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-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.     

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。     

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.     

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.     

Adhesive properties of platelets from different animal species

The use of large animals (e.g., pig and sheep) in human medicine, and the need to develop new therapeutic strategies for domestic animal diseases related to platelet disorders, require better characterization of the physiology of animal platelets. In this study, the ability of platelets from buffaloes, horses, pigs and sheep to adhere to immobilized autologous fibrinogen was compared with that of human platelets. Blood samples were collected from the jugular vein of six healthy subjects of each species and platelet-rich plasma (PRP) was obtained by centrifugation. Platelets, isolated by further centrifugation of PRP, were washed by gel-filtration on Sepharose-2B, counted and added to the wells of 96-well plates pre-coated with autologous fibrinogen. After different times of incubation, non-adherent platelets were removed, and the number of adherent platelets was assessed by measuring endogenous acid phosphatase activity. Horse platelets showed the strongest ability to adhere to autologous immobilized fibrinogen, being 1.7-, 3.1- and 2.3-fold more active than human, buffalo and porcine platelets, respectively. Sheep platelets were unable to adhere to autologous immobilized fibrinogen. Platelet activation by adenosine 5-diphosphate (ADP) increased both human and animal platelet adhesive response. ADP-stimulated sheep platelets were able to adhere to autologous immobilized fibrinogen, albeit to a lesser extent than platelets from the other animal species. The observed interspecies variability in adhesive properties of platelets may reflect structural differences, or differences in the availability of the fibrinogen receptor (glycoprotein IIb/IIIa) on the platelet surface.     

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.     

Platelet volume parameters as a diagnostic tool: The influence of anticoagulation and storage conditions on platelet impedance volume

Summary Rapid progress has been made in the design of aperture impedance cell counters, and parameters such as mean platelet volume and platelet distribution width have become routinely available to most physicians. Platelet volume is influenced by both platelet production in the bone marrow and platelet activation or sequestration in the circulation. In thrombocytopenic patients, it is often possible to differentiate between megakaryocytic and amegakaryocytic disease states on the basis of platelet volume analysis. In patients with thrombocytosis, a myeloproliferative disorder may be suspected if the platelet distribution width is high. However, the conditions of sample preparation and storage still give rise to considerable inaccuracy in the determination of platelet volume parameters. In this study, platelet impedance volume was strongly influenced by anticoagulation, storage time, and incubation temperature. Changes in platelet volume were more pronounced in whole blood than in platelet rich plasma. However, mainly large platelets were lost during the preparation of platelet rich plasma. Collecting blood directly into a mixture of citrate and low dose glutaraldehyde stabilized platelet volume for up to 2 h after venipuncture at room temperature. This method reduces platelet volume changes in vitro and is in this respect superior to the usual EDTA blood count or the use of platelet-inhibitory agents.Abbreviations CO₂ Carbon dioxide - EDTA Ethylene diamine tetraacetic acid - GTA Glutaraldehyde - LDH Lactate dehydrogenase - MPV Mean platelet volume - PRP Platelet rich plasma - WB Whole blood     

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.     

Activation of platelets upon contact with a vitamin E-coated/non-coated surface

The purpose of this study was to determine the effects of a vitamin E-coated surface on platelet activation, focusing on the interactions among the vitamin E-coated surface, platelets and leukocytes. Platelet-rich plasma (PRP) or PRP containing leukocytes (LPRP) was used. No difference was observed in platelet activation between PRP and LPRP for a vitamin E-coated membrane, meaning that platelet activation triggered by leukocytes was suppressed in plasma coming in contact with a vitamin E-coated membrane, while the membrane itself directly induced platelet activation. The antioxidant capacity of the vitamin E-coated membrane in contact with PRP or LPRP was partially reduced, but sufficient residual capacity remained. The in vitro experiments using an oxidized vitamin E-coated surface revealed that P-selectin expression and superoxide anion production in the platelets and platelet adhesion were induced by contact with the oxidized vitamin E-coated surface. We conclude that contact with a vitamin E-coated surface reduces platelet activation mediated by superoxide anions, probably by reducing superoxide anions, but during the process of the reduction, the vitamin E-coated surface itself becomes oxidized, which again causes platelet activation. The beneficial effects of a vitamin E-coated dialyzer in respect of platelet activation were counteracted by the formation of oxidized vitamin E.     

The role of plasma proteins and stress in the assessment of hemocompatibility.

The physiological and psychological conditions of subjects supplying blood for hemocompatibility tests significantly affect the behavior of platelets in terms of both adhesion and activation. The responses of platelets to a standard biomaterial, polyethylene (PE), were examined with blood collected from male rabbits both in basal conditions and after stress. Different media were utilized. First, platelet-rich plasma (PRP) was used to obtain a PE response to contact with platelets. Then platelets drawn from PRP were isolated and washed with Krebs-Ringer solution. One aliquot was suspended in serum (Pw-S) where fibrinogen was absent, another aliquot in Krebs-Ringer solution (Pw-KR) (in order to avoid the influence of the plasma proteins on platelets), and a third aliquot in the original plasma from which the platelets were drawn (Pw-PPP) (in order to restore the initial condition of the plasma but with washed platelets). The analysis of platelet adhesion and morphology was performed by Scanning Electron Microscopy (SEM). Differences in platelet adhesion and morphology were observed with four different media in nonstressed animals, with Pw-PPP showing a higher number and Pw-S and PW-KR lower numbers. Platelet morphology indicated low levels of activation. The platelets drawn from stressed subjects could not be counted in either PRP or PPP medium because they were fully aggregated and adhered; in contrast, in Pw-KR and Pw-S, no significant differences were found with respect to nonstressed conditions, and there was little difference in platelet morphology. All of these factors underline the role of plasma proteins, in particular fibrinogen, in the stress-induced activation of platelets.     

Augmented bone regeneration activity of platelet-rich plasma by biodegradable gelatin hydrogel

This study investigates the ability of platelet-rich plasma (PRP) combined with biomaterials to enhance in vivo bone-repairing activity. A biodegradable hydrogel was prepared from gelatin, which has an affinity for various growth factors. Rabbit PRP was conventionally prepared by blood centrifugation and dropped onto freeze-dried gelatin hydrogel to obtain gelatin hydrogel incorporating PRP. Gelatin hydrogel incorporating PRP was applied to a bone defect of rabbit ulna to evaluate bone formation at the defect in terms of soft X-ray and histological examinations. As controls, fibrin incorporating PRP, empty gelatin hydrogel, and free PRP were applied to the defect; in addition, defect without any application was examined. Successful bone regeneration was observed at bone defect treated with gelatin hydrogel incorporating PRP, in marked contrast to the control groups. When in contact with gelatin, growth factors, such as platelet-derived growth factor and transforming growth factor beta(1), were released from the PRP. PRP growth factors are immobilized in the hydrogel through physicochemical interaction with gelatin molecules. The immobilized growth factors are released from the hydrogel in concert with hydrogel degradation. It is likely that the gelatin hydrogel permitted the controlled release of bioactive growth factors, resulting in factor-induced promotion of bone regeneration.     

Platelet aggregation in platelet-rich plasma and whole blood in 120 patients with myeloproliferative disorders

In vitro platelet aggregation in platelet-rich plasma (PRP) and in whole blood (WB) was assessed in 31 patients with idiopathic myelofibrosis, 32 with essential thrombocytosis, 23 with polycythemia vera, and 34 with chronic myelogenous leukemia. In PRP most subjects showed normal or reduced platelet aggregation, whereas in WB the majority of patients showed increased platelet function. Spontaneous platelet aggregation (SPA) was observed frequently in WB, whereas it was seldom observed in PRP. SPA in WB was inhibited by in vitro addition of aspirin and apyrase, and SPA was only partially dependent on high platelet count because it also occurred in samples with normal platelet content (at variance with 13 subjects with reactive thrombocytosis, in which SPA was observed only in samples with high platelet concentration). Platelets from patients with idiopathic myelofibrosis had the highest tendency to undergo SPA.     

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.     

血小板凝胶的制备方法及其影响因素

背景：血小板凝胶制备方法繁多,分类标准不统一。 目的：总结血小板凝胶制备方法,并讨论影响因素。 方法：由第一作者检索1990至2011年 PubMed数据库及万方数据库。英文检索词为“Platelet gel,Classification,Parameters”,中文检索词为“血小板凝胶,分类,影响因素”。 结果与结论：依据凝胶产量与成分、凝胶中纤维蛋白结构两个主要影响因素可将血小板凝胶制备方法分为4大类,即纯富血小板血浆凝胶、富白细胞-血小板血浆凝胶、纯富血小板纤维蛋白凝胶和富白细胞-血小板纤维蛋白凝胶;根据制备流程不同,血小板凝胶的每一种制备方法还可以再分为手工制备方法和全自动制备方法,但各种分类方法均存在不足之处。     

兔富血小板血浆制备及其活性分析

背景：富血小板血浆是目前已知富含多种生长因子并能将其释放的自体提取物,并已应用于骨、软骨组织工程再生的研究。 目的：通过比较不同方法制备兔富血小板血浆中血小板浓度,并测定其血小板源性生长因子、转化生因子β1水平,探讨富血小板血浆制备方法及影响因素。 方法：采用Petrungaro法、Landesberg法、Aghaloo法制备新西兰大耳白兔富血小板血浆。检测3组富血小板血浆中血小板计数,以及3组富血小板血浆活化前后及正常血浆、贫血小板血浆中血小板源性生长因子、转化生因子β1水平。 结果与结论：3种方法制备的富血小板血浆中血小板计数、血小板回收率、血小板富集系数差异有非常显著性意义(P < 0.001),Landesberg法和Aghaloo法均可制备有效浓度的富血小板血浆,且Aghaloo法制备血小板浓度及活性高于Landesberg法(P < 0.05)。活化前3组富血小板血浆中血小板源性生长因子、转化生因子β1水平与正常血浆组、贫血小板血浆组比较差异无显著性意义。活化后,Landesberg法和Aghaloo法制备的血小板血浆中血小板源性生长因子、转化生因子β1水平明显高于活化前(P < 0.001),且Aghaloo法最高(P < 0.05)。富血小板血浆中血小板计数与血小板源性生长因子水平(r=0.872,P < 0.001),转化生因子β1水平(r=0.917,P < 0.001)呈正相关。     

Fibrinogen degradation products generation is the major determinant of platelet inhibition induced by plasminogen activators in platelet-rich plasma

The platelet function defect induced by thrombolytic agents has been referred either to the degradation of platelet surface receptors or to the anti-aggregatory effect of fibrinogen degradation products (FgDPs).In the present study we have evaluated platelet aggregation induced by ADP, collagen and ristocetin after incubation of washed platelets or platelet-rich plasma (PRP) with plasmin (1.1–3.4IU/ml), plasminogen activators (PAs) (streptokinase 250–1000 IU/ml; urokinase, 10–1000 IU/ml; t-PA 0.5–10 μg/ml) or FgDPs (0.062–2 mg/ml). In parallel the surface levels of platelet GP lb and IIb/IIIa complex were determined by fluorescence flow cytometry using specific monoclonal antibody.Washed platelets treated with plasmin (1.1IU/ml) for 10 to 90 min showed a progressive reduction of ristocetin-induced platelet agglutination and a progressive reduction of surface GP Ib. Surface expression of GP IIb/IIIa complex was significantly increased after plasmin exposure.The addition of PAs to PRP resulted in a marked reduction of ADP-induced platelet aggregation. Collagen-induced platelet aggregation was only slightly affected. Similar changes were observed when PRP was preincubated with high concentrations of FgDPs. In PRP treated with PAs platelet surface levels of GP Ib and GP IIb/IIIa complex did not show any significant changes.In conclusion our results show that in plasma no proteolysis of platelet adhesive receptors occurs after plasminogen activation. The platelet inhibition observed after incubation of PRP with PAs is likely to be caused by FgDPs generation.     

Functional C8 associated with human platelets

Haemolytic assay for C8 revealed its association in functionally active form with washed human platelets. Platelet-bound C8 haemolytic activity was inhibited by F(ab')2 anti-C8 and was undetectable in the platelet suspension obtained from three C8 deficient patients. Incubation of platelets from C8 deficient individuals in normal plasma did not restore C8 haemolytic activity, indicating that platelets do not absorb C8 from plasma in vitro during platelet preparation. Thrombin, a mediator of the platelet release reaction, did not induce the release of C8 from normal platelets. Conversely, lysis of EAC1-7.9 by platelet bound C8 was not accompanied by release of beta-thromboglobulin or serotonin from the platelets. C8 was detected in a homogenate prepared from platelets as well as in the supernatant collected after high speed centrifugation of the homogenate. The association of C8 with platelets as an individual component rather than as part of the C5b-9 membrane-attack complex was supported by the following evidence: platelet bound C8 eluted from a Sephacryl S-200 column at the same volume as C8 from normal human serum; F(ab')2 anti-C8, but not F(ab')2 anti-C5, inhibited platelet C8 activity; the platelet homogenate, which lysed EAC1-7.9, had no effect on EAC43 which are susceptible to the lytic activity of the C5b-9 complex.