Freeze-Etch Observations on the Plasma Membrane and Other Structures of Normal and Abnormal Platelets R. L. Reddick, MS and R. G. Mason, MD, PhD Human platelets in plasma were subjected to freeze fracture followed by etching. The outer surface of the platelet plasma membrane was exposed by etching but not by fracturing and was clearly identified by use of latex particles as markers. Surfaceassociated particles, apparently embedded in the plasma membrane, were found to measure from 83 to 332 A in diameter. Similar particles were associated with membranes lining the surface-connected canalicular system and with limiting membranes of storage granules. Fortuitous fractures exposed two inner faces of the plasma membrane, one of which contained greater numbers of surface-associated particles than did the outer surface of the plasma membrane. The second inner face of the plasma membrane contained numerous fibrillar structures measuring up to 770 A in length. Platelets from a congenitally afibrinogenemic patient appeared normal when examined by freeze-etch technics. Normal platelets exposed to a potent antiaggregating agent (REM 10,393) were found to have lost most of the particles associated with the outer surface of the plasma membrane and to have developed numerous defects in this membrane. A possible role for surface associated particles in platelet aggregation was further suggested by the finding that the nonaggregable platelets of two congenitally thrombasthenic sisters were nearly devoid of these structures (Am J Pathol 70:473-488, 1973). THE PLATELET PLASMA MEMBRANE is remarkable in both its origin and function. The plasma membrane of the platelet derives from coalescence of the membranes of the demarcation membrane system of vesicles which appear within the cytoplasm of the parent cell, the megakaryocyte.1 These demarcation vesicles have recently been shown to be invaginations of the plasma membrane of the megakaryocyte.2'3 The plasma membrane of the platelet appears largely responsible for the cell's primary functions of adhesion and aggregation.4 In hemostasis and thrombosis, platelet adhesion and aggregation are essential, basic events.5 No other cell population is called upon to respond almost instantaneously to specific stimuli by adhering to certain surfaces or by aggregating rapidly, only later perhaps to deaggregate. From the Department of Pathology, School of Medicine, University of North Carolina, Chapel Hill, NC. Supported in part by Grants HE-14228 (Thrombosis Center), HE-13296 and HE46351 Career Development Award (Dr. Mason) from the National Heart and Lung Institute. Accepted for publication Dec 3, 1972. Address reprint requests to Dr. R. G. Mason, Department of Pathology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27514. 473 474 REDDICK AND MASON American Journal of Pathology Past investigations of the ultrastructure of the platelet plasma membrane have been largely restricted to observations made by transmission or scanning electron microscopy."'7 Both of these technics are limited, in that a high resolution view of the various components of the platelet plasma membrane, especially the outer surface, seldom has been realized. The technic of freeze-etch affords an excellent means for observation of the external and internal surfaces of the plasma membrane as well as certain other structural components of cells.8 In the present study, the freeze-etch technic has been applied to an investigation of normal platelets, platelets exposed to a potent antiaggregating agent, and platelets from patients with congenital thrombasthenia or afibrinogenemia. Materials and Methods Blood was drawn from healthy donors or from patients using the two-syringe technic with one part 3.2% sodium citrate to anticoagulate 8 parts of whole blood. All blood samples were taken in plastic or silicone-coated syringes and processed using silicone-coated glassware.9 Platelet-rich plasma was obtained by centrifugation of blood at 600g for 10 minutes at 23 C.9 The thrombasthenic and afibrinogenemic patients have been reported previously.10'11 Platelets were examined, on two separate occasions, from the blood of one thrombasthenic patient and once from the sister of this patient. Platelets from the afibrinogenemic subject were examined on two separate occasions. Latex Spheres Polystyrene latex spheres (Dow Chemical Company) had an average diameter of 3000 A. Platelet-rich plasma was mixed gently with one-tenth volume of undiluted latex spheres. This mixture was allowed to incubate undisturbed for 5 minutes at 23 C and was subsequently processed for freeze-etch (vide infra). Antiaggregating Agent The antiaggregating agent, a soluble glycolate salt of a-(p(fluoren-9-ylidenemethyl) phenyl)-2-piperidineethanol (REM-10,393) (FYPE) was obtained from the William S. Merrill Company.12'13 FYPE was dissolved in 0.05 M tris(hydroxymethyl)aminomethane buffer (Tris), pH 7.35, and used at a final concentration of 12 ;1g/ml in platelet-rich plasma. Platelet preparations were incubated with FYPE for 10 minutes at 23 C. Fixative Glutaraldehyde (2%; Fisher Scientific Company) was prepared in 0.1 M phosphate buffer, pH 7.35. Glycerol (20%) was prepared using the same buffer. Freeze-Etch Technic Numerous preliminary studies demonstrated that the best preservation of morphologic detail of platelets was obtained by use of 0.1 M phosphate-buffered glutaraldehyde and glycerol. To an aliquote of platelet-rich plasma was added two tenths volume of phosphate-buffered 2% glutaraldehyde. This mixture was incubated for Vol. 70, No. 3 March 1973 FREEZE-ETCHED PLATELET MEMBRANES 475 5 minutes at 23 C. Subsequently, to this mixture was added an equal volume of phosphate-buffered 20% glycerol. The resulting phosphate-buffered glutaraldehydeglycerol-platelet-rich plasma mixture was incubated for 15 minutes at 23 C and then centrifuged to obtain a platelet button. The supernatant was discarded and the platelet button placed in 10 ml of a mixture of equal volumes of 2% phosphatebuffered glutaraldehyde and 20% phosphate-buffered glycerol until subjected to freeze-etch, usually 20 to 30 minutes later. The term freeze-etch is used to describe the technics of freeze-fracture followed by etching.8 Specimens were fractured, etched, and replicated in a Balzer's BA360M freeze-etch machine (Balzer's AG, Balzers, Liechtenstein). Platinum-carbon replicas of freeze-etch specimens were prepared according to the method of Moor.14 The cleaved specimen surfaces were etched at -100 C for 2 to 4.5 minutes. Transmission Electron Microscopy Samples were prepared as previously described and embedded in Epon 812.15 Ultrathin sections were cut with a diamond knife and observed in a JEM model T-7 electron microscope. Results Freeze-etch treatment of platelets exposes plasma membranes as well as cytoplasmic components for ultrastructural examination. The external or outer surface of the plasma membrane was demonstrated by etching deeply the previously fractured specimens. The fracture plane or plane of cleavage in many cases appeared to split the plasma membrane, although in some cleaved specimens it appeared to pass parallel to the outer surface as well as through various areas of the cell cytoplasm. Normal platelets, platelets exposed to an antiaggregating agent and platelets from patients with congenital thrombasthenia or afibrinogenemia were examined by freeze-etch for comparison of plasma membrane and cytoplasmic organelle ultrastructure. Transmission electron micrographic studies were used to supplement freeze-etch studies in all cases. Normal Platelets Freeze-Etch Studies The Platelet Plasmra Membrane. The outer surface of the plasma membrane of normal platelets as seen by freeze-etch is finely granular and is studded with spheroidal particles of 83 to 332 A diameter (Figures 1-3). In Figures 1 and 2 the fracture plane has passed through the platelet exposing areas of cytoplasm; the outer surfaces of these platelets were exposed by etching which followed the fracture procedure. When platelets were exposed briefly to latex spheres (Figure 3), some of the spheres adhered to the outer surface of the plasma membrane, serving as a marker for this surface. Collagen, colloidal iron, 476 REDDICK AND MASON American Journal of Pathology thoratrast and Sendai virus were found to be less useful than latex spheres as markers for the outer surface of the plasma membrane. Freeze-etch permitted not only visualization of the plasma membrane outer surface but also of inner areas of the membrane as well. In Figure 4, the fracture plane has exposed what appear to be two different fracture faces (FFa and FFb). FFa appears similar to the outer surface of the plasma membrane but contains many more surface associated particles than does the outer surface. The second fracture face (FFb) contains fibrillar structures measuring up to 770 A in length. These fibers are randomly oriented and in some areas overlie one another. Some fibers appear to have subunit structure. In addition to the fibrillar structures, FFb also contains scattered particles of 90 to 110 A diameter. Finally, it should be noted that the external surface of the platelet exhibits large, rounded elevations (Figures 3 and 4) probably produced by underlying cytoplasmic organelles. Platelet Cytoplasmic Organelles. The freeze-etch technic also makes possible a three dimensional study of the internal structure of the platelet. The cytoplasmic region of a platelet is shown in Figure 2. Examples of the stomata of the surface-connected system are revealed in several areas (Figure 2,S). Canaliculi of the surface connected system have a surface similar to the outer surfaces of the platelet. There is random arrangement of organelles within the platelet. Organelles with a finely granular rounded surface (Figure 2, G) studded with particles of a size similar to those observed on the outer surface of the plasma membrane are clearly evident. These organelles, which correspond to platelet storage granules and mitochondria, vary in size but for the most part are similar morphologically. Mitochondria were not demonstrated with certainty in any of these preparations since fracture planes seldom passed through these granulomere structures. Within the cytoplasm of the platelets are structures which closely resemble microtubules (Figure 1, arrow) when observed in longitudinal section. Thin Section Studies The normal human platelet has a discoid appearance when examined by thin sectioning (Figure 5). The plasma membrane is irregularly shown in this plane of section although it is trilaminar and encircles the cell. Cytoplasmic organelles including microtubules, storage granules, mitochondria and glycogen granules are randomly dispersed. The surface-connected system is barely distinguishable. FREEZE-ETCHED PLATELET MEMBRANES Vol. 70, No. 3 March 1973 477 Abnormal Platelets Freeze-Etch and Thin-Section Studies Platelets Treated uwth FYPE. Addition of the antiaggregating agent FYPE to normal platelet-rich plasma produced marked alterations in the platelet plasma membrane. In contrast to untreated platelets, which had a finely granular outer surface studded with particles, the outer surface of platelets treated with FYPE showed numerous small defects or holes as well as focal condensations of amorphous material (Figure 6). The membrane-associated particles normally present on the platelet surface are difficult to appreciate in FYPE-treated platelets due to the membrane alterations produced by the antiaggregant. Despite these alterations, the membrane-associated particles do appear to be reduced markedly in number. Low magnification thin-section studies of platelets treated with FYPE revealed relatively few changes (Figure 7). After exposure to FYPE, the platelets were rounded, but the plasma membrane appeared intact. Organelles were randomly arranged within the platelet cytoplasm. In many of the FYPE-treated platelets, a narrow rim of clear cytoplasm was present just beneath the plasma membrane. Dilated tubular structures, presumably components of the surface connected system, were also present throughout the cytoplasm (Figure 7, arrow). Intact microtubules were occasionally observed by thin section examination of these platelets, but the number of microtubules present appeared decreased in comparison to the normal. Examination of the plasma membrane of FYPE-treated platelets with higher magnification thin-sectioning revealed three types of membrane alteration (Figure 8). One type of alteration consisted of focal discontinuity of the outer lamina of the plasma membrane (Figure 8, A); a second type (Figure 8, B), of discontinuity of all layers of the membrane. In the third type of alteration (Figure 8, C), there was side by side overlapping of apparently previously discontinuous portions of the plasma membrane. These membrane alterations are illustrated schematically in Text-figure 1. N A TEXT-FIG 1-Schematic representation of the three types of plasma membrane alteration produced by ____ FYPE. The same labels (A,B,C) are used here as in ______B Figure 8; (N) refers to the normal plasma membrane. C 478 REDDICK AND MASON American Journal of Pathology Thrombasthenic Platelets. The freeze-etch appearance of thrombasthenic platelets differed significantly from that of the normal in one major respect. The outer surface of the plasma membrane of thrombasthenic platelets lacked most of the 83 to 332 A diameter particles characteristic of normal platelets (Figure 9). Examination of numerous freeze-etched specimens of thrombasthenic platelets from 2 patients revealed only a small number of membrane-associated particles. This marked reduction in the number of membrane-associated particles also was evident on the surface of cytoplasmic organelles and the membranes of the surface connected system. In other respects, thrombasthenic platelets appeared morphologically identical to normal platelets by both freeze-etch and thin-section technics. Afibrinogenemic Platelets. Platelets from a congenitally afibrinogenemic patient were not distinguishable from normal by either freezeetch or thin-section examinations. Discussion The freeze-etch technic affords an excellent means for ultrastructural examination of the outer surface and inner fracture faces of the platelet plasma membrane. Fortuitous fractures permitted subsequent exposure of the outer surface of the plasma membrane by etching, so that this interesting surface could be identified clearly. In addition, the outer surface was labeled with latex spheres to insure positive identification of this structure. The presence of small membrane-associated particles apparently embedded in the plasma membrane of platelets agrees with the findings of others who have studied platelets "I as well as additional cell types.17 That these particles may be protein that is embedded in a lipid matrix is one currently held view of cell membrane structure.18 The freeze-etch appearance of the outer surface of normal platelets is similar to that of the limiting membranes of platelet organelles and the membranes of the surface connected system as well as of the limiting membranes of other cells.19 This latter finding is of interest, since the platelet plasma membrane is thought to originate from the plasma membrane of the megakaryocyte. The ultrastructure of the inner fracture faces of the platelet plasma membrane suggests the presence of fibers and particles embedded in a finely granular matrix. The inner fracture faces are exposed by cleavage planes which pass parallel to the outer surface of the plasma membrane, a phenomenon of common occurrence in freeze-fracture of other cell types.8 The presence of increased numbers of membrane-associated particles on one of the fracture faces (FFa) as opposed to the outer surface Vol. 70, No. 3 March 1973 FREEZE-ETCHED PLATELET MEMBRANES 479 of the plasma membrane has been noted with other types of cells and in these cells is thought to correspond to an inner fracture face of the membrane.8 The function of the fibrillar structures present on the inner fracture face (FFb) is unknown, but similar structures have been described by others."' It is reasonable to speculate that these fibers may represent a type of structural protein, perhaps even the contractile protein thrombosthenin.20 However, since these inner fracture faces are seen relatively infrequently in freeze-etch of platelets, any conclusions regarding them at this time are purely speculative. The exact location of these inner fracture faces is uncertain at present, but they appear to lie within the plasma membrane and not the cytoplasm. The infrequent occurrence of fractures exposing one or more inner faces of the platelet plasma membrane compared to the increased frequency of such fractures in the other cell types may indicate basic differences between the plasma membrane of platelets and of other cells. Examination of platelet cytoplasmic organelles by freeze-etch is less rewarding than examination of the plasma membrane. Storage granules, the surface connected system and microtubules can be identified. Mitochondria were not identified with certainty in the present study, although they have been described in freeze-etch studies of other cell types.8 The use of freeze-etch as a tool to investigate effects of various agents on the platelet plasma membrane is well illustrated by the study of FYPE-treated platelets. Previously, the levels of FYPE employed in the present study had been thought not to alter the platelet plasma membrane, while much higher levels were known to produce marked membrane and organelle alterations.13 The freeze-etch study of FYPE-treated platelets revealed defects in the outer surface of the platelets (Figure 6), while there was little evidence of plasma membrane damage or organelle alteration in these same platelets by relatively low magnification thinsection examination (Figure 7). This paradox led to examination of FYPE-treated platelets by much higher magnification of thin-sections and the subsequent discovery of three types of plasma membrane change. These studies suggest that FYPE treatment results in the appearance of focal defects in the outer plasma membrane lamina as well as other defects extending through the entire thickness of the membrane. These fractures appear to be closed by side-to-side apposition of the membrane remnants. Plasma membrane alterations are present in platelets with only minor changes in cytoplasm or organelles. Perhaps it is only when membrane defects can no longer be sealed, as may occur with higher levels of FYPE, that internal changes appear in the platelet. 480 REDDICK AND MASON American Journal of Pathology Additionally, FYPE treatment produces a marked reduction of the number of particles associated with the outer surface of the plasma membrane and also inhibits aggregation.13 The freeze-etch appearance of the outer surface of the thrombasthenic platelets differed strikingly from that of the normal. There was near absence of particles from the outer surface of these nonadhesive, nonaggregable platelets. The primary defect in thrombasthenic platelets has been reported to lie in the platelet membrane.2' These platelets have been reported to show an abnormal protein pattern and to lack one of the normal platelet plasma membrane components.21 Further, a protein that is normally thrombin-labile has been reported to be thrombinresistant in thrombasthenic platelets.22 The greatly decreased numbers of particles associated with the outer surface of thrombasthenic platelets may be the first morphologic demonstration of the plasma membrane defect in these cells. Platelets from a congenitally afibrinogenemic patient appeared normal by freeze-etch and thin sectioning. Afibrinogenemic platelets are less aggregable than normal platelets." Despite this, the number and size of particles associated with the outer surface of afibrinogenemic platelets appeared normal. Unlike the thrombasthenic platelet, which is itself defective,23 the afibrinogenemic platelet aggregates and adheres normally when placed in normal plasma or when fibrinogen is added to the suspending medium.2425 These findings suggest that the particles which are associated with the outer surface of the platelet and which are present on normal and afibrinogenemic platelets, but are greatly decreased in number on FYPE-treated platelets and thrombasthenic platelets, may play a role in platelet adhesion and aggregation. These particles may possibly function by furnishing receptor sites for plasma fibrinogen, a necessary constituent of platelet aggregates formed by the action of a wide spectrum of aggregating agents. Freeze-etch affords a unique means of examination of the platelet plasma membrane. Not only can freeze-etch be employed to study normal or congenitally abnormal platelets, it is one of the most powerful tools available for detection of morphologic change in the plasma membrane. References 1. O'Brien JR: Platelet function: a guide to platelet membrane structure. Ser Haematol III, 4:68-72, 1970 2. Behnke 0: An electron microscope study of the rat megacaryocyte. II. Some aspects of platelet release and microtubules. J Ultrastruct Res 26:111-129, 1969 Vol. 70, No. 3 March 1973 FREEZE-ETCHED PLATELET MEMBRANES 481 3. Behnke 0: An electron microscope study of the megacaryocyte of the rat bone marrow. I. The development of the demarcation membrane system and the platelet surface coat. J Ultrastruc Res 24:412-433, 1968 4. Rodman NF: The morphologic basis of platelet function, The Platelet. Edited by KM Brinkhous, RW Shermer, FK Mostofi. Baltimore, Williams and Wilkins, Co, 1971, pp 55-70 5. Mustard JF, Packham MA: Factors influencing platelet function: adhesion, release, and aggregation. Pharmacol Rev 22:97-187, 1970 6. Behnke 0: The morphology of blood platelet membrane systems. Ser Haematol III, 4:3-16, 1970 7. Larrimer NR, Balcerzak SP, Metz EN, Lee RE: Surface structure of normal human platelets. Am J Med Sci 259:242-256, 1970 8. Muhlethaler K: Studies on freeze-etching of cell membranes. Int Rev Cytol 31:1-19, 1971 9. Brinkhous KM, LeRoy EC, Cornell WP, Hazelhurst JL, Vennart GP: Macroscopic studies of platelet agglutination; nature of thrombocyte agglutinating activity of plasma. Proc Soc Exp Biol Med 98:379-383, 1958 10. Pittman MA, Graham JB: Glanzmann's thrombopathy: an autosomal recessive trait in one family. Am J Med Sci 247:293-302, 1964 11. Rodman NF, Mason RG, Painter JC, Brinkhous KM: Fibrinogen-its role in platelet agglutination and agglutinate stability: a study of congenital afibrinogenemia. Lab Invest 15:641-656, 1966 12. MacKenzie RD, Blohm TR, Auxier EM, Henderson JG, Steinbach, JM: Effects of a. (p- (fluoren-9-ylidenemethyl)phenyl) -2-piperidineethanol (RMI 10,393) on platelet function. Proc Soc Exp Biol Med 137:662-667, 1971 13. Rodman NF, Mason RG, Brinkhous KM: Effect of a new antiaggregating chemical on the structure and function of the human platelet. Am J Pathol 65: 103-116, 1971 14. Moor H, Muhlethaler K: Fine structure in frozen-etched yeast cells. J Cell Biol 17:609-628, 1963 15. Saba SR, Rodman NF, Mason RG: Platelet ATPase activities II. ATPase activities of isolated platelet membrane fractions. Am J Pathol 55:225-233, 1969 16. Ruska C, Shulz H: Elektronenmicroskopische Darstellung von Thrombocyten mit der Gefrieratztechnik. Klin Wochenschr 46:689-696, 1968 17. Branton D: Membrane structure. Ann Rev Plant Physiol 20:209-238, 1969 18. Hendler RW: Biological membrane ultrastructure. Physiol Rev 51:66-97, 1971 19. Benedetti EL, Delbauffe D: The plasma membrane as a model of complex organization of biological structures, Cell Membranes: Biological and Pathological Aspects. Edited by GW Richter, DG Scarpelli. Baltimore, Williams and Wilkins, Co, 1971, pp 54-83 20. Luscher EF: Fibrinogen and thrombosthenin. Thromb Diath Haemorrh Suppl 26:135-142, 1967 21. Nachman RL, Marcus AJ: Immunological studies of proteins associated with the. subcellar fractions of thrombasthenic and afibrinogenemic platelets. Br J Haematol 15:181-189, 1968 22. Nachman RL: Platelet proteins. Sem Haematol 5:18-31, 1968 23. Caen JP, Castaldi PA, Leclerc JC, Inceman S, Larrieu MJ, Probst M, and Bernard J: Congenital bleeding disorders with long bleeding time and 482 REDDICK AND MASON American Journal of Pathology normal platelet count. I. Glanzmann's thrombasthenia (report of fifteen patients) Am J Med 41:4-26, 1966 24. Gugler E, Luscher EF: Platelet function in congenital afibrinogenemia. Thromb Diath Haemorrh 14:361-373, 1965 25. Mason RG, Read MS, Brinkhous KM: Effect of fibrinogen concentration on platelet adhesion to glass. Proc Soc Exp Biol Med 137:680-682, 1971 Acknowledgments Acknowledgment is made of the contributions of R. M. Brown Jr, PhD who permitted us use of his Balzer's apparatus and guided us in mastering the freeze-etch technic. Dr. Brown's numerous discussions of problems and interpretations of data are also gratefully acknowledged, as are the similar contributions of F. G. Dalldorf, MD. The technical assistance of Miss Barbara Pool in a portion of this work was most helpful. Dr. Mason is a Markle Scholar in Academic Medicine. Fig 1-Outer surface (OS) and cytoplasmic (C) area of a freeze-etched normal platelet. The OS is finely granular and is studded with particles. The arrow points to a cytoplasmic organelle that most likely is a microtubule. Four minute etch. Shadowing in all the freezeetched specimens is from the bottom toward the top; shadows are white (X 12,500). Fig 2-A normal platelet from citrated platelet-rich plasma. This cell was cleaved and later etched for 4 minutes. Shown within the platelet cytoplasm (C) are cytoplasmic granules (G) and glycogen particles (GLY). The exposed outer surface (OS) of the platelet plasma membrane is diffusely particulate. The arrow points to a structure considered to be one of the stomata (S) of the surface connected system (x 30,000). Fig 3-Freeze-etch view of the outer surface of the plasma membrane of a normal platelet. Membrane-associated particles are present. Latex spheres (L) are observed within the plasma milieu and as darkened spheres resting upon the outer surface of the platelet plasma membrane. Latex sphere stretching is also evident in this preparation as in the right center. Three minute etch (x 34,000). Fig 4-Two fracture faces of the plasma membrane of a normal platelet revealed by freeze-etch. The first or outer fracture face (FFa) contains particles similar in size but increased in number compared to those present on the outer surface of plasma membrane. The second or inner fracture face (FFb) contains fibrillar structures which are randomly oriented. Two minute etch (x 9000). aL, ,14 46 x n'Zil LN Vs .~ ~ A 6t.. - .t'# - Fig 5-Transmission electron micrograph of a normal platelet. The plasma membrane is present only in areas. Storage granules, mitochondria, microtubules, and glycogen granules are randomly distributed in the cytoplasm (x 9000). Fig 6-Freeze-etch view of the external surface of the platelet plasma membrane after treatment with FYPE. The arrows point to defects in the outer surface of the membrane (OS). Membrane-associated particles are markedly reduced in number. An area of cytoplasm is present (C). Two minute etch (x 16,000). N~~ .. * -Sl i , Fig 7-Transmission electron micrograph of a platelet treated with FYPE. Plasma membrane defects are not easily discernable at this magnification. The arrow points to dilated tubular elements present in the cytoplasm (x 28,000). Fig 8--Transmission electron micrograph of a platelet treated with FYPE. At this magnification three different types of plasma membrane alteration are apparent. The first of these alterations (A) is a defect in only the outer lamina of the membrane. The second (B), a defect in all three lamina. The third alteration (C) consists of side to side apposition of membrane remnants in what may be an effort to seal membrane defects (x 53,400). t. I-zwN .i|. '.-;.I -K'' . Pg:0: , 1 v ' F Fig 9-Freeze-etch view of the outer surface (OS) of a platelet from a thrombasthenic patient. Membrane-associated particles similar to those on the outer surface of normal platelets are absent. The outer surface background is slightly more irregular than that of the normal platelet. Two minute etch (x 1.1,000).