Human platelets are anucleate cellular derivatives of bone marrow megakaryocytes that circulate throughout the bloodstream at a concentration of 1.5 to 4.0 × 105/mm3. With a life span of only ~10 days, resting platelets maintain a disk-like shape unless activated, usually by exposure to a damaged vessel wall. Following activation, platelets undergo a rapid metamorphosis from nonadherent plate-shaped cells into highly adherent, pseudopod-containing, ameba-like entities, and participate in the formation of the so-called platelet plug at a site of vascular injury.
Platelet Adhesion and Aggregation
Platelets come equipped with a variety of membrane receptors and intracellular organelles that make them highly efficient “adhesion machines” for mediating the primary hemostatic process. The mechanisms by which platelets become activated are multiple and complex, and have a great deal of built-in redundancy to ensure rapid, efficient sealing of the wound. As illustrated in Fig. 177-1, platelets can be activated by numerous agonists, both soluble and insoluble. Damage to a vessel wall leads to disruption of the normal endothelial cell barrier, with exposure of extracellular matrix components, including fibronectin, collagen, and VWF. Platelets have receptors for these ligands, and rapidly initiate an adhesive cascade at the site of injury. Membrane-receptor complexes GPIb and α2β1 bind VWF and collagen, respectively, and mediate the primary adhesion of an initial layer of platelets that applies a thin bandage to the injury. Nearby platelets become secondarily exposed to extracellular agonists, such as thrombin, epinephrine, ADP, and various thromboxane metabolites that are released as a result of local tissue injury. Platelets become activated by one or more of these agonists, and mobilize still another adhesive receptor, the αIIbβ3 complex. On activation, this receptor complex undergoes conformational changes and binds fibrinogen, which, due to its symmetric structure, serves to crosslink platelets to each other, that is, platelet aggregation. The membrane receptors and their ligands that mediate these important adhesive interactions are summarized in Fig. 177-2.
Role of platelets in hemostasis. The transformation of platelets from a resting to an adherent state is depicted, together with several of the known soluble and insoluble agonists that can trigger the event. Adhesion to extracellular matrix components, exposed as a result of endothelial cell damage, is an activating event, and leads to granule release and the conversion of cell-surface receptors into an adhesive conformation. Additional soluble agonists, including thrombin, ADP, and epinephrine, generated at the site of the injury, serve to enhance the activation process, and recruit additional platelets into the developing platelet aggregate. TSP = thrombospondin.
Platelet adhesive interactions. Platelet receptors involved in platelet-platelet interactions are shown, together with the adhesive ligands with which they interact.
Adhesion is an activating event; it causes the secretion of several types of intracellular granules, the contents of which serve to embellish further the formation of the platelet plug by providing additional adhesive ligands that add to the local intercellular glue molecules. A number of soluble agonists that aid in recruiting other platelets to the site of injury are also secreted. The three major types of platelet granules, and some of their more interesting components, are listed in Table 177-1. The α-granules contain a large number of adhesive protein ligands, some of which, on secretion, bind to specific platelet plasma membrane receptors and help to crosslink platelets to each other during platelet aggregation. Dense, or δ-granules, contain most of the platelet calcium and stored ADP and serotonin. Although the function of serotonin in platelet function is not well understood, released calcium and ADP play important roles in mediating the platelet aggregation process. Lysosomal granules, as in other cell types, probably function in degrading various endocytosed plasma constituents, and may also play a role in receptor recycling.
Table 177-1: Platelet Granules and Their Contents |Favorite Table|Download (.pdf) Table 177-1: Platelet Granules and Their Contents
|α-Granules ||δ-Granules ||Lysosomes |
|Fibrinogen ||Serotonin || β-N-acetylglucosaminidase |
|VWF ||Calcium || β-Glucuronidase |
|Fibronectin ||ADP || β-Galactosidase |
|Thrombospondin ||Guanine ||Other acid hydrolases |
|Factor V ||nucleotides || |
|Platelet factor 4 ||ATP, AMP || |
| β-Thromboglobulin ||Inorganic || |
| ||pyrophosphate || |
Platelet Adhesion Receptors
Central to the role of platelets to adhere to both extracellular matrix components and to aggregation is an abundant supply of cell-surface adhesion molecules that exist in multiple states of activation. These cell adhesion receptors, in turn, are capable of transmitting signals to the cell interior by virtue of their ability to form cytoplasmic connections with G proteins, kinases, and cytoskeletal components. Many platelet cell-surface receptors have now been cloned and characterized, and while >50 platelet plasma membrane proteins are recognizable by two-dimensional electrophoresis, many of them fall into a limited number of well-recognized families. Five gene families, the integrins, the leucine-rich glycoproteins, the immunoglobulin superfamily, and the selectins, as well as their members that are present in human platelets, are summarized in Table 177-2. A brief description of the major distinguishing characteristics of each family follows.
Table 177-2: Cell Adhesion Molecule Families in Human Platelets |Favorite Table|Download (.pdf) Table 177-2: Cell Adhesion Molecule Families in Human Platelets
|Gene family ||Members on human platelets ||Functions |
|Intergrins ||αIIbβ3, αvβ3, α2β1, α5β1, α6β1 ||Cell-cell and cell-matrix interactions |
|Leucine-rich ||GPlbα, GPlbβ, GPV, GPIX ||Adhesion to subendothelium glycoprotein |
|Immunoglobulin ||FcγRIIA, Class I GMP, PECAM-1 ||Immunoregulation, adhesion gene (ITAM/ITIM) superfamily |
|Selectins ||P-selectin (HLA-140, PADGEM) ||Platelet-endothelial cell and platelet-leukocyte interactions |
The Leucine-Rich Glycoprotein Family.
Platelets contain four members of the leucine-rich glycoprotein family1,2 and, interestingly enough, these proteins (GPIbα, GPIbβ, GPIX, and GPV) exist on the platelet surface in a 2:2:2:1 stoichiometric complex,3,4 and are referred to in the remainder of this chapter as the GPIb complex. Each member of this receptor complex has been cloned.1,2,5 Each platelet expresses 2 to 3 × 104 copies of the GPIb complex on its surface, and all four components of the complex are missing from the platelet surface in patients with the Bernard-Soulier syndrome,2,6 a defect that is discussed in greater detail below.
The Immunoglobulin Superfamily.
The distinguishing feature of the immunoglobulin gene (Ig) superfamily is the presence of one or more immunoglobulin-like homology domains. Structurally, these homology domains resemble antibodies, containing 7 β-strands folded into 2 β-pleated sheets held together by 2 cysteines spaced approximately 50 residues apart. Human platelets are known to express three members of the Ig superfamily: class I histocompatibility antigens (HLA), FcγRIIA, and PECAM-1. Of these, the HLA molecules are extremely polymorphic, and probably account for the majority of cases of platelet transfusion refractoriness.
Seven Transmembrane Receptors.
There is a series of two transmembrane receptors present on platelets that bind to platelet agonists. For example, P242, P2X, and P2YAC are three such receptors for ADP binding and platelet activation. For thrombin, PAR1 and PAR4 are present on platelets. Epinephrine and thromboxane AZ also stimulate platelets through such receptors. The seven transmembrane receptors signal to the platelet via Gαβγ heterotrimers.
This family contains only three members, each named for the cell type in which it was first discovered: P-selectin, originally described in human platelets;7,8 E-selectin, which is present only on endothelium;9,10 and L-selectin, found on lymphocytes. Selectins share a common N-terminal lectin-like domain that is capable of interacting with carbohydrate moieties on nearby cellular receptors, and it is this structural motif that gives the family its name.10 P-selectin (previously called GMP-140 or PADGEM) is the only platelet selectin, and resides within the membrane of α-granules of resting platelets. On platelet activation, the α-granule membranes fuse with the plasma membrane, exposing 1.0 to 1.5 × 104 P-selectin molecules per platelet, where they participate in recruiting monocytes and neutrophils to the site of vascular injury.11
The integrins are composed of an αβ heterodimeric membrane complex that mediates cell-cell and cell-extracellular matrix adhesive interactions.12,13 At least 8 different β subunits and 11 different α-chains have been cloned and sequenced. It is now recognized that distinct α and β subunits can cross-pair in different cell types to form multiple, distinct functional entities. More than 15 members of this heterodimer family have been identified in species ranging from Drosophila to humans,12,13 with at least 5 members (α2β1, α5β1, α6β1, αIIbβ3, and αvβ3) expressed on the platelet surface. Of these, αIIbβ3 is uniquely expressed on platelets, and is by far the most abundant member of the integrin family to be found on the platelet surface, with 8 × 104 molecules/platelet.14 Other platelet integrins, although not as extensively studied as αIIbβ3, are thought to share similar overall structure due to the presence of multiple highly conserved cysteine residues.15,16 The integrin most closely related to αIIbβ3 is the receptor for vitronectin, αvβ3. This integrin uses the same β3 subunit, but forms the vitronectin receptor (VNR) by complexing with the αv subunit that shares 74 percent sequence similarity with αIIb.15 Although there are at most 50 to 100 αvβ3 molecules per platelet,17 there are nearly 105 VNR per endothelial cell,18 and they probably play an important adhesive function in this and other cell types. The other three platelet integrins share a different common β subunit (β1), which forms a complex with either an α2, α5, or α6 chain to form receptors for collagen, fibronectin, or laminin, respectively.19–22 Interestingly, of the three β1 integrin α-subunits that are present on platelets, only α2 is known to be naturally polymorphic in humans (see below). Of the β1 integrin α-chain subunits present on the platelet surface, α2 is unique in that it contains an inserted “I” domain of approximately 200 amino acids, which is also present in the α-chain subunits that pair with leukocyte β2 integrin subunits (LFA-1, p150,95, and MAC-1). High-resolution crystal structures of isolated I domains have established this motif as part of a unique metal coordination site designated the metal ion-dependent adhesion site or MIDAS domain.23 Interestingly, this motif has also been identified in integrin β-chain subunits and has been predicted to play a direct role in ligand binding function.24–26
Diseases Associated with the αIibβ3 Complex
The αIIbβ3 receptor is one of the most abundant receptors on the platelet surface, representing nearly 15 percent of total surface protein. This receptor is a key component in the pathway to platelet aggregation and consequently has become a target for therapeutic intervention.27 Platelet aggregation and fibrin formation are essential for the maintenance of normal hemostasis, but these processes can also be triggered by pathogenic events such as the rupture of an atherosclerotic plaque. A paradigm of antiplatelet therapy is found in the inherited bleeding disorder Glanzmann thrombasthenia. A key feature of this disease is that patients present with mucocutaneous bleeding and rarely demonstrate spontaneous central nervous system hemorrhage,28 a feared complication of anticoagulant and antiplatelet therapy. All of the mutations that have been identified in patients with Glanzmann thrombasthenia result in a functional deficiency of platelet αIIbβ3 receptors,29 and a hallmark of this disease is the absence of agonist-induced platelet aggregation. In addition to its role as the major receptor for adhesive ligands, αIIbβ3 is also highly immunogenic and is the target molecule for human auto-, allo-, and drug-dependent antibodies in immune-mediated platelet disorders. Because of its clinical significance in both immunologic, hemostatic, and thrombotic platelet disorders, αIIbβ3 is one of the most completely characterized membrane glycoproteins, and an abundant literature exists describing its biochemical, immunologic, and cell and molecular biologic properties (for reviews, see references29 to 32).
The αIIb and β3 subunits are derived from separate mRNA transcripts,33,34 although the genes encoding them are closely linked,35 on chromosome 17.36,37 The amino acid sequences of both these membrane glycoproteins have been determined in full from cloned cDNA, and a diagram of the complex is shown in Fig. 177-3. αIIb is ~145 kDa in size and contains 18 cysteine residues arranged in 9 disulfide bonds that are rather evenly spaced throughout its length.37 Like most other integrin α chains, αIIb contains four calcium-binding domains, similar in sequence to those found in calcium-binding proteins such as troponin C, tropomyosin, and calmodulin.37 Marguerie and coworkers have shown that all four sites must be occupied by divalent cations for proper functioning,38 and several groups have demonstrated that the ability of αIIb to associate with β3 depends on the continuous presence of at least micromolar levels of calcium.39–41 The four calcium-binding sites are located within the amino-terminal 450 amino acids of αIIb that have been identified as the minimal sequence required for ligand-binding.42 This region in all α-subunits contains seven homologous repeats that are comprised predominantly of β-strands43 predicted to fold into a β-propeller structure44 (Fig. 177-4). The β-propeller is highly conserved and contains seven repeats of β-sheets that are arranged similar to propeller blades around a central axis.44 Ligand binding has been proposed to occur on the face opposite the cation-binding sites that lie on the bottom of the structure.44 The biologic importance of the β-propeller domain of αIIb is illustrated below by examination of specific mutations that lead to dysfunctional platelet aggregation responses.
Representation of the major platelet integrin receptor αIIbβ3. The major features of β3 include the presence of five cysteine-rich regions, including one at the N-terminus, and four cysteine-rich repeats positioned closed to the membrane. Two long-range disulfide bonds (5 to 435, and 406 to 655) are depicted, as well as putative sites of interaction with fibrinogen (stippled areas). The four calcium-binding domains of αIIb are shown, as well as the site on this integrin α-subunit that was shown biochemically to bind the γ chain of fibrinogen (H12).
β-Propeller model of the N-terminus of αIIb. This representation of the β-propeller model of the N-terminus of αIIb depicts the known thrombasthenic mutations in this region of αIIb. The N- and C-termini of this region are shown, as are the seven propeller blades, each of which has four β-strands (depicted as arrows) and which are numbered 1 to 4 for the first propeller only. Those mutations that decrease surface expression and interfere with ligand binding are shown as stars, while the mutations that prevent intracellular processing of the αIIbβ3 receptor with no surface expression of the receptor are shown as circles.
Similar to other integrin β chains, β3 is ~90 kDa, and contains 762 amino acid residues in its mature form.45–47 β3 contains five cysteine-rich regions—one at the N-terminus and four located proximal to the transmembrane domain.46 β3 contains 56 cysteine residues in locations that are highly conserved in other human integrin β subunits. Each cysteine is involved in the formation of a disulfide bond that stabilizes the overall structure of the mature glycoprotein. Another distinguishing feature of β3 and other integrin β subunits is the presence of a large disulfide-bonded loop that extends from amino acids Cys5 to Cys435.48 At least one critical fibrinogen-binding functional domain of β3 has been localized within this loop49 (see below). Treatment of intact platelets with the proteolytic enzyme chymotrypsin results in cleavage of β3 at residues 121 and 348,50 effectively removing half the loop and leaving a nonfunctional 66-kDa fragment associated with the plasma membrane. The 66-kDa remnant is composed of two disulfide-linked chains—an N-terminal 17-kDa chain containing amino acids 1 to 120 and a larger fragment extending from residues 349 to 762. Immunochemical analyses of such proteolytic fragments have been useful in determining the antigenic epitopes that form the targets of alloimmune antibodies.
To understand the molecular nature of inherited disorders associated with αIIb and β3, it is necessary first to understand the mechanism by which these two glycoproteins are synthesized, associate in a complex, and are transported through the intercellular organelles of the megakaryocyte to reach the cell surface. The biosynthetic pathway for the formation of the αIIbβ3 complex is illustrated in Fig. 177-5. As shown, both αIIb and β3 are synthesized in the RER as single-chain precursors that associate to form a pre-αIIbβ3 complex. Both proteins are cotranslationally modified with high-mannose carbohydrate moieties that represent ~15 percent of each subunit's molecular mass.51–53 Following subunit association, pre-αIIbβ3 moves to the Golgi apparatus, where the pre-αIIb (1039 amino acids in length) is cleaved at amino acid 859 into a heavy and a light chain54 that remain linked by a disulfide bridge formed by residues Cys826 to Cys.880 The high-mannose carbohydrate residues of αIIb, but not β3, are converted to complex sugars, as indicated by a change in the susceptibility of αIIb to endoglycosidase H. There is evidence from endoglycosidase studies that O-linked sugars may be added to the protein backbone of αIIb as well.53 Following posttranslational processing, the mature αIIbβ3 complex is rapidly transported to the cell surface, where it is maintained in a resting conformation.
Flow chart of the biosynthetic pathway of the αIIbβ3 complex. Each subunit is translated from separate mRNA transcripts into single-chain precursors within the ER of the megakaryocyte, and assembled into a pre-αIIbβ3 complex before transport to the Golgi. Modification of αIIb carbohydrate chains and cleavage of αIIb into heavy and light chains takes place before eventual transport of the now mature complex to the cell surface.
There are ~8 × 104 αIIbβ3 receptors per platelet.14,56 Although >70 percent of these receptors are present on the platelet surface,14,57,58 the complex exists in a resting, nonadhesive state, and does not normally interact with its ligand fibrinogen, despite being bathed in 3 to 4 mg/ml concentrations of this abundant plasma protein. If, however, the platelet becomes activated by thrombin, ADP, thromboxane A2, or some other agonist, an inside-out signaling event takes place (for review see reference59) that results in the conversion of the complex into an active receptor that can bind ligand, resulting in platelet aggregation. Following ligand binding, “outside-in” signal transduction mechanisms59 mediate integrin-cytoskeleton interactions and receptor clustering. These are requirements for post-ligand occupancy events such as cell spreading and formation of focal adhesion sites (for review see reference60).
Ligands for many integrins contain the tripeptide sequence Arg-Gly-Asp (RGD).61 Thus, fibrinogen, fibronectin, VWF, and collagen contain one or more RGD sequences.61–64 Fibrinogen, which is an elongated, cigar-shaped, six-chain molecule composed of two Aα, two Bβ, and two γ chains held together in symmetrical fashion by a centrally located disulfide-dependent knot, contains four such RGD sequences, two on each Aα chain. On conversion of αIIbβ3 into the active state, one fibrinogen Aα chain binds,65 presumably through the RGD, at residues 572 to 575.66 Due to its symmetric nature, the other fibrinogen Aα chain is free to interact with an αIIbβ3 on a nearby platelet, thus serving to bridge the two cells and mediate platelet-platelet cohesion (see Fig. 177-2). Synthetic RGD peptides block fibrinogen binding and platelet aggregation,62,67,68 and photoaffinity-labeled RGD peptides interact directly with residues 109 to 171 of the β3 subunit.49 Another region on β3 also contributes to the interaction with fibrinogen, as a small linear synthetic peptide corresponding to amino acids 211 to 222 blocks fibrinogen binding to platelets and the subsequent platelet aggregation response.69 These findings are supported by mutations in patients with Glanzmann thrombasthenia (see below) and by mutagenesis studies,70–72 and define molecularly one mechanism of receptor-ligand interaction within the β3 MIDAS domain.25,26,42
The binding of fibrinogen to αIIbβ3 on the platelet surface, however, seems to be multivalent,65 as fibrinogen is also known to interact with its receptor via a dodecapeptide sequence (labeled γ12 in Fig. 177-3) located at residues 400 to 411 in a KGD recognition motif of the γ chain.73–76 As defined biochemically, the γ12 binds to amino acids 296 to 313 of the αIIb subunit, located in the fifth blade of the β-propeller model (Fig. 177-4).77,78 Due to the absence of a crystal structure of the αIIbβ3 receptor complex, precise information concerning the sites within integrin receptors that recognize ligands is not available, but the three putative regions on αIIbβ3 that interact with fibrinogen, as well as the corresponding site on the fibrinogen molecule, are summarized in Table 177-3.
Table 177-3: Sites of Interaction between αIIbβ3 and Fibrinogen |Favorite Table|Download (.pdf) Table 177-3: Sites of Interaction between αIIbβ3 and Fibrinogen
|Region of the αIIbβ complex ||Region on fibrinogen |
| β3 109–171 ||Aα Chain RGD residues 572 to 575 |
| β3 211–222 ||Unknown |
|αIIb 296–313 ||γ-Chain residues 400 to 411 |
Molecular Genetic Characterization of Platelet-Specific Defects
PCR technology79 combined with the discovery that human platelets contain enough RNA to generate cDNA fragments by reverse transcriptase (RT)-PCR80 revolutionized the field of platelet molecular biology. The αIIb and β3 Glanzmann thrombasthenia mutations are now routinely determined by sequence analysis of genomic DNA and platelet RNA using specific oligonucleotide primers and PCR.81 Briefly, the methodologies used include RT-PCR of platelet RNA, PCR of genomic DNA, and single-stranded conformational polymorphism (SSCP) analysis of PCR fragments containing intron and exon boundaries. PCR fragments are sequenced either directly, which provides the advantage of immediately determining homozygosity versus heterozygosity of the mutation, or from a cloned product. The mutations that affect RNA splicing are usually identified by sequence analysis of the patients' genomic DNA followed by analysis of the patients' platelet RNA. Using these methodologies, the molecular genetic defects responsible for the diseases Glanzmann thrombasthenia, Bernard-Soulier syndrome, von Willebrand disease, and the alloimmune thrombocytopenias have been identified, and our ability to diagnose these disorders, both prenatally and postnatally, is a reality. Much of the remainder of this chapter describes our current understanding of the molecular biologic basis of inherited platelet defects, and describes lessons learned from these findings.
In 1918, Glanzmann, a Swiss pediatrician, described a somewhat heterogeneous group of disorders that he termed “thrombasthenie” (“weak platelets”), that were characterized by normal platelet counts but abnormal clot retraction.82 Braunsteiner and Pakesch added to our understanding of what is now termed Glanzmann thrombasthenia (GT) by noting, in 1956, that platelets from these patients failed to spread onto a surface,83 while the laboratories of Hardisty84 and Zucker85 first described the failure of thrombasthenic platelets to stick to each other (aggregate). Glanzmann thrombasthenia is known to be a rare, inherited, autosomal recessive bleeding disorder, the hallmark of which is the failure of platelets to bind fibrinogen and aggregate following stimulation by physiological agonists such as ADP, thrombin, epinephrine, or collagen. In all patients with this disorder described to date, the underlying defect is an abnormality of the genes encoding either αIIb or β3, although it is theoretically possible that molecules involved in transmitting the signal to or from the αIIbβ3 receptor could also be affected.
GT is a lifelong disease characterized by repeat mucocutaneous bleeding beginning at an early age. Thus, epistaxis, gingival bleeding, and purpura or petechiae at sites of minor trauma are the most common features. Menorrhagia is a critical problem in teenage girls and younger women, when heavy bleeding during menstruation may need medical intervention.28 Bleeding that normally accompanies pregnancy, surgical procedures, tooth extractions, or physical trauma can be excessive in GT patients, although the severity of hemorrhage is not predictable, even in defined subtypes of the disease (see below). Severe unprovoked intracranial or gastrointestinal hemorrhages occur and account for a significant portion of the observed mortality in 5 to 10 percent of the patients. In addition, some patients experience joint bleeding or visceral hematomas more characteristic of coagulation protein disorders (the hemophilias).28 Treatment is limited to local measures although platelet transfusions may be useful until resistance to platelet infusions develop. Recent evidence suggests that recombinant activated Factor VII may be a useful addition in such a circumstance.86 An animal model for Glanzmann thrombasthenia was created by knocking out the β3 gene.87 The platelets from these animals do not express surface β3 as determined by flow cytometry and immunoblot of whole platelet lysates. Due to the absence of β3, αIIb is degraded and undetectable by immunoblot. Platelet aggregation in response to ADP is absent, but shape change is present. Platelet fibrinogen is markedly decreased. The mice have greatly prolonged bleeding times and suffer various degrees of anemia. Tests for occult blood show that more than 60 percent of null animals have blood in their stools. The mice are viable and fertile and some animals do die, either preweaning or later. Necropsies reveal a variety of abnormalities, including ulcerative dermatitis, enlarged spleens, and blood in the gastrointestinal tract.
Like many other rare disorders, GT is found at an unusually high incidence among certain isolated population clusters and/or those in which consanguinity is commonplace. Thus, in spite of its infrequent occurrence worldwide, a high carrier rate for thrombasthenia exists among Iraqi Jews,88–90 selected Arab populations,89–91 French gypsies,92–94 and individuals from southern India.95 Carriers or obligate heterozygotes of this disease have ~50 percent the normal number of αIIbβ3 receptors and these individuals do not have abnormalities of platelet function or clinically significant bleeding.28,90 The occurrence of abnormal αIIb and β3 genes in the human gene pool is low, but to date, ~40% of the patients with identified mutations are compound heterozygotes. Due to the autosomal recessive nature of this disorder, all of the identified homozygous mutations result from consanguinity (except one due to uniparental disomy96) and a putative founder effect has been described for two of the mutant genes identified in the Iraqi-Jewish population in Israel.97
Classification and Laboratory Diagnosis.
Despite several attempts to categorize Glanzmann thrombasthenia into subtypes, it will become apparent from the studies discussed below that there are almost as many molecular biologic etiologies of thrombasthenia as there are patient populations. In 1972, Caen proposed the first classification of this disease based on platelet intracellular fibrinogen content and the ability of platelets to retract a fibrin clot.98 Type I patients, representing 80 percent of those studied, lacked platelet fibrinogen and had absent clot retraction, whereas type II thrombasthenic platelets contained appreciable levels of platelet fibrinogen and maintained some clot retraction capability. Soon thereafter, the technique of SDS-PAGE became widespread, and Nurden and Caen used this method to detect an abnormal “glycoprotein II” pattern in three cases of GT.99,100 Phillips and Agin used two-dimensional SDS-PAGE analysis of radiolabeled platelets to show convincingly that both αIIb and β3 were specifically decreased in GT versus normal platelets.101 Several laboratories made the observation that whereas type I patients lacked detectable levels of αIIbβ3, type II GT platelets expressed moderate (15 to 25 percent) levels of this glycoprotein, as measured using immunochemical102–104 and electrophoretic105 techniques. Adding to the complexity of this classification was the identification of variant forms of thrombasthenia which are characterized by normal to near-normal levels of a dysfunctional form of αIIbβ3 present on the cell surface.106–109 It is important to note that platelets from these patients are functionally indistinguishable from type I and II platelets in that laboratory diagnosis for all three GT subgroups have in common the complete failure to aggregate in response to physiological agonists such as ADP, thrombin, or epinephrine, as illustrated in Fig. 177-6. The functional deficiency of platelet surface αIIbβ3 receptors and the failure to bind fibrinogen,110 VWF,111 and other adhesive ligands is the reason for the inability of platelets to cohere.
Typical platelet aggregation profiles of normal versus GT individuals. Whereas normal platelets aggregate readily when exposed to thrombin or other agonists, platelets from a patient with thrombasthenia fail to respond. Inset: Immunoblot of detergent lysates from normal control (C) and GT platelets. Rabbit polyclonal antibodies specific for αIIb, β3, and PECAM-1 (top band) were exposed to Immobilon strips containing 100 mg each of a platelet lysate, and developed with an alkaline phosphatase-conjugated second antibody. Platelets from the GT patient shown express less than 5 percent of normal β3 levels, and no αIIb can be detected, typical of an αIIb genetic defect.
As the genetic defects underlying the thrombasthenic phenotype have become defined, the rationale for maintaining these three traditional categories of GT becomes less convincing. Alternatively, more complicated classifications based on surface expression or on the fate of αIIb and β3 subunits as they traffic through the cell to reach the plasma membrane112 have been proposed. None of these are entirely satisfying, as biologic exceptions to each appear as readily as classification schemes are devised. Furthermore, no correlation exists between any of the proposed subtypes of GT and the severity of bleeding symptoms in patients.28 Some patients with absolutely no αIIbβ3 have relatively mild clinical symptoms, while others with a full complement of αIIbβ3, albeit dysfunctional, can have frequent bleeding episodes requiring multiple platelet transfusions. Nonetheless, even in the absence of predictive value, it remains instructive for both clinical diagnosis and research purposes to determine the level of αIIbβ3 complex surface expression in GT patients.
Currently, the most common method used for determining the levels of αIIbβ3 on thrombasthenic platelets involves flow cytometry,113 radiolabeled monoclonal antibody binding,56,115,116 and western blot (immunoblot) analysis,114 each of which offers the advantage of increased sensitivity over the previously used crossed immunoelectrophoretic technique.92,93 An immunoblot illustrating αIIbβ3 content of both a normal control and a GT patient platelet lysate is shown in the inset to Fig. 177-6, and demonstrates the severe deficiency of αIIbβ3 in this patient. In patients with <5 percent of the normal αIIbβ3 content, a trace of even one of the two integrin subunits can be instructive as to the nature of the molecular defect.117 In the example shown, a trace amount of β3 is apparent, while there is no detectable αIIb. This patient was later found to have a large deletion in the αIIb gene (see below), consistent with the immunoblot analysis. Because it is known that β3 does not survive intracellular trafficking in the absence of an integrin α-chain subunit, it is presumed that a small number of β3 molecules expressed on the platelet surface were “rescued” by their ability to form a complex with the VNR αv subunit (see “Biology of αIIbβ3” above). Analysis of surface expression of αvβ3 on either patient platelets118 or lymphocytes119 is a reliable indicator of αIIb and β3 defects. The decreased or undetectable binding of both αIIbβ3 and αvβ3-specific monoclonal antibodies indicates a defect in β3, whereas decreased or undetectable binding of αIIbβ3-specific, but not αvβ3-specific monoclonal antibody, indicates a defect in αIIb. This analysis has been utilized to identify defective αIIb or β3 subunits in numerous patients with GT and subsequent identification of the mutation in either gene has confirmed the reliability and usefulness of this assay.120–124
Molecular Abnormalities of αIIb and β3 Genes Resulting in Glanzmann Thrombasthenia.
Nearly 200 individuals with GT have been described in the literature. More than 61 of these have been solved at the molecular level. As in most genetic disorders, the molecular abnormalities have been found to range from major deletions and inversions to single-point mutations identified by nucleotide sequence analysis of genomic DNA or platelet mRNA-derived PCR products (for review see reference125). Forty-five of these mutations are in αIIb gene and 30 are in β3. In vitro studies indicate that production of both protein subunits is required for proper surface expression and function,125 and this concept has been nicely corroborated at the level of human biology through the molecular biologic analysis of GT defects. The mutations that have been identified in the αIIb and β3 genes result in qualitative and/or quantitative abnormalities of the platelet membrane proteins28–30,125 and the molecular characterization of GT in patients and their families has permitted DNA-based carrier detection and prenatal diagnoses to be performed.126,127 A worldwide interactive Internet database (http://med.mssm.edu/glanzmanndb) that includes all reported clinical, biochemical, and molecular information on patients with GT has been created from a published database125 in which the GT mutations are identified by amino acid number beginning with methionine of the ATG start codons and by nucleotide number beginning with the A of the ATG start codons in the reported cDNA sequences of αIIb37 and β3.46
Of the propositi kindreds with affected individuals described in the literature, ~40% were identified as compound heterozygotes and ~60% as homozygotes, in which one of the latter may have arisen from a rare uniparental disomy event.96 With an increasing number of mutations identified and characterized at the molecular level, the Glanzmann thrombasthenia subtypes can be categorized according to the biochemical consequences of their molecular genetic abnormality (Table 177-4). The αIIb and β3 mutations can be divided into two broad subsets of those that affect biosynthesis and those that affect function of the receptor complex, with some mutations resulting in both quantitative and qualitative defects.124,127,128 Mutations that result in biosynthetic defects of the receptor can be further divided into those that affect (a) RNA transcription, (b) subunit assembly or stability, and (c) complex maturation. A number of patients with point mutations or small deletions in αIIb or β3 genes leading to defects in the maturation of complex will be described below. Depending on the location of the mutation, the blockade can take place at the level of subunit association in the ER (complex formation), sorting to the Golgi apparatus, or trafficking to the cell surface (Fig. 177-5). Examples for each have been found, and each is characterized by absent to reduced levels of αIIbβ3 on the cell surface, although significant intracellular pools of the affected subunit can be recovered in transfection/immunoprecipitation studies of recombinant forms of the mutated subunit. Mutations that result in functional defects of the receptor complex can be further divided into those that affect (a) stability of the receptor complex and (b) the ability of the receptor complex to be activated.
Table 177-4: Classification of Glanzmann Thrombasthenia Subtypes by Biochemical Consequence of the Molecular Genetic Abnormality |Favorite Table|Download (.pdf) Table 177-4: Classification of Glanzmann Thrombasthenia Subtypes by Biochemical Consequence of the Molecular Genetic Abnormality
|Defect (examples) ||Characteristics ||mRNA ||Subunit synthesis ||Surface expressions |
| Transcriptional || || || || |
|(KW, GT3*) ||Major gene deletions, insertion, or rearrangements ||−/+ ||— ||— |
| Subunit stability || || || || |
|(I-J, Arab) ||Small deletions or insertions leading to a change in the number of amino acids, or substitution to a destablilizing amino acid. Binding of GPIIb-IIIa complex-specific antibodies and at least one subunit-specific antibody, even to intracellular pools are absent. ||+ ||Absent to reduced ||— |
| Complex maturation || || || || |
|(LM, FLD, KJ, SH) ||Point mutations affecting Ca2+-binding, subunit association, etc. that affect intracellular trafficking and surface expression. Binding of complex-specific antibodies is often affected, though subunit-specific antibodies bind to intracellular forms. ||+ ||Near normal ||Absent to reduced |
| Ligand binding || || || || |
|A. Complex stability (CAM, ET, Stras I) ||Normal to somewhat reduced levels of GPIIb-IIIa complex on the platelet surface. Often easily dissociable with EDTA. Binding of many subunit- and complex-specific antibodies is normal. ||+ ||Nornal to mildly reduced ||Normal to mildly reduced |
|B. Activatability (Paris I) || || || |
Patients with mutations resulting in defects affecting complex maturation, complex stability, ligand binding, and activation of the receptor complex are described below. These mutations are located within the α-chain β-propeller domain, the MIDAS domain of β3, and the cytoplasmic domain of β3 (Table 177-5). To view all of the mutations as well as clinical and biochemical information on the reported patients with Glanzmann thrombasthenia, the reader is referred to the Internet database (http://med.mssm.edu/glanzmanndb).
Table 177-5: Glanzmann Thrombasthenia Mutations* |Favorite Table|Download (.pdf) Table 177-5: Glanzmann Thrombasthenia Mutations*
|Mutations within the αIIb β-Propeller Domain || || || |
|Patient ||Genotype ||Mutation† ||Mutation phenotype ||Amino acid substitution‡ ||Biochemical defect |
|FLD ||Homozygote ||818G → A ||Missense ||G242D ||Complex maturation |
|Japanese-1 ||Compound ||959T → C ||Missense ||F289S ||Subunit assembly |
| ||heterozygote ||unknown ||Unknown ||Unknown ||or complex maturation? |
|FL ||Homozygote ||1063G → A ||Missense ||E324K ||Subunit assembly? |
|Swiss ||Compound ||1063G → A ||Missense ||E324K ||Subunit assembly? |
| ||heterozygote ||1787T → C ||Missense ||I565T || |
|Japanese-2 ||Compound ||1063G → A ||Missense ||E324K ||Subunit assembly? |
| ||heterozygote ||Unknown ||Unknown ||Unknown || |
|KJ ||Homozygote ||1073G → A ||Missense ||R327H ||Complex maturation ligand binding? |
|Mila-1 ||Homozygote ||1073G → A ||Missense ||R327H ||Complex maturation ligand binding? |
|LM ||Homozygote ||1346G → A ||Missense ||G418D ||Complex maturation |
|LeM ||Compound ||1366-1371del ||Del: In frame ||V425D426del ||Complex maturation |
| ||heterozygote ||Unknown ||Unknown ||Unknown || |
|Frankfurt I ||Homozygote ||620C → T ||Missense ||T176I ||Complex maturation ligand binding |
|LW ||Homozygote ||641T → C ||Missense ||L183P ||Complex maturation ligand binding |
|Mennonite ||Homozygote ||526C → G ||Missense ||P145A ||Complex maturation ligand binding |
|JF ||Compound ||526C → G ||Missense ||P145A ||Complex maturation |
| ||heterozygote ||2929C → T ||Nonsense ||R946X ||Premature termination |
|Chinese-14 ||Compound ||527C → T ||Missense ||P145L ||Complex maturation ligand binding? |
| ||heterozygote ||IVS15(–1)Gdel ||Unknown ||Unknown || |
| Mutations within the β3 MIDAS Domain || || || |
| Patient || Genotype || Mutation† || Mutation phenotype || Amino acid substitution‡ || Biochemical defect |
|Cam ||Homozygote ||433G → T ||Missense ||D119Y ||Ligand binding |
|NR ||Homozygote ||433G → A ||Missense ||D119N ||Ligand binding |
|Strasbourg I ||Homozygote ||718C → T ||Missense ||R214W ||Ligand binding complex stability |
|CM ||Homozygote ||718C → T ||Missense ||R214W ||Ligand binding complex stability |
|ET ||Homozygote ||719G → A ||Missense ||R214Q ||Ligand binding complex stability |
|SH ||Homozygote ||725G → A ||Missense ||R216Q ||Complex maturation ligand binding |
|MK ||Homozygote ||428T → G ||Missense ||L177W ||Complex maturation |
|BL ||Homozygote ||563C → T ||Missense ||S162L ||Ligand binding complex stability |
|LD ||Compound ||847delGC ||Del: Out of ||Premature ||Ligand binding complex stability |
| ||heterozygote ||863T → C ||frame missense ||termination L262P || |
| Mutations within the β3 Cytoplasmic Domain || || || |
| Patient || Genotype || Mutation† || Mutation phenotype || Amino acid substitution‡ || Biochemical defect |
|RM ||Compound ||1791delT ||Del: Out of ||Premature ||Receptor activation inside-out signaling |
| ||heterozygote ||2248C → T ||frame missense ||termination R724X || |
|Paris 1 ||Compound ||2332T → C ||Missense ||S752P ||Receptor activation |
| ||heterozygote ||Unknown ||Unknown ||No transcript || |
Mutations within the α-Chain β-Propeller Domain.
Different groups of Glanzmann thrombasthenia mutations located within the α-chain β-propeller are beginning to emerge. One group of mutations is located within and surrounding the calcium-binding domains, and another group is located within the vicinity of the third blade of the β-propeller structure. The mutations that are located within and surrounding the calcium-binding domains affect transport of the receptor complex to the cell surface. Six missense mutations and one in-frame deletion mutation in nine kindreds have been identified and their locations within the β-propeller model are shown in Fig. 177-4. These mutations include a G242D substitution (patient FLD)122 that precedes the first calcium-binding domain, an F289S substitution (Japanese-1) that precedes the second calcium-binding domain, an E324K (patients FL, Swiss, and Japanese-2)129,130 substitution located between the second and third calcium-binding domains, an R327H (patients KJ and Mila-1)131,132 substitution also located between the second and third calcium-binding domains, a G418D (patient LM)133 substitution that precedes the fourth calcium-binding domain, and a V425D426 (patient LeM)134 deletion at the beginning of the fourth calcium-binding domain. In patients FLD, LM, and LeM, the mutations lead to improperly folded pre-αIIb subunits that are stable but unable to assemble with β3 properly. In the cases of LM and LeM, the abnormal complexes accumulate within the ER. Thus, these patients' platelets do not express αIIbβ3 on their surface, as illustrated in the representative flow cytometric profile of patient LM shown in Fig. 177-7. The defects in patients KJ and Mila-1 differ somewhat from the others in that moderate amounts of their abnormal αIIbβ3 complex reach the cell surface (see Fig. 177-7). This group of GT patients can best be categorized as having maturational defects in the αIIbβ3 complex, as suggested by the decreased to absent levels that reach the cell surface, and by the inability of the residual surface receptor to bind fibrinogen or fibrinogen mimetics. Failure of complex, but not subunit-specific, monoclonal antibodies to bind to residual pools of abnormal subunits that accumulate intracellularly may also be a common feature.
Flow cytometric profile of two patients with GT as compared with a normal control individual. Both patients KJ and LM have mutations that affect the maturation of the αIIbβ3 complex, but differ in that the mutation in patient LM is within the calcium-binding domain of αIIb and perturbs maturation such that no part of the complex is able to traffic to the cell surface. The nature of the mutation in KJ is such that 20 to 30 percent levels of αIIbβ3 are expressed, but fail to function normally.
Another group of mutations has been localized to the vicinity of the third blade (W3) of the β-propeller. These mutations are located close to a predicted β-turn structure that has been implicated in ligand-binding of αIIbβ3 and other integrin receptors.135,136 Four missense mutations and one in-frame insertion mutation in five kindreds have been identified and their locations within the β-propeller model are shown in Fig. 177-4. These mutations include an RTins (patient KO) located in the connecting strand between W2 β-strand 4 and W3 β-strand 1, a T176I (Frankfurt I)137 located in the connecting strand between W3 β-strands 1 and 2, an L183P (patient LW)124 located at the end of the second β-strand in W3 near the 2-3 connecting strand, and a P145A (Mennonite and JF) and a P145L (Chinese-14)128 located within the 4-1 connecting strand between the second and third blades of the propeller. The L183P and P145A mutations both result in quantitative and qualitative defects of the receptor complex. The RTins mutation results in a quantitatively normal surface expressed receptor that is qualitatively defective. Independent support for the functional importance of this region has been shown by the D224V mutation,138 located within the 4-1 connecting strand between the third and fourth blades of the propeller. This mutation was identified from in vitro generated mutant αIIbβ3 receptors expressed in CHO cells72 and disrupts ligand-binding function of the receptor.
Mutations within the β3 MIDAS Domain.
This group of thrombasthenic defects is characterized by significant levels of αIIbβ3 surface expression, although the complex is not able to interact with its natural ligands. In contrast to the platelets of patients having integrin maturational defects, such as those described in the previous section, the binding of many αIIbβ3 complex-specific antibodies are normal in this group, indicating that subunit association and intracellular trafficking are largely unaffected by the nature of the mutation. That the αIIbβ3 complex is not normal is indicated by the fact that divalent-cation dependent regulation of its conformation can be affected and the complex may be easily dissociable by chelation of external calcium ions with EDTA (patient Cam, D119Y mutation).139 Nine missense mutations in twelve patients with Glanzmann thrombasthenia have been identified within the cation-binding sphere of the MIDAS domain (Fig. 177-8). Two mutations, the Cam variant and D119N (patient NR),140 are located within the conserved DXSXS amino acid motif; three mutations, R214W (Strasbourg I variant and patient CM),141,142 R214Q (patient ET),70 and R216Q (patient SH),143 are located within the putative coordinating sites;43 and four mutations, D117W (patient MK),144 S162L (patient BL),145 L262P (patient LD),146 and H280P (patients HJ, NT, TK) are located within the vicinity of the MIDAS domain. The mutations at residue D119 result in abnormalities of platelet αIIbβ3 function, but do not affect surface expression, while the mutation at D117 results in the intracellular retention of misfolded receptor complexes. The mutations at residue R214 result in surface expressed αIIbβ3 receptors that are abnormally sensitive to dissociation by calcium chelation, the mutation at R216 results in surface expressed αIIbβ3 that are unreactive with complex-dependent antibodies and cannot bind ligand, the mutations at residues S162 and L262 result in surface expression levels of ~30 percent of normal, but also show sensitivity to dissociation by calcium, and the mutation at residue H280P results in surface expression levels of ~17% of normal but does not appear to affect the function of the receptor complex. The importance of these sites is reinforced by the identification of a group of in vitro-generated mutant αIIbβ3 receptors expressed in CHO cells.72 The mutations D119N, R214W, D198N, E220Q, and E220K were identified as functional defects providing independent support for the importance of the MIDAS domain in ligand binding.
MIDAS model of the β3. This representation of the MIDAS model of the N-terminus of β3 depicts the known thrombasthenic mutations in this region of β3. The N- and C-termini of this region are shown. Thrombasthenic mutations are shown as circles; β-sheets are depicted as arrows; and α-helical regions are depicted as cylinders.
Mutations that Affect Receptor Activation.
The cytoplasmic domain of β3 is important for integrin activation and regulation of ligand binding.147–149 Two Glanzmann thrombasthenia mutations in two kindreds have been identified in this region. One is an R724X nonsense mutation (patient RM)150 that deletes the C-terminal 39 residues of β3, and the other is an S752P missense mutation (patient P or Paris I).147,148,151 These mutations do not affect surface expression of platelet αIIbβ3 complexes, but mutant receptors are unresponsive to agonist stimulation. Mammalian cell expression studies show normal adhesion to immobilized fibrinogen, but abnormal cell spreading. Cells expressing the S752P mutant receptors have reduced focal adhesion plaque formation and cells expressing the R724X mutant receptors have undetectable tyrosine phosphorylation of focal adhesion kinase pp125FAK. These mutations provide compelling evidence for the role of the β3 cytoplasmic tail in the function of the αIIbβ3 receptor complex.