Microfluidic model of the pathogenesis of heparin-induced thrombocytopenia

Valerie J. Tutwiler

doi:10.17918/00009984

HIT is an immune-mediated thrombocytopenia that is particularly prothrombotic often associated with life- and limb-threatening thrombosis caused by antibodies to a complex between platelet factor 4 (PF4) and heparin. Why this particular disorder results in a prothrombotic state relative to other immune thrombocytopenias is unclear. A passive immunization murine model of this disorder has provided important insights into the underlying pathogenesis of this disease, but is limited by its inability to study human cells and restricted potential to define the contribution of various hematopoietic and vascular cells to the prothrombotic state. We used a microfluidic system in conjunction with flow cytometry to further our understanding of the prothrombotic nature of HIT. The channels of the BioFlux system from Fluxion Bioscience were coated with species-specific von Willebrand factor (vWf), murine or human blood was perfused at a shear stress of 20 dyne/cm² at 37°C and time lapse and/or end point fluorescence light microscope images of the resulting clot were obtained. An increase in platelet adhesion (relative area covered by platelets), increased aggregate size, and increased fibrin generation was seen in the presence of the pathogenic HIT-like monoclonal antibody (moAb) KKO (50 [mu]g/ml) in conjunction with PF4 (10 [mu]g/ml) when compared to control samples with PF4 only or with PF4 plus a non-pathogenic anti-PF4 moAb RTO (p<0.01). We and others have shown that HIT not only is associated with platelet activation, but also involves activation of monocytes, which may influence the degree of the prothrombotic state. To understand the pathway by which monocytes contribute to the prothrombotic nature of HIT, monocyte-depletion/repletion studies as well as targeted disruption of potential downstream steps were studied. We found that monocytedepleted blood samples decreased platelet aggregation (p<0.0001). We believe that monocyte activation involved the Fc receptor on monocytes and using IV.3, an antibody that blocks activation through Fc [gamma] RIIA, and we found partial loss of monocyte activation which we interpreted as being due to other Fc receptors presence on monocytes (Fc [gamma] RI and Fc [gamma] RIII). A selective inhibitor of the Syk tyrosine kinase, PRT-060318, also decreased the prothrombotic contribution of monocytes. Activated monocytes release microparticles that express tissue factor on their surface and we found that inhibition of thrombin downstream of tissue factor also prevented the prothrombotic state. Coated platelets are prothrombotic and characterized by phosphatidylserine (PS) exposure and binding of FVa and FXa. This activated state requires dual stimulation via thrombin and ITAM receptors. Flow cytometric studies of annexin V and FXa binding showed extensive induction of coated platelets in whole blood by KKO plus PF4 in contrast to PF4 or PF4 plus RTO (annexin V: p<0.0001; Factor Xa p<0.01). To better understand the pathomechanistic development of HIT it is critical to investigate the structural properties of the resultant fibrin clot. We developed an analytical model that can extract the parts of the images representing the fibrin clot and characterize the resulting structure. Collectively these studies show that targeting the generation of coated platelets and/or fibrin may lead to new diagnostic tests and therapeutic interventions with respect to HIT.

Microfluidic model of the pathogenesis of heparin-induced thrombocytopenia

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Microfluidic model of the pathogenesis of heparin-induced thrombocytopenia

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