Introduction

The prevalence of thrombotic diseases in children has increased as the molecular processes involved in their pathophysiology have been better characterized. Several inherited disorders are described as triggers of these diseases, including deficiencies of the fibrinolytic system and anticoagulation factors, metabolic alterations, hemoglobinopathies, platelet hyperaggregation and various acquired autoimmune diseases, endocrine disorders, neoplastic diseases, infections, and chronic diseases (kidney, liver, among others.)¹

After the discovery of platelets and the description of their physiological function, research on these cells has focused predominantly on bleeding disorders² and they are now known to have multiple biological functions involved in hemorrhagic and thrombotic phenomena;³ however, it should be noted that platelet-associated thrombotic events are considered rare.⁴

Sticky platelet syndrome (SPS) is a familial thrombophilic condition of autosomal dominant inheritance characterized by an increase in platelet aggregation leading to arterial and venous thrombosis. SPS was first described by Holliday et al.⁵ at the Ninth International Joint Conference on Stroke and Cerebral Circulation in 1983, and later, in 1986, Rubenfire et al.⁶ published the first study describing patients with cardiovascular thrombotic events related to platelet hyperaggregation.

Then, in 1988, in a study describing cerebrovascular thrombotic events, Mammen et al.⁷ established the first two criteria for identifying SPS in venous blood samples, using sodium citrate as anticoagulant and three concentrations of two agonists: 1) tests with adenosine diphosphate (ADP) at concentrations of 2.34uM, 1.17uM and 0.58uM, with which aggregation ranges of 7.5-55%, 2-36% and 0-12%, respectively, were obtained in control subjects, and 2) tests with epinephrine at concentrations of 11uM, 1.1uM and 0.55uM, with which aggregation ranges of 39-80%, 15-27% and 9-20%, respectively, were obtained in control subjects. Furthermore, the researchers found that in patients with SPS, these values were significantly increased. Subsequently, Mammen⁸ described various thrombotic disorders related to platelet hyperaggregation in adults and in some pediatric cases; in addition, the diagnostic parameters of the SPS described above were confirmed in this study.

Finally, Bick⁹ published a study of 153 adults residing in Dallas, USA, who presented with thrombotic events, in which they found that 33% of participants met the diagnostic criteria for SPS. Based on the characteristics of these patients, the author recognized three types of the disease:

Bick⁹also established that the diagnosis is suggestive of SPS when, besides thrombosis, there is hyperaggregation along with a concentration of ADP or epinephrine higher than the normal ranges previously described in only one of the tests and that this is confirmed with one of the following possibilities: a) history of thrombotic disorder and presence of hyperaggregation with two concentrations of one of the two agonist reagents (ADP or epinephrine); b) history of thrombotic events and platelet hyperaggregation with one concentration of the two agonist reagents; and c) history of previous thrombosis and presence of hyperaggregation, with the same concentration of one of the agonists, that persists when the test is repeated.

The first case of SPS reported in Colombia in pediatrics was described in 2002 by Beltran et al.,¹⁰ who also stated that this entity is an important cause of thrombotic disorders during childhood. Later, also in Colombia, Velasquez et al.¹¹ published a series of cases of pregnant women diagnosed with SPS, in which they concluded that it is an unknown disorder despite being one of the most frequent causes of thrombotic events. Similarly, studies related to platelet hyperaggregation syndromes have been carried out in several countries, finding that the same inherited phenotype associated with SPS can be observed in certain metabolic conditions such as diabetes mellitus and atherosclerosis, or in some inflammatory and infectious diseases.^12-15

Other research, such as Kubisz et al.¹⁶ suggest that some thrombotic events without known etiology may be caused by SPS. Moreover, genetic studies have reported mutations and polymorphisms in the genes of the glycoprotein VI found in the platelet membrane, integrin beta 3 sub-unit, suggesting that variability in these genes may be related to SPS, although no significant differences have been found between patients with and without the disease.^17,18

Recent studies relate different mechanisms of hemostasis to activity-dependent intracellular calcium initiated by cell membrane receptors, which are activated by intracellular signals and modulated by calcium signaling receptors located on the membrane of the endoplasmic reticulum, with positive or negative feedback on receptors located on the cell membrane.^19,20

Furthermore, Barros-Garcia et al.,²¹ after analyzing the exome in an index case with SPS, identified a molecular alteration consisting of the deletion of 5 nucleotides of the ORAI1 gene (located in the long arm of chromosome 12), which resulted in an erroneous reading in protein synthesis. These authors also described that the protein encoded by this gene is the calcium channel subunit of the membrane activated by the calcium sensor STIM1 when calcium reserves are depleted in the cell.²¹

Similarly, although progress has been made in the understanding of platelet hyperaggregation in pediatric patients, many aspects remain to be clarified. Next-generation sequencing of the exome could be useful for this purpose since it has been possible to identify strategies to detect the genetic causes of different hereditary diseases.

Exome sequencing is a test used to identify DNA variants that cause genetic disease. It has been established that most known variants that cause disease occur within 1% of the genome containing exons and at the cleavage and splice junctions; therefore, studying this part of the genome is an effective and powerful clinical diagnostic tool in personalized medicine.^22-24 Thus, the objectives of this study were to identify common mutations by sequencing the exome in two patients from the same family diagnosed with SPS and, consequently, contribute to the molecular study of this disease.

Materials and methods

Descriptive study in which exome sequencing was performed on a pediatric patient (10-year-old index case at the time of the study) and in one of his first-degree adult relatives (mother). Both the index case and two of his relatives (mother and sister) had a history of thrombotic disease and had been diagnosed with SPS.

Results

The coverage data for the sequenced region of the samples taken from the patient and his relative are shown in Table 1:

Table 1 Coverage data for the sequenced region. 

Coverage: number of times the analyzed sequence is found.

* Average readings found in sequencing.

† Fraction of unidentified reading frames.

‡ Percentage of the target bases achieving that or greater coverage.

Source: Own elaboration.

Variants present in 176 candidate genes recorded in a list created by the authors include 42 genes possibly related to SPS and platelet aggregation. The number of variants resulting from sequencing is shown in Table 2:

Table 2 Number of variants resulting from sequencing. 

Source: Own elaboration.

Of the total number of variants of each type found, both in the patient and in his relative, 495 were observed in both participants, and a common variant related to platelet function was identified. Table 3 shows the variant resulting from the prioritization process of the participants' samples.

Table 3 Variant resulting from the prioritization process of patient and relative samples. 

Gene: gene involved; Exon: corresponding exon number; Chr: chromosome number; Point mutation: nucleotide that changed (cytosine to thymine); AA mutation: amino acid replaced (threonine to isoleucine).

Source: Own elaboration based on Richards et al.²⁸

The common variant identified in the index case and his relative is a cytosine to thymine transition at position c.236 (NM_000174.4) that causes the amino acid substitution from threonine to isoleucine at position p.79 of the extracellular domains of the leucine-rich repeat receptor (GPIba subunit of the glycoprotein Ib-IX-V complex), which could cause the conformational change in the main receptor of the Iba ligand, as well as platelet hyperaggregation and thrombosis. This variant has not been previously associated with SPS, although Alsahafi et al.²⁹ had already described mutations in this gene associated with other disorders.

Figure 1 presents the report generated by the Alamut Visual software for the variant found in the participants, which provides more data on a possible association between it and SPS. Figure 2 shows the cytosine to thymine transition in the samples from both the index case and his relative.

Source: Taken from the report generated by the Almut Visual software.

Figure 1 Report of the common variant found in the participants generated by the Alamut Visual software.  

Source: Document obtained during the course of the study.

Figure 2 Image of the genetic variant in the two samples analyzed using the IGV genomic viewer. Note: the image identifies the exact location of the string (at 41 bp).  

These results demonstrate the coincidences of molecular tests performed on both the patient and his relative.

Discussion

Platelet adhesion, activation, and aggregation are key events in the processes of hemostasis and thrombosis, in which platelet adhesion molecules, such as the GPIb-IX-V complex, are essential.

Diseases that cause an increase in platelet function may lead to thrombotic events, such as strokes, which can kill more people than respiratory diseases and cancer.

Antiplatelet agents such as aspirin and clopidogrel, which block thromboxane and ADP receptors, are vital for the treatment of thrombosis; however, thrombotic events persist in some patients despite treatment with these drugs, making it necessary to look for other effective activation pathways. Therapies targeting other ligands may also be considered in this process.³⁰

Brass et al.³¹ cite previous studies on physiological and genetic analyzes of platelet aggregation (normal and hyperaggregation states), including reports of possible genetic polymorphisms initiated by external and internal platelet stimuli, in which a relationship has been established between this process and membrane receptors of different structure, such as integrins, glycoproteins and ion channels with activation of metabolic calcium storage sequences.

The results of the present research showed a cytosine to thymine transition at position c.236 (NM_000174.4) that causes the substitution of the extracellular domains of the leucine-rich repeat receptor (GPIba subunit of the GPIb-IX-V complex) and that could also cause the conformational change of the main receptor of the Iba ligand, as well as platelet hyperaggregation and thrombosis.

It has also been described that the extracellular, transmembrane, and intracytoplasmic domains in the platelet membrane of the Ib-IX complex act as receptors responding to low thrombin concentrations and von Willebrand factor in inflammatory and infectious processes resulting in thrombus formation.³²

Likewise, Gardiner³³ reported that the Iba subunit is the receptor for the different ligands and acts through the N-terminal leucine-rich repeats (LRR), generating intracellular signals by itself, although binding to subunit IX promotes a fundamental conformational change to amplify these signals in thrombus formation. By the same token, Estevez et al.³⁴ demonstrated that, by binding to thrombin, the amplification of activation in the intracellular signal pathways mediated by GPIba and GP Ibp, and GPIX is synergized by protease-activated receptors, which also act as primary receptors for thrombin and which, in inflammatory states, increase their density in the platelet membrane, increasing the possibility of thrombosis

It is important to note that intracellular mediators such as G, Racl, LIMK1 and Syk proteins, and even other tyrosine kinases enzymes such as Toll-like receptors, originated by the binding of low thrombin concentrations and the platelet von Willebrand factor to the Ib-IX-V subunit of the platelet membrane and amplified in inflammatory or infectious states, are shared by other types of platelet receptors such as ADP, thromboxane, and fibrinogen to generate thrombosis. At this point, it should be noted that the binding of thrombin to the GPIb-IX-V complex is not continuous and ends at the catalytic site of glycoprotein V, and that states of recurrent thrombosis have been observed in genetic models of absence of function of the V-ATPase subunit.^35,36

It is evident that other platelet receptors should be explored given the importance of platelet activation; the relationship of platelets to inflammatory and infectious phenomena, angiogenesis, and recurrent thrombotic events despite the use of antiplatelet agents; and bleeding phenomena as side effects.³⁷

The results obtained in the present study have not been previously reported in the literature but may be related to SPS considering the importance of glycoprotein IX in platelet functions. In this sense, it is evident that research on blockers or modulators of the GPIb-IX-V-mediated agonist response as thrombosis generators is needed.³⁰ The main limitation of the present study is that it was carried out only in two patients diagnosed with SPS (index case and his relative); however, as a strength, this is the first time that a possible pathological variant of the disease has been described in Colombia by exome analysis.

Conclusions

In this study, it was established that both patients analyzed presented with a genetic variant consisting of a cytosine to thymine transition at position c.236 (NM_000174.4) that caused the substitution of threonine to isoleucine at position p.79 of the leucine-rich repetitive extracellular domain (GPIba subunit of the GPIb-IX-V complex). This variant has not been described previously as a finding associated with SPS, but it could be related considering the importance of glycoprotein IX in platelet functions.

In this sense, platelet hyperaggregation in SPS may not be caused by a specific mutation, but rather by the expression of several genetic polymorphisms found in patients with this disorder.

The results of the present study are highly relevant; however, further research is needed to establish the foundations of much more specific and effective treatments for the management of platelet hyperaggregation events such as SPS.