Platelet Ultrastructural Alterations
pH-Induced
A Possible Mechanism for Meir
Djaldetti, MD; Pnina Fishman;
Impaired Platelet Aggregation
Hanna
Bessler, MS; Chaim Chaimoff, MD
\s=b\ After the observation that lavages with alkaline solutions exert a beneficial effect on the bleeding tendency and increase platelet aggregation in patients with intragastric hemorrhage, studies were undertaken to find a relationship between changes of pH and platelet morphology. Transmission and scanning electron microscopy showed that at a pH lower than 7.4, normal human platelets change their internal structure as well as their shape, becoming spheres deprived of pseudopodia. On the other hand, a pH higher than 7.4 induces transformation of platelet internal organelles similar to that caused by thrombin. At an alkaline pH, the platelets develop multiple pseudopodia that facilitate their attachment to each other. These findings may explain the increased platelet aggregation in alkaline medium demonstrated in one of our previous works.
(Arch Surg 114:707-710, 1979) from the gastrointestinal tract is a hazardous condition that requires vigorous treat¬ ment. It has been shown that gastric lavage with alkaline solutions has a beneficial effect on the bleeding, giving opportunity to postpone or even to avoid surgery.' We have demonstrated in a previous study that change of the plasma pH in vitro may alter some of the plasma clotting properties, as well as some of the platelet functions.2 Of special interest were the changes in platelet aggregation that decreased at a lower pH and increased at a higher pH.
Massive bleeding
Accepted for publication Dec 21, 1978. From the Departments of Medicine B (Dr Djaldetti), Surgery A (Dr Chaimoff), and the Electron Microscopy Unit, Hasharon Hospital, Petah\x=req-\ Tiqva, Israel, and the Tel Aviv University Medical School, Tel Aviv, Israel (Mesdames Fishman and Bessler). Reprint requests to Department of Medicine B, Hasharon Hospital, Petah-Tiqva, Israel (Dr Djaldetti).
Our results support the
al.3
findings reported by
Green et
The present study was undertaken to evaluate by trans¬ mission (TEM) and scanning (SEM) electron microscopy the behavior of platelets at different pH. The existence of platelet ultrastructural alterations induced by changes in the PH may serve as an explanatory mechanism for the impaired platelet aggregation observed at a pH lower or higher than 7.4. SUBJECTS AND METHODS Platelet-rich plasma was obtained from 40 mL of venous blood withdrawn with a heparinized syringe from healthy donors. The platelets were separated by centrifugation, resuspended in mini¬ mal essential medium, and divided into five tubes. The cells of the first tube were fixed in cold 1% glutaraldehyde, pH of 7.4, and served as control. The pH of the medium in the remaining four tubes was brought to 7.4, 7.2, 5.5, and 9.0, respectively, and the tubes were kept at 37 °C for 30 minutes in a water bath. The platelets of these three tubes were fixed in cold 1% glutaraldehyde in phosphate buffer solution with a pH equal to that of the corresponding tube. For TEM, the cells were postfixed in 1% osmium tetroxide, dehydrated in graded alcohols, and embedded in an epoxy resin (Epon 812). Thin sections were cut with an ultratome and examined with a TEM. For SEM, the platelets were smeared on glass coverslips according to the method of Sanders et al,' dehydrated in graded fréons, critical-point dried using CO,, and coated with gold.
RESULTS TEM
Control platelets showed a normal ultrastructure of the specific granules, mitochondria, microtubules, and the rest of the cellular organelles (Fig 1). The platelet structure
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Fig 1.—Transmission electron micrograph of showing normal arrangement and ultrastructure nelles (original magnification 14,750).
control platelet of cellular orga-
Fig 2.—Platelets incubated in medium with pH of 7.4. Cellular organelles are well preserved, but tendency to central concentra¬ tion is visible (original magnification 14,820).
Fig 3.—Platelets
incubated at pH of 5.5. Cells are dispersed and almost completely deprived of pseudopodia (original magnifica¬ tion 14,750).
Fig 4—Platelets incubated at pH of 9.0. Cells aggregates (original magnification 24,350).
after incubation at a pH of 7.4 is shown in Fig. 2. The cells possessed a round or irregular shape with a noticeable tendency to aggregation. The granules were concentrated mostly in the center of the platelets, the organelles being well preserved. Platelets examined at a pH of 7.2 did not differ from those incubated at a pH of 7.4. At a pH of 5.5, the platelets appeared dispersed (Fig 3) and only rarely were aggregates of a few cells seen. In some of the cells, the number of the specific granules seemed to be reduced and very often they were dispersed throughout the hyalomere. Although the structure of the organelles was preserved, a certain degree of vacuolization of the cyto¬ plasm was noted. After incubation at a pH of 9.0, the platelets showed a noticeable tendency for aggregation (Fig 4). There was an increased number of granules
centripetally located. The platelet organelles were well preserved, but a slight degree of vacuolization was pres¬
are seen
in
ent. SEM
Control
platelets showed characteristic surface features consisting of irregular shapes with bulbous protuberations of different form and size, as well as short pseudopodia (Fig 5). After incubation at a pH of 7.4 and of 7.2, the platelets changed their shape showing less protuberations and more and longer pseudopodia. The tendency to aggre¬ gation was increased (Fig 6). Incubation at a pH of 5.5 transformed the platelets to oval or spheric cells, almost completely deprived of processes and pseudopodia (Fig 7). The cells seemed smaller in size, separated from each other, and no tendency to aggregation was observed. At a pH of
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Fig 5—Scanning electron micrograph of control platelets. Cells possess moderate amount of bulbous or needle-shaped pseudo¬ podia. Markers are 1 µ.
aggregate formation. Markers
Fig 7.—Platelets incubated at pH of 5.5. Round or oval shape and lack of pseudopodia is demonstrated. Markers are 1 µ.
Fig 8.-Platelets incubated at pH aggregation. Markers are 1 µ.
9.0, the platelet form changed again: the cells showed
amounts of thrombin, the cell shape is restored. When larger amounts of thrombin are used, a double wave of aggregation occurs, characterized by clumping of the orga¬ nelles at the cell center and the appearance of the platelet release reaction accompanied by cell destruction. It is evident, therefore, that any alteration of the platelet shape, irrespective of the inducing agent, may result in impairment of platelet aggregation.
multiple, long, needle-shaped pseudopodia. The tendency to aggregation was noticeably increased (Fig 8) and most of the cells
were seen
in clusters.
COMMENT In our previous work,2 we have shown the existence of a correlation between the pH level and platelet function. At a pH lower than 6.5 to 7.0, the platelet calcium and serotonin content decreased, whereas at a higher pH it noticeably increased. As for the platelet aggregation induced by epinephrine, a normal double-wave pattern was detected at a pH of 7.6. These findings are in agreement with previous reported observations.3·56 Changes of platelet shape and ultrastructure are observed during both the primary and secondary wave induced by thrombin.7 They consist in loss of the platelet discoid shape and appearance of multiple pseudopodia. The organelles move towards the cell center. These alterations may be reversible, depending on the amount of the added thrombin. Within ten minutes after the addition of small
Fig 6.—Platelets
incubated at
pH of
are
7.4. Note
1 µ
of 9.0. Note
tendency
to
tendency to
The present results demonstrate a correlation between the platelet shape and ultrastructure, on the one hand, and the pH of the incubation medium, on the other. Scanning electron microscopic observations showed that in addition to the alterations induced by changes in the pH, the simple process of incubation causes the appearance of pseudopo¬ dia and an increased tendency to aggregation. However, this tendency was much more pronounced when the pH of the incubation medium was increased to 9.0. The appear¬ ance of numerous, longer, and filamentous pseudopodia in alkaline medium facilitates the connection of the cells with each other and the precipitation of the aggregation process. The internal transformation of the platelet orga-
Downloaded From: http://archsurg.jamanetwork.com/ by a Johns Hopkins University User on 06/09/2015
nelles at
a pH of 9.0, which is similar to that observed in platelets exposed to thrombin, may be an additional trig¬ ger for increased aggregation. Moreover, since platelets incubated at a higher pH undergo internal transformation and surface changes similar to those observed in platelets exposed to thrombin, it is conceivable to assume that a higher pH exerts a thrombin-like effect on the platelets. On the other hand, the alterations of the platelet ultrastructure at a lower pH, such as observed with both TEM and SEM, serve as an explanation for the decreased platelet aggregation observed in acidified medium in vitro. It is evident that platelets transformed to spheres and deprived of pseudopodia cannot make firm contact with each other and they fail to aggregate. The role of the "decentralization" of the platelet orga-
nelles in the prevention of the aggregation process remains unclear. Changes of the platelet shape without platelet aggregation was observed after exposure of rabbit plate¬ lets to a lower pH.5 It is conceivable that similar changes of the platelet shape and ultrastructure occur during intra¬ gastric bleeding, when the platelets are exposed to the low pH of the gastric content, thus increasing the bleeding tendency in patients with intragastric hemorrhage. On the other hand, the increased tendency to aggregation induced by a higher pH may explain the beneficial effect of gastric lavage with alkaline solutions on the hemorrhagic diathesis in these patients. This work was supported by a grant from Ministry of Health, Israel. J. Sadovnik did the photographic work.
the Chief Scientist's Bureau,
References 1. Curtis LE, Simonian S, Buerk CA, et al: Evaluation of the effectiveness of controlled pH in management of massive upper gastrointestinal bleeding. Am J Surg 125:474-476, 1973. 2. Chaimoff C, Creter D, Djaldetti M: The effect of pH on platelet and coagulation factor activities. Am J Surg 136:257-259, 1978. 3. Green FW Jr, Kaplan ML, Curtis LE, et al: Effect of acid and pepsin on blood coagulation and platelet aggregation: A possible contributor to prolonged gastroduodenal mucosal hemorrhage. Gastroenterology 74:38-43, 1978.
4. Sanders SK, Alexander EL, Braylan RC: A high-yield technique for preparing cells fixed in suspension for scanning electron microscopy. J Cell
Biol 67:476-480, 1975. 5. McLean JR, Veloso H: Change of shape without aggregation caused by ADP in rabbit platelets at low pH. Life Sci 6:1983-1986, 1967. 6. Rogers AB: The effect of pH on human platelet aggregation induced by epinephrine and ADP. Proc Soc Exp Biol Med 139:1100-1103, 1972. 7. Williams WJ, Beutler E, Erslev AJ, et al: Hematology. New York, McGraw-Hill Book Co, 1972, p 1030.
Editorial Comment When platelets are activated by adenosine diphosphate, throm¬ bin, and many other stimuli, the first morphologic alteration is a change in shape, with transformation from disk to spiculated sphere. The possibility that such a change might facilitate platelet clumping was first suggested by Bangham and Pethica,' who pointed out that the mutually repulsive negative charges that prevent intimate contact of adjacent cells would be minimized at the tips of pseudopodia, because of their small radius of curvature and, therefore, reduced repellant forces. Thus, the observations by Djaldetti and associates describing spiculation of the platelet at elevated pH levels, at which aggregation is enhanced, and conver¬ sion to spheres at lower pH levels, at which aggregation is inhibited, fit into a rational framework. As usual, however, the story is more complex. Epinephrineinduced platelet clumping is not accompanied by a change in the
shape of the platelet, so formation of cytoplasmic processes seems not to be an absolute requirement for platelet-platelet propinqui¬ ty. Furthermore, platelets aggregated by thrombin can be disag¬ gregated, resume their normal shape, and circulate with a normal lif espan. Whether the change in shape observed by the authors at different pH levels accounts for the differences in platelet suscep¬ tibility to aggregation or simply reflects a parallel phenomenon is a question that will require additional investigation. Edwin W. Boston
Salzman, MD
1. Bangham AD, Pethica BA: The adhesiveness of cells and the nature of the chemical groups at their surfaces. Proc R Physic Soc Edinburgh 28:43-50,
1960.
Downloaded From: http://archsurg.jamanetwork.com/ by a Johns Hopkins University User on 06/09/2015
pH-Induced
A Possible Mechanism for Meir
Djaldetti, MD; Pnina Fishman;
Impaired Platelet Aggregation
Hanna
Bessler, MS; Chaim Chaimoff, MD
\s=b\ After the observation that lavages with alkaline solutions exert a beneficial effect on the bleeding tendency and increase platelet aggregation in patients with intragastric hemorrhage, studies were undertaken to find a relationship between changes of pH and platelet morphology. Transmission and scanning electron microscopy showed that at a pH lower than 7.4, normal human platelets change their internal structure as well as their shape, becoming spheres deprived of pseudopodia. On the other hand, a pH higher than 7.4 induces transformation of platelet internal organelles similar to that caused by thrombin. At an alkaline pH, the platelets develop multiple pseudopodia that facilitate their attachment to each other. These findings may explain the increased platelet aggregation in alkaline medium demonstrated in one of our previous works.
(Arch Surg 114:707-710, 1979) from the gastrointestinal tract is a hazardous condition that requires vigorous treat¬ ment. It has been shown that gastric lavage with alkaline solutions has a beneficial effect on the bleeding, giving opportunity to postpone or even to avoid surgery.' We have demonstrated in a previous study that change of the plasma pH in vitro may alter some of the plasma clotting properties, as well as some of the platelet functions.2 Of special interest were the changes in platelet aggregation that decreased at a lower pH and increased at a higher pH.
Massive bleeding
Accepted for publication Dec 21, 1978. From the Departments of Medicine B (Dr Djaldetti), Surgery A (Dr Chaimoff), and the Electron Microscopy Unit, Hasharon Hospital, Petah\x=req-\ Tiqva, Israel, and the Tel Aviv University Medical School, Tel Aviv, Israel (Mesdames Fishman and Bessler). Reprint requests to Department of Medicine B, Hasharon Hospital, Petah-Tiqva, Israel (Dr Djaldetti).
Our results support the
al.3
findings reported by
Green et
The present study was undertaken to evaluate by trans¬ mission (TEM) and scanning (SEM) electron microscopy the behavior of platelets at different pH. The existence of platelet ultrastructural alterations induced by changes in the PH may serve as an explanatory mechanism for the impaired platelet aggregation observed at a pH lower or higher than 7.4. SUBJECTS AND METHODS Platelet-rich plasma was obtained from 40 mL of venous blood withdrawn with a heparinized syringe from healthy donors. The platelets were separated by centrifugation, resuspended in mini¬ mal essential medium, and divided into five tubes. The cells of the first tube were fixed in cold 1% glutaraldehyde, pH of 7.4, and served as control. The pH of the medium in the remaining four tubes was brought to 7.4, 7.2, 5.5, and 9.0, respectively, and the tubes were kept at 37 °C for 30 minutes in a water bath. The platelets of these three tubes were fixed in cold 1% glutaraldehyde in phosphate buffer solution with a pH equal to that of the corresponding tube. For TEM, the cells were postfixed in 1% osmium tetroxide, dehydrated in graded alcohols, and embedded in an epoxy resin (Epon 812). Thin sections were cut with an ultratome and examined with a TEM. For SEM, the platelets were smeared on glass coverslips according to the method of Sanders et al,' dehydrated in graded fréons, critical-point dried using CO,, and coated with gold.
RESULTS TEM
Control platelets showed a normal ultrastructure of the specific granules, mitochondria, microtubules, and the rest of the cellular organelles (Fig 1). The platelet structure
Downloaded From: http://archsurg.jamanetwork.com/ by a Johns Hopkins University User on 06/09/2015
Fig 1.—Transmission electron micrograph of showing normal arrangement and ultrastructure nelles (original magnification 14,750).
control platelet of cellular orga-
Fig 2.—Platelets incubated in medium with pH of 7.4. Cellular organelles are well preserved, but tendency to central concentra¬ tion is visible (original magnification 14,820).
Fig 3.—Platelets
incubated at pH of 5.5. Cells are dispersed and almost completely deprived of pseudopodia (original magnifica¬ tion 14,750).
Fig 4—Platelets incubated at pH of 9.0. Cells aggregates (original magnification 24,350).
after incubation at a pH of 7.4 is shown in Fig. 2. The cells possessed a round or irregular shape with a noticeable tendency to aggregation. The granules were concentrated mostly in the center of the platelets, the organelles being well preserved. Platelets examined at a pH of 7.2 did not differ from those incubated at a pH of 7.4. At a pH of 5.5, the platelets appeared dispersed (Fig 3) and only rarely were aggregates of a few cells seen. In some of the cells, the number of the specific granules seemed to be reduced and very often they were dispersed throughout the hyalomere. Although the structure of the organelles was preserved, a certain degree of vacuolization of the cyto¬ plasm was noted. After incubation at a pH of 9.0, the platelets showed a noticeable tendency for aggregation (Fig 4). There was an increased number of granules
centripetally located. The platelet organelles were well preserved, but a slight degree of vacuolization was pres¬
are seen
in
ent. SEM
Control
platelets showed characteristic surface features consisting of irregular shapes with bulbous protuberations of different form and size, as well as short pseudopodia (Fig 5). After incubation at a pH of 7.4 and of 7.2, the platelets changed their shape showing less protuberations and more and longer pseudopodia. The tendency to aggre¬ gation was increased (Fig 6). Incubation at a pH of 5.5 transformed the platelets to oval or spheric cells, almost completely deprived of processes and pseudopodia (Fig 7). The cells seemed smaller in size, separated from each other, and no tendency to aggregation was observed. At a pH of
Downloaded From: http://archsurg.jamanetwork.com/ by a Johns Hopkins University User on 06/09/2015
Fig 5—Scanning electron micrograph of control platelets. Cells possess moderate amount of bulbous or needle-shaped pseudo¬ podia. Markers are 1 µ.
aggregate formation. Markers
Fig 7.—Platelets incubated at pH of 5.5. Round or oval shape and lack of pseudopodia is demonstrated. Markers are 1 µ.
Fig 8.-Platelets incubated at pH aggregation. Markers are 1 µ.
9.0, the platelet form changed again: the cells showed
amounts of thrombin, the cell shape is restored. When larger amounts of thrombin are used, a double wave of aggregation occurs, characterized by clumping of the orga¬ nelles at the cell center and the appearance of the platelet release reaction accompanied by cell destruction. It is evident, therefore, that any alteration of the platelet shape, irrespective of the inducing agent, may result in impairment of platelet aggregation.
multiple, long, needle-shaped pseudopodia. The tendency to aggregation was noticeably increased (Fig 8) and most of the cells
were seen
in clusters.
COMMENT In our previous work,2 we have shown the existence of a correlation between the pH level and platelet function. At a pH lower than 6.5 to 7.0, the platelet calcium and serotonin content decreased, whereas at a higher pH it noticeably increased. As for the platelet aggregation induced by epinephrine, a normal double-wave pattern was detected at a pH of 7.6. These findings are in agreement with previous reported observations.3·56 Changes of platelet shape and ultrastructure are observed during both the primary and secondary wave induced by thrombin.7 They consist in loss of the platelet discoid shape and appearance of multiple pseudopodia. The organelles move towards the cell center. These alterations may be reversible, depending on the amount of the added thrombin. Within ten minutes after the addition of small
Fig 6.—Platelets
incubated at
pH of
are
7.4. Note
1 µ
of 9.0. Note
tendency
to
tendency to
The present results demonstrate a correlation between the platelet shape and ultrastructure, on the one hand, and the pH of the incubation medium, on the other. Scanning electron microscopic observations showed that in addition to the alterations induced by changes in the pH, the simple process of incubation causes the appearance of pseudopo¬ dia and an increased tendency to aggregation. However, this tendency was much more pronounced when the pH of the incubation medium was increased to 9.0. The appear¬ ance of numerous, longer, and filamentous pseudopodia in alkaline medium facilitates the connection of the cells with each other and the precipitation of the aggregation process. The internal transformation of the platelet orga-
Downloaded From: http://archsurg.jamanetwork.com/ by a Johns Hopkins University User on 06/09/2015
nelles at
a pH of 9.0, which is similar to that observed in platelets exposed to thrombin, may be an additional trig¬ ger for increased aggregation. Moreover, since platelets incubated at a higher pH undergo internal transformation and surface changes similar to those observed in platelets exposed to thrombin, it is conceivable to assume that a higher pH exerts a thrombin-like effect on the platelets. On the other hand, the alterations of the platelet ultrastructure at a lower pH, such as observed with both TEM and SEM, serve as an explanation for the decreased platelet aggregation observed in acidified medium in vitro. It is evident that platelets transformed to spheres and deprived of pseudopodia cannot make firm contact with each other and they fail to aggregate. The role of the "decentralization" of the platelet orga-
nelles in the prevention of the aggregation process remains unclear. Changes of the platelet shape without platelet aggregation was observed after exposure of rabbit plate¬ lets to a lower pH.5 It is conceivable that similar changes of the platelet shape and ultrastructure occur during intra¬ gastric bleeding, when the platelets are exposed to the low pH of the gastric content, thus increasing the bleeding tendency in patients with intragastric hemorrhage. On the other hand, the increased tendency to aggregation induced by a higher pH may explain the beneficial effect of gastric lavage with alkaline solutions on the hemorrhagic diathesis in these patients. This work was supported by a grant from Ministry of Health, Israel. J. Sadovnik did the photographic work.
the Chief Scientist's Bureau,
References 1. Curtis LE, Simonian S, Buerk CA, et al: Evaluation of the effectiveness of controlled pH in management of massive upper gastrointestinal bleeding. Am J Surg 125:474-476, 1973. 2. Chaimoff C, Creter D, Djaldetti M: The effect of pH on platelet and coagulation factor activities. Am J Surg 136:257-259, 1978. 3. Green FW Jr, Kaplan ML, Curtis LE, et al: Effect of acid and pepsin on blood coagulation and platelet aggregation: A possible contributor to prolonged gastroduodenal mucosal hemorrhage. Gastroenterology 74:38-43, 1978.
4. Sanders SK, Alexander EL, Braylan RC: A high-yield technique for preparing cells fixed in suspension for scanning electron microscopy. J Cell
Biol 67:476-480, 1975. 5. McLean JR, Veloso H: Change of shape without aggregation caused by ADP in rabbit platelets at low pH. Life Sci 6:1983-1986, 1967. 6. Rogers AB: The effect of pH on human platelet aggregation induced by epinephrine and ADP. Proc Soc Exp Biol Med 139:1100-1103, 1972. 7. Williams WJ, Beutler E, Erslev AJ, et al: Hematology. New York, McGraw-Hill Book Co, 1972, p 1030.
Editorial Comment When platelets are activated by adenosine diphosphate, throm¬ bin, and many other stimuli, the first morphologic alteration is a change in shape, with transformation from disk to spiculated sphere. The possibility that such a change might facilitate platelet clumping was first suggested by Bangham and Pethica,' who pointed out that the mutually repulsive negative charges that prevent intimate contact of adjacent cells would be minimized at the tips of pseudopodia, because of their small radius of curvature and, therefore, reduced repellant forces. Thus, the observations by Djaldetti and associates describing spiculation of the platelet at elevated pH levels, at which aggregation is enhanced, and conver¬ sion to spheres at lower pH levels, at which aggregation is inhibited, fit into a rational framework. As usual, however, the story is more complex. Epinephrineinduced platelet clumping is not accompanied by a change in the
shape of the platelet, so formation of cytoplasmic processes seems not to be an absolute requirement for platelet-platelet propinqui¬ ty. Furthermore, platelets aggregated by thrombin can be disag¬ gregated, resume their normal shape, and circulate with a normal lif espan. Whether the change in shape observed by the authors at different pH levels accounts for the differences in platelet suscep¬ tibility to aggregation or simply reflects a parallel phenomenon is a question that will require additional investigation. Edwin W. Boston
Salzman, MD
1. Bangham AD, Pethica BA: The adhesiveness of cells and the nature of the chemical groups at their surfaces. Proc R Physic Soc Edinburgh 28:43-50,
1960.
Downloaded From: http://archsurg.jamanetwork.com/ by a Johns Hopkins University User on 06/09/2015
