Tissue Factor Enriched Microvesicles Plus Factor VIIa Induce Platelet Aggregation in the Absence of Coagulation Proteins: Evidence for the Involvement of Internalization and Trafficking Mechanisms.
Authors: Lopez-Vilchez Irene , Escolar Gines , Diaz-Ricart Maribel , Galan Ana M. , Cortadellas Nuria , White James G. 
Published: April 2020

Abstract
The favorable action of FVIIa in critically deterred hemostasis is not completely elucidated though it has been suggested that FVIIa could activate platelets through mechanisms independent of TF. Standard aggregometry, ultrastructural, and immunocytochemical methods were combined with other techniques to assess the interaction of human TF (hTF) enriched microvesicles (MVs) and rFVIIa in a system with repeatedly washed platelets in absence of plasma proteins. Sequential analysis of morphology and immunolocalization of TF on platelet cryosections were also performed. Modifications in the expression of activation antigens and exposure of anionic phospholipids (Annexin V binding) were evaluated by flow cytometry. Activation of tyrosine phosphorylation mediated mechanisms was also monitored. Several inhibitory strategies were used to characterize mechanisms involved in these interactions. After exposure to hTF bearing MVs (hTF-MVs), platelets became reversibly activated reaching a maximum peak in the first minute (41 ± 3% maximal aggregation). Ultrastructural studies revealed that hTF-MVs became rapidly internalized by platelets. This process induced changes in platelet shape, organelle centralization and enhanced platelet-platelet interactions, but did not result in the formation of compact aggregates. Immunocytochemical studies at ultrastructural level demonstrated hTF-MVs trafficking from the external membranes to the alpha granules via the open canalicular system. Flow cytometry studies detected a moderate increase in the expression of platelet activation antigens. In the absence of rFVIIa platelets always return to an apparent resting state in 5 min, recovering their discoid shape, though immunocytochemical studies still demonstrate TF presence in platelets. When rFVIIa was present together with hTF-MVs, platelets became irreversibly aggregated (81% ± 6%). Ultrastructural studies confirmed internal contraction, degranulation and formation of perfectly structured platelet aggregates. Flow cytometry confirmed a marked expression of activation dependent antigens and a significant enhancement of annexin V binding which was never detected in studies with hTF-MVs alone. Antibodies to GPIIb-IIIa blocked reversible and irreversible aggregation steps. Heparins or hirudin inhibited the irreversible aggregation that occurred in the presence of rFVIIa, but did not prevent the initial reversible aggregation caused by hTF-MVs. Antibodies to P-selectin reduced the initial reversible activation of platelets in response to hTF-MVs, but did completely block the aggregation that occurred in the presence of rFVIIa. This study demonstrates that platelets internalize TF when it is exposed on MVs and confirms the existence of TF trafficking from external sources into internal platelet storage pools. Our results prove that the complex rFVIIa-hTF activates platelets in the practical absence of plasma proteins. If TF can be stored in platelets, the hypothesis of a direct action of rFVIIa on platelets independent of TF should be reconsidered, since internal pools of TF could be exposed on the surface upon activation. Our studies indirectly suggest that TF stored in platelets could enhance the potential thrombogenic capacity of platelets under special pathologic situations.