Abstract The extent of platelet response to agonists varies between individuals and is to a large extent genetically controlled. Here we present the results of a systems biology study designed to define the association between sequence variation and platelet functional phenotype. The exons and flanking intronic regions of 98 genes, with known function in platelets, were sequenced in 48 Caucasoid CEPH DNA reference samples, identifying 1949 SNPs, approximately half of which were novel. From this enriched data set 1340 SNPs, comprising tagging and non-synonymous validated SNPs, were mounted on an Illumina Golden Gate array and typed in 500 DNA samples from a cohort of healthy subjects whose platelet functional phenotype was measured by flow cytometry, using fibrinogen binding and P-selectin expression in response to pathway-specific ADP (10−7M), or CRP-XL (0.1μg/ mL) stimulation. Thirty-six SNPs in 16 genes showed an association (p<0.005) with one or more platelet functional trait. To explore the mechanism by which sequence variation alters the platelet response, further tests were performed on platelets from individuals recalled on the basis of genotype for the SNPs with the lowest p-value for association in two of the genes, ITPR1 and VAV3. A SNP within the 3’UTR located in a putative transcription factor binding site of the VAV3 gene, for which the minor allele was associated with increased P-selectin expression following ADP stimulation (p=0.004), was associated with a higher VAV3 protein expression in subjects with the minor allele (p=0.03). Conversely, the minor allele of the lead SNP in ITPR1, associated with increased fibrinogen binding and P-selectin expression in response to ADP but not CRP-XL (p=0.003, >0.2, respectively), did not show significant effect on the level of ITPR1 protein, but there was increased mobilisation of [Ca2+]i in response to ADP (but not CRP-XL) with the minor allele (p=0.004). By applying systems biology to platelets we have identified SNPs in 16 genes which together account for up to 60% of the genetic variation underlying platelet response to ADP and CRP-XL. These data provide novel insights into key “hubs” in well-studied platelet signalling networks where sequence variation modifies the response phenotype.