Allogeneic natural platelet transfusions are clinically routinely required in hemostatic therapy of a variety of bleeding complications. However, natural platelets suffer from (i) limited supply, (ii pathogenic contamination risks resulting in short shelf-life (~3-5 days), and (iii) risks of multipe biological side-effects. Current photochemical pathogen reduction techniques extend the shelf-life of natural platelet products only to ~7 days. Consequently, there is a substantial clinical interest in artificial platelet analogs that can mimic platelet's hemostatic actions, while allowin large-scale production, longer shelf-life and safer in vivo applications. To this end, past approaches in artificial platelets have shown very limited efficacy, possibly because of a major design drawback: platelet's natural hemostatic action requires injury site-specific platelet adhesion and site- selective platelet aggregation to act in tandem, but none of the past approaches have integrated these two capabilities effectively on a single platform. Our research is the first to have successfully integrated these two key hemostatic functions via heteromultivalent vesicular assembly of adhesion-promoting and aggregation- promoting peptide-lipid conjugates. Our artificial platelet construct is simultaneously surface-decorated wit VWF-binding peptides (VBP) for shear-responsive VWF adhesion, collagen-binding peptides (CBP) for shear- independent collagen adhesion and fibrinogen-mimetic peptides (FMP) for enhancing the aggregation of active platelets onto the adhered constructs. This innovative artificial platelet design has exhibited superior hemostatic activity both in vitro and in vivo, an we hypothesize that this superior hemostatic efficacy is due to a combined effect of both primary hemostasis and secondary hemostasis mechanisms induced by our constructs. Our overall goal is to corroborate this hypothesis using three specific aims:
In Aim 1 we will establish a mechanistic model of the primary hemostatic action of our nanoconstructs, by first elucidating the domain-specific molecular mechanism of shear-responsive VBP interactions with VWF, and then combining this insight with the already established knowledge of shear-independent helicogenic interaction of CBP with fibrillar collagen and platelet activation-selective interactio of FMP with platelet integrin GPIIb-IIIa.
In Aim 2 we will investigate whether the construct-mediated direct enhancement of primary hemostasis, can also in effect enhance secondary hemostasis (coagulation) at the site of construct-induced platelet aggregation, due to pro- coagulant ability of the active platelet membrane. Thus, Aims 1 and 2 will help synergistically corroborate the mechanistic components of our hypothesis. Hence in Aim 3, we will determine whether these construct- induced mechanisms lead to superior hemostatic efficacy in a tail transection bleeding model in thrombocytopenic mice, compared to current clinical hemostat NovoSeven(R). Establishing the construct- induced hemostatic mechanisms in vitro and demonstrating its resultant superior therapeutic efficacy in the thrombocytopenia model in vivo, will lead to detailed evaluation in acute and chronic bleeding models in future.
Platelet transfusion is a clinically significant treatment modality for bleeding complications in patients with familial, disease-associated, or chemo/radiotherapy induced platelet disorders, as well as, in patients undergoing major surgery or traumatic injury. Natural platelet-based transfusion products suffer from supply shortage, very limited storage life and high risk of pathologic contamination and negative biologic side- effects, and hence there is a significant clinical interest in artificial platelet analogs that can mimic platelet's clot-promotig function while allowing long storage life and safe in vivo applications. To this end we have developed peptide-lipid assembly-based platelet-mimicking constructs and we propose to investigate the molecular mechanisms by which these constructs can promote safe injury site-selective clotting and evaluate their therapeutic efficacy in a small animal model, with a vision for future applications in transfusion therapy.
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