In the present study, we demonstrated that SAA exhibits an antiplatelet effect on patients with DM2. Notably, this antiplatelet effect is not less effective in DM2 patients with complications than in those without complications. All these findings enable a better understanding of the antiplatelet effect of SAA in DM2, which ultimately leads to the development of novel pharmaceutical strategies for the antiplatelet treatment of patients suffering from DM2.
Platelets obtained from DM2 are hyperactive and demonstrate exaggerated aggregation as well as thrombus generation . There are many different mechanisms that have been attributed to the diabetes-associated enhanced platelet activation , such as a loss of the antiplatelet effect of insulin , high blood glucose, oxidative stress , elevated vascular shear forces , increased binding of fibrinogen , altered expression of glycoprotein receptors and proteins attached to the platelet surface [21,22,23]. It is clear, from the literature, that there is an increased expression of platelet activation makers, such as CD62p and PAC-1, as measured by flow cytometry in DM2, which contributes to the progression of thrombotic and CVD events. Our study suggested that the expressions of CD62p and PAC-1 were elevated in DM2 under a resting condition, while the expressions of these two markers were evidently decreased, after preincubation with SAA in vitro, under a stimulating condition, compared to the control groups. Similar results were achieved in our previous study, which reported that platelet activation markers were at a low expression level in the SAA group in healthy volunteers . It is worth noting, in our present study, that the expressions of PAC-1 and CD62p in DM2 under the resting condition were not increased to as high a level as they did in the previous study . This might be associated with the dissimilarity of the participants. Most of the participants enrolled in this study were receiving insulin therapy under a glycemic control. Moreover, previous researches indicated that insulin treatment has a beneficial effect on platelet activation, and aggregation has a beneficial effect on “diabetic platelets”, which may be related to the direct action of insulin on erythrocyte deformability [25, 26]. This may explain why the level of platelet activation in our study is lower than that in other studies. Additionally, the present study revealed that the maximum platelet aggregation induced by ADP or Thrombin in the SAA group was also obviously inhibited. However, the inhibiting effect in this study was not as good as that in our previous study . In order to fully embody the functional status of diabetic platelets in plasma, this study chose PRP as the test specimen, instead of washed platelets. However, there are more small-molecule plasma proteins, which may interact with SAA and ultimately interfere with the antiplatelet effect of SAA in PRP, rather than washed platelets, which may account for the different result. Based on this point, the interaction between plasma proteins and SAA should be considered, and the use of SAA therapy in DM2 patients should be promoted in the future.
Recent researches on the action mechanisms of SAA have found that SAA can antagonize the activity of both P2Y1 and P2Y12 receptors in the low μM range . Moreover, pretreatment with SAA on platelets caused an increase in the cAMP level in platelets activated by ADP, which indicates that SAA might possess antithrombotic activities . In our study, we found that SAA inhibited platelet activation aroused by a variety of agonists, which indicates that SAA may intervene in a shared signaling molecule of platelet activation. However, a limited number of studies have investigated the interruption of signaling events by SAA. According to our previous study, SAA inhibits platelet activation and arterial thrombosis via the inhibition of phosphoinositide 3-kinase (PI3K) . Moreover, we found that SAA demonstrated a more potent inhibition of Rap1b activation than PI3KP inhibitors. As Rap1b is dually controlled by the PLC and PI3K pathways, SAA may affect other platelet signaling mechanisms, apart from PI3K pathways. Recent research has focused on the platelet-specific collagen receptor, glycoprotein VI (GPVI), as a potential antiplatelet target. Signaling events downstream from GPVI are influenced by hyperglycemia, oxidative stress, and shear stress . According to previous investigations, SAA has extensive pharmacological effects, including antidiabetic , antioxidant  and other effects. Therefore, the antiplatelet effect of SAA may be associated with the interruption of the GPVI signaling pathway. To confirm this hypothesis, further research will be needed to assess this possible signaling pathway.
In addition, we further investigated the diabetic complication-related difference in platelet aggregation and activation in response to SAA. Our findings indicate that the antiplatelet efficacy of SAA was not reduced in DM2 with diabetic complications, comparing to DM2 without complications. In other words, the antiplatelet effect of SAA did not abate diabetic complications. Besides, the baseline of the platelet aggregation and activation between DM2 with and DM2 without complications shows no statistical difference, which may explain their similar response to SAA.
Since SAA possesses a variety of bioactivities, including a defense from oxidative damage, improvement of remembrance , lowering of blood glucose and inhibition of platelet aggregation and activation, patients with diabetic complications treated with SAA could not only gain cardiovascular benefits, but also additional benefits relating to diabetic complications control. On the other hand, SAA could also play a preventive role, when SAA is treated as an antiplatelet agent in DM2 without complications. Based on the speculations above, SAA might be a novel and promising drug candidate for diabetes treatment, which may eventually contribute to the amelioration of the heavy burden of CVD among the population of DM2. Moreover, to verify all these speculations, further clinical trial investigations will be needed.
There are several limitations to this study. The low number of enrolled patients resulted in there being fewer than 20 in each of the three groups, with only 11 in the group consisting of DM2 patients without diabetic complications. A further limitation is that platelet reactivity was not measured in patients treated with SAA due to a lack of evidence that SAA is safe to use in a human body test.