Isolation and proteomic analysis of platelets by SELDI-TOF MS

Abstract

Many growth factors, leukotrines, and biological ligands are not circulating free in plasma or serum, except in the case of late or disseminated disease. During early tumor growth and angiogenesis, platelets actively and selectively sequester regulators of angiogenesis and, as such, the platelet protein content can be used as a marker of early tumor growth or angiogenesis. With the recent increase in the clinical use of biologic modifiers in cancer and chronic disease therapy, the search for markers of early disease, therapeutic response, and/or recurrence has suggested that analysis of platelet proteins may be more relevant and accurate. We provide a guideline for the proteomic analysis of platelet proteome, placing specific emphasis on angiogenesis regulators, even though other platelet proteins may serve as markers of disease in the future. The analysis of serum/plasma has been fraught with difficulties because of the extraordinarily large number of proteins and because some of the proteins are contained in extraordinarily large amounts, masking the less abundant proteins. Thus, platelets may provide a much more biologically relevant analyte for biomarker discovery.

Fig. 1

Graphical representation of protein levels of angiogenesis regulators in plasma and platelets. We present four angiogenic regulators and their respective pattern of expression. The levels of pro-angiogenic growth factors (VEGF, bFGF, and PDGF) tend to be higher in the platelets, but not plasma, of tumor-bearing mice as compared to tumor-free sham operated mice. Although at 30 days nonangiogenic tumors are ∼100 times smaller than their angiogenic counterparts, the platelet proteome in mice bearing nonangiogenic tumors shows detectable differences in the levels of angiogenesis regulators. In mice with nonangiogenic dormant tumors, the elevation of positive angiogenesis regulators tends to be counterbalanced by an elevation of the endogenous inhibitor endostatin. In contrast, in mice bearing the angiogenic clone, (red) platelets show a decrease in negative angiogenesis regulators such as endostatin. This suggests that, in the “angiogenic” tumors, the overall balance of angiogenesis regulators may be tipped toward a more pro-angiogenic phenotype in a manner reflected on the platelet proteome. Of note is the observation that all of the proteins are found at much higher concentrations in the platelets when compared to the plasma. The experiment was repeated on two separate occasions with five mice per each experiment, and the graph represents means ± standard error of means. This research was originally published in Blood. Klement GL, Yip TT, Cassiola F, Kikuchi L, et al. Platelets actively sequester angiogenesis regulators. Blood. 2009; 113:2835–2842. © American Society of Hematology.

Fig. 2

Morphological changes in platelets due to activation. In a resting state, platelets are discoid in shape with two types of granules, alpha and dense granules (a). Platelets also contain lysosomes and mitochondria. The membrane of the platelet is interspersed with open canallicular system (OCS), which represents direct communications of the cytoplasm with the exterior of the platelet. This tenuous balance of the platelet membrane is crucial in the activation of platelets, which results in retraction of the granular content to the center of the platelet, widening of the OCS, and extension of filopodia, lamellipodia, and pseudopodia (b). Image is courtesy of Dr. Flavia Cassiola (27)

Fig. 3

Pretreatment of platelets with prostaglandin E alters the level of a protein with a mass of 14,620 Da. Platelets in PRP were incubated with PGE₂ for 1 h at 37°C or untreated, but given vehicle control. Following incubation, platelets were isolated, lysed, and analyzed by SELDI-TOF MS. Only the peak with a mass of 14,620 was found to be significantly altered by treatment with PGE₂ (p-value < 0.05). This is an important observation as pharmacological pretreatment of platelets to prevent activation may alter the protein content of the platelets.

Fig. 4

Diagram of whole blood fractionation following centrifugation. Immediately following blood draw, the entire solution within the tube is homogenous and uniform in color (a). Following the first centrifugation at 150 × g, the whole blood separated into three distinct layers: PRP (top layer), buffy coat (middle layer), and the hematocrit (bottom layer). The hematocrit contains red blood cells, the buffy coat contains white blood cells, while the platelets are contained within the PRP. When removing the PRP, care should be taken to ensure that none of the buffy coat is taken up.

Fig. 5

Typical spectrum for Fraction 1 of a platelet lysate on CM10 surface arrays. A low laser energy was used to generate the spectrum resulting in mostly low molecular weight proteins being detected. One of the most abundant proteins in platelets is platelet factor 4 (PF-4) and this polypeptide is observed as the peak at approximately 7.8 kDa.

Fig. 6

Hemolysis alters the protein profile on CM10 arrays. Plasma samples obtained from 96 prostate cancer patients were segregated into four groups based on their level of hemolysis. Hemolysis was detected by eye and determined by the level of “redness.” Group 0 indicates samples in which no detectable red color was observable and hence contains no hemolysis (36 samples). Group 1 contains samples that were slightly “pink” (45 samples), group 2 contains samples that were more orange in color (12 samples), and group 3 contains samples that were “blood red” (3 samples). A protein with an approximate mass of 66 kDa (assumed to be serum albumin) was significantly decreased with increasing hemolysis levels. This most likely was the result of increased hemoglobin levels out-competing albumin for binding sites on the CM10-binding surface.