PROJECT SUMMARY Coagulation factor VIII (FVIII) and its carrier protein von Willebrand factor (VWF) play an essential role in blood coagulation. We have led genome-wide association studies that successfully identified loci contributing to plasma FVIII and VWF levels. Many of the identified genetic determinants affect both FVIII and VWF, while some loci affect only FVIII or only VWF. We performed Mendelian randomization (MR) and found associations of genetically determined FVIII and VWF levels with several arterial and venous thrombotic diseases. Despite these advances, knowledge gaps remain in understanding the genetics of FVIII and VWF levels, especially in understudied populations such as Hispanics. More complex regulatory mechanisms such as epistasis and epigenetics remain uncharacterized. Finally, because many of the identified loci are shared between FVIII and VWF, it has been difficult to determine the extent to which they play an independent or coordinated role in the pathology of thrombotic disease. The overall goals of the renewal of R01 HL139553 are to generate new biological knowledge about the genetic and epigenetic regulation of FVIII and VWF in multi-ancestry populations and gain a better understanding of how these hemostatic factors play a role in the development of thrombotic diseases. In Aim 1, we will identify new genomic loci and improve fine-mapping of existing loci by measuring FVIII and VWF in plasma from 14,000 Hispanics with whole genome sequence data from the Hispanic Community Health Study / Study of Latinos and combining it with existing multi-ancestry data from CHARGE and TOPMed. We will also evaluate epistatic effects with the ABO blood group. In Aim 2, we will perform an epigenome-wide association study to examine the association of FVIII and VWF levels with blood methylation levels at CpG sites across the genome and identify genetic variants associated with these CpG sites. We will characterize the function of known and newly identified loci from Aims 1 and 2 using in vitro and in vivo models. In Aim 3, we will use the genetic variants regulating FVIII and VWF identified in Aims 1 and 2 as genetic instruments in MR analyses to assess whether FVIII and/or VWF levels are causally related to the risk of several arterial and venous thrombotic diseases. The expected outcomes are to have identified and validated novel genetic and epigenetic determinants of FVIII and VWF levels, and to have elucidated the independent or coordinated effects of FVIII and VWF in thrombotic diseases. These results are anticipated to have an important positive impact because they will provide evidence of the relative importance of coagulation (i.e., via FVIII) or platelets (i.e., via VWF) in different thrombotic diseases, thereby informing the targeted use of existing and novel therapies.