Platelet Aggregation and Adhesion during Dietary Copper Deficiency in Rats

Abstract

SummaryThe role of dietary copper deficiency in platelet-to-endothelial cell adhesion and in platelet-to-platelet aggregation was studied in vitro. Platelets were obtained from male, weanling Sprague-Dawley rats fed purified diets which were either copper-adequate (CuA, 6.3 μg copper/g of diet) or copper-deficient (CuD, 0.3 μg copper/g of diet) for 4 weeks. The platelet adhesion study was performed by adding CuA or CuD platelets either suspended in homologous plasma or in Tyrode buffer salt solution (TBSS) to cultured rat endothelial cells. After a one hour incubation at 37° C non-adhered platelets were removed and counted in a microcytometer. Platelet aggregation in platelet rich plasma (PRP) samples was induced by adding ADP (2 × 10−4 M) and measured in a turbidometric platelet aggregometer. The content of von Willebrand factor (vWF) in platelets and in plasma and the content of fibrinogen in platelets was determined. Platelet adhesion to rat endothelial cells was significantly lower for platelets from CuD rats than for platelets from CuA rats. ADP induced platelet aggregation from CuD rats was significantly higher than platelet aggregation from CuA rats. The content of vWF in platelets and in plasma from CuD rats was significantly lower than in platelets and plasma from CuA rats. However, the amount of fibrinogen in platelets from CuD rats was about 4-fold higher than that in platelets from CuA rats while the plasma fibrinogen was lower in CuD rats than in CuA rats. These studies illustrate that copper deficiency diminishes platelet adhesion to endothelial cells but increases platelet aggregability. The results suggest that these physiological alterations may be the result of decreased platelet vWF and increased platelet fibrinogen during dietary copper deficiency.

In vitro platelet adhesion to endothelial cells at low shear rates during copper deficiency in rats

“…However, because in both the current study (Figs. 1-3) and in previous in vitro work [10], we have demonstrated a decreased adhesion of CuD platelets to cultured endothelial cells in the absence of subendothelial components, there appears to be another adhesion component that is dependent on copper. At present we have only studied the role of vWF in platelet adhesion during dietary copper restriction.…”

“…It is also synthesized and secreted from cultured endothelial cells [32]. We have previously shown that CuD platelets contain significantly less vWF than CuA platelets and that, independent of plasma and associated coagulation factors, the CuD platelets adhere less to cultured endothelial cells under static conditions than do the CuA controls [10]. In the current study, under two different shear rate conditions, CuA platelets adhere less to copper-chelated endothelial cells than to normal cultured endothelial cells ( Figs.…”

“…Our previous in vivo experiments have demonstrated decreased thrombogenesis and prolonged bleeding time in the microcirculation of copper-deficient rats [7][8][9]. Recently, we have reported that dietary copper deficiency causes increased ADP-induced platelet aggregation in vitro and decreased platelet adhesion to cultured rat endothelial cells [10] in a static measurement system. We have now examined the possibility that shear forces acting on platelet-endothelial adhesion may be a mechanism for the decreased thrombogenesis and hemostasis in copper deficiency.…”

“…In vitro platelet adhesion to cultured endothelial cells (static conditions) demonstrated that the adherence of platelets from copper-deficient rats to normal endothelial cells was lower [10], while ADP-induced platelet aggregation was greater for copper-deficient platelets than for copperadequate platelets-suggesting that platelet-to-platelet interaction is enhanced [10]. These changes in platelet reactivity from CuD animals were coincident with alterations in platelet vWF and fibrinogen production [10].…”

Fibrinogen and fragment D-induced vascular constriction

“…Endothelial cells from rat coronary, skeletal, and cremaster muscle microcirculations were a gift from Cynthia Meininger (Texas A&M University). The cells were cultured and grown according to the method described previously until they formed a complete monolayer (24). The cell lysates were collected, and expression of ICAM-1 was tested by Western blot analysis (35).…”