A comparative study of the effects of concentrated growth factors in two different forms on osteogenesis in vitro.

Extending the release cycle of growth factors to match the cycle of bone remodeling is difficult. When using concentrated growth factors (CGFs), the release of growth factors is excessively rapid. In the present study, CGF samples were prepared by centrifugation. CGF samples were then lyophilized and grinded into a powder, which was termed freeze­dried CGF. The freeze­dried CGF samples were mixed with chitosan­alginate composite hydrogels, and the mixture was lyophilized. The result was a chitosan­alginate composite CGF membrane, which was called sustained­release CGF. This study investigated whether freeze­dried CGF in a chitosan­alginate composite gel can release CGF steadily to achieve effective osteogenesis. The proliferation and osteogenic expression of MC3T3­E1 cells induced by the supernatants from incubation with freeze­dried CGF and sustained­release CGF were evaluated. The concentrations of the growth factors, transforming growth factor ß1 (TGF­ß1), insulin­like growth factor­1 (IGF­1), platelet­derived growth factor­AB (PDGF­AB) and vascular endothelial growth factor (VEGF), in these two experimental groups at different times were determined by ELISA kits. The freeze­dried CGF showed better osteogenic performance than the sustained­release CGF in the early stages. At later stages, the sustained­release CGF had significant advantages over freeze­dried CGF in terms of promoting osteogenic mineralization. By characterizing the biologic properties of the CGF in the two different forms in vitro, we obtained a better understanding of their clinical effects.

Platelet lysate as a substitute for animal serum for the ex-vivo expansion of mesenchymal stem/stromal cells: present and future.

The use of fetal bovine serum (FBS) as a cell culture supplement is discouraged by regulatory authorities to limit the risk of zoonoses and xenogeneic immune reactions in the transplanted host. Additionally, FBS production came under scrutiny due to animal welfare concerns. Platelet derivatives have been proposed as FBS substitutes for the ex-vivo expansion of mesenchymal stem/stromal cells (MSCs) since platelet-derived growth factors can promote MSC ex-vivo expansion. Platelet-derived growth factors are present in platelet lysate (PL) obtained after repeated freezing-thawing cycles of the platelet-rich plasma or by applying physiological stimuli such as thrombin or CaCl2.PL-expanded MSCs have been used already in the clinic, taking advantage of their faster proliferation compared with FBS-expanded preparations. Should PL be applied to other biopharmaceutical products, its demand is likely to increase dramatically. The use of fresh platelet units for the production of PL raises concerns due to limited availability of platelet donors. Expired units might represent an alternative, but further data are needed to define safety, including pathogen reduction, and functionality of the obtained PL. In addition, relevant questions concerning the definition of PL release criteria, including concentration ranges of specific growth factors in PL batches for various clinical indications, also need to be addressed. We are still far from a common definition of PL and standardized PL manufacture due to our limited knowledge of the mechanisms that mediate PL-promoting cell growth. Here, we concisely discuss aspects of PL as MSC culture supplement as a preliminary step towards an agreed definition of the required characteristics of PL for the requirements of manufacturers and users.

Functional and differential proteomic analyses to identify platelet derived factors affecting ex vivo expansion of mesenchymal stromal cells.

BACKGROUND: Multilineage differentiation, immunomodulation and secretion of trophic factors render mesenchymal stromal cells (MSC) highly attractive for clinical application. Human platelet derivatives such as pooled human platelet lysate (pHPL) and thrombin-activated platelet releasate in plasma (tPRP) have been introduced as alternatives to fetal bovine serum (FBS) to achieve GMP-compliance. However, whereas both pHPL and tPRP support similar proliferation kinetics of lipoaspirate-derived MSC (LA-MSC), only pHPL significantly accelerates bone marrow-derived MSC (BM-MSC) expansion. To identify functionally bioactive factors affecting ex vivo MSC expansion, a differential proteomic approach was performed and identified candidate proteins were evaluated within a bioassay. RESULTS: Two dimensional difference gel electrophoresis (2D-DIGE), MALDI-TOF analyses and complementary Western blotting revealed 20 differential protein species. 14 candidate proteins occured at higher concentrations in pHPL compared to tPRP and 6 at higher concentrations in tPRP. The candidate proteins fibrinogen and apolipoprotein A1 differentially affected LA- and BM-MSC proliferation.In a second set of experiments, reference cytokines known to foster proliferation in FBS were tested for their effects in the human supplements. Interestingly although these cytokines promoted proliferation in FBS, they failed to do so when added to the humanized system. CONCLUSIONS: The differential proteomic approach identified novel platelet derived factors differentially acting on human MSC proliferation. Complementary testing of reference cytokines revealed a lack of stimulation in the human supplements compared to FBS. The data describe a new coherent approach to combine proteomic technologies with functional testing to develop novel, humanized, GMP-compliant conditions for MSC expansion.

High level expression, efficient purification and bioactivity assay of recombinant human platelet-derived growth factor AA dimer (PDGF-AA) from methylotrophic yeast Pichia pastoris.

Platelet-derived growth factors (PDGFs) are important biochemical mediators regulating many physiological and pathophysiological processes, including promotion of the chemotactic recruitment and proliferation of cells involved in wound repair. Previously, homodimers of rhPDGF-AA protein were purified from Escherichia coli. However, eukaryotic proteins often contain posttranslational modifications, such as glycosylation, that are required for biological functions. In this study, an efficient method was established to purify a glycosylated rhPDGF-AA dimer from P. pastoris culture media by one step CM Sepharose ion exchange chromatography yielding about 20mg/L of over 95% highly purified rhPDGF-AA. Mass spectrometry analysis of the purified rhPDGF-AA displayed a molecular weight (MW) of 27,825.513Da, composed of a subunit with MW of 15,042.945Da and a subunit with MW of 12,904.374Da. The size difference is accounted for by differential glycosylation of the monomers. Biological activity of the rhPDGF-AA was confirmed by its ability to induce NIH/3T3 cells proliferation. The experimental procedure we have developed facilitates production of an active glycosylated rhPDGF-AA in large amounts for further research and drug development.

Aptamer-based colorimetric detection of platelet-derived growth factor using unmodified gold nanoparticles.

We developed a simple method for the detection of platelet-derived growth factors (PDGFs) based on base stacking effect coupled with an unmodified gold nanoparticle (AuNP) indicator. In the absence of a target, an aptamer probe and a capture probe stably co-exist in a solution, as it is difficult to sustain an interaction between both these probes due to the short 8bp duplex. However, when a target protein binds to the aptamer probe, the strong base stacking effect can lead to a favorable and stable interaction between the aptamer and capture probes. Hence, the capture probe dissociates from the AuNP surfaces, inducing AuNP aggregation. Compared with other AuNP-based aptasensors for PDGFs, using this base stacking effect can overcome a structured-aptamer method's limitation of requiring thiolated-aptamer-modified AuNPs. Under optimal detection conditions, this label-free colorimetric sensor could detect PDGFs down to 6nM with high selectivity in the presence of other interferring proteins. This simple detection approach provides viable methods for a structured-aptamer sensing protocol.

Platelet-rich plasma-derived growth factors promote osteogenic differentiation of rat muscle satellite cells: in vitro and in vivo studies.

PRP (platelet-rich plasma)-derived growth factors are a new application of tissue engineering and a developing area for researchers and clinicians. We have assessed the effects of PRP-derived growth factors on the proliferation and osteogenic differentiation of rMSCs (rat muscle satellite cells), and constructed a novel tissue engineering bone composed of PRP-derived growth factors and rMSCs. PRP were created by a freeze-thaw process. rMSCs were isolated from rat masticatory muscle using serial platings technique. Wst-1 assay, SEM (scanning electron microscopy), ALP (alkaline phosphatase) activity, total protein concentration, AR (Alizarin red S) staining, calcium analyses and RT-PCR (reverse transcription-PCR) of osteogenic-related genes were used to assess the effect of PRP-derived growth factors on proliferation and osteogenic differentiation of cultured rMSCs on scaffolds. The different composite scaffolds were implanted to the subcutaneous spaces of nude mice. H&E (haematoxylin and eosin) and Masson's trichrome staining were used to examine the ectopic bone formation. In vitro, we found that PRP-derived growth factors showed excellent cell compatibility and significantly enhanced cell proliferation over serum and control groups at 48 and 72 h. SEM, ALP activity, AR staining, calcium analyses and RT-PCR showed that PRP-derived growth factors significantly increased cells osteogenic differentiation when compared with other groups. In vivo examination showed that more fibrous tissue capsule and bone with lamellar structures appeared in PRP-derived growth factors groups. These results suggest that the PRP-derived growth factors significantly promote rMSCs proliferation, osteogenic differentiation compared with serum and scaffolds alone, and may be suitable for stem cell growth factors delivery and bone tissue engineering.

Optimized preparation method of platelet-concentrated plasma and noncoagulating platelet-derived factor concentrates: maximization of platelet concentration and removal of fibrinogen.

Platelet-rich plasma (PRP) has been clinically used as an easily prepared growth factor cocktail that can promote wound healing, angiogenesis, and tissue remodeling. However, the therapeutic effects of PRP are still controversial, due partly to the lack of optimized and standardized preparation protocols. We used whole blood (WB) samples to optimize the preparation protocols for PRP, white blood cell-containing (W-PRP), platelet-concentrated plasma (PCP), and noncoagulating platelet-derived factor concentrate (PFC). PRP and W-PRP were most efficiently collected by 10 min centrifugation in a 15-mL conical tube at 230-270 g and 70 g, respectively. To prepare PCP, platelets were precipitated by centrifugation of PRP at >2300 g, 90% of supernatant plasma was removed, and the platelets were resuspended. For preparation of noncoagulating PFC, the supernatant was replaced with one-tenth volume of saline, followed by platelet activation with thrombin. Platelet (before activation) and platelet-derived growth factor (PDGF)-BB (after activation) concentrations in PCP were approximately 20 times greater than those in WB, whereas PFC contained a 20-times greater concentration of platelets before platelet activation and a 50-times greater concentration of PDGF-BB without formation of a fibrin gel after platelet activation than WB. Surprisingly, total PDGF-BB content in the PFC was twice that of activated WB, which suggested that a substantial portion of the PDGF-BB became trapped in the fibrin glue, and replacement of plasma with saline is crucial for maximization of platelet-derived factors. As an anticoagulant, ethylene di-amine tetra-acetic acid disodium inhibited platelet aggregation more efficiently than acid citrate dextrose solution, resulting in higher nonaggregated platelet yield and final PDGF-BB content. These results increase our understanding of how to optimize and standardize preparation of platelet-derived factors at maximum concentrations.

Expression and production of therapeutic recombinant human platelet-derived growth factor-BB in Pleurotus eryngii.

Recombinant human platelet-derived growth factor-BB (rhPDGF-BB) is widely used in many therapeutic applications. Until now, there has been no report on rhPDGF-BB expressed in fungi. In this study, we tested whether Pleurotus eryngii could support the expression of human therapeutic rhPDGF-BB protein. A binary vector pCAMBIA1304 containing the hPDGF-BB gene was constructed and introduced into P. eryngii via Agrobacterium tumefaciens-mediated transformation. The transformation of hPDGF-BB gene was confirmed by Southern blot and PCR, whereas the expression was confirmed by Western blot analysis. The recombinant hPDGF-BB reached a maximum expression level of 1.98% of total soluble protein in transgenic mycelia and was in dimeric form. A bioassay revealed that hPDGF-BB expressed in P. eryngii increased proliferation of NIH-3T3 cells similarly to standard material. These results suggest that P. eryngii can be a robust system for the production of human therapeutic proteins including the hPDGF-BB.

Selection of aptamers for a non-DNA binding protein in the context of cell lysate.

Aptamer-facilitated Protein Isolation from Cells (AptaPIC) is a recently introduced method that allows, in particular, generation of aptamers for a protein target in a context of a crude cell lysate. The approach enables efficient, tag-free, affinity purification of target proteins which are not available in a pure form a priori, and for which no affinity ligands are available. In the proof-of-principle work, AptaPIC was used to develop aptamers for and purify MutS, a DNA mismatch repair protein. The DNA-binding nature of MutS raised concerns that AptaPIC was not a generic technique and could be inapplicable to protein targets that do not possess native nucleic acid-binding properties. Here we prove that these concerns are invalid. We used AptaPIC to generate pools of aptamers for human Platelet-Derived Growth Factor chain B (PDGF-B) protein, a non-DNA binding protein, in the context of a bacterial cell lysate, and subsequently purify it from the same lysate. Within a small number of rounds, the efficiencies of aptamer selection were similar in conventional Systematic Evolution of Ligands by Exponential Enrichment (SELEX) for pure protein and in AptaPIC for protein in the cell lysate. The conventional selection approach resulted in an aptamer pool with an EC(50) value of 2.0±0.1 µM, while the AptaPIC selection approach resulted in a pool with an EC(50) value of 3.9±0.4 µM. Our results clearly demonstrate that selection of aptamers for proteins in the cell lysate is not only realistic but also efficient.

A novel virally inactivated human platelet lysate preparation rich in TGF-beta, EGF and IGF, and depleted of PDGF and VEGF.

There is emerging interest in the use of standardized virally inactivated human platelet lysate preparations rich in GFs (growth factors) for cell cultures, cell therapy and clinical applications. In the present paper, we report a simple process to prepare a virally inactivated platelet lysate preparation rich in TGF-beta1 (transforming growth factor-beta1), EGF (epidermal growth factor) and IGF (insulin-like growth factor) and depleted of PDGF (platelet-derived growth factor) and VEGF (vascular endothelial growth factor). Apheresis platelet concentrates were treated by the S/D (solvent/detergent) viral inactivation procedure, then subjected to an oil extraction followed by adsorption with activated charcoal and finally sterile-filtered. The resulting preparation contained a mean of 368.4, 2.4 and 54.7 ng/ml of TGF-beta1, EGF and IGF respectively. PDGF-AB and VEGF were essentially completely removed by the charcoal treatment. The mean albumin, IgG, IgM and IgA and fibrinogen contents were approx. 40.0, 8.5, 0.87, 1.66 and 2.65 mg/ml respectively, cholesterol and triglycerides were at 15 and 20.7 mg/ml respectively and TnBP (tri-n-butyl phosphate) and Triton X-45 were at 8.7 and 8.8 p.p.m. respectively. Supplementing MEM (minimum essential medium) with 1-10% of this S/D-treated platelet lysate promoted the proliferation of MG63 and SIRC cell lines as well as, or better than, 10% (v/v) FBS (fetal bovine serum), as based on the MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay. The process used to prepare such S/D-treated platelet lysates is easily scalable for industrial production. Our results open up the possibility to evaluate the potential of this new preparation for stem cell expansion and/or bone tissue engineering and regeneration.

A virally inactivated platelet-derived growth factor/vascular endothelial growth factor concentrate fractionated from human platelets.

BACKGROUND: Human platelet concentrates (PCs) may be a source material to produce purified growth factors (GFs) for clinical use or cell therapy. However, no fractionation process of therapeutic-grade GF from PCs has ever been developed. STUDY DESIGN AND METHODS: PCs were virally inactivated by solvent/detergent (S/D) treatment, subjected to oil extraction to remove part of the S/D agents, and fractionated on a SP-Sepharose (SP) chromatographic column equilibrated in a phosphate-buffered saline (PBS) buffer, pH 7.5. The breakthrough was recovered, and the column was washed with the PBS buffer and then eluted by a 0.7 mol/L NaCl-PBS buffer pH 7.5 (SP-eluate). The SP-breakthrough and SP-eluate were characterized for their content in GF, proteins, lipids, and S/D agents. The MTS value of three cell lines cultivated in a medium containing 10% fetal bovine serum supplemented with 1% to 3% of SP-eluate or recombinant human (rHu) platelet-derived growth factor (PDGF)-BB was compared. RESULTS: The SP-eluate contained a mean of 47, 17, and 6 ng/mL PDGF-AB, -BB, and -AA, respectively, and 0.26 ng/mL vascular endothelial growth factor (VEGF). It was largely depleted of transforming growth factor-ß1 (2.33 ng/mL), epidermal growth factor (0.09 ng/mL), insulin-like growth factor (3.40 ng/mL), albumin, immunoglobulin (Ig)G, IgM, IgA, and fibrinogen, which were mostly in the breakthrough. tri-n-butyl phosphate and Triton X-45 were less than 2 ppm. Cell growth-promoting activity of the SP-eluate was at least as good as that of rHu-PDGF-BB. CONCLUSION: Human PC can be fractionated into a purified, virally inactivated PDGF and VEGF concentrate, opening perspectives for the development of a new range of blood products for clinical use and cell therapy procedures.

Preparation of pooled human platelet lysate (pHPL) as an efficient supplement for animal serum-free human stem cell cultures.

Platelet derived growth factors have been shown to stimulate cell proliferation efficiently in vivo(1,2) and in vitro. This effect has been reported for mesenchymal stromal cells (MSCs), fibroblasts and endothelial colony-forming cells with platelets activated by thrombin(3-5) or lysed by freeze/thaw cycles(6-14) before the platelet releasate is added to the cell culture medium. The trophic effect of platelet derived growth factors has already been tested in several trials for tissue engineering and regenerative therapy.(1,15-17) Varying efficiency is considered to be at least in part due to individually divergent concentrations of growth factors(18,19) and a current lack of standardized protocols for platelet preparation.(15,16) This protocol presents a practicable procedure to generate a pool of human platelet lysate (pHPL) derived from routinely produced platelet rich plasma (PRP) of forty to fifty single blood donations. By several freeze/thaw cycles the platelet membranes are damaged and growth factors are efficiently released into the plasma. Finally, the platelet fragments are removed by centrifugation to avoid extensive aggregate formation and deplete potential antigens. The implementation of pHPL into standard culture protocols represents a promising tool for further development of cell therapeutics propagated in an animal protein-free system.

High-level secretory production of recombinant human platelet-derived growth factor--BB by Saccharomyces cerevisiae under the non-selective conditions.

Recombinant human platelet-derived growth factor-BB (rhPDGF-BB) was produced by Saccharomyces cerevisiae. A two-stage cultivation strategy with mixture of glucose and galactose was developed to enhance rhPDGF-BB production, with its concentration being 32 mg/l fermentation broth under optimal conditions: corn steep powder as nitrogen source, 2 g/1 of glucose concentration at the beginning of induction phase, a pH of 5.0 and 6.5 for cell growth and rhPDGF-BB accumulation. The purification process consisted of yeast supernatant ultrafiltration followed by ion exchange chromatography and molecular sieve, and the recovery of rhPDGF-BB was estimated to be 20.28%. Biological activity of the purified rhPDGF-BB was 3.05 ng/ml.

Preliminary separation of the growth factors in platelet-rich plasma: effects on the proliferation of human marrow-derived mesenchymal stem cells.

BACKGROUND: Platelet-rich plasma (PRP) as a storage vehicle of growth factors has been successfully used in clinical applications, but in most cases the platelets were autologous. However, the large volume of blood withdrawn has detrimental effects on patients with anemia or poor general health. To overcome these limitations, this study was designed to separate the growth factors in homologous platelet-rich plasma. METHODS: The gel chromatography with Superdex-75 column was applied to separate PRP supernatants into 4 major fractions. Then the four fractions were vacuumed freeze-dried and re-dissolved in phosphate buffered saline. Proteins concentrations in PRP and in four fractions were detected by bicinchoninic acid protein assay; platelet derived growth factor-AB (PDGF-AB) and transforming growth factor beta1 (TGF-beta1) levels were determined by sandwich enzyme-linked immunosorbent assays. The effects of fractions on the proliferation of human marrow-derived mesenchymal stem cells (MSCs) were determined by 3-(4, 5- dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. RESULTS: PRP supernatants were separated into four major fractions by gel chromatography. The proteins recovery was 96.72%. Of the four fractions, fraction B contained the highest TGF-beta1 and PDGF-AB levels, and the highest proteins concentrations. Cell proliferation curves of MSC demonstrated that fraction B and C induced a remarkable increase of MTT values compared to the untreated culture (P < 0.05), and the effects of fraction B and C showed no significant difference compared to the PRP group (P > 0.05). Fraction A and D showed no significant difference to the negative control group (P > 0.05). CONCLUSIONS: The growth factors in PRP supernatants could be preliminarily separated into four fractions by gel chromatography, and the freeze-drying fractions retained the biological activity of growth factors. The growth factors were mostly presented in fraction B and C, and they promoted cell proliferation effectively.

The molecular cloning of platelet-derived growth factor-C (PDGF-C) gene of Gekko japonicus and its expression change in the spinal cord after tail amputation.

The platelet-derived growth factor-C (PDGF-C) gene of Gekko japonicus was obtained from a brain and spinal cord cDNA library. The results of Northern blot showed that transcript of PDGF-C gene of gecko is 2.8 kb in length, and it was abundantly expressed in tissues of heart, lung, kidney, and ovary. In situ hybridization (ISH) revealed that positive hybridization signals were present in both gray matter and white matter of the spinal cord. The change of PDGF-C expression in the spinal cord after tail amputation was examined by reverse transcription polymerase chain reaction (RT-PCR) and Western blot. The expression of PDGF-C in the spinal cord showed highest level at 1 day after tail amputation, and gradually decreased until 2 weeks, which indicated that the expression level of PDGF-C might be associated with the process of spinal cord injury and regeneration.

Platelet-rich plasma and platelet gel preparation using Plateltex.

BACKGROUND: The platelet gel is made by embedding concentrate platelets within a semisolid (gel) network of polymerized fibrin. It is believed that this blood component will be used more and more in the treatment of several clinical conditions and as an adjunctive material in tissue engineering. Several systems are available to produce platelet-rich plasma (PRP) for topical therapy. Recently, a new system became commercially available, Plateltex. Here we report the technical performance of this system in comparison with the performance of other commercially available systems: PRGF, PRP-Landesber, Curasan, PCCS, Harvest, Vivostat, Regen and Fibrinet. MATERIAL AND METHODS: Both the PRP and the gel were prepared according to the manufacturer's directions. The blood samples of 20 donors were used. The yield, the efficiency, and the amount of platelet-derived growth factor AB (PDGF-AB), transforming growth factor beta, vascular endothelial growth factor and fibroblast growth factor were measured in the resulting PRP. The feature of the batroxobin-induced gelation was evaluated. RESULTS: The yield, the collection efficiency and the growth factor content of Plateltex were comparable to those of most of the other available systems. The gelation time was not dependent on the fibrinogen concentration; however, it was strongly influenced by the contact surface area of the container where the clotting reaction took place (P < 0.0001). CONCLUSIONS: Plateltex provided platelet recovery, collection efficiency and PDGF-AB availability close to those provided by other systems marketed with the same intended use. Batroxobin, the enzyme provided to induce gelation, acts differently from thrombin, which is used by most other systems. Platelets treated with thrombin become activated; they release their growth factors quickly. Furthermore, thrombin-platelet interaction is a physiological mechanism that hastens the clot-retraction rate. On the contrary, platelets treated with batroxobin do not become activated; they are passively entrapped within the fibrin network, and their growth factor release occurs slowly. In these conditions, the clot retraction takes longer to occur. According to these differences between thrombin and batroxobin, it is expected that batroxobin-induced PRP activation will tailor slow release of the platelet content, thus, providing longer in loco availability of trophic factors. In selected clinical conditions, this durable anabolic factor availability might be preferable to quick thrombin-induced growth factor release.

[The effect of serum rich in platelet-released growth factors on proliferation of rat osteoblast in vitro].

OBJECTIVE: To study the effect of serum rich in platlelet-released growth factors on the biological functions of rat osteoblast. METHODS: Rat osteoblasts were isolated from parietal bone of fetal Spraque-Dawley (SD) rat. Platelet-rich plasma(PRP) and platelet-poor plasma (PPP) obtained from healthy SD rat were activated by thrombin to get serum rich in growth factors (SRGF) or serum poor in growth factors (SPGF). The cells were treated with 1.25%, 2.5%, 5% SRGF or SPGF, and cellular mitogenic activity was evaluated by thiazoly blue (MTT) colorimetric assay. Results When compared with SPGF at the same concentration, SRGF promoted the proliferation of rat osteoblast significantly (P = 0.002 - 0.004), and the proliferation seemed to correlate with the concentration of SRGF (Spearman's correlation coefficient, r(s) = 0.834). CONCLUSION: SRGF is capable of upregulating the proliferation of rat osteoblast.

Light-switching excimer probes for rapid protein monitoring in complex biological fluids.

Quantitative protein bioanalysis in complex biological fluids presents considerable challenges in biological studies and disease diagnosis. The major obstacles are the background signals from both the probe and the biological fluids where the proteins reside. We have molecularly engineered light-switching excimer aptamer probes for rapid and sensitive detection of a biomarker protein, platelet-derived growth factor (PDGF). Labeled with one pyrene at each end, the aptamer switches its fluorescence emission from approximately 400 nm (pyrene monomer) to 485 nm (pyrene excimer) upon PDGF binding. This fluorescence wavelength change from monomer to excimer emission is a result of aptamer conformation rearrangement induced by target binding. The excimer probe is able to effectively detect picomolar PDGF in homogeneous solutions. Because the excimer has a much longer fluorescence lifetime (approximately 40 ns) than that of the background (approximately 5 ns), time-resolved measurements were used to eliminate the biological background. We thus were able to detect PDGF in a cell sample quantitatively without any sample pretreatment. This molecular engineering strategy can be used to develop other aptamer probes for protein monitoring. Combined with lifetime-based measurements and molecular engineering, light-switching excimer aptamer probes hold great potential in protein analysis for biomedical studies.

Unique in vivo modifications of coagulation factor V produce a physically and functionally distinct platelet-derived cofactor: characterization of purified platelet-derived factor V/Va.

Platelet- and plasma-derived factor Va (FVa) serve essential cofactor roles in prothrombinase-catalyzed thrombin generation. Platelet-derived FV/Va, purified from Triton X-100 platelet lysates was composed of a mixture of polypeptides ranging from approximately 40 to 330 kDa, mimicking those visualized by Western blotting of platelet lysates and releasates with anti-FV antibodies. The purified, platelet-derived protein expressed significant cofactor activity such that thrombin activation led to only a 2-3-fold increase in cofactor activity yet expression of a specific activity identical to that of purified, plasma-derived FVa. Physical and functional differences between the two cofactors were identified. Purified, platelet-derived FVa was 2-3-fold more resistant to activated protein C-catalyzed inactivation than purified plasma-derived FVa on the thrombin-activated platelet surface. The heavy chain subunit of purified, platelet-derived FVa contained only a fraction ( approximately 10-15%) of the intrinsic phosphoserine present in the plasma-derived FVa heavy chain and was resistant to phosphorylation at Ser(692) catalyzed by either casein kinase II or thrombin-activated platelets. MALDI-TOF mass spectrometric analyses of tryptic digests of platelet-derived FV peptides detected an intact heavy chain uniquely modified on Thr(402) with an N-acetylglucosamine or N-acetylgalactosamine, whereas Ser(692) remained unmodified. N-terminal sequencing and MALDI-TOF analyses of platelet-derived FV/Va peptides identified the presence of a full-length heavy chain subunit, as well as a light chain subunit formed by cleavage at Tyr(1543) rather than Arg(1545) accounting for the intrinsic levels of cofactor activity exhibited by native platelet-derived FVa. These collective data are the first to demonstrate physical differences between the two FV cofactor pools and support the hypothesis that, subsequent to its endocytosis by megakaryocytes, FV is modified to yield a platelet-derived cofactor distinct from its plasma counterpart.

Modificadores biológicos: técnica de obtención y manejo clínico del plasma rico en plaquetas / Biologic modifiers: technique of obtention and clinical manipulation of plasma-enriched platelets

Los modificadores biológicos, son sustancias de estructura proteica que participan en los procesos de diferenciación y proliferación celular. Las plaquetas contienen por lo menos dos de estas sustancias, el factor de crecimiento derivado de las plaquetas (PDGF-platelet derived growth factor) y el factor de crecimiento transformador beta (TGF b-transforming growth factor beta). El plasma rico en plaquetas es un preparado autólogo que se utiliza en técnicas de regeneración e injertos óseos. Una vez que las plaquetas liberan su contenido en la zona quirúrgica, los factores de crecimiento contenidos en ellas estimulan el proceso de cicatrización normal, logrando un hueso de mayor calidad en períodos más cortos. En este trabajo se hace una síntesis de los procesos de proliferación y diferenciación celular y de la influencia que en dichos procesos tienen los factores de crecimiento. Se describe una técnica simplificada para la obtención de un plasma rico en plaquetas (PRP) cuya activación promueve la liberación de los factores de crecimiento plaquetarios en el lecho quirúrgico. La textura y adherencia de este preparado facilitan la manipulación y esculpido del material a injertar