Abstract
Background: A subset of patients with mild bleeding disorders have may have an underlying platelet dysfunction that cannot be evaluated with current lab tests. Among this subset of patients are those with mild type 1 von Willebrand Disease (vWD) which comprises a population that was recently more broadly redefined to capture patients with von Willebrand Factor (vWF) levels between 30-50 IU/dL and abnormal bleeding. There is no diagnostic test or biomarker that can accurately identify which patients are at risk for bleeding within this population which comprises a broad spectrum of patients with varying degrees of bleeding. There are uncertainties surrounding the clinical relevance of current diagnostic lab results based on vWF levels alone as the broadly defined range for this diagnosis manifests with a spectrum of bleeding complications. There may be a subset of patients with type 1 vWD that also have an undefined platelet dysfunction that is contributing to their bleeding phenotype. In order to fill this diagnostic gap in disorders of primary hemostasis, there is a clinical need for more assays of platelet function.
Methods: To that end, we have engineered two new assays to assess disorders of primary hemostasis and apply these assays of platelet function testing to potentially define new bleeding disorders, characterize platelet phenotypes in patients with mild bleeding disorders and refine the definition of type 1 vWD. Our assays enable us to simultaneously assess different facets of platelet spreading and interaction with the microenvironment, thereby capturing various aspects of platelet function with a single blood sample. Platelet spreading area is measured quantitatively through an automated software we developed that is able to detect area spread on images obtained by confocal microscopy. Collagen or fibrinogen is used as the substrate to quantify platelet spreading area on these surfaces. The platelet geometry-sensing assay detects how platelets secrete granules and spread in a spatially dependent manner. This assay is engineered such that the area of the adhesive matrix encompassed by a microdot is less than the spreading area of a platelet and as such, it assesses the capability of the platelet to secrete granular contents to enable adhesion beyond the boundaries of microdot. A platelet can spread on microdots of collagen or fibrinogen substrate and on a range of microdot sizes as platelets sense the surrounding microenvironment. This area of spread within and beyond the microdots is quantitatively measured. We have now established that our assays can be used to study blood samples from patients with disorders including type 1 vWD, Hermansky-Pudlak Syndrome, FLI-1 gene mutation, and other platelet function disorders, demonstrating the clinical utility of our platelet function assays.
Results: Here we demonstrate the translational application of our platelet function assays in providing a deeper understanding of platelet adhesion and geometry-sensing as it relates to hematologic conditions, with implications for investigation of disorders of primary hemostasis. Leveraging the platelet geometry-sensing assay on collagen substrate, we observed greater platelet spreading on collagen in patients with type 1 vWD (panel A) compared with control, suggestive of a possible platelet dysfunction related to granule secretion or adhesion. Patient 1 optimally spread on microdot diameters of 5 μm and 7 μm, demonstrating 59% and 45% more spreading respectively when compared with patient 2. Ongoing accrual of patients with type 1 vWD will determine the significance of these differences as this may be suggestive of a abnormal platelet function. Across mild bleeding disorders, the quantitative adhesion assay demonstrated differences in platelet spreading with most platelets in bleeding of unknown cause (BUC) spreading 44% less compared with FLI-1 gene mutation platelets (panel B). Further investigation of these platelets on the platelet geometry-sensing assay may further elucidate if there are storage pool defects or cytoskeletal changes in BUC platelets that hinder spreading ability. Ongoing research is being conducted using our platelet function assays to investigate platelet properties in type 1 vWD and mild bleeding disorders and correlate these assays with bleeding phenotypes.
Disclosures
Brown:Genentech: Membership on an entity's Board of Directors or advisory committees. Meeks:Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: advisory board; CSL Behring: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Novo Nordisk: Consultancy, Other: advisory board; Shire: Consultancy, Other: advisory board; Octapharma: Research Funding; Biomarin: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: advisory board; Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: advisory board, Research Funding; Spark: Consultancy; Pfizer: Consultancy.
