THE EFFECTS OF CYTOCHALASIN A AND cwocumsm a on HUMAN P-LATELET ULTRASTRUCTURE ' . AND FUNCTION Thesis forthe' Degree of M., S. - MiGHIGAN- STATE ’UfilVERSlTY ' PHiLUP £0N. BORGERDING-- : . ~Wilt-"Y". ._ ' ‘F '(b:o“3fl-'M f7 "’ ..- . 1“ In" “\ 0' “ ‘9 7 Y) 1 :- ‘ ~s Is Li... M ..__ . ‘ for-"v. .w ' I " ._ “ ‘a BINDING By I HDAG & sour , ABSTRACT THE EFFECTS OF CYTOCHALASIN A AND CYTOCHALASIN B ON HUMAN PLATELET ULTRASTRUCTURE AND FUNCTION BY Phillip Jon Borgerding Human platelets have been shown to undergo marked changes during the performance of their functions in hemostasis. When stimulated by a break in the circu- latory system, platelets first adhere to exposed col- lagen and then clump together in aggregates. The normally discoid platelet shape becomes swollen and irregular, with a surface studded with pseudopods. At the same time, the platelets release ADP, serotonin, and platelet factor 3 from their granules. After a hemostatic plug has formed, platelets also have a role in clot retraction. These various functions are thought to be mediated by contractile microfilaments within the platelets. It is the role of these microfilaments that is studied in this research. In 1967, a family of new drugs, the Cytochalasins, was discovered, which would selectively inhibit the \Q 09\\Q (é functioning of contractile elements within cells without Phillip Jon Borgerding destroying the cells. One variant of this drug, Cyto- chalasin B (CB), has been studied most extensively on many different types of cells including platelets. A sister compound, Cytochalasin A (CA), has received little attention. This study was undertaken to compare and contrast the effects of the two drugs on human platelet ultrastructure and function, using transmission electron microscopy and aggregation studies. The first part of the study dealt with the effects of the two drugs on "resting" nonaggregated platelets. CA and CB appeared to preserve the resting discoid shape of the platelets, making them resistant to even the minor stimulations of the preparation procedure. Microfilaments, which are not normally Visible in resting platelets, were also not seen in the CA and CB treated unstimulated platelets. The second part of the study concerned changes in platelet aggregation following CA and CB treatment. ADP was used as the aggregating agent. In general, both CA and CB inhibited ADP-induced aggregation, but CA did so completely and at very low concentrations while CB was only partially effective, and then only at very high levels. Both drugs seemed to potentiate aggregation when used in low concentrations. This potentiated aggregation was associated with large dilatations of Phillip Jon Borgerding the surface connecting system and enhanced degranulation. The resulting aggregates were very large even though they appeared to contain the same number of platelets as the control aggregates. Thus, the effects of CA and CB are similar. Both agents can inhibit normal platelet aggregation and both produce platelets with large dilatations. The CA was almost 20 times more effective as an inhibitor. THE EFFECTS OF CYTOCHALASIN A AND CYTOCHALASIN B ON HUMAN PLATELET ULTRASTRUCTURE AND FUNCTION BY Phillip Jon Borgerding A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Pathology 1975 ACKNOWLEDGMENTS A special word of appreciation is due: Joann, my wife, Joan Mattson MD, Donna Ladd BS, and my many blood donors. Financial assistance was received through Public Health Service Grant #l-AOZ-AH00237-02 ii TABLE OF CONTENTS Page INTRODUCTION . . . . . . . . . . . . . . 1 MATERIALS AND METHODS . . . . . . . . . . . 4 Preparation of Platelet-rich Plasma . . . . 4 Drug Treatment . . . . . . . . . . . 4 Aggregation Studies. . . . . . . . . . 5 Tissue Processing . . . . . . . . . . 6 Results 0 O O O O O O O O O O O O O O 7 Non-stimulated Platelet Series . . . . . . 7 Stimulated Platelet Series . . . . . . . 12 Discussion. . . . . . . . . . . . . . 21 Summary. . . . . . . . . . . . . . . 29 APPENDIX C O O O O O O O O O O O O O 0 3o BIBLIOGRAPHY O O O O O O O O O O O O O O 32 iii LIST OF FIGURES Figure Page 1. Unstimulated control platelets showing general structure of normal platelets . . . . . . 8 2. Unstimulated control platelets, side view, showing granules, glycogen (gly), and the surface connecting system (s) . . . . . . 8 3. Solvent control platelets with 0.10% DMSO x 9000 O O O O O O O O O O O O 0 lo 4. Solvent control platelets with 0.36% DMSO X 9000 O O O O 0 O O O O O O O O 10 5. Platelets treated with 10 ug/ml CA showing smooth "relaxed" shapes . . . . . . . . ll 6. Platelets treated with 16 ug/ml CB showing presence of pseudopods . . . . . . . . 11 7. Platelets treated with 36 ug/ml CB showing smooth rounded shapes. . . . . . . . . 13 8. ADP—stimulated untreated control platelets showing normal aggregation . . . . . . . 13 9. ADP-stimulated untreated control platelets showing how tightly packed the normal aggre- gates become. . . . . . . . . . . . 14 10. ADP-stimulated solvent control platelets showing tightly packed aggregate of normal appearance . . . . . . . . . . 14 ll. Aggregometer tracings showing normal (top) and CB-modified aggregation. . . . . . . 16 12. Aggregometer tracings showing normal (top) and CA-modified aggregation . . . . . . . . 17 iv Figure 13. 14. 15. 16. 17. l8. 19. 20. ADP-stimulated 2 ug/ml CA . ADP-stimulated 6 ug/ml CA . ADP-stimulated 16 ug/ml CA. ADP-stimulated 5 ug/ml CB . ADP-stimulated 36 ug/ml CB. ADP-stimulated 16 ug/ml CB. ADP-stimulated 100 ug/ml CB platelets platelets platelets platelets platelets platelets platelets pre-treated with pre-treated with pre-treated with pre-treated with pre-treated with pre-treated with pre-treated with Epiniphrine-stimulated platelets pre-treated with 20 ug/ml CB . . Page 18 18 20 20 22 22 23 23 INTRODUCTION Blood platelets are essential in the maintenance of vascular integrity through hemostasis. In response to injury, platelets immediately clump together in aggregates to plug the injured site, and then release specific factors into the blood which are necessary for continued coagulation. After a clot has formed, the platelets also function in clot retraction. In the performance of these duties, platelets have been shown to undergo very definite changes in shape. These changes are thought to originate in the action of contractile microfilaments within the cells (1). This study was undertaken to analyze the role of microfilaments in platelet aggregation and shape change by using the Cytochalasin drugs as microfilament inhibitors. The five Cytochalasin drugs, A, B, C, D, and E, are metabolites of two different species of molds. They were first studied by Carter in 1967 (2). Following this initial work, the responses of many different types of cells to the Cytochalasin drugs were studied. One of the general effects was found to be on the permeability of cell membranes. Very low levels of Cytochalasin B (CB) were shown to block the active transport of glucose into many different cell types (3, 4) including rabbit platelets (5). This effect appears to be largely reversible. The lesser studied Cytochalasin A (CA) was also found to inhibit glucose transport into rabbit platelets but its effect was only about 25 to 65% as great as that of CB (5). Another general effect of the Cytochalasin drugs was found to be an inhibition of contractile processes in cells. CB inhibited phagocytosis in rabbit Leuko- cytes (6), inhibited surface contractility in XenOpus eggs (7), and prevented clot retraction in mamalian blood (8). Wessels lists fourteen different cellular processes which are contractile in nature and also inhibited by CB (9). Studies with CA revealed that it has a similar effect (10, S). It is this second general property of the Cytochalasin drugs which is utilized in this study. Given that the Cytochalasin drugs inhibit con- tractile processes, and that microfilaments are the con- tractile elements within platelets, it follows that any functional changes seen in Cytochalasin treated platelets would be due to a loss of microfilament function. Thus, these drugs are useful in the study of the exact role of platelet microfilaments in the normal activities of these cells. While studies of this nature on platelets using CB have been reported (11), little if any data have been reported concerning the effects of CA. Therefore, this study will compare the effects of both drugs on human platelet ultrastructure and function, using both resting platelets and platelets stimulated to aggregate with ADP . MATERIALS AND METHODS Pre aration of Platelet- ricE Plasma Blood was drawn from healthy adult donors. All donors were required to abstain from all medications and drugs for at least four days. All labware used was plastic unless otherwise noted. All experimental pro- cedures were completed within four hours of blood col- lection. Immediately after a blood specimen was drawn, 3.8% sodium citrate anticoagulant was added in a ratio of 1 part citrate to 9 parts blood. The citrated blood was then centrifuged at 200xG for five minutes to remove the red blood cells. The supernatant citrated platelet- rich plasma (C-PRP) was then carefully decanted into another test tube and stored at 37 C until needed. Drug Treatment CA and CB were obtained through the Imperial Chemical Industries Ltd., Cheshire, England. The drugs were dissolved in dimethyl sulfoxide (DMSO) in their original containers. When dissolved, the concentration of each drug was 10 mg. per ml. or 10 ug per uL. This 10 mg. per m1. stock solution was stored in the dark at 4 C until needed. These special precautions were taken to prevent exposure of the light-sensitive CA. The following amounts of dissolved CA were added to 12 by 70 mm reaction tubes with a 10 uL syringe: 0.5, 0.8, 1.3, and 1.8 uL. To each tube was added 0.5 ml C-PRP to give a final drug concentration of 10, 16, 26, and 36 ug per ml. respectively. This same procedure was followed using CB, and in addition, a control series was processed using the same amount of DMSO solvent but with- out the drugs. Finally, a Specimen of C-PRP was left completely untreated to serve as an additional control. All these specimens were then mixed well and incubated at 37 C for 30 minutes. Aggregation Studies Dissolved CA was added as before to siliconized glass cuvettes in the following amounts: 0.1, 0.3, 0.5, 0.8, 1.3, and 1.8 uL. To each was then added 0.5 ml C-PRP to yield final CA concentrations of 2, 6, 10, 16, 26, and 36 ug per ml respectively. After mixing, the tubes were incubated for 30 minutes at 37 C before aggregation studies were initiated. A similar series was tested using CB but with final concentrations of 5, 10, 16, 20, 26, 36, and 100 ug per m1. As before, two controls were performed: C-PRP + DMSO and C-PRP alone. All aggregation studies were carried out in a Chronolog Aggregometer with an attached continuous strip recorder. An increase in the transmission of light through the sample of C-PRP occurs as aggregation pro- ceeds. Cuvettes with preincubated C-PRP were placed in the aggregometer and allowed to stand until a stable baseline tracing was obtained. At this point, 20 uL of 1,25x10-4 M ADP dissolved in imidazole-buffered saline was added to stimulate aggregation. The final ADP con- centration was then leO_6 M. The resulting aggregation was allowed to continue for 3 or 4 minutes at which time the reaction was stopped with the addition of the fixa- tive. Some aggregation studies were also performed using 20 uL of epinephrine. Tissue Processing All the treated platelets were fixed using a procedure specially designed for use with cell suspen- sions. To the C-PRP was added an equal amount of 0.1% glutaraldehyde in 0.2M cacodylate buffer. After mixing, the fixed C-PRP was poured into labeled Beem capsules and capped. The Beem capsules were placed in specially adapted screw-capped test tubes and centrifuged at 900xG for 10 minutes in a swing-head centrifuge. Follow- ing this, the capsules were removed and placed in racks, and the 0.1% glutaraldehyde was aspirated off the now compacted button of platelets. 3.0% glutaraldehyde in 0.1M cacodylate buffer was carefully added. After 2 hours at room temperature, this was aspirated off and replaced by a 0.2M cacodylate buffer wash. After 15 minutes, the wash was removed and replaced with 1% osmium tetroxide in 0.1M cacodylate buffer. After 30 minutes, this final fixative was replaced with two 0.2M buffer washes. The platelets were then dehydrated with graded acetone solutions up to 100% acetone. This was followed by a 1:3 acetone-Epon Araldyte plastic mixture for 4-6 hours, then pure Epon Araldyte embedding plastic. The capsules were incubated for 12-24 hours at 60 C to poly— merize the resin. After cooling, the capsules were cut away to expose the tissue block. The platelet blocks were sectioned on an LKB Ultratome microtome to yield silver sections. These sections were stained with uranyl acetate and lead citrate for 30 and 5 minutes respectively. Microscopic analysis and photography were performed with a Phillips 201 transmission electron microscope. Results Non—stimulated Platelet Series Untreated control platelets appear as flat discoid-shaped structures (Figures 1, 2). Microtubules are evident in a circumferential band at the platelets' FIGURE 1. Unstimulated control platelets showing general structure of normal platelets. Microtubules (mt) and a few pseudopods are evident. X 9000. FIGURE 2. Unstimulated control platelets, side view, showing granules, glycogen (gly), and the surface connect- ing system (s). X 30,000. periphery. A few pseudopods and buds are present. Scattered granules are prominent along with masses of glycogen particles. Most of the platelets also have a well-developed surface connecting system (Figure 2). There is no evidence of any microfilaments in these unstimulated platelets, nor is there any tendency of the platelets to adhere to each other. These platelets appear similar in all respects to normal platelets as reported widely in the literature. Control platelets treated only with DMSO (Figure 3) are essentially identical to the untreated control pla- telets. The general shape and substructure of both are the same. Even atthe highest concentration, DMSO does not appear to alter platelet structure or shape. Random pseudOpods and buds are evident on occasion (Figure 4). Platelets treated with CA alone show preservation of the "relaxed" state. In general, the minor stimulatory effects of the experimental procedures are not present in the CA treated platelets. Even at the lowest concen- tration of CA, the platelets assume a definite ”relaxed" circular shape with few if any pseudOpods (Figure 5). The effect is the same for all platelets treated with CA from 2 to 36 ug per ml. No swellings, vacuole formation, degranulation or sphering was noted and the platelet substructures appear unchanged. 10 FIGURE 3. 9000. FIGURE 4. Solvent control platelets with 0.36% DMSO X 9000. th 10 ug/ml CA sh smooth "relaxed" shapes. X 9000. owing FIGURE 6. Platelets treated with 16 ug/ml CB showing presence of pseudopods. X 9000. 12 CB treated platelets also had a similar "relaxed" appearance although this protective effect was not as apparent at lower concentrations. With CB levels of 10 and 16 ug per ml, the platelets still retain some pseudo- pods (Figure 6) but with 26 and 36 ug per ml. these dis- appeared (Figure 7). Stimulated Platelet Series Untreated control platelet samples aggregated with ADP gave a typical two-wave curve (Figure 11 top). When the same amount of ADP (5x10-6M) was added to DMSO treated control platelets, the same two-wave curve resulted. Even at levels of 1% DMSO, the ADP-aggregation curve remained the same. Electron microscopic studies of these control ADP stimulated platelets reveal large tightly packed aggregates (Figure 8). Granules are still present in some platelets although many show complete degranulation. Prominent as well is the surface connecting system. Most of the platelets are of irregular shape with many inter— twining pseudOpods. Higher magnification shows some disruption of platelets at the center of the aggregate. At the periphery, the platelets mold to each other tightly while still retaining their individuality (Figure 9). Also visible are scattered microtubules and microfilaments (Figure 9 insert). Glycogen stores are scattered throughout the cytoplasm. The 13 2(1III; FIGURE 7. Platelets treated with 36 ug/ml CB showing smooth rounded shapes. X 9000. FIGURE 8. ADP-stimulated untreated control platelets showing normal aggregation. X 6000. am: I ...‘ , ”c“; FIGURE 9. ADP-stimulated untreated control platelets showing how tightly packed the normal aggregates become. Microtubules (mt) and microfilaments (mf) are evident. X 30,000. Inset X 67,000. FIGURE 10. ADP-stimulated solvent control platelets showing tightly packed aggregate of normal appearance. X 9000. 15 DMSO-treated platelets are similar and appear to be unaffected by the solvent (Figure 10). Platelets pretreated with CA show definite alterations upon ADP stimulation. Two ug per ml of CA did not inhibit ADP aggregation but tended to increase the recorder oscillation as aggregation prOgressed (Figure 12). With 6 ug per ml, the platelets just barely started to aggregate and then apparently dis- engaged (Figure 12). Levels of CA of 10 ug per ml and above completely inhibited ADP-induced aggregation. The only effect noted was a slight step in the recorder tracing due to dilution with ADP. Electron microscopic studies of these CA-treated platelets reveal rather loose aggregates in the 2 ug per ml Specimen. Most of the platelets have distorted shapes with pseudopods typical of aggregated platelets (Figure 13). The majority still contain granules. Microfilaments are visible within the cytoplasm (Figure 13 insert). In addition many platelets have large vacuole-like spaces distending their cytoplasm. These appear to be membrane-bound sacs and are usually empty but occasionally contain floccular material. Platelets treated with 6 ug per ml CA are not clumped at all and appear relatively unstimulated in spite of the ADP added to them (Figure 14). Most platelets carry a full complement of granules. Large 16 EFFECTS OF CB ON ADP-INDUCED PLATELET AGGREGATION .z- -. :1 - ._ ’ 1" L‘_. -Ln-’ , ¢ 4 , w— * ,_, v , - - . . FIGURE 11. Aggregometer tracings showing normal (top) and CB-modified aggregation. 17 (3N EFFECTS or CA ON ADP-INDUCED PLATELET AGGREGATI ._ ‘ ' '1 ,7. FIGURE 12. Aggregometer tracings showing normal (top) and CA-modified aggregation. \, . . ~ _'_v ' . _, . J‘ n": C .' FIGURE 13. ADP-stimulated platelets pre-treated with 2 ug/ml CA. Note the loose aggregate and the large dila- tations (arrows). X 6000. Inset X 45,000. P 3] FIGURE 14. ADP-stimulated platelets pre-treated with 6 ug/ml CA. No aggregation is evident but some dila- tations remain. X 6000. 19 vacuole-like structures are still present in their cyto- plasm although not in the same numbers as in the 2 ug per ml specimens. The 10, 16, 26, and 36 ug per ml CA speci- mens are all similar to the unstimulated platelets of the previous section, being ovoid in shape and devoid of pseudopods, microfilaments or evidence of aggregation. They look as if they were never stimulated at all (Figure 15). All of these, however, have occasional large distentions of their cyctOplasm similar to those of the 2 ug per m1 CA platelets. Platelets pretreated with CB show no effects until a concentration of 10 ug per ml or greater is used. Concentrations of 10, 20, and 30 ug per m1 did not inhibit aggregation but did produce dramatic oscil- lations of the recorder tracings similar to those seen with CA at 2 ug per ml (Figure 11). Only at 100 ug per ml did CB have any inhibitory effect on ADP-induced aggregation. With this level the pronounced oscil- lations of the tracing were no longer present. Ultrastructural studies of these CB-treated platelet aggregates reveal some startling changes as compared to control aggregates. While the 5 and 10 ug per ml CB specimens are similar to controls (Figure 16), the 16, 20, 26, and 36 ug per m1 platelets show many of the large distentions seen in some of the low con- centration CA platelets. With these CB-treated FIGURE 15. ADP-stimulated platelets pre-treated with 16 ug/ml CA. No aggregation is evident; platelets appear "relaxed" and carry numerous granules. X 9000. 1 “"55 335%; . i I. - , ‘ fl~n’~)~*';iff l :.., L - . 4 .7 A- ab) )> ' FIGURE 16. ADP-stimulated platelets pre-treated with 5 ug/ml CB. No inhibition of aggregation is apparent. X 14,000. 21 platelets, however, the distentions are much more numerous and larger (Figure 17). The platelets are dominated by the spaces, which appear to be membrane bound (Figure 18). Microfilaments are visible in the pseudopods (Figure 18 insert). Most of the granules have disappeared and what granules remain are displaced to the cell periphery. In spite of these many distentions, the general structure of the aggregates appears to be tightly compact with few extracellular spaces. Only in the 100 ug per ml CB platelets did the aggregates seem to be loose and breaking up (Figure 19). This corresponds to the inhibition of aggregation documented on the aggregometer tracing at this concentration of CB. Here the platelets seem to be "relaxed" in shape with few pseudopods, few disten- tions, but many granules. By comparison, the 100 ug per ml CB platelets were very similar to the 6 ug per ml CA platelets, both showing few of the signs of ADP stimu- lation. Finally, CB-treated platelets stimulated with epinephrine showed no inhibition of aggregation but did show the presence of large vacuoles identical to ADP stimulated CB-treated platelets (Figure 20). Discussion Exactly how the Cytochalasins are able to inhibit contractile events is unclear. Some feel that the effect is due to an actual disruption of the contractile 22 ““197; FIGURE 17. ADP-stimulated platelets pre-treated with 36 ug/ml CB. Aggregation is not inhibited, but many dilatations are apparent. Few granules remain. X 20,000. E...- 16 ug/ml CB. The dilatations appear to be membrane bound (arrows). X 20,000. Inset X 45,000. FIGURE 19. ADP-stimulated platelets pre-treated with 100 ug/ml CB. Little aggregation is apparent. X 6000. FIGURE 20. Epiniphrine-stimulated platelets pre-treated with 20 ug/ml CB. Aggregation is not inhibited and many dilatations are present. X 6000. 24. filaments (14). White (11) feels that, instead of destroying the filaments, the Cytochalasins effectively disconnect them from their points of attachment so that, while the actual contraction is not prevented, the results are the same as if it had been. He has demonstrated the presence of what appear to be contracted masses of micro- filaments in the centers of CB-treated platelets stimu- lated with ADP. In this study, we were unable to find any such masses in CB-treated platelets. Another theory prOposes that, by denying the passage of glucose into the cells, the Cytochalasins inhibit cellular contractility by blocking the energy source (3). Other studies, however, have shown that cells cultured in glucose-free media do not behave the same as Cytochalasin-treated cells at all (15). Indeed, our studies indicate that CA, the weaker inhibitor of glucose tranSport, is a very effective aggregation inhibitor. Wessels et a1. prOpose that the Cytochalasins act upon calcium-dependent phases of microfilament activity (16). This is feasible since it is thought that platelet contractile filaments, like skeletal muscle, possess a troponin-tropomyosin-like system (18) and require calcium to function (17). The effect of CA/CB on unstimulated platelets seems to be entirely consistent with the effects seen in other cells (2, 6, 7). High concentrations of CA/CB 25 appear to have a stabilizing or protective effect on platelets. The result is an inability of CA/CB-treated platelets to change shape or form pseudopods when stimu- lated. Boyle and Fudenberg (13) reported similar results with platelets stimulated with glass contact. In their study, the pre-existing pseudopods and buds were forced to regress with the application of the Cytochalasin drugs. Evidence seen thus far in this study indicates that the CA is stronger in its effect than the CB. This difference in effect is greatly amplified when the platelets are stimulated to aggregate with ADP. With normal aggregation, the clumping of platelets begins immediately after ADP is added. These initial aggregates consist of granulated platelets loosely bound together. Apparently, externally added ADP somehow stimulates the platelet membrane to become "sticky" (19) while reducing the intercellular repulsive forces (20). The ADP also stimulates the initial contractions of the microfilaments. These activities make up the first wave of aggregation during which the platelets begin to change their shapes as described in the introduction. As these shape changes intensify, the constricting microfilaments draw the granules to the platelet centers where their contents are released (17). The ADP and serotonin released from the granules further stimulate the platelets to aggregate while the platelet factor 3 is essential to fibrin 26 formation. This release reaction produces a second wave of aggregation (Figure 11 top). When the platelets are pre-treated with CA and CB as described, the changes are obvious. The results clearly indicate that small amounts of CA inhibit both primary and secondary aggregation very effectively. While CB also inhibits, it does so only partially and then only at very high concentrations. This inhibition of aggregation correlates with the absence of pseudopods and visible microfilaments at the ultrastructural level. It is thus apparent that without microfilament activity, the platelets are essentially paralyzed. This is all in total agreement with the findings of Haslam on pig platelets (5) and of Weiss on ascites cells (10). White, however, found that he could completely inhibit ADP- induced platelet aggregation with only 20 ug per m1 CB (11). He was careful to point out, however, that a slight increase in the amount of ADP would overcome the inhibition of the CB and stimulate normal aggregation. In this study, the ADP levels were just strong enough to produce a two-waved aggregation curve on the control platelets, but no higher so that any inhibition from the CA/CB would not be masked with overstimulation. One might speculate that White's CB may have been con- taminated with the more powerful inhibitor, CA. 27 Another interesting effect noted was the presence of the vacuole-like swellings within the treated platelets. These ultrastructural changes brought about with low con- centrations of the Cytochalasins have not been described before. The large dilatations result in platelets of increased size and therefore in platelet aggregates of enormous size. The large aggregates cause the erratic oscillations seen in the CA-2, CB-20, and CB-30 tracings (Figure 11, 12). Although the mechanism of their for- mation is not apparent, it is clear that both CA/CB and ADP are necessary to produce them. Neither ADP nor CA/CB alone will cause swellings in platelets; only the combination appears to work this way. In this apparent interaction, the ratio of Cytochalasin to ADP may be important. Low levels of CB do not produce swellings nor do the very high levels. Only when CB is present at between 16 and 36 ug per ml does it interact with ADP in this way. Likewise, with CA, only low levels interact; inhibitory doses of CA yield platelets with only a few dilatations each. It is interesting to note that epinephrine-stimulated platelets pre-treated with CB deve10p the same kind of swelling. In this case, the cause is still probably ADP in combination with the CB, only here, the ADP comes from the platelets themselves during their release reaction. 28 One might speculate that these large vacuole-like dilatations represent segments of the surface connecting system. They are membrane bound, and, while connections with the surface could not be demonstrated, they can be seen to communicate with smaller typical portions of the surface connecting system. Also, they often contain floccular material similar to that seen in the surround- ing medium. If granule contents are released into the surface connecting system as Day et al. (17) speculate, then the large dilated vacuoles seen here might represent a surface connecting system which has been overdistended during the release reaction. It is possible that, under the influence of CA/CB-ADP, the tube-like surface connecting system constricts at various points and that the resulting segments balloon out as observed. It has been demonstrated that both CA and CB have profound effects on platelet structure and function. Treatment with low concentrations of either drug followed by ADP stimulation results in platelets with large vacuoles and platelet aggregates of enormous size. This effect of CA and CB may not be related to contrac- tile microfilaments since, at these low levels, aggre- gation is not inhibited and microfilaments are still demonstrable. Higher concentrations of CA and CB clearly inhibit ADP aggregation of platelets. Clumping does not occur, nor do the usual shape changes or pseudopod 29 formation. At these higher concentrations the resulting changes appear to be directly related to an inhibition of contractile microfilament function. Summary That contractile microfilament activity is necessary for normal platelet function has been clearly seen in this study. Platelets without microfilament function such as those treated with CA or CB would obviously be useless in vivo. They neither could aggregate to plug vascular injuries nor could they release from their granules the factors necessary for further coagulation to proceed. The cornerstone of hemostasis rests on a foundation of contractile platelet microfilaments. APPENDIX APPENDIX Two technical aspects of these experiments deserve further elaboration. First of all, the use of Beem cap- sules as containers for the platelets throughout pro- cessing was a definite aid in this study and would be in any electron microscOpic study involving particulate samples. The initial 0.1% glutaraldehyde fixation stops cellular activity but does not coagulate the plasma so that the platelets can still be easily spun down. The second fixation with 3.0% glutaraldehyde after centrifu- gation serves to bind the platelets to each other and to the tip of the capsule. Once fixed in this manner, the platelet button remains firmly attached in the capsule tip through all the subsequent changes of solutions. This greatly reduces the time consumed in processing as well as the hazard of losing one's specimen in the aSpirator. The capsules may be rotated to facilate the penetration of the platelets by the various solutions. Care, however, must be taken to limit the size of the platelet button since too large a specimen will not be prOperly fixed or embedded. In this connection, some late work has shown that better infiltration and 30 31 embedding occur if ethanol solutions are used in dehy- dration followed by propylene oxide. Secondly, the importance of the various buffer concentrations cannot be overestimated. If, for example, the 0.1% glutaraldehyde is dissolved in 0.1M cacodylate buffer, most of the platelets are lost, probably through osmotic rupture. With 0.2M buffer, the platelets survive and have a normal appearance. On the other hand, if the 3.0% glutaraldehyde is dissolved in 0.2M cacodylate buffer, the platelet survival is good but the exces- sively dark appearance of the platelets under the microscope renders them useless. BIBLIOGRAPHY BIBLIOGRAPHY Specific References White, J. G., "Microtubules and Microfilaments in the Contractile Mechanism of Human Platelets" J. Cell Biol. 33: 141A (1969). 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