THE YALE JOURNAL OF BIOLOGY AND MEDICINE 63 (1990), 469-475
Platelet Membrane Glycoproteins: Role in Primary Hemostasis and Component Antigens DIANA S. BEARDSLEY, M.D., Ph.D. Division of Pediatric Hematology, Yale University School of Medicine, New Haven, Connecticut Received May 2, 1990
The biochemical details of the platelet surface as they relate to normal platelet function have been elucidated through study of labeled membranes from both normal platelets and those with congenitally defective function. Several cytoadhesive glycoprotein complexes which are integral components of the platelet membrane have been demonstrated to act as important receptors for extracellular matrix macromolecules. Glycoproteins Ia/Ila (collagen receptor), Ic/IIa (fibronectin receptor), and Ilb/IlIa (fibrinogen receptor) belong to a family of cytoadhesive complexes called the integrins, while glycoprotein Ib/IX, the major von Willebrand receptor, has different features. These same major glycoproteins comprise all of the alloantigens and most of the autoantigens that have been characterized. Glycoprotein Ilb/IIIa contains the alloantigens, pIA (Zw), Bak (Lek), and Pen (Yuk), as well as the most frequent target antigenic sites for anti-platelet autoantibodies. Because a number of platelet alloantigens were discovered independently by more than one group, nomenclature is confusing at present, although a system analogous to that used for histocompatibility antigens has been proposed. Precise identification of the antigenic epitopes has not yet been accomplished for all of the platelet antigens. Current research efforts include characterization of antigenic epitopes, elucidation of mechanisms by which platelet immunization occurs, and determination of the clinical implications of the presence of various platelet antibodies.
BACKGROUND Platelets are the key blood cells which participate in primary hemostasis. Under baseline conditions, they glide smoothly over the intact vascular interior, yet, upon interruption of the vascular endothelium, they interact promptly to form an intertwined platelet plug. Abnormalities of this primary hemostatic system can lead to a serious hemorrhagic state or to excessive thrombosis and vascular insufficiency. Advances in understanding platelet function and thrombopathic syndromes have resulted from studies of the major component glycoproteins of the platelet plasma membrane. This report will summarize our current view of platelet glycoproteins and their role in clinically important processes. The early investigations in this area were aimed at identifying specific component proteins after proteolytic digestion of intact platelets [1,2] or gel electrophoresis and staining of the carbohydrate-containing components [3,4]. The three groups of glycoproteins first noted were termed glycoproteins (GPs) I, II, and III. Approaches which have contributed to our current understanding of platelet membrane proteins have 469 Abbreviations: GP: glycoprotein ITP: idiopathic thrombocytopenic purpura Aided by a Clinical Grant from the March of Dimes Birth Defects Foundation and an Anna and Argall Hull Award from the Yale Comprehensive Cancer Center Address reprint requests to Diana S. Beardsley, M.D., Ph.D., Division of Pediatric Hematology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06510 Copyright © 1990 by The Yale Journal of Biology and Medicine, Inc. All rights of reproduction in any form reserved.
470
_R0.gt~ ~nal.
DIANA S. BEARDSLEY
Nonreduced
Reduced
I~~i
kD
-
FIG. 1. Autoradiogram of surface radioiodinated platelet membranes separated by two-dimensional SDS polyacrylamide gel electrophoresis. Glycoprotein IlIa appears above the diagonal since its apparent molecular weight increases after disulfide reduction. Glycoproteins such as lb lose a
(GPIb,) 1|11 1111 _ Idisulfide-linked fragment tion; therefore, GPIba appears belowupon the reducdiago-
~;IE
included radiolabeling of intact platelets, followed by gel electrophoresis to separate the labeled components, characterization of defective platelet membranes from patients with congenital thrombopathies, and creation of monoclonal antibodies directed against platelet surface components. Figure 1 is an autoradiogram of radioiodinated platelet membranes which have been separated by two-dimensional SDS gel electrophoresis. Separation in the first dimension was performed with disulfide bonds intact. After reduction of these linkages, separation in the second dimension resulted in improved resolution of nearby bands. The major components of the platelet membrane are listed in Table 1 and shown in diagrammatic form in Fig. 2. Considerable refinement of the details of platelet surface structure has been accomplished over the past 20 years, and the system of nomenclature has been expanded from the original GPI, GPII, and GPIII to include the glycoproteins listed in Table 1. For reviews in more depth, see [6-9]. This summary will focus primarily on the state of our current understanding of the platelet membrane, particularly in relation to normal platelet function, congenital defects of platelets, and the role of antibodies against platelet surface proteins in disease and as they are important to transfusion medicine. TABLE 1 Platelet Membrane Glycoproteins
MW (kDa)
Complexes Existing in
Nonreduced
After Disulfide Reduction
GPIa GPIb GPIc GPIIa
155 170 150 130
170 170 150 145
GPIIb GPIIIa GPIV (or GPIIIb) GPV GPIX
145 95 95
145 115 95
GPIa/IIa GPIb/IX GPIc/IIa GPIa/IIa; GPIc/IIa GPIIb/IIIa GPIIb/IIIa
80 20
80 20
Name
Membrane
Function
Collagen receptor von Willebrand factor receptor
Fibronectin receptor Collagen receptor; Fibronectin receptor
Fibrinogen receptor Fibrinogen receptor Thrombospondin receptor Cleaved by thrombin
GPIb/IX
von Willebrand factor receptor
PLATELET MEMBRANE GLYCOPROTEINS
Ilia
471
Iba
llbazp~ Ba
FIG. 2. A. Glycoprotein IIb/IIIa. The approximate locations of epitopes defining the alloantigen systems, plA, Pen, and Bak, are indicated, as are sites of cleavage by trypsin (T) and chymotrypsin (Cl). Reprinted with permission from [5]. B. Glycoproteins Ib-IX and V. The approximate locations of epitopes defining the pIE and pIT alloantigen systems are indicated, along with sites of cleavage by serratia protease (SP), trypsin (T), and platelet calpain (Cip). Reprinted with permission from [5].
THE PLATELET MEMBRANE GLYCOPROTEINS: CHARACTERISTICS AND DEFICIENCIES Platelet Adhesion, Activation, and Aggregation The main role of platelets in the hemostatic system is to provide the initial protection from hemorrhage upon interruption of vascular integrity. Thus, whenever the continuous interior layer of vascular endothelium is interrupted, platelets become attached to the exposed subendothelium. The linkage site on the platelet is GPIb, a 170 kDa platelet protein with a large amount of sialic acid. A plasma protein, von Willebrand's factor, acts as a bridge between platelet GPIb and the subendothelial matrix. This initial phase of primary hemostasis is specifically called "adhesion." After platelet adhesion occurs, the platelets contract their shape, extending pseudopods of plasma membrane, and become activated. Platelet "activation" refers to the transferral of the surface signal to generation of arachadonic acid within the platelet, production of prostaglandins, and the release of granule contents (including ADP, Ca++, and serotonin) into the exterior milieu. These substances act as agonists to draw more platelets into the area and facilitate the platelet-to-platelet interactions called platelet "aggregation." The two glycoproteins, GPIIb and GPIIIa, exist as a complex within the platelet membrane. In the presence of calcium, the GPIIb/IIIa complex undergoes a conformational change to become a receptor for fibrinogen. The biochemical bridging interaction of platelet-fibrinogen-platelet via the GPIIb/IIIa receptors is the essential step in platelet aggregation. Bernard-Soulier syndrome is a rare autosomal recessive defect in platelets, leading to recurrent hemorrhage from mucous membranes and at sites of trauma. The associated moderate to severe thrombocytopenia can suggest a diagnosis of idiopathic thrombocytopenic purpura (ITP), unless the peripheral blood smear is examined, for the platelets in Bernard-Soulier syndrome are giant platelets with a diameter similar to that of a small lymphocyte. These platelets lack the ability to adhere to exposed endothelium [10], and are deficient in the platelet receptor for adhesion, GPIb [1 1].
472
DIANA S. BEARDSLEY
GPIb exists in the normal platelet membrane as a heterodimeric complex with GPIX; this glycoprotein is also absent from Bernard-Soulier platelets, as is GPV. A diagnosis of Bernard-Soulier syndrome is supported by the failure of these platelets to undergo ristocetin-induced agglutination. Confirmation should be made by failure to interact with monoclonal antibodies specific for GPIb or GPIX. Glanzmann's thrombasthenia is another congenital abnormality of platelet function which has an autosomal recessive pattern of inheritance. Clinical manifestations are those of recurrent, often severe, hemorrhage. Platelet count and appearance are normal, but the bleeding time is prolonged and clot retraction is absent. These platelets fail to aggregate in response to an agonist and are lacking in the GPIIb/IIIa complex, the fibrinogen receptor involved in normal platelet aggregation.
The Integrin Family The GPIIb/IIIa complex serves as a model member of an important group of cytoadhesive proteins, the integrin family. These receptor protein complexes occur on a wide variety of cell types, including endothelial cells, fibroblasts, lymphocytes, and a number of cultured cell lines. The proteins in this family are composed of two (alpha and beta) subunits. The number of glycoprotein complexes belonging to the integrin family continues to expand as the cytoadhesive properties of other cells are characterized. In addition to GPIIb/IIIa, two other platelet glycoprotein complexes belong to the integrin family. Glycoprotein Ia/IIa forms a receptor for collagen. The GPIa portion has been shown to be absent from a patient with defective platelet aggregation in the presence of collagen [12]; this patient had a normal bleeding time and no history of excessive bleeding. The GPIc/IIa complex forms a receptor for fibronectin.
Other Glycoproteins Other proteins of the platelet membrane to which a role in platelet function have been ascribed include GPIV, a receptor for thrombospondin [13], and a 40 kDa protein which acts as a platelet Fc receptor [ 14]. The platelet receptors for complement, ADP, and epinephrine have not yet been identified. Thrombin activation of platelets cleaves GPV, but there may be a different receptor for thrombin mediated platelet aggregation. The glycoprotein, GMP 140 [15], also called PADGEM protein [ 16], can serve as a marker for platelet activation. Present in the alpha granules of resting platelets, this protein is found on the platelet surface only after activation, when granule membranes fuse with the plasma membrane. Initially identified on platelets which were activated in vitro, quantitation of GMP 140 on freshly drawn platelets is now being studied as a possible marker for vascular disease. PLATELET GLYCOPROTEIN TARGETS OF ANTI-PLATELET ANTIBODIES
Introduction and Terminology Antibodies against platelet surface components are important in the etiology of thrombocytopenias, in contributing to refractoriness to platelet transfusion, and as tools in basic platelet research. Xenoantigens, those identified by antibodies raised in an animal, include a large number of murine monoclonal antibodies whose specificities
473
PLATELET MEMBRANE GLYCOPROTEINS
TABLE 2 Platelet Glycoprotein-Associated Alloantigens Name
PlA, Zw Ko, Sib Bak, Lek Pen, Yuk Br, Zav, Hc
PIE PIT
Glycoprotein
Proposed HPA System
GPIIIa GPIb GPIIb GPIIIa GPIa GPIb GPV
(HPA-1) (HPA-2) (HPA-3) (HPA-4) (HPA-5) ? ?
have been well defined, as well as rabbit polyclonal antisera which have been valuable as precipitins for crossed immunoelectrophoresis. Isoantibodies are formed by an individual whose platelets are congenitally deficient in the target protein upon exposure to normal platelets. For example, a patient with Bernard-Soulier syndrome may form anti-GPIb/IX isoantibodies after transfusion with normal platelets. Alloantibodies, in contrast, are those which identify different allelic epitopes present on normal platelets. plAl and plA2 are allelic forms of GPIIIa. If an individual with pjA2 (i.e., PlAl-negative) platelets is exposed to platelets that are PlAl-positive, anti-PlAl alloantibodies may form. The antibodies which are involved in the etiology of ITP are autoantibodies. They are directed against the individual's own platelets; their antigenic targets are autoantigens. Alloantigens and autoantigens are those most involved in clinically important thrombocytopenias; they will be summarized here. Xenoantigens and isoantigens will not be discussed further. Alloantigens The clinically important alloimmune thrombocytopenias include neonatal alloimmune thrombocytopenia and post-transfusion purpura. The clinical features and immunology of both have been reviewed recently [17] and will not be included here. Antibodies against the plA1 antigen have been implicated most frequently in both of these syndromes; however, the other alloantigens listed in Table 2 may be involved in at least an equal number of cases. Note that the reported terminology is very cumbersome. With the exception of plA, plE, and pIT, these antigens have been named after the last name of the patient in whom the incompatibility was first observed. Not surprisingly, separate reports of antigens which were later demonstrated to be identical have led to synonymous terminology for plA (Zw), Bak (Lek), Pen (Yuk), and Br (Zav). At a recent international platelet immunology symposium [ 18], a simplified nomenclature system was proposed, based upon the HLA terminology. These tentative names are indicated in Table 2 in parentheses. Autoantigens The targets of antibodies identified from patients with autoimmune thrombocytopenia are autoantigens. These antigens have been found to be "public" antigens, present upon normal platelets from many individuals. The GPIIb/IIIa complex [19], particularly GPIIIa [20], has been the most frequently observed target protein for the autoantibodies; however, GPIb, GPV, GPIIb, and other platelet glycoproteins have also been reported (for a review, see [21]). Studies are currently under way to
474
DIANA S. BEARDSLEY
determine whether antibodies against a particular platelet glycoprotein will be correlated with a specific clinical course.
CLINICAL IMPLICATIONS OF OUR UNDERSTANDING OF THE PLATELET GLYCOPROTEINS Our understanding of the platelet glycoproteins has grown rapidly since the early 1970s, when GPI, GPII, and GPIII were first identified by the new technique of gel electrophoresis. Knowledge of the molecular defects which lead to Bernard-Soulier syndrome and Glanzmann's thrombasthenia open the possibility of anti-thrombotic therapies designed to produce a very mild version of one of these hemorrhagic states. The fact that GPIb/IX and GPIIb/IIIa are so important for normal platelet function suggests that the preparation and storage processes involved in providing platelet products for transfusion should preserve the presence of these glycoproteins in order to assure functional platelets for the transfusion recipient. Characterization of the platelet alloantigens makes possible typing of platelets and other blood products for those patients who are demonstrated to suffer from one of the syndromes of alloimmunization. It is also theoretically possible to create a panel of typed platelets for use in platelet cross-matching; however, the importance of these platelet glycoprotein-associated alloantigens in transfusion refractoriness has not yet been established. Anti-HLA antibodies are certainly a more important cause of transfusion resistance. The complete biochemical composition of the platelet membrane has not yet been determined. As our understanding increases, we will certainly find new possibilities for maintaining a normal hemostatic state in spite of the many perturbations which can arise in diseases involving blood platelets. REFERENCES 1. Pepper DS, Jamieson GA: Studies on glycoproteins. III. Isolation of sialylglycopeptides from human platelet membranes. Biochemistry 8:3362-3369, 1969 2. Pepper DS, Jamieson GA: Isolation of a macroglycopeptide from human platelets. Biochemistry 9:3706-3713, 1970 3. Phillips DR: Effect of trypsin on the exposed polypeptides and glycoproteins in the human platelet membrane. Biochemistry 11:4582-4588, 1972 4. Nachman RL, Ferris B: Studies on the proteins of human platelet membranes. J Biol Chem 247:44684475, 1972 5. Beardsley DS: Platelet autoantigens. In Platelet Immunobiology. Edited by TJ Kunicki, J George. Philadelphia, JB Lippincott Co, 1989, pp 111-112 6. George JW, Nurden AT, Phillips DR: Molecular defects in interactions of platelets with the vessel wall. N Engl J Med 311:1084-1098, 1984 7. George JN, Nurden AT, Phillips DR: Platelet Membrane Glycoproteins. New York, London, Plenum Press, 1985 8. Nurden AT: Platelet glycoproteins: Clinical relevance. In Thrombosis and Haemostasis 1987. Edited by M Verstraete, J Vermylen, R Lijnen, J Arnout. Leuven, Belgium, Leuven University Press, 1987, pp 93-125 9. Fitzgerald LA, Phillips DR: Structure and function of platelet membrane glycoproteins. In Platelet Immunobiology. Edited by TJ Kunicki, J George. Philadelphia, JB Lippincott Co, 1989, pp 9-30 10. Weiss HJ, Tschopp TB, Baumgartner HR, Sussman II, Johnson MM, Egan JJ: Decreased adhesion of giant (Bernard-Soulier) platelets to subendothelium-further implications on the role of von Willebrand factor in hemostasis. Am J Med 57:920-925,1974 11. Nurden AT, Caen JP: Specific roles for platelet surface glycoproteins in platelet function. Nature 255:720-722, 1975
PLATELET MEMBRANE GLYCOPROTEINS
475
12. Nieuwenhuis HK, Akkerman JWN, Houdyk WPM, Sixma JJ: Human blood platelets showing no response to collagen fail to express surface glycoprotein Ia. Nature 318:470-472, 1985 13. Asch AS, Barnwell J, Silverstein RL, Nachman RL: Isolation of the thrombospondin membrane receptor. J Clin Invest 79:1054, 1987 14. Rosenfeld SI, Looney RJ, Leddy JP, Phipps, DC, Abraham GN, Anderson CL: Human platelet Fc receptor for IgG: Identification as a 40 RD membrane protein shared by monocytes. J Clin Invest 76:2317, 1985 15. McEver RP, Martin MN: A monoclonal antibody to a membrane glycoprotein binds only to activated platelets. J Biol Chem 259:9799, 1984 16. Hsu-Lin SC, Berman CL, Furie BC, August D, Furie B: A platelet membrane protein expressed during platelet activation and secretion. Studies using a monoclonal antibody specific for thrombin-activated platelets. J Biol Chem 259:9121, 1984 17. Kunicki TJ, Beardsley DS: The alloimmune thrombocytopenias: Neonatal alloimmune thrombocytopenia and post transfusion purpura: Prog Hemost Thromb 9:203-232, 1989 18. von dem Borne AEGKr, Beardsley DS, Mueller-Eckhardt C: Unpublished communication from International Conference on Immune Thrombocytopenia, Lucerne, Switzerland, August 1988 19. Woods VL Jr, Oh EH, Mason D, McMillan R: Autoantibodies against the platelet glycoprotein Ilb/lIla complex in patients with chronic idiopathic thrombocytopenid purpura. Blood 63:368, 1984 20. Beardsley DJS, Spiegel JE, Jacobs MM, Handin RI, Lun SE: Platelet membrane glycoprotein Ila contains target antigens that bind anti-platelet antibodies in immune thrombocytopenias. J Clin Invest 74:1701, 1984 21. Beardsley DS: Platelet autoantigens. In Platelet Immunobiology. Edited by TJ Kunicki, J George. Philadelphia, JB Lippincott Co, 1989, pp 121-131
Platelet Membrane Glycoproteins: Role in Primary Hemostasis and Component Antigens DIANA S. BEARDSLEY, M.D., Ph.D. Division of Pediatric Hematology, Yale University School of Medicine, New Haven, Connecticut Received May 2, 1990
The biochemical details of the platelet surface as they relate to normal platelet function have been elucidated through study of labeled membranes from both normal platelets and those with congenitally defective function. Several cytoadhesive glycoprotein complexes which are integral components of the platelet membrane have been demonstrated to act as important receptors for extracellular matrix macromolecules. Glycoproteins Ia/Ila (collagen receptor), Ic/IIa (fibronectin receptor), and Ilb/IlIa (fibrinogen receptor) belong to a family of cytoadhesive complexes called the integrins, while glycoprotein Ib/IX, the major von Willebrand receptor, has different features. These same major glycoproteins comprise all of the alloantigens and most of the autoantigens that have been characterized. Glycoprotein Ilb/IIIa contains the alloantigens, pIA (Zw), Bak (Lek), and Pen (Yuk), as well as the most frequent target antigenic sites for anti-platelet autoantibodies. Because a number of platelet alloantigens were discovered independently by more than one group, nomenclature is confusing at present, although a system analogous to that used for histocompatibility antigens has been proposed. Precise identification of the antigenic epitopes has not yet been accomplished for all of the platelet antigens. Current research efforts include characterization of antigenic epitopes, elucidation of mechanisms by which platelet immunization occurs, and determination of the clinical implications of the presence of various platelet antibodies.
BACKGROUND Platelets are the key blood cells which participate in primary hemostasis. Under baseline conditions, they glide smoothly over the intact vascular interior, yet, upon interruption of the vascular endothelium, they interact promptly to form an intertwined platelet plug. Abnormalities of this primary hemostatic system can lead to a serious hemorrhagic state or to excessive thrombosis and vascular insufficiency. Advances in understanding platelet function and thrombopathic syndromes have resulted from studies of the major component glycoproteins of the platelet plasma membrane. This report will summarize our current view of platelet glycoproteins and their role in clinically important processes. The early investigations in this area were aimed at identifying specific component proteins after proteolytic digestion of intact platelets [1,2] or gel electrophoresis and staining of the carbohydrate-containing components [3,4]. The three groups of glycoproteins first noted were termed glycoproteins (GPs) I, II, and III. Approaches which have contributed to our current understanding of platelet membrane proteins have 469 Abbreviations: GP: glycoprotein ITP: idiopathic thrombocytopenic purpura Aided by a Clinical Grant from the March of Dimes Birth Defects Foundation and an Anna and Argall Hull Award from the Yale Comprehensive Cancer Center Address reprint requests to Diana S. Beardsley, M.D., Ph.D., Division of Pediatric Hematology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06510 Copyright © 1990 by The Yale Journal of Biology and Medicine, Inc. All rights of reproduction in any form reserved.
470
_R0.gt~ ~nal.
DIANA S. BEARDSLEY
Nonreduced
Reduced
I~~i
kD
-
FIG. 1. Autoradiogram of surface radioiodinated platelet membranes separated by two-dimensional SDS polyacrylamide gel electrophoresis. Glycoprotein IlIa appears above the diagonal since its apparent molecular weight increases after disulfide reduction. Glycoproteins such as lb lose a
(GPIb,) 1|11 1111 _ Idisulfide-linked fragment tion; therefore, GPIba appears belowupon the reducdiago-
~;IE
included radiolabeling of intact platelets, followed by gel electrophoresis to separate the labeled components, characterization of defective platelet membranes from patients with congenital thrombopathies, and creation of monoclonal antibodies directed against platelet surface components. Figure 1 is an autoradiogram of radioiodinated platelet membranes which have been separated by two-dimensional SDS gel electrophoresis. Separation in the first dimension was performed with disulfide bonds intact. After reduction of these linkages, separation in the second dimension resulted in improved resolution of nearby bands. The major components of the platelet membrane are listed in Table 1 and shown in diagrammatic form in Fig. 2. Considerable refinement of the details of platelet surface structure has been accomplished over the past 20 years, and the system of nomenclature has been expanded from the original GPI, GPII, and GPIII to include the glycoproteins listed in Table 1. For reviews in more depth, see [6-9]. This summary will focus primarily on the state of our current understanding of the platelet membrane, particularly in relation to normal platelet function, congenital defects of platelets, and the role of antibodies against platelet surface proteins in disease and as they are important to transfusion medicine. TABLE 1 Platelet Membrane Glycoproteins
MW (kDa)
Complexes Existing in
Nonreduced
After Disulfide Reduction
GPIa GPIb GPIc GPIIa
155 170 150 130
170 170 150 145
GPIIb GPIIIa GPIV (or GPIIIb) GPV GPIX
145 95 95
145 115 95
GPIa/IIa GPIb/IX GPIc/IIa GPIa/IIa; GPIc/IIa GPIIb/IIIa GPIIb/IIIa
80 20
80 20
Name
Membrane
Function
Collagen receptor von Willebrand factor receptor
Fibronectin receptor Collagen receptor; Fibronectin receptor
Fibrinogen receptor Fibrinogen receptor Thrombospondin receptor Cleaved by thrombin
GPIb/IX
von Willebrand factor receptor
PLATELET MEMBRANE GLYCOPROTEINS
Ilia
471
Iba
llbazp~ Ba
FIG. 2. A. Glycoprotein IIb/IIIa. The approximate locations of epitopes defining the alloantigen systems, plA, Pen, and Bak, are indicated, as are sites of cleavage by trypsin (T) and chymotrypsin (Cl). Reprinted with permission from [5]. B. Glycoproteins Ib-IX and V. The approximate locations of epitopes defining the pIE and pIT alloantigen systems are indicated, along with sites of cleavage by serratia protease (SP), trypsin (T), and platelet calpain (Cip). Reprinted with permission from [5].
THE PLATELET MEMBRANE GLYCOPROTEINS: CHARACTERISTICS AND DEFICIENCIES Platelet Adhesion, Activation, and Aggregation The main role of platelets in the hemostatic system is to provide the initial protection from hemorrhage upon interruption of vascular integrity. Thus, whenever the continuous interior layer of vascular endothelium is interrupted, platelets become attached to the exposed subendothelium. The linkage site on the platelet is GPIb, a 170 kDa platelet protein with a large amount of sialic acid. A plasma protein, von Willebrand's factor, acts as a bridge between platelet GPIb and the subendothelial matrix. This initial phase of primary hemostasis is specifically called "adhesion." After platelet adhesion occurs, the platelets contract their shape, extending pseudopods of plasma membrane, and become activated. Platelet "activation" refers to the transferral of the surface signal to generation of arachadonic acid within the platelet, production of prostaglandins, and the release of granule contents (including ADP, Ca++, and serotonin) into the exterior milieu. These substances act as agonists to draw more platelets into the area and facilitate the platelet-to-platelet interactions called platelet "aggregation." The two glycoproteins, GPIIb and GPIIIa, exist as a complex within the platelet membrane. In the presence of calcium, the GPIIb/IIIa complex undergoes a conformational change to become a receptor for fibrinogen. The biochemical bridging interaction of platelet-fibrinogen-platelet via the GPIIb/IIIa receptors is the essential step in platelet aggregation. Bernard-Soulier syndrome is a rare autosomal recessive defect in platelets, leading to recurrent hemorrhage from mucous membranes and at sites of trauma. The associated moderate to severe thrombocytopenia can suggest a diagnosis of idiopathic thrombocytopenic purpura (ITP), unless the peripheral blood smear is examined, for the platelets in Bernard-Soulier syndrome are giant platelets with a diameter similar to that of a small lymphocyte. These platelets lack the ability to adhere to exposed endothelium [10], and are deficient in the platelet receptor for adhesion, GPIb [1 1].
472
DIANA S. BEARDSLEY
GPIb exists in the normal platelet membrane as a heterodimeric complex with GPIX; this glycoprotein is also absent from Bernard-Soulier platelets, as is GPV. A diagnosis of Bernard-Soulier syndrome is supported by the failure of these platelets to undergo ristocetin-induced agglutination. Confirmation should be made by failure to interact with monoclonal antibodies specific for GPIb or GPIX. Glanzmann's thrombasthenia is another congenital abnormality of platelet function which has an autosomal recessive pattern of inheritance. Clinical manifestations are those of recurrent, often severe, hemorrhage. Platelet count and appearance are normal, but the bleeding time is prolonged and clot retraction is absent. These platelets fail to aggregate in response to an agonist and are lacking in the GPIIb/IIIa complex, the fibrinogen receptor involved in normal platelet aggregation.
The Integrin Family The GPIIb/IIIa complex serves as a model member of an important group of cytoadhesive proteins, the integrin family. These receptor protein complexes occur on a wide variety of cell types, including endothelial cells, fibroblasts, lymphocytes, and a number of cultured cell lines. The proteins in this family are composed of two (alpha and beta) subunits. The number of glycoprotein complexes belonging to the integrin family continues to expand as the cytoadhesive properties of other cells are characterized. In addition to GPIIb/IIIa, two other platelet glycoprotein complexes belong to the integrin family. Glycoprotein Ia/IIa forms a receptor for collagen. The GPIa portion has been shown to be absent from a patient with defective platelet aggregation in the presence of collagen [12]; this patient had a normal bleeding time and no history of excessive bleeding. The GPIc/IIa complex forms a receptor for fibronectin.
Other Glycoproteins Other proteins of the platelet membrane to which a role in platelet function have been ascribed include GPIV, a receptor for thrombospondin [13], and a 40 kDa protein which acts as a platelet Fc receptor [ 14]. The platelet receptors for complement, ADP, and epinephrine have not yet been identified. Thrombin activation of platelets cleaves GPV, but there may be a different receptor for thrombin mediated platelet aggregation. The glycoprotein, GMP 140 [15], also called PADGEM protein [ 16], can serve as a marker for platelet activation. Present in the alpha granules of resting platelets, this protein is found on the platelet surface only after activation, when granule membranes fuse with the plasma membrane. Initially identified on platelets which were activated in vitro, quantitation of GMP 140 on freshly drawn platelets is now being studied as a possible marker for vascular disease. PLATELET GLYCOPROTEIN TARGETS OF ANTI-PLATELET ANTIBODIES
Introduction and Terminology Antibodies against platelet surface components are important in the etiology of thrombocytopenias, in contributing to refractoriness to platelet transfusion, and as tools in basic platelet research. Xenoantigens, those identified by antibodies raised in an animal, include a large number of murine monoclonal antibodies whose specificities
473
PLATELET MEMBRANE GLYCOPROTEINS
TABLE 2 Platelet Glycoprotein-Associated Alloantigens Name
PlA, Zw Ko, Sib Bak, Lek Pen, Yuk Br, Zav, Hc
PIE PIT
Glycoprotein
Proposed HPA System
GPIIIa GPIb GPIIb GPIIIa GPIa GPIb GPV
(HPA-1) (HPA-2) (HPA-3) (HPA-4) (HPA-5) ? ?
have been well defined, as well as rabbit polyclonal antisera which have been valuable as precipitins for crossed immunoelectrophoresis. Isoantibodies are formed by an individual whose platelets are congenitally deficient in the target protein upon exposure to normal platelets. For example, a patient with Bernard-Soulier syndrome may form anti-GPIb/IX isoantibodies after transfusion with normal platelets. Alloantibodies, in contrast, are those which identify different allelic epitopes present on normal platelets. plAl and plA2 are allelic forms of GPIIIa. If an individual with pjA2 (i.e., PlAl-negative) platelets is exposed to platelets that are PlAl-positive, anti-PlAl alloantibodies may form. The antibodies which are involved in the etiology of ITP are autoantibodies. They are directed against the individual's own platelets; their antigenic targets are autoantigens. Alloantigens and autoantigens are those most involved in clinically important thrombocytopenias; they will be summarized here. Xenoantigens and isoantigens will not be discussed further. Alloantigens The clinically important alloimmune thrombocytopenias include neonatal alloimmune thrombocytopenia and post-transfusion purpura. The clinical features and immunology of both have been reviewed recently [17] and will not be included here. Antibodies against the plA1 antigen have been implicated most frequently in both of these syndromes; however, the other alloantigens listed in Table 2 may be involved in at least an equal number of cases. Note that the reported terminology is very cumbersome. With the exception of plA, plE, and pIT, these antigens have been named after the last name of the patient in whom the incompatibility was first observed. Not surprisingly, separate reports of antigens which were later demonstrated to be identical have led to synonymous terminology for plA (Zw), Bak (Lek), Pen (Yuk), and Br (Zav). At a recent international platelet immunology symposium [ 18], a simplified nomenclature system was proposed, based upon the HLA terminology. These tentative names are indicated in Table 2 in parentheses. Autoantigens The targets of antibodies identified from patients with autoimmune thrombocytopenia are autoantigens. These antigens have been found to be "public" antigens, present upon normal platelets from many individuals. The GPIIb/IIIa complex [19], particularly GPIIIa [20], has been the most frequently observed target protein for the autoantibodies; however, GPIb, GPV, GPIIb, and other platelet glycoproteins have also been reported (for a review, see [21]). Studies are currently under way to
474
DIANA S. BEARDSLEY
determine whether antibodies against a particular platelet glycoprotein will be correlated with a specific clinical course.
CLINICAL IMPLICATIONS OF OUR UNDERSTANDING OF THE PLATELET GLYCOPROTEINS Our understanding of the platelet glycoproteins has grown rapidly since the early 1970s, when GPI, GPII, and GPIII were first identified by the new technique of gel electrophoresis. Knowledge of the molecular defects which lead to Bernard-Soulier syndrome and Glanzmann's thrombasthenia open the possibility of anti-thrombotic therapies designed to produce a very mild version of one of these hemorrhagic states. The fact that GPIb/IX and GPIIb/IIIa are so important for normal platelet function suggests that the preparation and storage processes involved in providing platelet products for transfusion should preserve the presence of these glycoproteins in order to assure functional platelets for the transfusion recipient. Characterization of the platelet alloantigens makes possible typing of platelets and other blood products for those patients who are demonstrated to suffer from one of the syndromes of alloimmunization. It is also theoretically possible to create a panel of typed platelets for use in platelet cross-matching; however, the importance of these platelet glycoprotein-associated alloantigens in transfusion refractoriness has not yet been established. Anti-HLA antibodies are certainly a more important cause of transfusion resistance. The complete biochemical composition of the platelet membrane has not yet been determined. As our understanding increases, we will certainly find new possibilities for maintaining a normal hemostatic state in spite of the many perturbations which can arise in diseases involving blood platelets. REFERENCES 1. Pepper DS, Jamieson GA: Studies on glycoproteins. III. Isolation of sialylglycopeptides from human platelet membranes. Biochemistry 8:3362-3369, 1969 2. Pepper DS, Jamieson GA: Isolation of a macroglycopeptide from human platelets. Biochemistry 9:3706-3713, 1970 3. Phillips DR: Effect of trypsin on the exposed polypeptides and glycoproteins in the human platelet membrane. Biochemistry 11:4582-4588, 1972 4. Nachman RL, Ferris B: Studies on the proteins of human platelet membranes. J Biol Chem 247:44684475, 1972 5. Beardsley DS: Platelet autoantigens. In Platelet Immunobiology. Edited by TJ Kunicki, J George. Philadelphia, JB Lippincott Co, 1989, pp 111-112 6. George JW, Nurden AT, Phillips DR: Molecular defects in interactions of platelets with the vessel wall. N Engl J Med 311:1084-1098, 1984 7. George JN, Nurden AT, Phillips DR: Platelet Membrane Glycoproteins. New York, London, Plenum Press, 1985 8. Nurden AT: Platelet glycoproteins: Clinical relevance. In Thrombosis and Haemostasis 1987. Edited by M Verstraete, J Vermylen, R Lijnen, J Arnout. Leuven, Belgium, Leuven University Press, 1987, pp 93-125 9. Fitzgerald LA, Phillips DR: Structure and function of platelet membrane glycoproteins. In Platelet Immunobiology. Edited by TJ Kunicki, J George. Philadelphia, JB Lippincott Co, 1989, pp 9-30 10. Weiss HJ, Tschopp TB, Baumgartner HR, Sussman II, Johnson MM, Egan JJ: Decreased adhesion of giant (Bernard-Soulier) platelets to subendothelium-further implications on the role of von Willebrand factor in hemostasis. Am J Med 57:920-925,1974 11. Nurden AT, Caen JP: Specific roles for platelet surface glycoproteins in platelet function. Nature 255:720-722, 1975
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12. Nieuwenhuis HK, Akkerman JWN, Houdyk WPM, Sixma JJ: Human blood platelets showing no response to collagen fail to express surface glycoprotein Ia. Nature 318:470-472, 1985 13. Asch AS, Barnwell J, Silverstein RL, Nachman RL: Isolation of the thrombospondin membrane receptor. J Clin Invest 79:1054, 1987 14. Rosenfeld SI, Looney RJ, Leddy JP, Phipps, DC, Abraham GN, Anderson CL: Human platelet Fc receptor for IgG: Identification as a 40 RD membrane protein shared by monocytes. J Clin Invest 76:2317, 1985 15. McEver RP, Martin MN: A monoclonal antibody to a membrane glycoprotein binds only to activated platelets. J Biol Chem 259:9799, 1984 16. Hsu-Lin SC, Berman CL, Furie BC, August D, Furie B: A platelet membrane protein expressed during platelet activation and secretion. Studies using a monoclonal antibody specific for thrombin-activated platelets. J Biol Chem 259:9121, 1984 17. Kunicki TJ, Beardsley DS: The alloimmune thrombocytopenias: Neonatal alloimmune thrombocytopenia and post transfusion purpura: Prog Hemost Thromb 9:203-232, 1989 18. von dem Borne AEGKr, Beardsley DS, Mueller-Eckhardt C: Unpublished communication from International Conference on Immune Thrombocytopenia, Lucerne, Switzerland, August 1988 19. Woods VL Jr, Oh EH, Mason D, McMillan R: Autoantibodies against the platelet glycoprotein Ilb/lIla complex in patients with chronic idiopathic thrombocytopenid purpura. Blood 63:368, 1984 20. Beardsley DJS, Spiegel JE, Jacobs MM, Handin RI, Lun SE: Platelet membrane glycoprotein Ila contains target antigens that bind anti-platelet antibodies in immune thrombocytopenias. J Clin Invest 74:1701, 1984 21. Beardsley DS: Platelet autoantigens. In Platelet Immunobiology. Edited by TJ Kunicki, J George. Philadelphia, JB Lippincott Co, 1989, pp 121-131
