PTGS1 compound heterozygosity impairs gene expression and platelet aggregation and is associated with severe bleeding complications.

Dear Sirs, The prostaglandin endoperoxide synthase 1 (PTGS1, COX1) that is deposited in the Online Mendelian Inheritance in Man (OMIM) database (accession no. 176805) catalyses the conversion of arachidonic acid (AA) into prostaglandin H2 intermediates and thromboxane A2 (TxA2). Polymorphisms of genes involved in AA metabolism are potential modifiers in platelet function (1-4). The functional consequences of most allelic variants, in terms of their influence on basal PTGS1-mediated AA metabolism and bleeding risk, are not well understood (5). We here describe a 36-year-old male German patient without bleeding history prior surgical interventions but with postoperative recurrent disseminated bleeding complications after hemorrhoidectomy and atherectomy. The patient was neither on any medication nor physiotherapeutica and there was no known history of bleeding disorder. Prompt and detailed coagulation studies were performed that showed normal platelet count (mean: 187 x109/l), normal PT value (111 %), normal aPTT (38 seconds [sec]) and a normal fibrinogen concentration (mean 267 mg/dl). The lupus-sensitive PTT and dilute Russell’s viper venom time (dRVVT) revealed no pathological findings (PTT-LA 31.7 sec; dRVVT 0.9). Broad factor assay analysis was performed to rule out a clotting factor deficiency (all results revealed normal activities between 68 % 119 %), including Ristocetin Cofactor (51 %), von Willebrand factor antigen (57 %) and normal von Willebrand multimer patterns. Initial PFA-100® test revealed significant impaired platelet function compared to healthy controls (collagen/ADP: 150 sec vs 83 ± 21 sec; p=0.161 and collagen/epinephrine 226 sec vs 126 ± 44 sec; p=0.016; unpaired t-test). Moreover, different platelet function tests such as light transmission aggregation (AA (1 mM): 7 % vs 96 ± 15 %, p<0.001; epinephrine (50 μM): 11 % vs 84 ± 15 %, p<0.014; ADP (10 μM): 47 % vs 81 ± 13 %; p=0.016; collagen (1.9 mg/ml) and ristocetin (1.5 mg/ml): 81% and 93%; not significant) and platelet impedance aggregation in whole blood (AA (0.5 mM): 893 AU*min vs 1,215 ± 40 AU*min, p=0.013; ADP (5 μM): 384 AU*min vs 1,087 ± 16 AU*min, p=0.047) showed pathological platelet function (6, 7). Normal aggregation to the TxA2 analogue U46619 (980 AU*min vs 1,180 ± 228 AU*min, p=0.279) suggested no TxA2-receptor defect. In summary, the patient had an aspirin-like defect (ALD) phenotype which could indicate a defect in the AA metabolism (8). The patient provided written consent to perform DNA studies on his blood samples. DNA was extracted from peripheral blood leukocytes using a DNA extraction kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. The entire coding regions of the ALD-relevant genes PTGS1, TBXAS1, TBXA2R and P2RY12 were sequenced according to standard procedures. PCR with sequence-specific primers (PCR-SSP) for genotyping the PTGS1 22C>T and 50C>T single nucleotide polymorphisms (SNPs) was performed using standard protocols as described before (9) and primers as follows: forward PTGS1-22C 5’-tgcagggagtctcttgctcc-3’; forward PTGS1-22T 5’-ctgcagggagtctcttgctct-3’; reverse PTGS1-50C 5’-aggacggggagcggcg-3’; reverse PTGS1-50T 5’-. caggacggggagcggca-3’; internal control (HBB) forward 5’-ggttggccaatctactcccagg-3’, reverse 5’-gctcactcagtgtggcaaag-3’. PCR products were separated on 3% agarose gels. Molecular DNA analysis of the ALD relevant genes in the patient revealed wildtype sequence of the TBXAS1 gene. The TBXA2R and P2RY12 genes were homozygous for the minor 1026A (silent; rs5757) and 18T (silent; rs6785930) variants, respectively, and showed no further alteration. For the PTGS1 gene we found heterozygosis of two known SNPs 22C>T (R8W; rs1236913) and 50C>T (P17L; rs3842787). These SNPs are located in the coding region and affect both alternative spliced variants of the PTGS1 gene that were reported previously (10). Using PCRSSP for both SNPs we could confirm the compound heterozygous genotype and exclude a transconstellation of the 22T and 50T variants (▶ Figure 1 A). However, in vitro function studies showed no significant effect on the PTGS1 metabolic activity for the two variants (1). Gene expression analyses were then focused on the PTGS1 mRNA levels in platelets of the patient and of healthy controls with different PTGS1 genotypes. Total RNA was extracted from leukocyte-depleted platelets as described before (11). Relative quantification of PTGS1, TBXAS1, TBXA2R and P2RY12 gene expression was done by reverse transcriptase real-time polymerase chain reaction (qRT-PCR) using Universal Probe assays and a LightCycler 480 system (Roche, Basel, Switzerland). All mRNA levels were quantified relative to the levels of the reference genes GAPDH and YWHAZ using the deltaCp method. Primers and probes for qRT-PCR were: PTGS1 forward 5’-tccatgttggtggactatgg-3’, reverse 5’-gtggtccatgttcctgcc-3’ PTGS1 compound heterozygosity impairs gene expression and platelet aggregation and is associated with severe bleeding complications