A Humanin Analog Attenuates Platelet Responses to Vascular Injury

Abstract 1131

Humanin (HN), a 24-amino acid endogenous antiapoptotic peptide, was initially shown to protect against neuronal cell death by Alzheimer's disease-related insults. It has recently been found that an exogenous analog of HN (HNG) in which the 14^th amino acid serine is replaced with glycine protected against cerebral and cardiac ischemia reperfusion (I/R) injury in cortical neurons and cardiomyocytes, respectively. Platelet activation and thrombus formation has been shown to play an important role during I/R injury by exacerbating the extent of the infarct size. However, it is presently unknown whether HNG affects platelet function and the subsequent arterial thrombus formation. We thus examined whether HNG affects platelet activation and thrombus formation both in vitro and in vivo. Human platelets were isolated from healthy adults. Preincubation of washed human platelets with HNG (4μM) reduced collagen- or convulxin-induced platelet aggregation by 56.8% (P<0.05) and 71.9% (P<0.001), respectively. Similarly, HNG significantly reduced ATP release stimulated by collagen or convulxin. Convulxin-induced P-selectin expression and fibrinogen binding on single platelet was inhibited by HNG, as measured by flow cytometry. Moreover, HNG reduced platelet spreading on the fibrinogen coated surface by 62.9 % (P <0.05). Western blot revealed a reduction of platelet AKT phosphorylation by HNG upon collagen stimulation, implying the involvement of PI3K pathway. In addition, MAPK P38 phosphorylation by collagen and convulxin was also reduced by HNG. HNG effects on thrombus formation were tested in vivo in a ferric chloride-induced carotid artery injury model in mice. The intraperitoneal injection of HNG (25μg/kg) to male C57BL6/J mice significantly extended the first occlusion time (7.3±0.4 min, N=10), when compared to the saline injected littermates (5.4±0.7 min, N=12) (P <0.05). Furthermore, the number of mice that formed stable thrombus was less in the HNG–treated group (3/13) than the control group (6/13), while the non-occlusion mouse number was more in the HNG-treated group (3/13) than the control group (1/13). Together, these data show that HNG inhibits platelet activation and arterial thrombus formation. This might suggest that the protective effects of HNG against ischemia reperfusion injury could be, in part, via attenuating platelet activation. Therefore, HNG could be a potential therapeutic agent in thrombotic and cardiovascular disorders.