Heparin-induced thrombocytopenia (HIT) is a prothrombotic, thrombocytopenic disorder that occurs in patients exposed to heparin due to the development of antibodies directed against complexes of platelet factor 4 (PF4) with heparin with other polyanions . We have recently shown that the perithrombus endothelium is targeted in HIT. Using a photochemical injury model and an endothelialized microfluidic system, we now show that PF4 binds to elongated strands of von Willebrand factor (VWF) released from injured endothelium. This binding appears to be periodic along the VWF strand and to favor larger VWF strands, suggesting formation of oligomers that may cross-link individual VWF subunits. KKO, a monoclonal HIT-like antibody, and HIT patient plasma form PF4-VWF-HIT antibody complexes that may stabilize the complexes which, in turn, avidly bind platelets, exacerbating thrombus growth in a human whole blood microfluidic system. This increase in platelet binding is attenuated by blocking either the platelet FcRIIA or the glycoprotein Ib/IX receptor. Charge-based as well as VWF-binding drugs can disrupt the PF4-VWF-HIT complexes. Dynamic light scattering studies provide additional support for the concept that PF4 and VWF form complexes in vitro. In vivo studies in a passive immunization model of HIT indicate a potential role for VWF in the prothrombotic nature of HIT. Thus, these studies newly identify VWF as a potentially important polymer involved in the formation of HIT antigenic complexes that contribute to the prothrombotic nature of HIT and offer a potential new therapeutic target to mitigate the thrombotic complications of affected patients.