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-beta), 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-beta but only when PRP had not been activated during the preparation process in vitro.     

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.     

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.     

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。      

Human platelet inhibition of Toxoplasma gondii growth

The human platelet contribution against the intracellular growth of the parasite in vitro in human pulmonary fibroblasts was explored. It was observed that tachyzoites of Toxoplasma gondii induced activation of human platelets and additionally that platelets mediated inhibition of intracellular growth in a virulent T. gondii strain. A prominent role for platelet-derived growth factor (PDGF) was demonstrated in this phenomenon, by testing human recombinant PDGF-AA, -AB and -BB and antibodies to human PDGF-AB that partially reversed its effects. Moreover, the effect of PDGF was significantly higher if the host cells were treated 2 h before parasite infection. PDGF was not directly ‘toxic’ to free tachyzoites, but only affected parasites within host cells. PDGF-mediated inhibition may involve the cyclooxygenase cycle of the fibroblasts being partially reversed by the cyclooxygenase inhibitors, acetylsalicylic acid and indomethacin. However, a thromboxane synthetase pathway was not implicated. PDGF action against intracellular tachyzoites may also include increased IL-6 production in fibroblasts. Finally, transforming growth factor-beta 1 (TGF-β1), another component of α-granules released at the same time as PDGF, may not be antagonistic to the PDGF parasite inhibitory effect in confluent host cells.     

Blood-derived biomaterials: fibrin sealant,platelet gel and platelet fibrin glue

Blood-derived biomaterials include fibrin sealant (FS) (also called fibrin glue), platelet gel (PG), and platelet fibrin glue. They are used in many surgical fields because of their functional properties and unique physical advantages compared to synthetic products. FS can be made industrially by the fractionation of large plasma pools, or from single plasma donations. Thanks to a high content in fibrinogen, FS exhibits, after activation by thrombin and formation of a strong fibrin clot, tissue sealing and haemostatic properties. PG and platelet fibrin glue are made from single blood donations (platelet concentrates combined or not with cryoprecipitate). Owing to their richness in platelet, PG and PFG can release, upon thrombin activation, a myriad of growth factors that can stimulate cell growth and differentiation, generating much interest for hard and soft tissues regeneration and healing, as well as, increasingly, cell therapy protocols to replace fetal bovine serum. Blood-derived biomaterials have the advantages, over synthetic glues and other biomaterials, of being physiologically compatible with human tissues, and of not inducing tissue necrosis or other tissue reactions. They can be readily colonized by cells and are totally biodegradable in a matter of days to weeks. These blood-derived biomaterials are used increasingly as tissue engineering tools, allowing surgeons to influence and improve the in vitro or in vivo cellular environment to enhance the success of tissue grafting. We review here the three main types of biomaterials that can be made from human blood and describe their biochemical and physiological properties as well as their clinical applications.     

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.     

Effect of mitosis inhibitors on blood platelet microtubules and aggregation

1. An inhibitory action of the mitosis inhibitors colchicine, colcemid and vinblastine on blood platelet aggregation has been demonstrated.

2. The inhibitory action of low concentrations of these drugs was surmountable by increasing the concentration of the aggregating agents adenosine diphosphate (ADP) or thrombin. High concentrations completely inhibited aggregation.

3. The presence of a circumferential bundle of microtubules has been demonstrated in rabbit platelets.

4. After exposure to aggregating doses of ADP and thrombin the microtubules move to the centre of the cell, `trapping' the platelet intracellular organelles in a relatively clear cytoplasm.

5. Concentrations of the antimitotic agents which reduce platelet aggregation depolymerize the platelet microtubules and prevent the rearrangement of the platelet organelles during aggregation.

6. It is suggested that the microtubules are not essential for platelet aggregation but are intimately involved in the movement of intracellular granules. This may be a preliminary stage in the eventual discharge of the granules during the platelet `release reaction'.

     

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.     

At low extracellular calcium concentration platelet activating factor induces beta-thromboglobulin release from human platelets through thromboxane-endoperoxides formation

Summary The aim of this study was to investigate the mechanism involved in -thromboglobulin (BTG) release induced by platelet activating factor (PAF) in human platelet-rich plasma (PRP) and washed platelets (WP) during aggregation. PAF was used in PRP at increasing concentrations starting at its threshold concentration for irreversible aggregation (TAC: 90–150 nM). In citrated PRP, PAF induced release of BTG (80–95% of total content) and thromboxane B₂ (TXB₂) formation (30–40 pmol/ml). At low PAF concentrations aggregation and BTG release were blocked by apyrase (a scavenger of ADP), by ASA (an inhibitor of cyclooxygenase) and by BM 13177 (a thromboxane receptor antagonist). Higher concentrations of PAF overcame the effect of apyrase, but only induced reversible aggregation and minor release in the presence of ASA or BM 13177. In heparinized PRP, PAF induced full irreversible aggregation, but only very low BTG release (about 25% of total content) and thromboxane synthesis (2–3 pmol/ml). WP resuspended in the presence of 2 mmol/l Ca²⁺ seldom responded to PAF alone, as previously shown by others, but full aggregation could be induced by concomitant addition of subthreshold concentrations of PAF (25–50 nM) and epinephrine (1 M). In these conditions average BTG release from WP was less than 20% of the amount releasable by thrombin. In contrast, when WP were resuspended in the absence of Ca²⁺, stimulation by PAF+EPI induced sustained BTG release (40–50% of total content) and TXB₂ synthesis (15–20 pmol/ml). We conclude that at low Ca²⁺ concentration PAF induces BTG release mainly through thromboxane-endoperoxides formation. In contrast, when [Ca²⁺] is normal, PAF does not or weakly induces thromboxane formation and BTG release.     

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 &#45 SD platelet count in whole blood from these donors was 262,000 &#45 58,000/ &#119 l, while in PC produced by discontinuous cell separation it was 1,419,000 &#45 333,000/ &#119 l. The mean growth factor concentrations in PC preparations in ng/ml were as follows: PDGF-AB 125 &#45 55 ng/ml; TGF- &#103 1 221 &#45 92 ng/ml; IGF-I 85 &#45 25 ng/ml; PDGF-BB 14 &#45 9 ng/ml; TGF- &#103 2 0.4 &#45 0.3 ng/ml. These growth factor concentrations typically covered a 3-10 fold range: PDGF-AB 29-277 ng/ml; PDGF-BB 2-33 ng/ml; TGF- &#103 1 32-397 ng/ml; TGF- &#103 2 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.     

Effect of nicotine infusion in humans on platelet aggregation and urinary excretion of a major thromboxane metabolite

The objective of this study was to evaluate further a possible role of nicotine as a stimulator of platelet aggregability and platelet arachidonic acid metabolism in vivo. In six healthy, non-smoking males, platelet aggregability was assessed by filtragometry and impedance aggregometry before, during and after an intravenous infusion of nicotine at two different doses (0.25 and 0.5 μg kg^-1 min^-1) for 30 min. The aggregatory response was also measured after the addition of nicotine at final concentrations ranging from 10^-11 mol L^-1 to 10^-5 mol L^-1 directly to the aggregating blood. The synthesis of thromboxane A₂ (TxA₂) in platelets was estimated by quantitating the urinary excretion of 2,3-dinor-thromboxane B₂ (Tx-M). Despite the plasma concentrations of nicotine, cotinine and catecholamines in the range of those occurring during acute cigarette exposure, the excretion of Tx-M (204±36 pg mg^-1 creatinine) remained unaltered during nicotine infusion. Similarly, platelet aggregatory response to collagen was not influenced by nicotine when infused or added in vitro. However, an enhanced aggregability was detected by filtragometry during the infusion of nicotine at the higher dose employed. The results indicate that nicotine, infused at moderate doses, produces a weak platelet stimulation that is not accompanied by significant release of thromboxane A₂, as monitored by urinary excretion of Tx-M. Although a direct action of nicotine on platelets cannot be excluded, it appears more likely that the enhancement of platelet function is mediated by other, secondary mechanisms.     

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.     

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.     

Surface and bulk effects on platelet adhesion and aggregation during simple (laminar) shear flow of whole blood

This study attempts to clarify the role of the artificial surface and the fluid bulk on platelet adhesion and aggregation events during simple shear flow of whole blood. The experimental approach involved the shearing of fresh whole blood samples over the shear rate range of 720-5680 s^-1, which corresponded to a shear stress maximum of about 150 dyn cm^-2. Results on platelet adhesion, measured as surface coverage by platelets, and platelet aggregation, measured in terms of reduction in platelet count and adenosine diphosphate (ADP) release, were determined as a function of the surface to volume ratio (S/V); and artificial surface used. Both shear-induced platelet adhesion and platelet count reduction showed significant variation over the range of S/V employed. The ratios between the three different S/V values used in this system (10:6:4) were about the same as the ratio of the shear rate-averaged results obtained. Also, for shear-induced hemolysis, an increase in the release of hemoglobin from red blood cells was found as S/ V was increased, again with ratios between the shear rate-averaged values similar to the ratio of S/V values employed. The shear-induced release of ADP, presumably from platelets and from red blood cells indicated a different dependence of ADP release on S/ V than was observed for the other parameters reported. Irreversible platelet aggregation was expected to occur because the amount of ADP that was released as a result of the shear was substantial. Models proposed to explain the experimental results were found to support a surface-controlled mechanism.     

Assessment of soluble platelet CD40L and CD62P during the preparation process and the storage of apheresis platelet concentrates: Absence of factors related to donors and donations

Platelet transfusions may be associated with certain adverse effects in recipients, potentially caused by the presence of biological response modifiers contained in the platelet concentrates. The aim of this study is to identify the parameters that reflect platelet activation during both the preparation process and the storage of platelet concentrates. A total of 3,949 apheresis platelet concentrate samples were studied with regard to parameters related to the donor as well as to the preparation process and their storage. Key glycoproteins characteristic of platelet activation, i.e. soluble CD40L and CD62P, were quantified in platelet concentrate supernatants on completion of their processing and during storage, using Luminex technology. We observed an increase in soluble factors over time. However, the different parameters studied in connection either with the donors or with the donations, such as (i) donor gender, (ii) donor blood group, (iii) time of collection and (iv) type of apheresis separator, do not seem to have any effect on platelet activation or the release of soluble CD40L and CD62P.     

An optimized growth factor cocktail for ovine mesenchymal stem cells

Growth factors that regulate proliferation, migration, and invasion of ovine mesenchymal stem cells (oMSCs) are not well defined. In this study, we have evaluated five growth factors for their ability to initiate and support in vitro proliferation, migration, and invasion of oMSCs. oMSCs were exposed to different doses and combinations of the growth factors: basic fibroblast growth factor (bFGF), transforming growth factor-β (TGF-β), epidermal growth factor (EGF), insulin growth factor-I (IGF-I), connective tissue growth factor, and platelet-derived growth factor-AB (PDGF-AB). Cellular proliferation, motility, and invasiveness were assayed. The most proliferative stimulating growth factors are PDGF-AB+TGF-β and PDGF-AB+IGF-I. Combinations EGF+bFGF and EGF+bFGF+PDGF-AB demonstrated the greatest ability to stimulate migration. Moreover, the triple cocktail EGF+bFGF+TGF-β has the most significant effect on invasion. Different growth factor cocktails are required to enhance proliferation, migration, and invasion. These results may be useful for the development of a tissue-engineered heart valve by stimulating cellular repopulation.     

Leucocyte-dependent platelet activation: an alternative pathway for initiation of blood clotting in inflammation

Fibrin deposition is a prominent feature of several inflammatory diseases but the extract mechanism(s) leading to fibrinogen-fibrin conversion has not been completely clarified. We describe here a new cellular pathway for initiation of blood clotting resulting from a leucocyte-platelet interaction. Human washed platelet suspensions, free of leucocytes, isolated from whole blood or leucocyte-enriched platelet-rich plasma (PRP) after four hours' incubation with bacterial endotoxin, had strong procoagulant activity (40–100-fold that of control platelets). When platelets were challenged with endotoxin in the absence of white blood cells (i.e. in PRP) the subsequently washed platelets were devoid of procoagulant activity indicating that leucocytes are essential mediators in the development of platelet coagulant activity induced by endotoxin. This property is mostly confined to the mononuclear fraction. Stimulated platelets have the peculiarity that they trigger blood coagulation by activating factor X independently of both the intrinsic and extrinsic pathways. These findings add a new function to circulating mononuclear cells and provide experimental evidence for an unrecognized cellular pathway of fibrin formation in inflammatory diseases.     

Computational Simulation of Platelet Deposition and Activation: I. Model Development and Properties

To better understand the mechanisms leading to the formation and growth of mural thrombi on biomaterials, we have developed a two-dimensional computational model of platelet deposition and activation in flowing blood. The basic formulation is derived from prior work by others, with additional levels of complexity added where appropriate. It is comprised of a series of convection-diffusion-reaction equations which simulate platelet-surface and platelet-platelet adhesion, platelet activation by a weighted linear combination of agonist concentrations, agonist release and synthesis by activated platelets, platelet-phospholipid-dependent thrombin generation, and thrombin inhibition by heparin. The model requires estimation of four parameters to fit it to experimental data: shear-dependent platelet diffusivity and resting and activated platelet-surface and platelet-platelet reaction rate constants. The model is formulated to simulate a wide range of biomaterials and complex flows. In this article we present the basic model and its properties; in Part II (Sorensen et al., Ann. Biomed. Eng. 27:449–458, 1999) we apply the model to experimental results for platelet deposition onto collagen. © 1999 Biomedical Engineering Society. PAC99: 8719Uv, 8380Lz, 8717Aa, 8710+e, 8768+z