Multiparameter phenotyping of platelet reactivity for stratification of human cohorts

Abstract

Accurate and comprehensive assessment of platelet function across cohorts of donors may be key to understanding the risk of thrombotic events associated with cardiovascular disease, and hence help personalise the application of antiplatelet drugs. However, platelet function tests can be difficult to perform and analyse, unreliable or uninformative and poorly standardised across studies. The Platelet Phenomic Analysis (PPAnalysis) assay and associated open-source software platform was developed in response to these challenges. PPAnalysis utilises pre-prepared freeze-dried microtitre plates to provide a detailed characterisation of platelet function. The automated analysis of the high-dimensional data enables the identification of sub-populations of donors with distinct platelet function phenotypes. Using this approach we identified that the Sensitivity of a donor's platelets to an agonist and their Capacity to generate a functional response are distinct independent metrics of platelet reactivity. Hierarchical clustering of these metrics identified six subgroups with distinct platelet phenotypes within healthy cohorts, indicating that platelet reactivity does not fit into the traditional simple categories of 'high' and 'low' responders. These platelet phenotypes were found to exist in two independent cohorts of healthy donors and were stable on recall. PPAnalysis is a powerful tool for stratification of cohorts on the basis of platelet reactivity which will enable investigation of the causes and consequences of differences in platelet function and drive progress towards precision medicine.

A guide to molecular and functional investigations of platelets to bridge basic and clinical sciences

“…generate a platelet-response profile of each individual on the basis of surface marker expression 36 . Although this technique requires a more careful and elaborate assay design compared with that of conventional flow cytometry, this analysis could be potentially helpful in studying platelet function variability in a cohort with better reproducibility.…”

Platelet reactivity and platelet count in women with iron deficiency treated with intravenous iron

“…Following collection into sodium citrate, whole blood was diluted into HT buffer (1:5) and aliquoted into a set of tubes preloaded with fluorophore‐conjugated antibodies (ie, PAC1‐FITC, CD62‐APC) and platelet agonists, including crosslinked collagen‐related peptide (CRP‐XL), 16 ADP, and thromboxane analog U46619. Previous studies from our group and others have established standardized protocols, where platelet α‐granule secretion and integrin α IIb β 3 activation increases in an agonist concentration–dependent manner 17‐20 . At baseline, surface levels of platelet P‐selectin—a marker of platelet degranulation and α‐granule secretion—significantly decreased on unstimulated platelets in whole‐blood samples after iron repletion (Figure 3A,B).…”

“…Previous studies from our group and others have established standardized protocols, where platelet α‐granule secretion and integrin α IIb β 3 activation increases in an agonist concentration–dependent manner. 17 , 18 , 19 , 20 At baseline, surface levels of platelet P‐selectin—a marker of platelet degranulation and α‐granule secretion—significantly decreased on unstimulated platelets in whole‐blood samples after iron repletion (Figure 3A,B ). There was no significant change in platelet integrin activation in resting or agonist‐stimulated platelets following iron repletion.…”

Heterogeneity of platelets and their responses

“…Platelets derived from platelet-rich plasma have shown higher levels of P-selectin and annexin V as compared with those shown by platelets isolated from a buffy coat or using apheresis [ 53 ]. In recent work using a novel platelet phenomics assay [ 54 ], platelets from healthy individuals were stratified into 6 distinct platelet phenotypes based on their reactivity and shown to be consistent between different cohorts and across time. Storage of platelets has also been reported to introduce subpopulations, including a predominance of degranulating platelets after cryopreservation [ 55 ] and populations arising over 7 days of storage from stress-induced platelet activation and cell death [ 56 ], with potential functional consequences [ 57 ].…”