The results of these experiments supported the predictions that the variant allele caused differential RNA splicing. Two single nucleotide variations were detected inF2R; a novel c. -67G> C transversion which reducedF2Rtranscriptional activity and a rare c. 1063C> T transition predicting a p. L355F substitution which did not interfere with PAR1 expression or signalling. Two synonymous single nucleotide variations were identified inF2RL3(c. 402C> G, p. A134 =; c. 1029 G> C p. V343 =), both of which introduced less commonly used codons and were predicted to be deleterious, though neither of them affected PAR4 receptor expression. A third single nucleotide variation inF2RL3(c. 65 C> A; p. T22N) was co-inherited with a synonymous single nucleotide variance inTBXA2R(c. 6680 C> T, p. S218 =). Expression and signalling of the p. T22N PAR4 variant was similar to wild-type, while theTBXA2Rvariation introduced a cryptic splice site that was predicted to cause premature termination of protein translation. The enrichment of single nucleotide variations in G protein-coupled receptor genes among type 1 von Willebrand disease patients supports the view of type 1 von Willebrand disease as a polygenic disorder. == Introduction == Type 1 von Willebrand disease is a highly heterogeneous bleeding disorder that is characterised by a partial quantitative deficiency of functionally normal von Willebrand factor (VWF) [1]. Its diagnosis is complicated by the incomplete penetrance of the disease and the wide variability in plasma VWF levels which are influenced by environmental (e. g. age, stress, Xanthopterin (hydrate) exercise) and genetic factors (e. g. ABO blood group) [1]. While the pathogenetic mechanisms underlying the disease remain to be fully resolved, data from molecular epidemiological studies indicate that mutations in the VWF gene (VWF)are present in approximately 65% of index cases diagnosed with the disorder [24]. The majority ofVWFsequence variations predict missense substitutions in VWF and the likelihood of identifying a variation is greater in those patients having more severe deficiency of the protein (3). The search for genetic factors that may explain the bleeding tendency in the 35% of patients with no recognisableVWFmutations has focused mainly on the identification of modifier genes that have a role in influencing plasma VWF levels. Thus, in addition to theABOlocus which is an established contributor to variation in plasma VWF levels, genome wide relationship Xanthopterin (hydrate) studies have identified several other genes encoding proteins that have roles in trafficking and clearance which have been shown to influence VWF levels [5, 6]. The co-existence of other mild bleeding diatheses might also modify the expression of the bleeding tendency in patients diagnosed with type 1 VWD. Given the role of VWF in primary haemostasis, and the clinical similarities of patients with type 1 VWD and platelet bleeding disorders, the bleeding tendency in patients with type 1 VWD may be influenced by variance in the genes encoding the receptors and signalling proteins that mediate platelet adhesion and collectiong. Indeed, we have previously identified two mutations in the platelet P2Y12ADP receptor Xanthopterin (hydrate) gene (P2RY12) in patients with type 1 VWD who were recruited through the EuropeanMolecular and Xanthopterin (hydrate) Clinical Markers for the Diagnosis and Management of type 1 von Willebrand Disease(MCMDM-1VWD) study and showed that they could contribute to the bleeding phenotype in these patients [7, 8]. P2Y12is one of several GPCRs expressed on the cell surface of platelets which, when activated, generate stimulatory or inhibitory signals that serve to both amplify and limit platelet recruitment and collectiong at sites of vessel injury. Platelet activation through GPCRs is mediated primarily via ADP, which, in addition to P2Y12, elicits its response through the P2Y1ADP receptor; thromboxane A2(TxA2), which elicits its response through the thromboxane receptor TP; and thrombin, which activates platelets through protease-activated receptors-1 and -4 (PAR1 and PAR4) [9]. The receptors P2Y1, TP, PAR1 and PAR4 are coupled via Gq EBR2 to phospholipase C2 (PLC2), activation of which results in an increase in cytosolic Ca2+levels, leading to activation of phospholipase A2(PLA2) and generation of TxA2which, when released, activates additional platelets though TP [9, 10]. Interaction of TP with TxA2, and of PAR1 and PAR4 with thrombin, also prospects, via G13, to activation of Rho kinase and the cytoskeletal responses resulting in platelet shape change [11]. In contrast to the stimulatory effects of thrombin, ADP and TxA2, the major endothelium-derived inhibitor of platelet activation, prostacyclin (PGI2), mediates its protective effects through the platelet Gs-coupled IP receptor to stimulate adenylyl cyclase, raising cAMP levels and leading to activation of protein kinase A (PKA) [12]. In this study, we have investigated the possible contribution of variations in the genes encoding these five GPCRs, to the bleeding tendency of patients diagnosed with type 1 VWD and recruited to the MCMDM-1VWD study. Our results reveal enrichment of rare and novel GPCR gene variations among patients with type 1 VWD, which would.