PB0748 - Identification of key regulators of procoagulant COAT platelet generation by quantitative temporal phosphoproteomic analysis

Background: Abnormal generation of procoagulant platelets may lead to bleeding or thrombotic events. The intracellular mechanisms underlying the dichotomous generation of aggregating (AGG) and procoagulant (COAT) platelets upon combined activation by Collagen-And-Thrombin are only partially described.

Aims: Here, we investigated the utility of temporal phosphoproteomics to identify regulators of the procoagulant response.

Methods: Human platelets from healthy donors were activated with convulxin-plus-thrombin in presence/absence of calcium, generating procoagulant/aggregating phenotypes, respectively. Platelets were sampled at different timepoints up to 8 min after activation. The phosphoproteomes of resting, AGG, and COAT platelets were analysed by isobaric Tandem-Mass-Tag Mass-Spectrometry. Differentially regulated phosphosites between AGG and COAT platelets were identified using Kruskal-Wallis test.

Results: At 8 min after stimulation, we identified 4223 significantly differently regulated phosphosites, corresponding to 1643 unique proteins (Fig. 1A). Up to 1 min after activation, most platelet proteins hyperphosphorylated, while some underwent differential phosphorylation in AGG versus COAT platelets, such as STIM1, SLC9A1, PDE3A, MRVI1, FLNA, MYCT1, UBE2O, and TNIK. Then, up to 8 min, most proteins in COAT platelets experienced gradual dephosphorylation (Fig. 1B). This occurred in five different clusters of phosphorylation (Fig. 1C), involving major response transducers (e.g., BTK, Fyn, PLCγ2, PKCD), small GTPases (e.g., ARHGEF12, ARHGAP6) and other actin components (e.g., zyxin, vinculin). Additionally, the levels of some vesicle-associated proteins (e.g., VAMP8, SNAP23) were gradually reduced upon COAT platelet generation.

Conclusion(s): The present study highlights the utility of temporal phosphoproteome analysis for identifying proteins, potentially underlying the generation of the procoagulant phenotype. For instance, early differential phosphorylation of proteins involved in sodium/calcium mobilisation and actin components may initiate the procoagulant dichotomy. Additionally, we observed that the procoagulant response is accompanied with an important dephosphorylation compared to the aggregatory response. Based on our data, we hypothesize that activated proteolytic pathway and membrane phosphatidylserine scrambling concomitantly to actin remodelling may uncouple major kinase complexes.