A Targeted Photochemical Microfluidic Vascular Injury Model for in Vitro Thrombosis Studies: Usage in Heparin-Induced Thrombocytopenia (HIT)

Replicating the complexities of the human blood vessel include the establishment of a 3-D confluency of viable endothelial cells (ECs) on an appropriate matrix, use of human whole blood or specific components of blood, varied shear stresses, and the induction of a localized and controlled injury within the observable field to understand and intervene in hemostatic events. This array of complexities have made vascular modeling an important unmet challenge. Such a model would enhance our understanding of the pathogenesis of diverse coagulation disorders, such as the prothrombotic disorder HIT. In HIT, the platelet-rich clots lead to its other designation as the “white clot syndrome”. To provide an improved injury model, we adapted a hematoporphyrin-photochemical injury using a Fluxion Bioflux microfluidic system. When illuminated with 405 nm light, hematoporphyrin releases reactive oxygen species, inducing localized EC injury within the exposed field, but without denuding ECs. Unlike rose bengal, hematoporphyrin does not cause fluorescent interference with quantitative analysis of the developing thrombus. We propose that the model permits refined analysis of ECs transitioning from areas of quiescence to injury to quiescence, allowing us to localize and quantify the contribution of various components to the growing thrombus. In HIT, patients form antibodies to complexes of the platelet-specific chemokine, platelet factor 4 (PF4), and negatively-charged molecules such as infused heparin and heparans found on the surface of platelets and monocytes. ECs also bind PF4 immune complexes due to their highly negatively charged glycocalyx-rich surface. Prior murine cremaster laser injury studies showed that HIT antibodies bind predominantly to the EC surface rather than platelet within the thrombus itself. Was this observation related to the nature of the cremaster injury? Would antibody binding to the EC lining also be important in a wholly human HIT detection system? Using the described photochemical microfluidic system, we created a localized injury in human umbilical vein EC-lined channels through which we flowed whole human blood. Activated platelets established growing aggregates at the site of EC injury, releasing more PF4 that then bound to non-activated ECs downstream of the injury. Whole blood containing a HIT-like antibody to simulate the prothrombotic state of HIT was then flowed over this injured vasculature. HIT antibodies and then platelets bound sequentially to this new site of HIT antigen, allowing the thrombus to propagate downstream. We have named this phenomenon “rolling barrage” and suggest that a key part of the prothrombotic nature of HIT lies in antibody-mediated activation of downstream EC with subsequent thrombus propagation. HIT often occurs in the setting in surgery, which might prime EC dysfunction. We therefore treated the ECs (TNF-α) prior to injury and introducing the HIT-like antibody. TNF-α activated the EC lining, leading to loss of the anticoagulant EC surface, which enhanced clot formation downstream of the site of photochemical injury. Therefore, in a prothrombotic state such as HIT, we propose that a local injury acts as a nidus for thrombus initiation. The procoagulant process spreads distally in part because of released PF4 adhering to the downstream EC glycocalyx, which is exacerbated by mediators of inflammation. We anticipate that the described model can be used to study novel interventions to block this cycle in a wholly human system with control over the contribution of individual cellular elements, and will further understanding of the importance of this mechanism in other prothrombotic disorders.