Platelet Glycoprotein VI: An Adenine Insertion in Exon 6 Generates a Truncated Form of the Protein Associated with Bleeding Disorder in 4 Chilean Unrelated Patients

Abstract 3302

Glycoprotein VI (GPVI) is a trans-membrane collagen receptor expressed in blood platelets and megakaryocytes. Collagen, a major extracellular matrix constituent is a distinctive platelet ligand that mediates platelet adhesion and triggers platelet activation. Thus, GPVI has a central role in platelet adhesion and signal transduction. GPVI is a 64kDa member of the immunoglobulin receptor family, with an extracellular portion of 247 Aa harboring two immunoglobulin-type domains, followed by a mucin-like, presumably O-glycosylated Ser-Thr-rich region, a short peptide linker sequence, a 21 Aa trans-membrane domain and a short 51 Aa cytoplasmic tail. This receptor is also activated by laminin, collagen-related peptide (CRP) and snake toxins, i.e., convulxin and alborrhagin. Inherited defects of major platelet GP′s, e.g, α_IIbβ₃ integrin or GPIb-IX-V complex cause severe disorders of primary hemostasis, namely Glanzmann thrombasthenia and Bernard-Soulier syndrome, respectively. Another collagen receptor in platelets is integrin α₂β₁, with scarce reports of molecular defects clinically expressed as mild bleeding disorder. Acquired platelet GPVI defects, characterized by absent platelet aggregation with collagen, have been described in patients with immune thrombocytopenia, in whom autoantibodies are directed against GPVI causing shedding of the receptor from the platelet membrane. Two recent reports have described single patients with abnormal bleeding associated with compound heterozygous mutations of GPVI. In the first case, the patient is compound heterozygous for an out-of-frame 16-bp deletion and a missense mutation S175N in a highly conserved residue of the 2nd Ig-like GPVI domain. GPVI DNA sequencing of the second patient also showed 2 genetic abnormalities, an R38C mutation in exon 3 of one allele and an insertion of 5 nucleotides in exon 4 of the other allele, leading to a premature nonsense codon and absence of the corresponding mRNA.

Now we report 4 unrelated Chilean patients living in different places along the country, with a unique GPVI mutation clinically manifested as mild or moderate bleeding disorders. All the patients suffered from mucous and skin bleeding and one of them had received platelet transfusions to treat heavy menorrhagias. All these patients had null platelet aggregation and ¹⁴C-serotonin secretion when stimulated with collagen types I and II, convulxin and collagen-related peptides (CRP). Platelet responses to arachidonate and ADP were normal, and 2 of these patients had only first-wave aggregation with epinephrine. The mutation in these patients consists in an homozygous adenine insertion at position 712 of the cDNA, which causes a translation frameshift, creating a premature stop codon which predicts a truncated form of the protein. The mutation detected in the patient's gDNA was reflected in their platelet mRNA. The sequencing of the RT-PCR products obtained from platelets of homozygous carriers of the mutation showed the adenine insertion as well. Heterozygous relatives had no bleeding disorder, and normal aggregation and ¹⁴C-serotonin secretion with collagen and convulxin, but decreased aggregation with CRP. Flow cytometry and immunofluorescence-confocal microscopy revealed absence of membrane GPVI in the 4 homozygous carriers of the mutation, whereas heterozygous relatives exhibited a decreased expression of the GP on their platelets.

Antibodies recognizing the extracellular portion of the receptor were used for Western blots of platelet membrane fractions or platelet lysates. Blots showed a single band of approximately 49 KDa in patients, whereas heterozygous relatives expressed both the normal 64kDa integral receptor and the abnormal protein band. The size of the truncated protein was that predicted by the mutation. This is the first report describing a point mutation in the GPVI gene with functional and clinical consequences. The collection of 4 of these non-related patients suggests that this defect may be more common than previously suspected.