Morphology Of Thrombi Research Articles

Severe platelet dysfunction in NHL patients receiving ibrutinib is absent in patients receiving acalabrutinib.

The Bruton tyrosine kinase (Btk) inhibitor ibrutinib induces platelet dysfunction and causes increased risk of bleeding. Off-target inhibition of Tec is believed to contribute to platelet dysfunction and other side effects of ibrutinib. The second-generation Btk inhibitor acalabrutinib was developed with improved specificity for Btk over Tec. We investigated platelet function in patients with non-Hodgkin lymphoma (NHL) receiving ibrutinib or acalabrutinib by aggregometry and by measuring thrombus formation on collagen under arterial shear. Both patient groups had similarly dysfunctional aggregation responses to collagen and collagen-related peptide, and comparison with mechanistic experiments in which platelets from healthy donors were treated with the Btk inhibitors suggested that both drugs inhibit platelet Btk and Tec at physiological concentrations. Only ibrutinib caused dysfunctional thrombus formation, whereas size and morphology of thrombi following acalabrutinib treatment were of normal size and morphology. We found that ibrutinib but not acalabrutinib inhibited Src family kinases, which have a critical role in platelet adhesion to collagen that is likely to underpin unstable thrombus formation observed in ibrutinib patients. We found that platelet function was enhanced by increasing levels of von Willebrand factor (VWF) and factor VIII (FVIII) ex vivo by addition of intermediate purity FVIII (Haemate P) to blood from patients, resulting in consistently larger thrombi. We conclude that acalabrutinib avoids major platelet dysfunction associated with ibrutinib therapy, and platelet function may be enhanced in patients with B-cell NHL by increasing plasma VWF and FVIII.

Abstract 529: Assessing Hemostasis and Platelet Function Using a Microfluidic Device Lined with Fixed Human Endothelium

Mutual signaling between endothelium and platelets is widely recognized as critical for regulation of hemostasis and thrombotic disorders associated with various diseases, such as, arteriosclerosis, sepsis, and diabetes. Yet no reliable and practical assays exist that can measure the effects of cross-talk between platelets and inflamed vessel walls in the presence of physiological shear. Flow chambers have been applied previously to study basic science, but they have not been used in practical settings due to the difficulty in maintaining living cells in devices. Specifically, because it is extremely difficult to maintain living cell cultures for extended times, it would be impossible to maintain the robustness of these assays. Here, we describe a microfluidic device lined by a chemically fixed human endothelium that retains its ability to support thrombus formation in the presence of blood flow for multiple days under standard storage conditions. We coat the inner surface of a rectangular device with collagen and then culture with endothelial cells on all four walls in order to create a vascular tube. Under activation by increasing doses of TNF-α followed by fixation, the device continues to exhibit multiple proinflammatory and adhesion molecules. Correspondingly, a dose-dependent increase in platelet adhesion to the endothelial layer is also observed. Importantly, we do not observe any significant difference in platelet adhesion between living and fixed endothelium. Furthermore, the morphology of thrombi appears similar to that of thrombi that form on living endothelium of a laser-injured mouse in vivo . To establish clinical utility, we also perfused whole blood of patients taking antiplatelet drugs, and found that these subjects showed a significant reduction in platelet adhesion in our devices. In contrast, conventional aggregometry analysis was very time consuming and tedious. Also, on a collagen-coated flow chamber we found no significant difference in platelet coverage between normal controls and patients. Taken together, this study confirms that this microdevice-incorporating fixed endothelium, whole blood and shear stress-can be used as a global hemostasis assay in research as well as many practical and clinical settings.

Blood/Material Interactions on Silicone Rubber-Hydrogels

The composition and structure of a polymer surface in contact with flowing blood are major factors determining the extent of thrombus and embolus formation. Use of scanning electron microscopy (SEM) provides temporal information on morphology of thrombi and cells on polymer surfaces exposed to blood, and associated Fourier transform infrared/attenuated total reflectance (FT-IR/ATR) measurements aid in characterizing protein buildup.Medical grade silicone rubber-filler formulations (0,2,12 and 24 weight percent cristobalite filler contents; source material Silastic 382) were polymerized to form substrates. These were subsequently coated with five different hydrogels by radiation grafting (y-rays) to provide samples with a range of hydrophilicity characteristics.