Absent Collagen-Induced Platelet Activation in a Patient Double Heterozygous for Two GPVI Mutations

The physiological role of platelet GPVI is to initiate thrombus formation in haemostasis. A few patients with GPVI-related defects have been identified. In most cases, a defect in GPVI expression and function is associated to an immunological abnormality with evidence for antibody-induced shedding of the GPVI extracellular domain or/and GPVI-internalisation. In a few cases of GPVI dysfunction without GPVI deficiency the abnormality was attributed to deficient intracellular signals. Remarkably, only one patient with a congenital GPVI deficiency and a proven gene mutation has so far been reported (

). Here, we describe a 10 years old female presenting a mild bleeding tendency with only ecchymoses since infancy. She has a prolonged bleeding time (>15 min), a normal platelet count and no morphological abnormalities on blood smears. Coagulation and VWF studies are normal. The PFA100 closure time is prolonged on collagen/epinephrine (= 210 s.) but normal on collagen/ADP cartridges. Platelet aggregation is normal in response to usual agonists except collagen: aggregation is absent in response to Horm collagen up to 4 μg/mL both in PRP and washed platelets. In addition, thrombin generation measured by thrombinography on collagen-activated PRP is profoundly impaired. There is no evidence in favour of an antiplatelet antibody and the patient’s parents do not present bleeding tendency and their platelet aggregation is normal. GPVI expression and function was analysed. Flow cytometry shows a 50% decreased expression of GPVI while other platelet membrane glycoproteins are normally expressed. Platelets aggregates in response to convulxin, a GPVI-specific snake venom protein, but with a decreased sensitivity for washed platelets, suggesting a GPVI dysfunction. GPVI is co-expressed with the signalling Fc receptor g-chain (FcRg) and associated deficiencies in GPVI and FcRg have been reported. GPVI and FcRg expression were analysed by immunoblot. Using monoclonal antibodies, GPVI is undetectable in platelet extracts whereas a low intensity doublet at 58 and 55 kDa is detected using a polyclonal antibody suggesting quantitative and qualitative GPVI abnormalities. The band corresponding to FcRg is of comparable intensity in platelets extracts from the patient and a normal subject. To get further insight into the GPVI defect, the GP6 gene was sequenced. The eight exons and flanking regions were amplified from genomic DNA. The patient was homozygous for the “a” allele of the common GPVI haplotype (SKTH/PEALN). Two sequence abnormalities were identified: one substitution in the 3^rd exon (C172T) resulting in a missense mutation R58C and duplication of 5 nucleotides in exon 4 producing a frame shift with the appearance of a nonsense codon 30 bases downstream. RT-PCR analysis of platelet mRNA showed that only the allele bearing the C172T substitution was present suggesting that the mRNA with a premature nonsense codon was unstable. These data indicate that the patient is heterozygous for 2 GPVI abnormalities: the first results in a premature nonsense codon that presumably leads to nonsense mediated mRNA decay explaining the observed 50% deficiency in GPVI. The second leads to a severe modification of one residue, R58, in the 1^st Ig-like domain of GPVI known to play a critical role in collagen binding but a minor role in convulxin binding. The Arg/Cys substitution could produce a miss-paired disulfide bridge and a large conformational change. The production of the mutant recombinant GPVI is ongoing. This is the first case of a patient with two genetic GPVI defect resulting in impaired platelet functions and a mild bleeding disorder.