3-3--dihexyl-2-2--oxacarbocyanine and Thrombocytopenia

Prototypes of platelet (PLT) substitutes have been studied and the focus was on a dodecapeptide, HHLGGAKQAGDV (H12), which is a fibrinogen gamma-chain carboxy-terminal sequence (gamma 400-411) and exists only in the fibrinogen domain.. H12 was conjugated to the surface of polymerized albumin particles (polyAlb) as biocompatible and biodegradable particles with a mean diameter of 260 +/- 60 nm, and the hemostatic ability of H12-conjugated polyAlb (H12-polyAlb) under flow conditions and thrombocytopenic rats have been studied.. H12-polyAlb enhanced the in vitro thrombus formation of activated PLTs on a collagen-immobilized plate when exposed to the flowing thrombocytopenic imitation blood. Furthermore, the analysis of the tail bleeding time of rats that were made thrombocytopenic by busulfan injection showed that H12-polyAlb had a hemostatic effect. Based on the bleeding time and the amount injected, the hemostatic capacity of 20 H12-polyAlb was estimated to correspond to that of one PLT.. These results were important first steps toward the development of PLT substitutes and indicated that H12-polyAlb may be a suitable candidate for an alternative to human PLT concentrates transfused into thrombocytopenic patients in the future.

Topics: Albumins; Animals; Bleeding Time; Blood Platelets; Blood Substitutes; Busulfan; Carbocyanines; Cell Adhesion; Coated Materials, Biocompatible; Collagen Type I; Dose-Response Relationship, Drug; Fibrinogen; Fluorescent Dyes; Hemostatics; Humans; Male; Metabolism; Microspheres; Oligopeptides; Platelet Activation; Platelet Aggregation; Platelet Aggregation Inhibitors; Platelet Count; Rats; Rats, Wistar; Solutions; Thrombocytopenia

The mechanism(s) involved in the clearance of senescent platelets are largely unknown. We have recently demonstrated that platelet aging in vivo is associated with loss of membrane phospholipid asymmetry, a universal phenomenon in cells undergoing apoptosis. Thus, we postulated that senescent platelets may exhibit programmed cell death changes. which may trigger their removal from circulation. Since platelets contain the apoptosis machinery as well as mitochondria, a key organelle in the regulation of apoptosis, we studied the appearance of apoptotic-like changes during platelet aging in vivo. To investigate this, we assessed changes in mitochondrial membrane potential (deltapsi) in circulating canine platelets during decline in platelet count after suppression of thrombopoiesis by estradiol injection, a validated model to obtain circulating platelets of increasing mean age. Phosphatidylserine (PS) exposure was determined by flow cytometry by binding of FITC-labeled annexin V. Mitochondrial deltapsi was studied with the cationic lipophilic dye DIOC6 (3) and the J-aggregate-forming cation JC-1 and analysis by flow cytometry. The proportion of platelets with exposed PS rose significantly with age, from 2.88% before to 6.7%, 8 days after estradiol injection. By flow cytometry it was demonstrated a significant decreased in DIOC6 (3) fluorescence (median fluorescence intensity 791+/-98 vs 567+/-102 day 0 vs day 8 post injection of estradiol, respectively; n: 11; p <0.01), consistent with mitochondrial deltapsi collapse. JC-1 has the unique property of forming J-aggregates under high mitochondrial deltapsi (red fluorescence, FL2) whereas the monomeric form fluoresces in green (FL1). Aged platelets in vivo, loaded with JC-1, exhibited a significant increase in FL1/FL2 ratio (2.5+/-1.7 vs 4.7+/-1.6, day 0 vs day 8 post injection of estradiol, respectively; n: 13; p <0.05), confirming the mitochondrial deltapsi alteration. The results show that platelet aging in vivo is associated with a decrease in mitochondrial deltapsi and PS exposure. In conclusion, our data provide for the first time, evidence that platelet senescence is associated with changes characteristics of apoptosis, which may promote their removal from circulation.

Topics: Animals; Apoptosis; Blood Platelets; Carbocyanines; Cattle; Cellular Senescence; Depression, Chemical; Estradiol; Fluorescent Dyes; Hematopoiesis; Intracellular Membranes; Membrane Lipids; Membrane Potentials; Mitochondria; Models, Animal; Phosphatidylserines; Thrombocytopenia