Abstract
In hemostasis and thrombosis, the complex process of thrombus formation involves different molecular pathways of platelet and coagulation activation. These pathways are considered as operating together at the same time, but this has not been investigated. The objective of our study was to elucidate the time-dependency of key pathways of thrombus and clot formation, initiated by collagen and tissue factor surfaces, where coagulation is triggered via the extrinsic route. Therefore, we adapted a microfluidics whole-blood assay with the Maastricht flow chamber to acutely block molecular pathways by pharmacological intervention at desired time points. Application of the technique revealed crucial roles of glycoprotein VI (GPVI)-induced platelet signaling via Syk kinase as well as factor VIIa-induced thrombin generation, which were confined to the first minutes of thrombus buildup. A novel anti-GPVI Fab EMF-1 was used for this purpose. In addition, platelet activation with the protease-activating receptors 1/4 (PAR1/4) and integrin αIIbβ3 appeared to be prolongedly active and extended to later stages of thrombus and clot formation. This work thereby revealed a more persistent contribution of thrombin receptor-induced platelet activation than of collagen receptor-induced platelet activation to the thrombotic process.
Highlights
Thrombosis, and thrombo-inflammation, multiple platelet and coagulation activation processes interact to establish the formation of a thrombus composed of aggregated and contracted platelets connected by a fibrin network or clot [1,2,3]
We used a novel in-house developed intervention method for acutely switching blood samples during perfusion through a microfluidic chamber in order to resolve the time-dependency of platelet activation and coagulation processes via collagen (GPVI-induced ITAM and Syk tyrosine kinase signaling) and tissue factor (TF)/thrombin
We have demonstrated that the current multiparameter approach of wholeblood thrombus formation under flow over collagen- and TF-coated surfaces provides detailed insights into the importance of specific platelet and coagulation pathways, and that this method can be used as a proxy assessment for hemostasis to explain the bleeding phenotypes of patients with certain platelet or coagulation defects [10,26,41]
Summary
The complex process of thrombus formation involves different molecular pathways of platelet and coagulation activation. These pathways are considered as operating together at the same time, but this has not been investigated. The objective of our study was to elucidate the time-dependency of key pathways of thrombus and clot formation, initiated by collagen and tissue factor surfaces, where coagulation is triggered via the extrinsic route. The binding of fibrinogen to activated integrin αIIbβ on adjacent platelets generates a scaffold for the formation of platelet aggregates and, in flowing blood, for the buildup of a thrombus [2,11,13]
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