3.1. Large Platelets and Thrombocytopenia 3.1.1. Bernard-Soulier Syndrome
Bernard-Soulier syndrome (BSS) is a widely studied, albeit rare, disorder whose incidence is approximately less than one in one million. (2, 21, 22). This autosomal recessive genetic disorder is characterized by the presence of abnormally large platelets seen on peripheral blood smears, mild thrombocytopenia, and prolonged bleeding time. The hallmark of BSS is a defect or congenital deficiency of the platelet glycoprotein Ib/V/IX complex (GPIb/V/IX). GPIb is the receptor for von Willebrand factor (vWF) whereby platelets adhere to the exposed vascular endothelium, and also binds thrombin (2, 21-23).
The GPIb/IX/V complex is composed of four transmembrane polypeptide sub-units. GPIb consists of disulfide-linked a and p subunits (GPIba and GPIb^). GPIX and GPV are noncovalently associated subunits (21). A short 24-amino acid motif containing highly conserved leucine residues is found in all four polypeptide subunits of the GPIb/IX/V complex (24, 25). These leucine-rich repeats (LRRs) might have a critical role in the assembly of the receptor. Indeed, several mutations found in BSS have been located in the LRRs of the GPIba and GPIX genes (24). BSS arises from a heterogeneous array of genetic defects leading to decreased or absent glycoprotein expression on the platelet surface. The disorder can be characterized broadly by localization of the defect to the GPIba gene (chromosome 17ptr-p12), the GPIb^ gene (chromosome 22q11.2), and the GPIX gene (chromosome 3) (24, 26). Several point mutations in the GPIba coding sequence resulting in BSS have been reported (27, 28). In addition, a di-nucleotide deletion in the GPIba gene resulting in a deficiency of GPIba on the surface of the platelet membrane was reported in one patient who was homozy-gous for this deletion mutation (29). Deficiency of GPIba could also arise from a deletion in a chromosomal region (22q11.2), causing BSS (30). Thus, while many mutations in the GPIba gene have been reported since the initial report by Nurden and co-workers (31), only five mutations in the GPIX gene, one in the GPIb^ gene, and none in the GPV gene have been reported (31-33). Apparently, the GPV gene is not essential for the expression of a functional GPIb/IX complex (32).
BSS is characterized by a long bleeding time and defective platelet aggregation in response to ristocetin. A variant form of BSS in which the GPIb/V/IX complex is present, but platelets nonetheless demonstrate impairment of ristocetin-induced aggregation (34), has been described. A point mutation in the GPIba leucine tandem repeat region was noted in this variant form (34).
The severity of thrombocytopenia in BSS is variable, with platelet counts ranging from less than 30 to as much as 200 x 103 ^L (<30 to 2000 x 109/L). The platelet lifespan is reduced compared to normal. Clot retraction as well as platelet aggregation in response to agents such as collagen, arachidonic acid, ADP, epi-nephrine, and thrombin is normal (21, 24). The bleeding time, however, is prolonged. Ristocetin-induced platelet aggregation, as noted earlier, is reduced or absent due to abnormalities in the platelet GPIb/V/X complex (34). The laboratory findings in BSS correlate with molecular defects affecting the expression or function of the platelet GPIb/V/IX complex (21, 35).
In addition, ultrastructural changes have been observed in megakaryocytes of patients with BSS, including an irregularly spaced demarcation membrane system (DMS) and disorganization of microtubules (21, 36). Ultrastructural changes in platelets impart a Swiss-cheese appearance to some of the BSS platelets (37). These changes are strikingly prominent and are reflected in an increase in the number of the surface-connected, dense tubular systems (21, 37). Apparently, normal megakaryocyte maturation and platelet size are dependent on a functional GPIb/IX complex (38). The reduced platelet lifespan and, in turn, the thrombocytopenia seen in BSS may be related to decreased platelet-surface sialic acid expression due to decreased expression of the sialic acid-rich GPIb subunit (39).
Markedly severe thrombocytopenia with platelet counts in the range of 5-40 x 103/^L (5-40 x 109/L) has been reported in a condition called Montreal platelet syndrome (MPS), which has been reported in three generations of Canadian families (40). In addition, patients have prolonged bleeding time and display spontaneous platelet aggregation with reduced response to thrombin (41) and normal platelet membrane glycoproteins. The calcium-activated neutral proteinase, calpain, which is normally involved in the cleavage of the cytoskeleton proteins, especially the actin-binding protein and talin, may be responsible for spontaneous platelet aggregation by exposure of platelet binding sites to adhesive proteins (41). However, the exact mechanism of the relationship between spontaneous platelet aggregation and severe thrombocytopenia seen in MPS remains to be established.
Lack of alpha granules in platelets results in a condition called gray platelet syndrome (42, 43). Patients have variable degrees of thrombocytopenia, with platelet counts ranging from as low as 20 x 103/^L (20 x 109/L) to normal values. Patients also have a prolonged bleeding time, and large agranular platelets, grayish to gray-blue in appearance, are seen in the peripheral blood smear stained with Wright-Giemsa stain (21). Apparently, there is a defect in megakaryocytes interfering with the ability to package the constituents commonly seen in alpha granules such as thrombospondin, platelet factor 4, vWF, beta thromboglobulin, and fibrinogen (44). Although, dense granule contents such as ADP and serotonin are normal, their release following platelet activation with collagen or thrombin is reduced to varying degrees, suggesting that alpha granules may be involved in modulating the release of dense granule contents (45-48).
Ultrastructural changes and giant platelets are seen in May-Hegglin anomaly. An alteration in the platelet microtubule system affects platelet structure, although platelet aggregation and interaction with vWF is normal. The lack of platelet spreading and defective pseudopod formation seen in this condition can be explained by the fact that the platelet microtubules play a critical role in maintaining the discoid form of platelets and controlling platelet shape and pseudopod formation (21,49).
A characteristic of the May-Hegglin anomaly is the presence in platelets (stained with Wright-Giemsa) of neutrophil inclusions called Dohle bodies, which are spindle-shaped and appear bright blue. These inclusions are found in the periphery of the cytoplasm (50). Thrombocytopenia is mild to moderate, with platelet counts in the range of 60-100 x 103/^L (60-100 x 109/L). With some exceptions, most patients have a mild bleeding tendency, with the amount of bleeding dependent on the extent of thrombocytopenia (21). Myocardial infarction due to coronary artery thrombosis has been reported (51). Large platelets may compensate for thrombocytopenia in terms of hemostasis (52).
This is another condition in which cytoplasmic inclusions are noticeable in platelets on Wright-Giemsa stained peripheral blood smears. Fechtner syndrome is also characterized by renal disease (nephritis) and deafness (53). The cyto-plasmic inclusions appear smaller and pale blue in contrast to those seen in the May-Hegglin anomaly. Large platelets are seen in Fechtner syndrome, with thrombocytopenia in the range of 30-90 x 103/^L (30-90 x 109/L) (21). Ultrastructural changes are seen in megakaryocytes characterized by abnormal accumulation of demarcation membranes, leading to ineffective platelet production and resulting thrombocytopenia (54).
Thus, a spectrum of molecular defects in platelets can lead to the production of giant platelets with varying degrees of thrombocytopenia. Platelet glycoprotein complexes such as GPIb/IX complex are crucial in ensuring normal megakaryocyte maturation and regulating platelet size. As noted earlier, the lack of GPIb/IX complex leads to the production of giant platelets in Bernard-Soulier syndrome. In contrast, a deficiency of GPIIb/IIIa complex does not result in the production of giant platelets as noted in Glanzmann's disease (55).
Table 5 lists causes of platelet pathology.
3.2. Therapeutic Approaches to Treat Severe Thrombocytopenia
While platelet transfusion is effective in replacing platelets, high-dose recombinant factor VIIa therapy has recently been used to correct bleeding in patients with severe thrombocytopenia, as well as congenital platelet function defects (56). The mechanism of action of factor VIIa in this setting is under investigation. Conceivably, VIIa may act on platelets in the absence of tissue factor to increase thrombin generation by activating factors IX and X. This production of thrombin on the surface of a few platelets at the site of vessel wall injury may result in production of fibrin, which could recruit additional platelets either directly or indirectly via interaction with vWF. The efficacy of factor VIIa in the treatment of bleeding resulting from intrinsic platelet defects requires further investigation.
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