Abstract 216: Cyclophilin A Regulates Hemostasis by Affecting Multiple Platelet Functions

Abstract

Background: Cyclophilin A (CyPA) is ubiquitously expressed and regulates multiple cellular functions including protein folding, intracellular trafficking and signal transduction. Extracellular CyPA is an inflammatory mediator that contributes to atherosclerosis, aortic aneurysms, vascular remodeling and myocardial ischemic reperfusion injury. Recent studies have shown that interactions among platelets, endothelial cells and leukocytes play important roles in inflammation. However, the role of CyPA in platelet function and thrombosis remains unclear. We hypothesized that CyPA is involved in platelet activation, resulting in an impact on hemostasis and arterial thrombosis. Methods: Hemostasis was assessed by determining tail bleeding times in CyPA -/- mice (28±3d of age) compared to age matched wild-type (WT) littermates. Blood loss during the bleeding time assays was quantified by determination of hemoglobin concentration as absorbance at 575nm. Blood was collected in EDTA tubes from the retro-orbital plexus and platelet counts were measured using an automatic hemocytometer. Flow cytometric analysis was used to determine the expression of P-selectin and fibrinogen binding in response to various agonists. Washed platelets from WT and CyPA -/- mice were allowed to spread on immobilized fibrinogen and visualized by differential interference contrast (DIC) microscopy. Results: Tail bleeding times were significantly prolonged in CyPA -/- mice (n=13) compared to WT mice (n=20) (median bleeding time: 1025 s vs. 456 s). Accordingly, blood loss during the bleeding time assay was also significantly increased in CyPA -/- mice (OD575: 0.54±0.22 vs. 0.08±0.01). CyPA -/- mice have similar platelet counts as the age matched WT mice. CyPA deficiency impaired P-selectin expression in response to thrombin and ADP plus U46619 , and fibrinogen binding in response to thrombin. Furthermore, CyPA -/- platelets had decreased filopodia and lamellipodia formation on immobilized fibrinogen. CyPA -/- platelets exhibited a decrease of 41% in spreading area as compared to WT platelets. Conclusion: These data indicate for the first time that CyPA regulates hemostasis through affecting multiple platelet functions including α-granule secretion, platelet activation and spreading.