This report describes a mass spectrometry based approach to platelet proteomics and additional investigational strategies into the role of transfusion storage, including donor glucose dysregulation (diabetes) effects on the platelet proteome. Technically, our approach enabled detection with great sensitivity of a variety of molecular species in stored and/or platelets from diabetics. However as a caution it should be remembered that our MS approach involves a bottoms-up characterization of trypsin digested proteins and that it is not a direct measurement of biological activity. Previous studies of platelet storage have employed other approaches, including sonication rather than detergent for lysing platelets, and restricting proteomic methods to those that are gel based.45
These approaches have the limitation that they cannot be employed to characterize membrane molecules. Nonetheless, it is reassuring that previously reported increased abundance of septin and actin with storage was replicated in our findings. In a more recent publication, use of mass spectrometry and detergent lysis confirmed the original description of increases in septin with storage and also described increases in superoxide dismutase, Rho-GDP dissociation inhibitor, and zyxin, as well as enzymes involved in glucose metabolism (G6PD, hexokinase).8
New findings in our study show an increase in detectable molecular species, almost doubling the number previously reported. The variety of cell membrane species showing abundance changes in our study is noteworthy (, and ). Furthermore, the presence of altered abundance of molecular species of nuclear origin confirms our previous observation that transcription factors are present in platelets, and play previously unsuspected roles in platelet biology. Our group recently demonstrated that platelets contain and release in platelet microparticles (PMPs), the transcription factor, peroxisome proliferator-activated receptor gamma (PPARγ) and its heterodimeric partner retinoid X receptor (RXR). PPARγ ligands attenuate platelet release of pro-inflammatory and pro-coagulant mediators including soluble CD40L (sCD40L) and thromboxane A2
), and elicit transcellular effects.46, 47
This provides evidence that transcription factors are capable of biologic activity and have important roles in platelet physiology.
Other novel findings include the first data on differences between the proteome of platelets from diabetics versus those from healthy individuals, as well as the effects of diabetes on the proteome of platelets prepared and stored for blood transfusion. Most notable is that fresh platelets from diabetic patients in otherwise good health demonstrated almost as many abundance changes as stored platelets from healthy donors (well over 100). Most of these changes are in the same direction, namely a decrease from normal abundance. However, the most striking deviations quantitatively are increases in the abundance of hemoglobin subunits. While these may represent contaminants due to sample red cell lysis during preparation, this increased abundance of hemoglobin species was seen in all platelet sample from diabetics and was not seen in the three platelet samples from non-diabetics. Consistent with our findings, globin and ferritin mRNAs have been identified as abundant transcripts in platelets by two independent groups.35, 48
While these two facts taken together imply that platelets from diabetics have increased synthesis of these proteins, further investigation will be necessary to determine whether this is true or whether they take up increased amounts of hemoglobin from plasma or internalize it from interactions with red cells in vivo
Interestingly, platelets from diabetics had lower abundance of several adhesion molecules including a number of integrins and PECAM. Diabetic and stored platelets were both characterized by an increased abundance of an endopeptidase inhibitor and decreased abundance of fibrinogen receptors and thrombospondin. Our supporting experiments reinforced these findings as diabetic and stored platelets had impaired ability to spread on fibrinogen (). In contrast, increased abundance has been observed for some key collagen receptors. For example, the hemostatic cascade is initiated at sites of vascular injury by exposed extracellular matrix (ECM) and collagen, a key constituent. We found that extracellular matrix protein (ECM)-1 is significantly elevated in platelets from diabetics. Interestingly, ECM1 functions in the regulation of blood vessel formation and maintenance. It has been suggested that the influence on angiogenesis exerted by ECM1 may be important in wound repair49
and diabetic retinopathy.50
Moreover, it was previously shown that expression of the collagen-binding GPVI, a major platelet receptor involved in the development of thrombosis and atherosclerosis, is elevated in diabetics.43
Excitingly, our supporting studies herein provided corroborating data showing that integrin α2
central for stable adhesion to collagen following GPVI activation,42, 51
has diabetic-specific effects ().
Platelet storage over a five day period prior to transfusion, the FDA permissible duration, was characterized by some shared abundance changes with platelets from diabetics, and other unique changes in abundance. GPIX, an adhesion molecule, increased in abundance with storage, but most other receptors and adhesion molecules decreased in abundance, including GPIb, GPV, and GPIV. These findings suggest storage may decrease the functional capabilities of both diabetic and stored platelets, and impair interactions with endothelium and hemostatic molecules such as fibrinogen. These findings will need to be confirmed by techniques employing functionally viable platelets as we demonstrated here with our initial two supporting studies ( and ). This combined approach will assist in constructing a thorough understanding of the differences between diabetic and normal platelet physiology, and the role of storage in the “platelet storage lesion”.