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Laminar Flow-based Assays to Investigate Leukocyte Recruitment on Cultured Vascular Cells and Adherent Platelets

The recruitment of leukocytes upon injury or inflammation to sites of injury or tissue damage has been investigated during recent decades and has resulted ...

Maastricht University

This paper provides an overview of the various types of microfluidic devices that are employed to study the complex processes of platelet activation and blood coagulation in whole blood under flow conditions. We elaborate on how these devices are used to detect impaired platelet-dependent fibrin formation in blood from mice or patients with specific bleeding disorders. We provide a practical guide on how to assess formation of a platelet-fibrin thrombus under flow, using equipment that is present in most laboratories. In addition, we describe current insights on how blood flow and shear rate alter the location of platelet populations, von Willebrand factor, coagulation factors, and fibrin in a growing thrombus. Finally, we discuss possibilities and limitations for the clinical use of microfluidic devices to evaluate a hemostatic or prothrombotic tendency in patient blood samples.

Use of microfluidics to assess the platelet-based control of coagulation.

Linking non-coding genetic variants associated with the risk of diseases or disease-relevant traits to target genes is a crucial step to realize GWAS potential in the introduction of precision medicine. Here we set out to determine the mechanisms underpinning variant association with platelet quantitative traits using cell type-matched epigenomic data and promoter long-range interactions. We identify potential regulatory functions for 423 of 565 (75%) non-coding variants associated with platelet traits and we demonstrate, through ex vivo and proof of principle genome editing validation, that variants in super enhancers play an important role in controlling archetypical platelet functions.

Platelet function is modified by common sequence variation in megakaryocyte super enhancers.

In patients with dysfunctions of the Cachannel ORAI1, stromal interaction molecule 1 (STIM1) or integrin-regulating kindlin-3 (FERMT3), severe immunodeficiency is frequently linked to abnormal platelet activity. In this paper, we studied platelet responsiveness by multiparameter assessment of whole blood thrombus formation under high-shear flow conditions in 9 patients, including relatives, with confirmed rare genetic mutations of ORAI1, STIM1 or FERMT3. In platelets isolated from 5 out of 6 patients with ORAI1 or STIM1 mutations, store-operated Caentry (SOCE) was either completely or partially defective compared to control platelets. Parameters of platelet adhesion and aggregation on collagen microspots were impaired for 4 out of 6 patients, in part related to a low platelet count. For 4 patients, platelet adhesion/aggregation and procoagulant activity on von Willebrand Factor (VWF)/rhodocytin and VWF/fibrinogen microspots were impaired independently of platelet count, and were partly correlated with SOCE deficiency. Measurement of thrombus formation at low shear rate confirmed a greater impairment of platelet functionality in the ORAI1 patients than in the STIM1 patient. For 3 patients/relatives with a FERMT3 mutation, all parameters of thrombus formation were strongly reduced regardless of the microspot. Bone marrow transplantation, required by 2 patients, resulted in overall improvement of platelet function. We concluded that multiparameter assessment of whole blood thrombus formation in a surface-dependent way can detect: i) additive effects of low platelet count and impaired platelet functionality; ii) aberrant ORAI1-mediated Caentry; iii) differences in platelet activation between patients carrying the same ORAI1 mutation; iv) severe platelet function impairment linked to a FERMT3 mutation and bleeding history.

Article	Year
Use of microfluidics to assess the platelet-based control of coagulation. Platelets\| PubMed ID: 28358995	2017
Platelet function is modified by common sequence variation in megakaryocyte super enhancers. Nature communications\| PubMed ID: 28703137	2017
Variable impairment of platelet functions in patients with severe, genetically linked immune deficiencies. Haematologica\| PubMed ID: 29242293	2018

Magdolna Nagy

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