The PIs propose to test whether spontaneous nucleation of fibrinogen fibers occurs in these hydrophobic areas, possibly explaining this link. They propose to simulate the arterial wall by producing lipid bilayers, where cholesterol and other fatty acids are added. They will then use SPM, in conjunction with Raman and FTIR to determine the role of the rafts on fibrinogen adsorption, fibrilogenesis , and thrombosis. They will also experiment with different types of fats, and the findings will be used to screen different therapies to prevent their accumulation. The work proposed will be conducted mostly at the FDA, White Oak Facility, in collaboration with Drs. Katherine Vorvolakos and Dinesh Patwardhan. Human fibrinogen will be isolated and purified at Stony Brook University, as part of a collaboration with Dennis Gallanakis, MD, Director of the SBU Hospital Blood Bank and recognized expert on fibrinogen. Dr. Gallanakis keeps an inventory of different fibrinogen fractions and antibodies specific to different segments of the molecule. Functionalized polymers will be synthesized at Stony Brook, brought to White Oak where the spun cast substrates will be prepared and the adsorption experiments conducted. Lipid bilayer films with incorporated fat rafts will be prepared at SBU, and subsequent analysis which will be conducted at White Oak.
This grant enabled the publication of two manuscripts in peer reviewed journals and two more are in preparation. Two graduate students worked on this project. One completed his PhD, while the second student is finalizing some of the experiments. Engineering surfaces which may suppress thrombogenesis and promote endothelialization: Wound healing is a complex process initiated by the formation of fibrin fibers and endothelialization. Normally, this process is triggered in a wound by thrombin cleavage of fibrinopeptides on fibrinogen molecules, which allows them to self spontaneously-assemble into large fibers that provide the support structure of the clot and promote healing. We have found that the fibrous structures can also form without thrombin on most polymer or metal surfaces, including those commonly used for stents. We show that the relatively hydrophobic E and D regions of the fibrinogen molecule are adsorbed on these surfaces, exposing the αC domains, which in turn results in the formation of large fiber structures that promote endothelial cell adhesion. We show that the entire process can be suppressed when stents or other substrates are coated with polymers that are functionalized to bind the αC domains, leading to the development of potentially nonthrombogenic implant materials. Thromboelastography of fibrinogen variants : Role of hydrophobic surface on shear modulus and clot lysis Thrombelastography (TEG) is widely used for clinical coagulation assessment, but is poorly understood. We investigated the effect of fibrinogen/fibrin adsorption to the TEG cup and pin hydrophobic surface on the shear modulus and clot lysis parmeters. Among them 18 variant clots with decreased clot turbidity maxima (range 12%-57% of controls) also displayed decreased TEG maximum signal amplitude (MA), ranging from 2-38% of normal controls. Two with increase clot turbidity (165% and 173% of controls) also displayed increased MA, 158% and 153% of controls. To assess effects on hydropohobic surface anchoring of clots, normal clots were formed in the TEG cup and pin pre-coated with variant fibrinogen (83.5 pM/cm2). The results disclosed little or no effect on normal clot MA. The clearly abnormal MA of variant clots indicates for the first time a major clot stiffness role by each affected fibrin polymerization site. Lack of effect by each surface-coated variant implies that unaffected polymerization sites are sufficient for surface anchoring of the clot. For tPA-induced clot lysis in afibrinogenemic plasma, Lysis was accelerated by 10, delayed by 1, and unaffected by 7 variants. The novel and precise clot lysis measurement reflects the susceptibility of each adsorbed variant to tPA- or plasmin-induced fibrinolysis.
