[1]
PLATELET
STRUCTURAL AND FUNCTIONAL
ORGANIZATION
3
[1] I n t r o d u c t i o n to P l a t e l e t S t r u c t u r a l and Functional Organization
By JACEK
HAWIGER
The function of platelets evolves from their structure. When one observes platelets at work sealing the cut in a hemorrhaging vessel, their ability to adhere to nonendothelialized surfaces and to aggregate is such an arresting phenomenon that the impression of single-minded functionality cannot be ignored. 1 Platelets circulate in blood as smooth disks of an average volume of 5 to 7.5/zm 3, 14 times smaller than that of erythrocytes. Each day an estimated 3.5 x 10l° platelets per liter of blood are produced by cytoplasmic fragmentation of their mother cell, a megakaryocyte, through two proposed mechanisms. 2'3 Their expected life span is 10 days. 4 The first encounter of platelets with a damaged vessel wall results in a change of shape from smooth disks into spiny spheres. Pseudopods promote contact with a zone of vascular injury and the subsequent spreading of platelets completes their adhesion. 5 A contractile apparatus harnesses and conveys the energy for this process by rearranging the membrane skeleton and the cytoplasmic actin filaments. In addition to actin and myosin, platelets contain at least nine distinct cytoskeletal proteins involved in assembly, disassembly, and regulation of the contractile apparatus. 6 As in other cells, adhesion is mediated through specific contact sites formed by fibronectin on the outside and extends via integrin receptors to the inside of platelets. Therein, the integrin receptor is clutched by protein P235, analogous to talin. 7 Another adhesive receptor, the GPIb-IX complex, communicating with von Willebrand factor in the extracellular matrix, is linked to short actin filaments that are kept together by an actinbinding protein on the inside of the platelet membrane. 8 An "experiment of nature," in which a deficiency of the GPIb-IX complex in Bernard-Soulier syndrome results in apparently giant platelets, illustrates the role of the J. J. Sixma and J. Wester, Semin Hematol. 14, 265 (1977). 2 M. Shaklai and M. Tavassoli, J. UItrastruct. Res. 62, 270 (1978). 3 j. M. Radley and G. Scurfield, Blood 56, 996 (1980). 4 L. A. Harker and C. A. Finch, J. Clin. Invest. 48, 963 (1969). 5 K. S. Sakariassen, R. Mugli, and H. R. Baumgartner, this series, Vol. 169, p. 37. 6 j. E. B. Fox, in "Thrombosis and Hemostasis" (M. Verstate, J. Vermylen, R. Lijnen, and J. Arnout, eds.), p. 175. Leuven Univ. Press, 1987. 7 T. O'Halloran, M. C. Beckerle, and K. Burridge, Nature (London) 317, 449 (1985). s j. E. B. Fox, J. Biol. Chem. 260, 11977 (1985).
METHODS IN ENZYMOLOGY, VOL. 215
Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
4
ISOLATION OF PLATELET COMPONENTS
[1]
membrane skeleton in maintaining platelet size.9 The coil of microtubules polymerized circumferentially to the inside of the membrane skeleton provides an additional scaffolding that maintains the discoid shape of platelets, l0 The surface membrane area increases by an estimated 60% in activated platelets. 11 Pseudopods facilitate the contact of platelets with a vascular surface stripped of endothelium and with each other to form an aggregate. Whereas the predicted radius of pseudopods (0.1 /~m) has the "right" dimensions for penetrating the electrostatically repulsive forces on the opposite surface, and for the Ca 2+-mediated bonding,~2 The larger protrusions would require the help of complex adhesive molecules. On close inspection of congeries of platelets heaped together, their paleness due to degranulation and their membranous appearance are particularly striking. 1 Nonactivated platelets carry a load of storage granules. Their contents (nucleotides, serotonin, Ca 2+ in dense granules, growth factors, adhesive glycoproteins in a granules, and hydrolytic enzymes in lysosomes) are discharged following fusion of the granule membrane with the platelet membrane. Activated platelets acquire glycoprotein constituents from the membrane of secretory granules. For example, platelet activation-dependent granule to external membrane protein (PAGEM) or granule membrane protein 140 (GMP-140) is detected on activated platelets only) 3,14 Inward systems of membrane invaginations, called the surfaceconnected open canalicular system, provide convoluted channels for transit of secreted constituents. One should be aware that in some species, e.g., oxen, platelets lack the surface-connected open canalicular system involved in membrane-mediated interactions. ~5 A dense tubular system, likened to sarcoplasmic reticulum in muscle cells, is prominently involved in Ca 2÷ storage and mobilization. 16 Wholesale homogenization of platelets by the "brute" force of ultrasound, or by freezing and thawing, will result in a conglomerate of membranes derived from different compartments. Fortunately, gentler techniques such as N 2 cavitation permit isolation of enriched membrane fractions and preserved storage granules for biochemical studies. Such studies are particularly useful in regard to isolation and characterization of membrane receptors and of material stored in granules. Among several 9 j. G. White, Hum. Pathol. 18, 123 (1987). 10 j. G. White, this volume [11]. 11 G. V. R. Born, J. Physiol. (London) 209, 487 (1970). 12 B. A. Pethica, Exp. Cell Res., Suppl. 8, 123 (1961). 13 C. L. Berman, E. L. Yeo, B. C. Furie, and B. Furie, this series, Vol, 169, p. 311. 14 R. P. McEver and M. N. Martin, J. Biol. Chem. 259, 9799 (1984). 15 K. M. Meyers, H. Holmsen, and C. L. Seachord, Am. J. Physiol. 243, R454 (1982). 16 L. Cutler, G. Rodan, and M. B. Feinstein, Biochim. Biophys. Acta 542, 537 (1978).
[2]
P L A T E L E T MEMBRANE ISOLATION
5
classes of membrane receptors reviewed in [12] in this volume, those involved in binding agonists that generate a signal to evoke a response, e.g., secretion, are still not completely understood. Analysis of platelets, nonactivated and activated, is followed by their dissection to obtain a closer view of their component parts: membranes, cytoskeleton, granules. Another procedure involves studying permeabilized platelets allowing insertion of messenger molecules. Thus, despite their minuscule size, platelets are accessible to more than one method of structural analysis to shed light on their omnipotent functionality in hemostatic and thrombotic processes.
[2] I s o l a t i o n a n d C h a r a c t e r i z a t i o n o f P l a t e l e t M e m b r a n e s Prepared by Free Flow Electrophoresis
By
NEVILLE
CRAWFORD,
KALWANT
S. AUTHI,
and NASHRUDEEN HACK The blood platelet, the smallest of the circulating blood cells (-2- to 3-/zm diameter), has always presented difficulties for biochemical studies involving subcellular fractionation. First, although in the circulation it is a relatively quiescent discoid-shaped cell (Fig. la-c), once removed it is readily activated by contact with foreign surfaces or by the generation of excitatory agonists during subsequent handling procedures. Such activation can produce profound morphological changes (from disks to spheres and the formation of filopodia arising from the surface), much reorganization of surface membrane constituents, movement of intracellular granules and their fusion with the surface membrane for release of stored constituents, and, more severely, irreversible aggregation and loss of single cell identity. All these events, which are part of the profile of activation of platelets, present major difficulties for the researcher in the choice of the right anticoagulant and in deciding on the most innocuous isolation and washing procedures, so that changes due to activation are minimized and any analytical or enzymatic data generated can be confidently interpreted in the context of the normal circulating platelet. If this were the extent of the problem it would be formidable, but further difficulties arise in subcellular studies due to the small size of the platelet and its particular resistance to the mechanical shear forces required to rupture the cell, such as one would use in conventional cell homogenization procedures. Moreover, if the physical insult is too severe the integrity of the more sensitive intracellular organelles (lysosomes, mitochondria, and storage granules, etc.) is METHODS IN ENZYMOLOGY, VOL. 215
Copyright © 1992 by Academic Press. Inc. All rights of reproduction in any form reserved.
PLATELET
STRUCTURAL AND FUNCTIONAL
ORGANIZATION
3
[1] I n t r o d u c t i o n to P l a t e l e t S t r u c t u r a l and Functional Organization
By JACEK
HAWIGER
The function of platelets evolves from their structure. When one observes platelets at work sealing the cut in a hemorrhaging vessel, their ability to adhere to nonendothelialized surfaces and to aggregate is such an arresting phenomenon that the impression of single-minded functionality cannot be ignored. 1 Platelets circulate in blood as smooth disks of an average volume of 5 to 7.5/zm 3, 14 times smaller than that of erythrocytes. Each day an estimated 3.5 x 10l° platelets per liter of blood are produced by cytoplasmic fragmentation of their mother cell, a megakaryocyte, through two proposed mechanisms. 2'3 Their expected life span is 10 days. 4 The first encounter of platelets with a damaged vessel wall results in a change of shape from smooth disks into spiny spheres. Pseudopods promote contact with a zone of vascular injury and the subsequent spreading of platelets completes their adhesion. 5 A contractile apparatus harnesses and conveys the energy for this process by rearranging the membrane skeleton and the cytoplasmic actin filaments. In addition to actin and myosin, platelets contain at least nine distinct cytoskeletal proteins involved in assembly, disassembly, and regulation of the contractile apparatus. 6 As in other cells, adhesion is mediated through specific contact sites formed by fibronectin on the outside and extends via integrin receptors to the inside of platelets. Therein, the integrin receptor is clutched by protein P235, analogous to talin. 7 Another adhesive receptor, the GPIb-IX complex, communicating with von Willebrand factor in the extracellular matrix, is linked to short actin filaments that are kept together by an actinbinding protein on the inside of the platelet membrane. 8 An "experiment of nature," in which a deficiency of the GPIb-IX complex in Bernard-Soulier syndrome results in apparently giant platelets, illustrates the role of the J. J. Sixma and J. Wester, Semin Hematol. 14, 265 (1977). 2 M. Shaklai and M. Tavassoli, J. UItrastruct. Res. 62, 270 (1978). 3 j. M. Radley and G. Scurfield, Blood 56, 996 (1980). 4 L. A. Harker and C. A. Finch, J. Clin. Invest. 48, 963 (1969). 5 K. S. Sakariassen, R. Mugli, and H. R. Baumgartner, this series, Vol. 169, p. 37. 6 j. E. B. Fox, in "Thrombosis and Hemostasis" (M. Verstate, J. Vermylen, R. Lijnen, and J. Arnout, eds.), p. 175. Leuven Univ. Press, 1987. 7 T. O'Halloran, M. C. Beckerle, and K. Burridge, Nature (London) 317, 449 (1985). s j. E. B. Fox, J. Biol. Chem. 260, 11977 (1985).
METHODS IN ENZYMOLOGY, VOL. 215
Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
4
ISOLATION OF PLATELET COMPONENTS
[1]
membrane skeleton in maintaining platelet size.9 The coil of microtubules polymerized circumferentially to the inside of the membrane skeleton provides an additional scaffolding that maintains the discoid shape of platelets, l0 The surface membrane area increases by an estimated 60% in activated platelets. 11 Pseudopods facilitate the contact of platelets with a vascular surface stripped of endothelium and with each other to form an aggregate. Whereas the predicted radius of pseudopods (0.1 /~m) has the "right" dimensions for penetrating the electrostatically repulsive forces on the opposite surface, and for the Ca 2+-mediated bonding,~2 The larger protrusions would require the help of complex adhesive molecules. On close inspection of congeries of platelets heaped together, their paleness due to degranulation and their membranous appearance are particularly striking. 1 Nonactivated platelets carry a load of storage granules. Their contents (nucleotides, serotonin, Ca 2+ in dense granules, growth factors, adhesive glycoproteins in a granules, and hydrolytic enzymes in lysosomes) are discharged following fusion of the granule membrane with the platelet membrane. Activated platelets acquire glycoprotein constituents from the membrane of secretory granules. For example, platelet activation-dependent granule to external membrane protein (PAGEM) or granule membrane protein 140 (GMP-140) is detected on activated platelets only) 3,14 Inward systems of membrane invaginations, called the surfaceconnected open canalicular system, provide convoluted channels for transit of secreted constituents. One should be aware that in some species, e.g., oxen, platelets lack the surface-connected open canalicular system involved in membrane-mediated interactions. ~5 A dense tubular system, likened to sarcoplasmic reticulum in muscle cells, is prominently involved in Ca 2÷ storage and mobilization. 16 Wholesale homogenization of platelets by the "brute" force of ultrasound, or by freezing and thawing, will result in a conglomerate of membranes derived from different compartments. Fortunately, gentler techniques such as N 2 cavitation permit isolation of enriched membrane fractions and preserved storage granules for biochemical studies. Such studies are particularly useful in regard to isolation and characterization of membrane receptors and of material stored in granules. Among several 9 j. G. White, Hum. Pathol. 18, 123 (1987). 10 j. G. White, this volume [11]. 11 G. V. R. Born, J. Physiol. (London) 209, 487 (1970). 12 B. A. Pethica, Exp. Cell Res., Suppl. 8, 123 (1961). 13 C. L. Berman, E. L. Yeo, B. C. Furie, and B. Furie, this series, Vol, 169, p. 311. 14 R. P. McEver and M. N. Martin, J. Biol. Chem. 259, 9799 (1984). 15 K. M. Meyers, H. Holmsen, and C. L. Seachord, Am. J. Physiol. 243, R454 (1982). 16 L. Cutler, G. Rodan, and M. B. Feinstein, Biochim. Biophys. Acta 542, 537 (1978).
[2]
P L A T E L E T MEMBRANE ISOLATION
5
classes of membrane receptors reviewed in [12] in this volume, those involved in binding agonists that generate a signal to evoke a response, e.g., secretion, are still not completely understood. Analysis of platelets, nonactivated and activated, is followed by their dissection to obtain a closer view of their component parts: membranes, cytoskeleton, granules. Another procedure involves studying permeabilized platelets allowing insertion of messenger molecules. Thus, despite their minuscule size, platelets are accessible to more than one method of structural analysis to shed light on their omnipotent functionality in hemostatic and thrombotic processes.
[2] I s o l a t i o n a n d C h a r a c t e r i z a t i o n o f P l a t e l e t M e m b r a n e s Prepared by Free Flow Electrophoresis
By
NEVILLE
CRAWFORD,
KALWANT
S. AUTHI,
and NASHRUDEEN HACK The blood platelet, the smallest of the circulating blood cells (-2- to 3-/zm diameter), has always presented difficulties for biochemical studies involving subcellular fractionation. First, although in the circulation it is a relatively quiescent discoid-shaped cell (Fig. la-c), once removed it is readily activated by contact with foreign surfaces or by the generation of excitatory agonists during subsequent handling procedures. Such activation can produce profound morphological changes (from disks to spheres and the formation of filopodia arising from the surface), much reorganization of surface membrane constituents, movement of intracellular granules and their fusion with the surface membrane for release of stored constituents, and, more severely, irreversible aggregation and loss of single cell identity. All these events, which are part of the profile of activation of platelets, present major difficulties for the researcher in the choice of the right anticoagulant and in deciding on the most innocuous isolation and washing procedures, so that changes due to activation are minimized and any analytical or enzymatic data generated can be confidently interpreted in the context of the normal circulating platelet. If this were the extent of the problem it would be formidable, but further difficulties arise in subcellular studies due to the small size of the platelet and its particular resistance to the mechanical shear forces required to rupture the cell, such as one would use in conventional cell homogenization procedures. Moreover, if the physical insult is too severe the integrity of the more sensitive intracellular organelles (lysosomes, mitochondria, and storage granules, etc.) is METHODS IN ENZYMOLOGY, VOL. 215
Copyright © 1992 by Academic Press. Inc. All rights of reproduction in any form reserved.
