Fibrin Generation in Heparin-Induced Thrombocytopenia (HIT): Pathomechanistic Background for Novel Therapy and Prophylaxis

Abstract 635

HIT is an immune mediated prothrombotic disorder often associated with life- and limb-threatening thrombosis caused by antibodies to a complex between platelet factor 4 (PF4) and heparin. Platelet activation and clearance are considered key to the pathogenesis. Direct thrombin inhibitors, the most commonly used anticoagulant therapy in the treatment of HIT, provide incomplete prevention against development of new thrombi and little effect on the incidence of loss of limb and life. Thus, there is a need for a better understanding of pathogenesis of HIT and new approaches to therapy. We and others have shown that HIT not only is associated with platelet activation, but also involves activation of monocytes and endothelial cells, which together increase thrombin generation that may affect both the amount and structural properties of the resultant fibrin clot. However, this proposed increase in fibrin formation though suspected, has never been directly investigated. Previously, we have shown in the cremaster muscle laser-injury model of thrombosis induced by the HIT-like murine monoclonal anti-hPF4/heparin antibody KKO that transgenic mice expressing both human PF4 (hPF4+) and hFcγRIIA developed larger, more fibrin-rich occlusive thrombi than in control mice expressing hPF4 or hFcγRIIA alone. To quantify fibrin formation in a more controlled setting, we simulated HIT in a BioFlux microfluidic channel system coated with von Willebrand factor by perfusing whole blood at a venule shear stress of 20 dyne/cm² at 37°C for 10 min. Platelets were labeled by adding Calcein-AM (3 μM) and fibrin was visualized by adding Alexa 647 labeled fibrinogen (1.5 μg/ml) to the whole blood prior to the perfusion. Using NaCitrate-anticoagulated human blood, we observed that recalcified human blood samples exposed to KKO plus hPF4 formed large platelet thrombi and an extensive fibrin network, with fibers radiating from the platelet aggregates and often organized along the direction of flow. In contrast, control samples exposed to a combination of the non-pathogenic anti-hPF4 monoclonal antibody RTO plus hPF4 showed little fibrin and less organization. Quantitative fluorescence analysis showed nine times more fibrin formed after stimulation with KKO plus PF4 than RTO plus PF4. Inhibition of KKO-mediated platelet activation by blocking FcgRIIA with Fab fragments of monoclonal antibody IV.3 in whole blood suppressed platelet adhesion by > 80%, but decreased fibrin formation by only ∼40%. On the other hand, addition of a selective inhibitor of the Syk tyrosine kinase PRT-060318 (Reilly et al., Blood 2011:117:2241–6; kindly provided by Dr. Uma Sinha, Portola Pharmaceuticals) to whole blood at a concentration of 3 μM suppressed both platelet adhesion and fibrin formation by 80% and 70%, respectively. IV.3 inhibits platelet activation alone, while we have shown that PRT-060318 inhibits both platelet activation and monocyte activation with the subsequent release of tissue factor-rich microparticles. These results provide a mechanistic basis for the use of novel therapies in HIT such as fibrinolytic agents. To do so, we studied a novel chimeric pro-fibrinolytic composed of a C-terminal thrombin-specific activatable low molecular weight urokinase (uPA-T) that has its plasmin-activation site replaced by a thrombin cleavage site and linked at its N-terminus to a single-chain variable region (scFv) that binds with high affinity to human platelet aIIb, designed to deliver the agent to sites of incipient thrombosis. Preliminary results show that uPA-T profoundly suppressed fibrin accumulation in both in vitro and in an in vivo model of HIT. This novel approach to therapy takes advantage of our growing understanding of the pathogenesis of the prothrombotic nature of HIT including monocyte activation and formation of fibrin-rich clots. Such therapeutics may be especially effective as targeted therapy in HIT.