Platelet-rich plasma preparation using three devices: Implications for platelet activation and platelet growth factor release

Background: In this study, three commercial systems for the preparation of platelet-rich plasma (PRP) were compared and platelet growth factors release was measured.

Methods: Ten healthy volunteers donated whole blood that was fractionated by a blood cell separator, and a table-top centrifuge to prepare PRP. Furthermore, an autologous growth factor filter was used to concentrate PRP fractionated by the blood cell separator. PRP was subsequently activated with autologously produced thrombin to degranulate the platelets to measure platelet-derived growth factor-AB (PDGF-AB), transforming growth factor-beta (TGF-β), insulin-like growth factor-1 (IGF-1), and vascular endothelial growth factor (VEGF).

Results: PRP contained significantly higher platelet counts compared with baseline values (p < 0.001). PDGF-AB concentrations were increased more than 18-fold in the platelet gel supernatant when the cell-separator and GPS were used, whereas only a 3-fold increase was seen with the AGF.

Conclusion: The three PRP devices enable the preparation of PRP for the release of high concentrations of platelet growth factor, but showed different harvesting capacities for the collection of concentrated platelets. The administration of thrombin for PRP activation resulted in the release of high concentrations of PDGF-AB and TGF-β but only when PRP had not been activated during the preparation process in vitro.     

Effect of different bone substitutes on the concentration of growth factors in platelet-rich plasma

This study is conducted to determine the effect of different kinds of bone substitutes and collagen on the concentration of platelet-derived growth factor (PDGF) and transforming growth factor beta-1 (TGF beta-1) in platelet-rich plasma (PRP). PRP is treated with thrombin, hydroxyapatite (HA), and thrombin, HA alone, collagen-grafted HA, calcium metaphosphate (CMP), and collagen-grafted CMP. The concentrations of PDGF-AB and TGF beta-1 are measured. After PRP treated with HA and CMP, the concentrations of PDGF and TGF beta-1 are not significantly different from the concentration of them in PRP alone. The concentrations of PDGF in PRP with collagen-grafted HA and collagen-grafted CMP are significantly higher than that of PRP with HA and CMP. The concentrations of PDGF and TGF beta-1 in PRP with collagen-grafted CMP are higher than with collagen-grafted HA. The results of multiple regression analysis show that PDGF increased with the use of collagen and thrombin, and is higher in native whole blood with higher platelet counts. However, PDGF decreased with the use of HA. In conclusion, HA and CMP do not seem to be able to activate platelets by themselves. However, if they had collagen grafted onto them, they could activate platelets and release growth factors.     

Platelet and growth factor concentrations in activated platelet-rich plasma: a comparison of seven commercial separation systems

Platelet-rich plasma (PRP) is blood plasma that has been enriched with platelets. It holds promise for clinical use in areas such as wound healing and regenerative medicine, including bone regeneration. This study characterized the composition of PRP produced by seven commercially available separation systems (JP200, GLO PRP, Magellan Autologous Platelet Separator System, KYOCERA Medical PRP Kit, SELPHYL, MyCells, and Dr. Shin’s System THROMBO KIT) to evaluate the platelet, white blood cell, red blood cell, and growth factor concentrations, as well as platelet-derived growth factor-AB (PDGF-AB), transforming growth factor beta-1 (TGF-β1), and vascular endothelial growth factor (VEGF) concentrations. PRP prepared using the Magellan Autologous Platelet Separator System and the KYOCERA Medical PRP Kit contained the highest platelet concentrations. The mean PDGF-AB concentration of activated PRP was the highest from JP200, followed by the KYOCERA Medical PRP Kit, Magellan Autologous Platelet Separator System, MyCells, and GLO PRP. TGF-β1 and VEGF concentrations varied greatly among individual samples, and there was almost no significant difference among the different systems, unlike for PDGF. The SELPHYL system produced PRP with low concentrations of both platelets and growth factors. Commercial PRP separation systems vary widely, and familiarity with their individual advantages is important to extend their clinical application to a wide variety of conditions.     

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.     

不同抗凝剂和激活剂联合应用对富血小板血浆凝胶释放生长因子影响的比较

背景：富血小板血浆凝胶生物效应的发挥受多种因素的影响,如富血小板血浆制备的方法、血小板的完整性、抗凝剂及激活剂的选择等。 目的：比较不同抗凝剂与激活剂联合应用对富血小板血浆凝胶释放生长因子影响的差异。 方法：抽取新西兰兔全血制备富血小板血浆,再用牛凝血酶和Ⅰ型胶原激活,实验共分4组：依地酸钠钙-凝血酶组,依地酸钠钙-Ⅰ型胶原组,肝素-凝血酶组,肝素-Ⅰ型胶原组。分别计数各组富血小板血浆血小板数目。在激活富小板血浆后2 h,1 d,3 d,5 d使用酶联免疫吸附法测定空白对照组(全血)和各组富血小板血浆凝胶中转化生长因子β1及血小板源性生长因子AB的浓度,比较各组间2种生长因子释放方式和浓度的差异。  结果与结论：依地酸钠钙-Ⅰ型胶原组合制备的富血小板血浆凝胶中转化生长因子β1和血小板源性生长因子AB累积释放量最大(P < 0.05);使用Ⅰ型胶原作为激活剂的富血小板血浆凝胶中上述2种生长因子的释放方式均为持续缓慢,并且转化生长因子β1的释放与激活时间呈正相关关系(r=0.873);而凝血酶激活的富血小板血浆凝胶释放生长因子的速度则较为快速(P > 0.05)。结果证实,依地酸钠钙与Ⅰ型胶原制备的富血小板血浆凝胶所释放生长因子的浓度较大。      

Effects of calcium and thrombin on growth factor release from platelet concentrates: kinetics and regulation of endothelial cell proliferation

Platelet concentrates (PCs) constitute new biological mediators used in osseous reconstructive surgery. In this study, we assessed (i) the effects of various concentrations of calcium and thrombin on the kinetics of platelet-derived growth factor (PDGF-BB), transforming growth factor-beta1(TGF-beta 1), basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF) release by PCs and (ii) the contribution of PC supernatants to endothelial cell proliferation. Our results indicate that high concentrations of calcium (Ca) and thrombin (Thr) trigger an immediate and significant increase in bFGF, TGF-beta 1 and PDGF-BB concentrations. Thereafter, PDGF-BB, VEGF and TGF-beta 1 levels remained generally constant over a 6-day period while a decrease in bFGF concentrations was noted after 24h. Lower Ca and Thr concentrations tended to reduce and delay growth factors release from PCs. Endothelial cell proliferation was greatly enhanced with PC supernatants (mean: 20-fold increase). This was especially evident when endothelial cells were treated with supernatants harvested early after PC treatment with high concentrations of Ca and Thr or later after PC treatment with low Ca and Thr concentrations. Additional research aiming to measure the effects of Ca and Thr on bone formation in vivo is needed.     

Effects of poly(ethyleneglycol)-modified hemoglobin vesicles on agonist-induced platelet aggregation and RANTES release in vitro

We studied the effects of hemoglobin-vesicles modified with PEG (PEG-HbV), a type of liposome-encapsulated hemoglobin (LEH), on human platelet functions in vitro. The effect of a low concentration of PEG-HbV (Hb; 5.8 mg/dl) was assessed by examining an agonist-induced aggregation response, and that of relatively high concentrations of PEG-HbV (Hb; 0.29, 1 and 2 g/dl) by measuring the release of RANTES (Regulated upon activation, normal T-cell expressed and presumably secreted) from platelets, which is regarded as a marker of platelet activation. The preincubation of platelets with PEG-HbV at 5.8 mg/dl of Hb did not affect platelet aggregation induced by collagen, thrombin and ristocetin. The pretreatment of platelet-rich plasma (PRP) with PEG-HbV at concen trations up to 2 g/dl of Hb had no aberrant effects on the collagen-induced RANTES release. Furthermore, the collagen-induced release of RANTES from PRP was not affected by longer incubation with PEG-HbV at 2 g/dl of Hb. The basal levels of RANTES from PRP were unchanged in the presence of PEG-HbV. These results suggest that PEG-HbV, at the concentrations studied, have no aberrant effects on platelet functions in the presence of plasma.     

Spontaneous platelet aggregation in a hereditary giant platelet syndrome (MPS)

The characteristics of spontaneous platelet aggregation (SPA) in a hereditary giant platelet syndrome (Montreal platelet syndrome, MPS) are examined. SPA was quantitated by microscopy from the decrease in single platelets in platelet-rich plasma (PRP). In contrast to normal donors, a significant proportion (20-50%) of platelets in MPS whole blood and PRP occurred in microaggregates typically containing 2-6 disk-shaped platelets. Stirring MPS-PRP at 1000 rpm for 10 minutes further increased the fraction of platelets in aggregates by 10-170%, the percentage increase not being correlated to the donor's platelet count (5000-220,000 microliters-1). Normal platelets resuspended in MPS platelet-poor plasma (PPP) did not undergo SPA, whereas MPS platelets resuspended in normal PPP or Ca2+-free, fibrinogen-free Tyrode's continued to show SPA. The increase in SPA could be inhibited by 10 microM prostaglandin (PG) E1, 150 mM ASA or glutaraldehyde or formaldehyde fixation; however, it was not inhibited by 10 nM PGI2 and was only partially inhibited by 1 microM 2-chloroadenosine and 1-10 units/ml apyrase. SPA in Acid-citrate-dextrose-PRP was much less than in PRP; however, SPA reoccurred on returning the platelets to platelet-free plasma or Tyrode's. Platelet aggregation (PA) could be increased over that due to SPA alone by the addition of adenosine diphosphate, adrenaline, collagen, ionophore A-23187, arachidonic acid and ristocetin, with results suggesting that the response to these agents is normal. The ristocetin-induced increase in PA was completely blocked by an IgG specific for Bernard-Soulier syndrome. In contrast, MPS platelets had a reduced sensitivity to thrombin, which appeared to be more pronounced at low platelet counts. There was no correlation between the thrombin insensitivity and the extent of SPA. Total adenosine triphosphate (ATP) and thrombin-induced release of ATP and platelet factor 4 appeared normal for MPS platelets. The ultrastructural features of MPS platelets were within normal limits except for an increased frequency of giant granules. SPA was observed for 5/5 MPS donors, but only one of three MPS donors' platelets evaluated for glycoprotein I and sialic acid content showed any measurable reduction as compared with normal controls. The above observations point to the existence of an as yet undetermined anomaly of MPS plasma membrane related to a fibrinogen and Ca2+ independent form of platelet aggregation.     

Quantification of thrombocyte growth factors in platelet concentrates produced by discontinuous cell separation

Platelet concentrates (PC) are increasingly used to increase bone regeneration in pre-prosthetic surgery. Although it is generally appreciated that certain growth factors (PDGF, TGF, EGF, and ECGF) are present in thrombocyte preparations, relatively little is known about these components in quantitative terms. The study reported here analysed the amounts of growth factors in PC produced under standard conditions from healthy volunteers. All the blood samples (237 in total) were analysed using Quantikine ELISA kits (R and D). The mean +/- SD platelet count in whole blood from these donors was 262,000+/-58,000/microl, while in PC produced by discontinuous cell separation it was 1.419,000+/-333,000/microl. The mean growth factor concentrations in PC preparations in ng/ml were as follows: PDGF-AB 125+/-55 ng/ml; TGF-beta1 221+/-92 ng/ml; IGF-I 85+/-25 ng/ml; PDGF-BB 14+/-9 ng/ml; TGF-beta2 0.4+/-0.3 ng/ml. These growth factor concentrations typically covered a 3-10 fold range: PDGF-AB 29-277ng/ml; PDGF-BB 2-33ng/ml; TGF-beta1 32-397ng/ml; TGF-beta2 0.1-1.2 ng/ml; IGF-I 40-138 ng/ml. Platelet counts in PC were slightly higher for women (Mann-Whitney Test all p < 0.001) than for men, while the concentrations of growth factors in PC exhibited no gender-related difference of any statistical significance.     

Platelet augmentation of IgE-dependent histamine release from human basophils and mast cells

A factor(s) from human platelets enhances IgE-mediated histamine release from human basophils and mast cells. This effect is directly related to the platelet number; at physiological platelet/leukocyte ratios (40:1), the enhancement was 66 +/- 11%. Platelet stimulation by thrombin more than doubled the enhancement, to 172 +/- 10% at 40:1. Mast cell release was also enhanced by platelets although the magnitude was more limited (86 +/- 13% at 40:1 with thrombin). Direct basophil/platelet contact was unnecessary in that platelet supernatants were fully active; a direct platelet factor/basophil interaction is suggested, however, by the fact that basophils purified 100-fold with respect to other leukocytes were enhanced by the platelet factors. The appearance of platelet-enhancing activity is associated with the release of an alpha-granule marker (PF4) rather than with products of arachidonic acid metabolism (thromboxane B2). The platelet factor(s) responsible for these effects are not dialyzable, are heat stable and do not appear to be identical to PF4 or platelet-derived growth factor (PDGF). Since anti-IgE-stimulated basophils cause PF4 release and this correlates with the release of enhancing factor, we suggest that a pro-inflammatory feed forward relationship exists. Together with our previous data showing that platelets are activated in vivo during antigen challenge of allergic asthmatic subjects, these results suggest that platelets may be important in modulating IgE-mediated allergic reactions in man.     

Platelet-rich plasma: quantification of growth factor levels and the effect on growth and differentiation of rat bone marrow cells

Platelet-rich plasma (PRP) is a new application of tissue engineering and a developing area for clinicians and researchers. It is a storage vehicle of growth factors (GFs) such as platelet-derived growth factor (PDGF)- AA, -BB, -AB; transforming growth factor (TGF)-beta1 and -2; platelet-derived epidermal growth factor (PDEGF); platelet-derived angiogenesis factor (PDAF); insulin growth factor-1 (IGF-1); and platelet factor- 4 (PF-4), which are known to influence bone regeneration. However, animal and clinical studies reveal different results with the use of PRP and its effect on bone healing. This could be due to the differences between species, that is, differences between species in GF concentrations or variation in presence of GFs between the various PRPs. In this study, rat bone marrow cells were cultured in PRP-coated wells or in uncoated wells for 16 days in osteogenic medium, and analyzed on cell growth (DNA content) and cell differentiation (alkaline phosphatase [ALP] activity, calcium content, scanning electron microscopy, and QPCR). The concentrations of TGF-beta1, PDGF-AA, PDGF-AB, and PDGF-BB in rat, goat, and human PRP were subsequently determined. The results showed that PRP stimulated initial cell growth and had no effect on ALP activity. The calcium measurements showed a significant increase in calcium at days 8, 12, and 16. The real-time PCR results showed that PRP upregulated osteocalcin at day 1 and collagen type I at day 8. Overall, the immunoassays revealed that human PRP contained higher concentrations of growth factors per platelet compared to rat and goat PRP. Goat PRP showed higher concentrations of growth factors per platelet as compared to rat PRP except for PDGF-BB, which had a higher concentration in rat PRP. TGF-beta1 was the most abundant growth factor in all 3 PRPs. On the basis of our results, we conclude that platelet-rich plasma contains osteo-inductive growth factors, which are probably species related. However, we cannot generalize the results because of large intraspecies variations. Further, we conclude that rat PRP gel stimulates initial growth and differentiation of rat bone marrow cells in vitro.     

富血小板血浆临床应用及其生物材料性能

背景：富血小板血浆的制备方法、设备及应用手段等方面有了长足发展,但是相对于国内临床工作者而言富血小板血浆技术仍是一项相对较新的生物技术。 目的：对富血小板血浆分类等相关概念和存在问题加以说明和论述,使其制备和应用技术等更加清晰和明确。 方法：以“platelet rich plasma,preparation,富血小板血浆,制备”为检索词,检索PubMed数据库及维普数据库1995-09/ 2010-09相关文章。 结果与结论：根据所含白细胞的多少可以将富血小板血浆分为贫白细胞的富血小板血浆和富白细胞的富血小板血浆;根据应用形式又可以分为未激活富血小板血浆和激活的富血小板血浆,后者又可以分为富血小板血浆凝胶和富血小板血浆释放物。被视为第二代血小板浓缩物的富血小板纤维蛋白与富血小板血浆都含有高浓度的血小板,但两者在制备方法,凝胶形成方式等方面有根本的区别。由于其安全性和易于制备,富血小板血浆在医学领域的应用将会越来越多,但临床应用还要保持谨慎,因为富血小板血浆在多个方面还缺乏相关研究。     

The disappearance of adenosine from blood and platelet suspension in relation to the platelet cyclic AMP content

Adenosine exerts anti-aggregatory effects on human platelets in vitro, probably by increasing intraplatelet levels of cyclic AMP. In addition, adenosine prevents platelet loss in vivo. We have studied the relationship between the concentration of adenosine in the platelet media and the level of cAMP. In PRP, exogenous adenosine (2-16 microM) was eliminated with a half-life close to 5 min. Approximately half of the added adenosine was deaminated (blocked by 1-2 microM EHNA), and half was eliminated by uptake into platelets (blocked by 2 microM dipyridamole). In whole blood the half-life for adenosine was much shorter, about 15 s. Addition of adenosine deaminase (0.3 microgram ml-1) to PRP resulted in a measured half-life for adenosine approximating that of whole blood. In PRP where adenosine was eliminated as quickly as in whole blood, the adenosine-mediated stimulation of cAMP was 35% lower than in PRP, and the cAMP response lasted 2 min versus 15 min in normal PRP. These results suggest that the magnitude and duration of adenosine's effect on platelets are markedly overestimated by studying platelet suspensions. In blood, the effect of adenosine is smaller in magnitude and very transient. The possibility is discussed that the action of adenosine in vivo on blood platelets can therefore be quite local.     

Enhanced angiogenesis by multiple release of platelet-rich plasma contents and basic fibroblast growth factor from gelatin hydrogels

The objective of this study is to evaluate the angiogenic effects induced by biodegradable gelatin hydrogel granules incorporating mixed platelet-rich plasma (PRP) growth factor mixture (PGFM) and bioactive basic fibroblast growth factor (bFGF). The PRP was prepared by a double-spinning technique for isolating animal bloods, followed by treatment with different concentrations of calcium chloride (CaCl(2)) solution. The CaCl(2) solution treatment activated the platelets of PRP, allowing the release of various growth factors, such as platelet-derived growth factor (PDGF)-BB, vascular endothelial growth factor (VEGF), transforming growth factor (TGF)-β(1), and epithelial growth factor (EGF). In the PRP treated with different CaCl(2) solutions, high amounts of representative platelet growth factor, PDGF-BB, VEGF, EGF, and TGF-β(1) were detected in the CaCl(2) concentrations of 1, 2, and 4 wt.% compared with higher or lower ones. The PRP treated was impregnated into gelatin hydrogel granules freeze-dried at 37°C for 1h, and then the percentage of PGFM desorbed from the gelatin hydrogel granules was evaluated. The percentages of PDGF-BB, VEGF, EGF, and TGF-β(1) desorbed tended to decrease with decreasing CaCl(2) concentration. Taken together, the CaCl(2) concentration to activate PRP for PGFM release was fixed at 2 wt.%. In vitro release tests demonstrated that the PGFM was released from the gelatin hydrogel granules with time. For the gelatin hydrogels incorporating PGFM and bFGF, the time profile of PDGF-BB or bFGF release was in good correspondence with that of gelatin hydrogel degradation. The gelatin hydrogel granules incorporating mixed PGFM and bFGF were prepared and intramuscularly injected to a mouse leg ischemia model to evaluate the angiogenic effects in terms of histological and laser Doppler perfusion imaging examinations. As controls, hydrogel granules incorporating bFGF, PGFM, and platelet-poor plasma were used for the angiogenic evaluation. The number of blood vessels newly formed and the percentage of anti-α-smooth muscle actin antibody-positive cells increased around ischemic sites injected with the gelatin hydrogel granules incorporating mixed PGFM and bFGF, in marked contrast to other control groups. The blood reperfusion level of ischemic tissues was enhanced by the hydrogel granules incorporating mixed PGFM and bFGF, whereas no enhancement was observed for other groups. It is concluded that the dual-release system of PGFM and bFGF from gelatin hydrogel granules shows promise as a method to enhance angiogenic effects.     

The role of Fbg in platelet adhesion to polymeric materials in conditions of psychological stress.

The effect of psychological stress on platelet adhesion to five polymeric materials (polyurethane, polyurethane filled with BaSO4, polyethyleneterephthalate, silicone and low-density polyethylene) was studied. The platelets were obtained from non-stressed and stressed rabbits as platelet-rich plasma (PRP) and, once washed (Pw), were suspended in different media, i.e. in platelet poor plasma (Pw-PPP), in serum (Pw-S) and in Krebs-Ringer solution (Pw-KR). Scanning electron microscopy of platelet adhesion and morphology revealed differences in the platelet activating power of the various materials. The washing procedure and resuspension in PPP generally resulted in an increased number of adherent platelets, compared with the number of platelets adherent to the same material in PRP. However, platelets washed and suspended in Pw-KR or Pw-S showed the same shape distribution as in PRP. When platelets from stressed rabbits were used, there was very strong aggregation and activation of the platelets in both PRP and Pw-PPP, independent of the chemical nature and surface structure of the material. In contrast, in Pw-KR and Pw-S (in which Fbg is absent) a general picture of single, not very modified platelets was observed. Their number and shapes changed according to the nature of the different materials. On the whole, the present results confirm our original hypothesis of a key role of the psychological condition of the blood donor and strongly indicate Fbg as the determinant factor in the pattern of platelet adhesion.     

Glomerular mesangial cell migration. Response to platelet secretory products.

Glomerular mesangial cells migrate in response to platelet-derived growth factor (PDGF), but to date these cells have not been examined for migratory behavior in response to other platelet secretory products. Because migration might provide an additional mode of cell redistribution and local mesangial hypercellularity in certain forms of glomerular disease, we examined, in vitro, the potential of isolated rat mesangial cells to migrate toward gradients of platelet releasate and selected platelet secretory proteins. Chemotaxis assays were performed in two compartment blind well chambers, each compartment separated by a porous membrane. Releasate of activated platelets was added in incremental concentrations (25, 50, and 100 micrograms/ml) to lower compartments, and mesangial cells were placed in upper compartments. The chambers were then incubated at 37 degrees C for 4 hours. Mesangial cell migration through the membranes was quantitated by scanning electron microscopy. Mesangial cells migrated toward platelet releasate in a linear dose-response, achieving cell numbers of approximately 40 times those of controls. Examination of specific platelet alpha granule secretory proteins disclosed a potent mesangial cell migratory response to platelet-released fibronectin (Fn), but not to transforming growth factor-alpha (TGF-alpha), -beta (TGF-beta), epidermal growth factor (EGF), or platelet factor 4 (PF4). Secretory levels of platelet Fn (1 to 25 micrograms/ml) induced a maximum migratory response of approximately 60-fold over controls. Mesangial cell migration in response to both platelet Fn and platelet releasate was abrogated by blocking the integrin receptor for Fn with RGDS tetrapeptide. Thus, platelet Fn appears to be a prominent component of platelet releasate responsible for mesangial cell migration.     

The role of whole blood in thrombin generation in contact with various titanium surfaces

Understanding of the thrombotic response (activation of the intrinsic coagulation system followed by platelet activation) from blood components upon contact with a titanium dental implant is important and not fully understood. The aims of this study were to evaluate: (1) the thrombogenic response of whole blood, platelet-rich plasma (PRP) and platelet-poor plasma (PPP) in contact with a highly thrombogenic surface as titanium, (2) the thrombogenic response of clinically used surfaces as hydroxyapatite (HA), machined titanium (mTi), TiO2 grit-blasted titanium (TiOB) and fluoride ion-modified grit-blasted titanium (TiOB-F). An in vitro slide chamber model, furnished with heparin, was used in which whole blood, PRP or PPP came in contact with slides of the test surfaces. After incubation (60 min rotation at 22 rpm in a 37 degrees C water bath), blood/plasma was mixed with EDTA or citrate, further centrifuged at +4 degrees C (2200 g at 10 min). Finally, plasma was collected pending analysis. Whole blood in contact with Ti alloy resulted in the binding of platelets to the material surface and in the generation of thrombin-antithrombin (TAT) complexes. With whole blood TAT levels increased 1000-fold compared with PRP and PPP, in which both almost no increase of TAT could be detected. In addition, the platelet activation showed a similar pattern with a 15-fold higher release of beta-TG in whole blood. In the in vitro chamber model with the clinically relevant materials, the fluoride-modified surface (TiOB-F) showed pronounced TAT generation compared with TiOB, mTi and HA. Similar results were achieved for platelet consumption and activation markers of the intrinsic coagulation system. Taken together these results implicate first that whole blood is necessary for sufficient thrombin generation and platelet activation during placement of implants. Second, a fluoride ion modification seems to augment the thrombogenic properties of titanium.     

Morphological characteristics of bovine platelets activated with platelet activating factor or thrombin

The morphological alterations induced by the activation of bovine platelet rich plasma suspensions with the inflammatory mediator, platelet activating factor (PAF), and following the activation of washed bovine platelet suspensions with thrombin are described. The unstimulated bovine platelet exhibits a smooth oval or discoid shape and granules are randomly dispersed throughout the cytoplasm. The initial activation response to PAF is the development of irregularly shaped cells, the migration of granules to the periphery of the cell and the appearance of large pseudopodia devoid of membrane organelles. As activation continues and large platelet aggregates form, two zones of irregularly shaped, discrete platelets are evident within each large aggregate: an outer zone in which the cells are devoid of granules and an inner zone in which many of the platelets exhibit the typical ultrastructural features of a non-activated cell. In washed platelet preparations activated with thrombin, virtually all platelets undergo shape change and yet many cells retain their alpha granules. In addition, discrete irregularly shaped agranular platelets are also found. The distinctive morphological alterations observed in activated bovine platelets are likely associated with the absence of an open canalicular system, characteristic of many other types of mammalian platelets, and with the ability of the cytoplasmic microtubule coil to reorganise into a linear array following thrombin activation. It is postulated that the bovine platelet has evolved as a cell that can respond to various stimuli, for example inflammatory mediators, by releasing active metabolites from its granular stores without forming platelet-platelet bridges that can serve as a foci for thrombus formation. In this manner the bovine platelet can effectively function as an inflammatory cell without acting as a potent thrombogenic agent.     

A rapid method to isolate platelets from human blood by density gradient centrifugation.

Platelets can be damaged easily or activated during isolation, making them unsuitable for functional studies. The most common technique for isolating platelets involves centrifugation. Although gentler methods have been devised to isolate platelets by density gradient centrifugation or electrophoresis, these techniques either result in a relatively dilute platelet preparation or are time-consuming. A simple, gentle technique for isolating concentrated platelet preparations for experimental or clinical use is reported. Freshly drawn whole blood was spun over a commercially available density gradient medium for 30 minutes. The mononuclear cell layer (which also contains most of the platelets) was collected and nucleated cells were pelleted by centrifugation. The recovery of platelets was about 60%. Contamination with leukocytes was less than 1%, and the platelet concentration was about 130% of blood concentration. Higher concentrations can be obtained if more whole blood is layered onto the Mono-Poly Resolving Medium (MPRM; Flow Laboratories, McLean, VA). About 10% of the platelets expressed the activation marker GMP-140 by flow cytometric analysis. They could be activated by thrombin so that 70% to 90% of the platelets expressed GMP-140. Thus, this technique can rapidly and easily yield a functionally intact platelet preparation. This preparation can be purified again if needed. No specialized skills or equipment are needed. A significant advantage of the method is that platelets can be obtained from thrombocytopenic patients in final concentrations that are high enough to use for platelet function testing.     

Critical evaluation of platelet aggregation in whole human blood

Platelet aggregation studies generally are performed in platelet-rich plasma (PRP) by the turbidometric method. The authors compared this technic with the recently introduced impedance aggregometry in PRP and whole blood (WB). In healthy controls there was a good correlation between the two technics when aggregation was induced by ADP or collagen. As compared with PRP, platelets in WB were more sensitive to the aggregating effect of thrombin, ristocetin, and arachidonic acid. Platelet sensitivity to prostacyclin was increased in WB. The anti-platelet effect of a single oral dose of aspirin could be detected for a longer period in WB than in PRP. Platelet aggregation tests in WB from patients with platelet dysfunctions showed the same response pattern to different aggregating agents as in PRP. In contrast to turbidometry, the impedance method in PRP and WB enabled registration of platelet aggregation in a dose-dependent fashion in a sample from a patient with severe hyperlipoproteinemia. It is concluded that platelet aggregation can be studied conveniently with the impedance method in the more physiologic medium of WB. Providing the same information as the well-established turbidometry, the time-sparing impedance method needs less citrated blood. Moreover, our results show an increased sensitivity of the WB system to some aggregating and anti-platelet agents.