New Insights into the Prothrombotic State of Heparin-Induced Thrombocytopenia (HIT) Using a Novel Microfluidic System: The Endothelial Lining and “Rolling Barrage” of Activation

The most feared feature of HIT is antibody-mediated thrombosis. We have shown that this prothrombotic state is related to binding of platelet factor 4 (PF4), a chemokine densely packaged into platelet alpha-granules, to surface glycosaminoglycans (GAGs) expressed on hematopoietic and vascular cells. PF4/GAG surface complexes are recognized by HIT antibodies, activating the targeted cells. Unlike platelets that express only low-affinity chondroitin sulfate surface GAGs, endothelial cells (EC) express a glycocalyx enriched in heparan sulfate, which has higher affinity for PF4, potentially increasing their propensity to become a target for immune injury leading to thrombosis. We examined the details of the development of in situ thrombi using the cremaster arteriole laser injury model beginning with transgenic mice expressing only human PF4 (hPF4+), but lacking FcγRIIA. These mice do not develop thrombocytopenia or thrombosis when injected with the HIT-like monoclonal antibody KKO or IgGs isolated from patients with HIT. In these mice, antigenic PF4/GAG complexes were recognized by KKO at sites of vascular injury even in the absence of infused heparin. In fact, infusion of sufficient heparin dissociated PF4 from sites of injury, consistent with its higher affinity for PF4 than cell surface GAGs. This suggests that antigenic PF4/GAG complexes normally develop intravascularly whenever thrombus formation occurs, yet these complexes do not typically initiate antibody-mediated thrombosis. Real-time confocal imaging of injured vessels revealed that PF4 first bound almost exclusively to the peri-injury endothelium. This was especially evident immediately upstream of the thrombus where turbulent blood flow may lead to platelet degranulation and subsequent adherence of the released PF4 to the glycocalyx. Beginning approximately two minutes post-injury, binding of KKO, presumably to PF4/GAG complexes on platelets, is seen at the interface between the shell and core of the thrombus. We then repeated these same studies in hPF4+/FcγRIIA+ mice, where infused KKO or HIT IgGs leads to significant thrombocytopenia and widespread development of thrombi as in HIT. Similar adherence of PF4 to the peri-injury EC and then to the core/shell interface of the thrombus as seen in the hPF4+ mice, but the changes were more extensive in hPF4+/FcγRIIA+ mice after KKO infusion and often lead to vascular occlusion. To further define the basis of the prothrombotic state in HIT and to extend our studies to a human system, we examined thrombus formation in HIT in a novel microfluidic system in which vascular injury was induced in an upstream portion of a human umbilical vein EC-lined channel by reactive oxygen species generated through excitation of infused hematoporphyrin by blue light (490 nm). Following infusion of human blood, platelets accumulated and released PF4, which bound the injured endothelium, while the downstream endothelium remained quiescent. Addition of KKO to the infused whole blood lead to a HIT-like state with marked increase in platelet adhesion and binding of PF4 to the injured endothelium, but binding of PF4 now spread downstream of the boundary between injured and uninjured endothelium. This was followed by downstream platelet adhesion and often occlusion of the channel. We proposed that this spread in EC injury was a result of a “rolling barrage” of PF4 released from platelets binding to the injured patch of EC complexing to the downstream glycocalyx on the non-injured endothelium followed by KKO binding and subsequent endothelial activation. The newly activated ECs bound additional platelets and the process repeats, rolling downstream and extending thrombus growth. Thus, these studies provide important new insights into the local sequence of events that propagate clots in HIT: Targeting of the endothelial glycocalyx by HIT antibodies is a major contributor to the prothrombotic state. Platelets adherent to the site of original injury release PF4, which then binds to downstream EC glycocalyx and initiates repetitive cycles of PF4 binding, EC activation and platelet adherence, and further release of PF4 that propagates growth of thrombi to previously uninvolved vasculature.