson for his gift of the lymphoblasts and Pat Pileggi for typing the manuscript. although at low prothrombin concentration the rates lower are than would be predicted from Michaelis-Menten kinetics, 12. Painter, R. G., and Ginsberg, M. (1982) J. Cell Biol. 92, suggesting that positive cooperativity may be occurring at 13. Geiger, B. (1979) Cell 18, higher substrate concentrations or these rate effects may be 14. Bennett, V., and Stenbuck, P. J. (1980) J. Biol. Chem. 255,2540- due to the adsorption of substrate onto a surface. However, it Tuszynski, G. P., Walsh, P. N., Piperno, J. R., and Koshy, A. does appear that cytoskeletons and platelets potentiate pro- (1982) J. Biol. Chem. 257, thrombin activation by similar mechanisms. This conclusion 16. Tangen, O., Berman, H. J., and Marfey, P. (1971) Thromb. Diath. is strengthened by our earlier observations that platelets and Haemorrh. 25, cytoskeletons bind equal amounts of exogenously added Fac- 17. Timmons, S., and Hawiger, J. (1978) Thromb. Res. 12, tor Xa with equal affinity (15). The reason cytoskeletons 18. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. possess only 38% of the platelet activity even in the presence (1951) J. Biol. Chem. 193, of saturating levels of exogenously added lipid is unknown. Lewis, M. L., and Ware, A. G. (1953) Proc. SOC. Exp. Biol. Med. 84, We have previously shown that the Factor Xa receptor sites 20. Laemmli, U. K. (1970) Nature (Lord.) 227, on both cytoskeletons and platelets are equivalent (15) as 21. Miletich, J. P., Broze, G. J., and Majerus, P. W. (1980) Anal. revealed by Factor V assays and Factor Xa binding studies. Biochem. 105, It could be that some factor important in prothrombin con- 22. DiScipio, R. G., Hermodson, M. A., Yates, S. G., and Davie, E. version is lost during isolation of cytoskeletons or that some W. (1977) Biochemistry 16, denaturation has occurred during preparation of the cyto- 23. Kane, W. H., and Majerus, P. W. (1981) J. Biol. Chem. 256, skeletons. We favor the latter explanation since the cytoskel- 24. Bligh, E. G., and Dyer, W. J. (1959) Can. J. Biochem. Physiol. eta1 activity is relatively unstable over time, losing 80% of its 37, activity if stored overnight at -20 C. Nevertheless, these 25. Hauser, G., and Eichberg, J. (1973) Eiochim. Biophys. Acta 326, results suggest that the major functional components of the prothrombinase complex essential for catalysis on the platelet 26. Mauco, G., Chap, H., and Douste-Blazy, L. (1983) FEES Lett. surface are preserved on the platelet cytoskeleton after Triton 153, Hauser, G., and Eichberg, J. (1975) J. Biot. Chem. 250, extraction. Having shown that the cytoskeletal activity is in 28. Bottcher, C. J. F., VanGent, C. M., and Pries, C. (1961) Anal. part due to lipid, one is lead to postulate that the important Chim. Acta 24, platelet surface components of the prothrombinase complex 29. Schick, P. K., Kurica, K. B., and Chacko, G. K. (1976) J. Clin. are retained on the platelet cytoskeletons. These lipid com- Inuest. 57, ponents do not appear to form an organized membrane 30. Schick, P. K., Tuszynski, G. P., and VanderVoort, P. W. (1983) bilayer on the cytoskeleton; therefore, would it appear likely Blood 61, that these lipids may function as boundary lipids (32) in 31. Zucker, M. B., and Masiello, N. C. (1983) Blood 61, Singer, S. J., and Nicolson, G. L. (1972) Science (Wash. D. C.) 175, Bevers, E. M., Comfurius, P., VanRijn, J. L. M. L., Hemker, H. C., and Zwaal, R. F. A. (1982) Eur. J. Biochem. 122,