Inherited Platelet Disorders

Biology of the α _IIb β ₃ Receptor

The α _IIb and β ₃ subunits are encoded by separate genes ( ITGA2B and ITGB3 ) that are closely linked on chromosome 17q21-23. α _IIb is approximately 145 kD in size and contains 18 cysteine residues arranged into 9 disulfide bonds that are rather evenly spaced throughout its length ( Fig. 30-1 ). The α _IIb prochain complexes with the β ₃ subunit in the endoplasmic reticulum. During maturation in the Golgi body, the α _IIb prochain is cleaved into the α _IIb heavy fragment and the α _IIb light fragment, with the two fragments remaining linked by a disulfide bond. Like many other integrin α chains, α _IIb contains four calcium-binding domains near its N-terminal. This region in all α subunits contains seven homologous repeats that fold into a β-propeller structure ; portions of the surface loops of this structure are critical for ligand binding (in interaction with the βA domain of the β ₃ subunit).

Similar to other integrin β chains, β ₃ is approximately 90 kD and contains 762 amino acid residues in its mature form (see Fig. 30-1 ). β ₃ contains five cysteine-rich regions for a total of 56 cysteine residues, including a large disulfide-bonded loop, termed the βA region, that extends from amino acids Cys5 to Cys435. This loop participates in fibrinogen binding and contains three divalent cation-binding domains that appear to be important in ligand binding. One of these cation-binding sites (the βA MIDAS–metal ion–dependent adhesion site) interacts directly with ligand, and binding of cations to the βA domain may stabilize the ligand-occupied conformation.

There are about 8 × 10 ⁴ α _IIb β ₃ receptors per platelet, making this receptor the most abundant one on platelets (see Table 30-1 ). Most of these receptors are located on the platelet surface, although a portion are found on the inner surface of alpha granules and are involved in the localization of fibrinogen to these granules. On resting platelets, α _IIb β ₃ exists in a folded, inactive state that does not interact with its ligand. However, upon platelet activation, an “inside-out” signaling event takes place in which the α _IIb β ₃ complex is activated and unfolded like a switch blade, thereby resulting in binding of fibrinogen at the N-terminals of the two proteins and platelet aggregation. After ligand binding, an “outside-in” signal mediates integrin-cytoskeleton interactions and platelet spreading.

Many integrin ligands contain an arginine-glycine–aspartic acid (RGD) motif that participates in integrin binding. Conversely, peptides containing RGD act as competitive inhibitors of ligand binding. For example, the RGD motif located in the C1 domain of VWF appears to be necessary for binding of VWF to α _IIb β ₃ . In contrast, deletion of the two RGD sequences in the fibrinogen α chain does not impair its ability to bind to α _IIb β ₃ . Rather, binding of fibrinogen to α _IIb β ₃ requires a KQAGD sequence located at the carboxyl-terminal of the fibrinogen γ chain.

Whereas expression of the α _IIb chain is restricted to megakaryocytes, the β ₃ subunit is expressed more widely as a component of the α _V β ₃ (vitronectin) receptor. Mouse studies have shown that deletion of α _V β ₃ function results in placental defects and osteosclerosis, although to date no differences have been noted between patients with GT caused by an α _IIb or β ₃ defect.

Clinical Features

Thrombasthenia is characterized by repeated mucocutaneous bleeding beginning at an early age ( Fig. 30-2, A ). Epistaxis and gastrointestinal bleeding are frequent issues in early childhood that often require intervention, as well as early iron supplementation. Menorrhagia is a critical problem in teenage girls. The bleeding that nor­mally accompanies pregnancy, surgical procedures, tooth extraction, or physical trauma can be excessive in GT patients. Unprovoked intracranial or gastrointestinal hemorrhage occurs and accounts for a significant por­tion of the observed 5% to 10% premature mortality rate. In addition, some patients experience joint bleeding or muscle hematomas more characteristic of the hemophilias.

Most frequent is recurrent and problematic epistaxis often complicated by prior nasal cauterizations that damage the local nasal architecture. Frequently, local compression strategies or topical applications of thrombin are useful when bleeding occurs. DDAVP (1-deamino-8-δ-arginine vasopressin) has been used as well, and oral contraceptive management of menorrhagia is generally sufficient. Platelet transfusions may be useful until resistance to platelet infusions develops, although patients do not necessarily form antibodies to the α _IIb β ₃ receptor. Antibodies may be particularly problematic because they may not only result in increased platelet clearance or platelet refractoriness but may also interfere with platelet function. Case reports and small case series suggest that recombinant activated factor VII may be a useful supplement to platelet transfusions. We have also successfully used embolization of arterioles feeding the nose and uterus in cases of recurrent, life-threatening hemorrhage.

Incidence

Although infrequent worldwide, GT is more prevalent among certain isolated populations and in the setting of consanguineous relationships, particularly among Arab populations, Iraqi Jews, French gypsies, and individuals from southern India. Heterozygotes for this disease have approximately 50% of the normal number of α _IIb β ₃ receptors, but generally no evidence of platelet dysfunction or clinically significant bleeding. Although most cases are observed in specific populations or in the setting of consanguinity and are thus homozygous for a shared mutation inherited from both parents, about one third of patients with identified mutations are compound heterozygotes.

Classification and Laboratory Diagnosis

In 1972, Jacques Caen classified GT according to platelet intracellular fibrinogen content and the ability of platelets to retract a fibrin clot. Type I patients, representing 80% of those studied, lacked platelet fibrinogen and had an absence of clot retraction. Type II thrombasthenic platelets contained appreciable levels of platelet fibrinogen and maintained some clot retraction capability. Soon thereafter, the technique of sodium dodecyl sulfate–polyacrylamide gel electrophoresis became widespread, and it became clear that there were three different patterns seen with thrombasthenic platelet membrane glycoproteins. Type I platelets lacked detectable levels of α _IIb β ₃ , whereas type II platelets expressed moderate (15% to 25%) levels of these glycoproteins. Adding to the complexity of this classification was the identification of variant forms of thrombasthenia characterized by normal to nearly normal levels of a dysfunctional form of α _IIb β ₃ (type III). Finally, a related integrin dysfunction syndrome was identified involving a GT-like platelet defect along with a white cell disorder termed leukocyte adhesion deficiency type III . These patients have a deficiency of Kindlin-3 (encoded by the FERMT3 gene on chromosome 11), an intracytoplasmic signaling molecule involved in the function of β ₂ - as well as β ₃ -based integrins. Patients have both GT-like bleeding manifestations and the infectious complications and leukocytosis of leukocyte adhesion deficiency.

GT patients fail to aggregate in response to physiologic agonists such as adenosine diphosphate (ADP), thrombin, or epinephrine (see Fig. 30-2, B ) because they all have a functional deficiency of platelet surface α _IIb β ₃ receptors. The failure to bind fibrinogen and other adhesive ligands is the reason for the inability of platelets to aggregate. No correlation exists between any of the proposed subtypes of GT and the severity of bleeding symptoms in patients. Some patients with no α _IIb β ₃ have relatively mild clinical symptoms, whereas others with a full complement of α _IIb β ₃ , albeit dysfunctional, can have frequent bleeding episodes requiring repeated transfusions.

Currently, the most common method used for determining levels of α _IIb β ₃ on thrombasthenic platelets involves flow cytometry and immunoblot analysis. Figure 30-2, C illustrates flow cytometric analysis of the α _IIb β ₃ content of both a normal control and a GT patient with a severe deficiency of α _IIb β ₃ secondary to a mutation in one of the Ca ²⁺ -binding domains of her α _IIb chain. This mutation blocks export of the α _IIb β ₃ receptor out of the endoplasmic reticulum and into the Golgi body for final processing, as indicated by failure to cleave the α _IIb prochain into an α _IIb heavy chain.

Several hundred individuals with GT have been described in the literature. To date, more than four hundred different mutations have been described. As in most genetic disorders, the molecular abnormalities have been found to range from major deletions and inversions to single point mutations. Some of the better-characterized subgroups of mutations are described in the following text.

The earliest and largest group of thrombasthenic mutations described failed to express α _IIb β ₃ on the cell surface. Several of these patients had major deletions or inversions in their α _IIb or β ₃ genes. In addition, a number of point mutations or small deletions in the α _IIb or β ₃ genes have been described with no surface expression of α _IIb β ₃ ; both chains are formed intracellularly and may assemble the complex but fail to undergo intracellular processing or reach the cell surface. These defects result in the type 1 form of thrombasthenia. One major subgroup of these patients involves missense mutations in the N-terminal β-propeller repeats of α _IIb near the proposed Ca ²⁺ -binding sites on its lower surface as exemplified by the patient in Figure 30-2 .

Another group of mutations occur in the same β-propeller region of α _IIb but take place on the upper surface and have expressed surface receptor that cannot bind its ligand. These mutations are located within three upper loops of the β-propeller, the connecting loop between the second and third blades, the loop in the middle of the third blade, and the loop connecting the third and fourth blades. These mutations occur within the pocket of α _IIb that binds RGD ligand.

Other thrombasthenic patients with significant levels of nonfunctional α _IIb β ₃ surface expression have mutations that involve the β ₃ chain. These mutations result in a surface α _IIb β ₃ that is easily dissociable by chelation of external calcium ions. Many of these missense mutations have been identified within the cation-binding MIDAS domain. The importance of these sites was reinforced by the identification of a group of in vitro–generated mutant α _IIb β ₃ receptors expressed in Chinese hamster ovary cells, which provided independent support for the importance of the MIDAS domain in ligand binding.

A second group of dysfunctional mutations with normal levels of surface α _IIb β ₃ receptors are localized to the cytoplasmic domain of β ₃ , thus demonstrating the importance of this domain for integrin activation and regulation of ligand binding. These mutations do not affect surface expression of platelet α _IIb β ₃ complexes, but mutant receptors are unresponsive to agonist stimulation. These mutations provide compelling evidence for the role of the β ₃ cytoplasmic tail in downstream platelet activation by α _IIb β ₃ .

Biology of the Glycoprotein Ib/IX Complex

Adhesion through the GPIb/IX complex involves binding of VWF to the subendothelium. Platelet membranes contain two binding sites for VWF (see Table 30-1 ). One of these sites requires previous platelet activation and is located on the platelet membrane α _IIb β ₃ complex. The second binding site involves the GPIb/IX complex, and it is this membrane complex that is crucial for initial attachment and proper adhesion to the extracellular matrix of a damaged vessel wall. It should be pointed out that the GPIb/IX complex may also bind to other ligands, including P-selectin, thrombospondin-1, high-molecular-weight kininogen, and Mac-1. Furthermore, GPIb/IX also binds thrombin, and its role in the activation of platelets by thrombin is discussed later.

The GPIb/IX complex is the second most abundant receptor on the platelet membrane surface, with approximately 25,000 copies per platelet. This complex actually consists of four different proteins ( Fig. 30-3 ), all of which have one or more leucine-rich repeats composed of a 24–amino acid motif with 7 conserved leucine residues. Other proteins have been described with this leucine-rich repeat, and these regions mediate ligand binding.

GPIbα is the largest subunit (135 kD, 610 amino acids, chromosome 17p12) and has 7 leucine repeats. It is susceptible to cleavage by trypsin or calpain, which gives rise to a heavily glycosylated 135-kD fragment known as glycocalicin . In addition to containing the binding site for VWF, the glycocalicin portion of GPIbα also binds thrombin. Under normal flow in vivo, plasma VWF does not bind to the GPIb complex, but under shear stress conditions, VWF simultaneously binds to collagen and the GPIb/IX complex. In clinical assays, the antibiotic ristocetin or the venom-derived protein botrocetin is used to mimic this effect by inducing conformational changes in VWF that promote binding to GPIb/IX in stirred platelet-rich plasma.

GPIbα is disulfide-bonded to GPIbβ (25 kD, 181 amino acids, chromosome 22q11.2) through a single cysteine residue located in each subunit near the transmembrane domains of GPIbα and GPIbβ (see Fig. 30-3 ). This peptide has only one leucine repeat. The cytoplasmic tail of GPIbβ contains a filamin-binding site that links the receptor complex to F-actin below the membrane and a binding site for 14-3-3ζ. The disulfide-bound GPIbα-β is noncovalently associated with platelet GPIX and GPV. GPIX is the smallest member of the GPIb complex (22 kD, 160 amino acids, chromosome 3q29), with only one leucine repeat. GPV (82 kD, 344 amino acids, chromosome 3q21) is a transmembrane protein with 15 leucine repeats. This protein is a proteolytic substrate for thrombin and releases a 69-kD soluble fragment. GPV is present in only a single copy per complex, whereas there are two copies of the other three subunits in a single receptor (see Fig. 30-3 ). Furthermore, cleavage of the GPV extracellular domain by thrombin bound to GPIbα activates platelets. Consistent with this observation, the GPV-knockout mouse has a mild prothrombotic state.

Binding of activated VWF to the GPIb/IX complex activates platelets through activation of phospholipase C (PLC) and mobilization of protein kinase C, which, together with increases in [Ca ²⁺ ] _i , promotes platelet secretion and potentiates platelet aggregation. The GPIb-IX complex also appears to activate the cell by binding to the cytoskeleton protein 14-3-3ζ and by interacting with the FcγRIIA receptor on platelets, where it activates an intracellular tyrosine–based activation motif receptor (see Fig. 30-3 ).