Native Platelet-rich Plasma Research Articles

The use of platelet-rich plasma for activation of tissue regeneration in otosurgery

The article describes the effect of platelet-rich plasma (PRP) on the processes of tissue regeneration in operative treatment of the patients with chronic suppurative otitis media with cholesteatoma with the simultaneous plastic surgery of postoperative cavity and ossiculotympanoplasty. In the main group, in the children patients (n = 59), the operative cavity was filled with bone chips with ceftriaxone powder and plasma gel. The adults (n = 16) were additionally injected the native PRP on both sides of the developing cicatrix and parameatally. In the comparison group, PRP was not used. The study conducted has proven the efficiency of PRP usage for regeneration of organ-specific tissues and restoration of the organ function.

Treatment of a diaphyseal long-bone defect with autologous bone grafts and platelet-rich plasma in a rabbit model.

Large bone defects are a therapeutic challenge to surgeons and are often associated with a high morbidity. The use of autologous cancellous bone graft represents an essential therapeutic option and is considered the gold standard. However, the use of platelet-rich plasma (PRP) for improving bone defect healing has been discussed controversially. The aim of this study was to evaluate the treatment of a diaphyseal long-bone defect in a rabbit model with a combination of PRP and autologous cancellous bone. A monocortical long-bone defect in the radial diaphysis of 24 New Zealand white rabbits was filled either with autologous cancellous graft as a control group or with autologous cancellous graft combined with autologous PRP. Histomorphometrical and radiological analysis as well as quantification of platelets and growth factors were performed. The animals were euthanatized after three and six weeks according to the study arms. A significant improvement in bone healing was observed histomorphometrically in the PRP group in the central area of the defect zone (p <0.01) as well as the cortical defect zone (p <0.01). The radiological findings were in accordance with the histomorphometrical results. Comparing native blood and PRP, an enrichment of growth factors and platelets was detectable in the PRP. Within this animal study, the combination of PRP and autologous cancellous bone grafts improved bone healing significantly compared to the sole application of autologous bone. Therefore, further efforts should be initiated to establish the composite of PRP and autologous bone for bone healing disorders in clinical use.

A Prospective Study Involving the Use of Platelet Rich Plasma in Enhancing the Uptake of Bone Grafts in the Oral and Maxillofacial Region

Platelet-rich plasma (PRP) is an autologous product that contains highly concentrated number of platelets in a small volume of plasma, derived from whole blood by gradient density centrifugation. It has been speculated that local growth factors in human platelets (insulin-like growth factor, IGF; transforming growth factor, TGF-β; platelet derived growth factor, PDGF) would enhance healing of grafts and also counteract resorption. The aim of this study was to evaluate efficacy of PRP on early healing after autogenous bone grafting. Of the twenty patients selected ten were treated with autogenous bone graft and PRP (PRP group) and other ten with autogenous bone graft alone (non-PRP group). PRP group consisted of two benign tumor of mandible, one post surgical defect, two unilateral alveolar cleft, one bilateral alveolar cleft with skeletal class III malocclusion, one maxillary hypoplasia, one oronasal fistula, one recurrent tumor of mandible, one multiple impacted mandibular teeth. Non-PRP group consisted of seven benign tumor of jaw, one keratocyst odontogenic tumor, one orbital blow out fracture, one residual traumatic defect. Biopsies were taken in the native bone, PRP treated grafted bone, grafted bone without PRP at 3months to assess the maturity of bone. Radiographic imaging was performed by panoramic radiography at 3 and 6months to evaluate bone opacity of grafted bone on comparison with native bone and computerized tomography at 6months to evaluate grafted bone morphologically and to measure bone density in Hounsfield units. Microscopic results showed that significantly more matured bone was formed at PRP treated sites as that of native bone and immature bone in controls after 3months of healing. Bone opacity of PRP treated bone grafts was close to that of native bone than that of non-PRP treated bone grafts on panoramic radiograph at 3 and 6months. There was graft loss in three cases and graft resorption in one case of non-PRP treated bone grafts at 6months. In PRP group the compact bone was clearly differentiated from cancellous bone as in native bone and thick in five cases, thin in five cases. In non-PRP group the compact bone was thin as a whole. Comparing native bone group and PRP group the CT value of PRP treated bone graft was more or less close to native bone group and comparing native bone group and non-PRP group CT value was low in non-PRP treated bone graft. Whereas when comparing PRP and non-PRP group CT value was higher in PRP group. Autologous PRP was a safe, biocompatible, effective, source for growth factors and carries no risk of transmissible diseases. It enhances and accelerates bone regeneration of autogenous bone grafts.

A prospective cohort study of light transmission platelet aggregometry for bleeding disorders: Is testing native platelet-rich plasma non-inferior to testing platelet count adjusted samples?

Light transmission platelet aggregometry (LTA) is important to diagnose bleeding disorders. Experts recommend testing LTA with native (N) rather than platelet count adjusted (A) platelet-rich plasma (PRP), although it is unclear if this provides non-inferior, or superior, detection of bleeding disorders. Our goal was to determine if LTA with NPRP is non-inferior to LTA with APRP for bleeding disorder assessments. A prospective cohort of patients, referred for bleeding disorder testing, and healthy controls, were evaluated by LTA using common agonists, NPRP and APRP (adjusted to 250 x 10⁹ platelets/l). Recruitment continued until 40 controls and 40 patients with definite bleeding disorders were tested. Maximal aggregation (MA) data were assessed for the detection of abnormalities from bleeding disorders (all causes combined to limit bias), using sample-type specific reference intervals. Areas under receiver-operator curves (AUROC) were evaluated using pre-defined criteria (area differences: < 0.15 for non-inferiority, > 0 for superiority). Forty-four controls and 209 patients were evaluated. Chart reviews for 169 patients indicated 67 had bleeding disorders, 28 from inherited platelet secretion defects. Mean MA differences between NPRP and APRP were small for most agonists (ranges, controls: -3.3 to 5.8; patients: -3.0 to 13.7). With both samples, reduced MA with two or more agonists was associated with a bleeding disorder. AUROC differences between NPRP and APRP were small and indicated that NPRP were non-inferior to APRP for detecting bleeding disorders by LTA, whereas APRP met superiority criteria. Our study validates using either NPRP or APRP for LTA assessments of bleeding disorders.

Variability of non-response to aspirin in patients with peripheral arterial occlusive disease during long-term follow-up

Non-responsiveness to aspirin as detected by laboratory tests may identify patients at high risk for future vascular events. The aim of this prospective study was to evaluate whether non-responsiveness to aspirin is stable over time. Ninety-eight patients with stable peripheral arterial occlusive disease (PAOD) treated with 100 mg/d aspirin were followed over a median timeframe of 17 months. Platelet function tests were performed initially and at follow-up using arachidonic acid-induced light transmittance aggregometry (LTA) in native platelet-rich plasma with the Behring Coagulation Timer and by measuring the collagen-epinephrine closure time (CT) on a Platelet Function Analyzer (PFA-100). When determining platelet function using LTA, four patients (4.1%) had residual platelet function (i.e., MaxAggr > or =78%) despite aspirin treatment, whereas, according to the PFA-100 results, 12 patients (12.2%) were identified as non-responders (i.e., CT <192 s). Fifty-seven patients who were still under treatment with 100 mg/d aspirin at the time of follow-up provided a second blood sample. Further platelet function tests with the PFA-100 system identified a persistent non-responsiveness to aspirin over time in three patients (5.3%) whereas four (7.0%) and 15 (26.3%) patients had changes in response status when platelet function was assessed by LTA and on the PFA-100(R), respectively. We conclude that true non-responsiveness to aspirin is a rare phenomenon in stable PAOD patients. Furthermore, we conclude that in a number of patients, aspirin non-responsiveness is not stable over time.

To adjust or not to adjust the platelet count in light transmission aggregometry in patients receiving dual aspirin/clopidogrel treatment

We evaluated whether the results of light transmittance aggregometry (LTA) differ when “native” platelet-rich plasma (PRP) or adjusted (to a standard platelet count of 250.000/µL) PRP is used in patients on dual antiplatelet therapy with aspirin and clopidogrel. LTA has been performed on the blood of 142 stable angina pectoris patients who were adequately pretreated with aspirin and clopidogrel. Platelet aggregation was significant higher in native PRP as compared to platelet count adjusted PRP (P<0.0001) for all four concentrations of adenosine-5′-diphosphate (ADP) (2, 5, 10 and 20 μmol/L). The interindividual variability was significantly higher in platelet count adjusted PRP as compared to native PRP when stimulated with 10 and 20 μmol/L of ADP. The absolute magnitude of aggregation in non-adjusted PRP is clearly dependent on platelet number. These observations are important since several studies have used empirically defined cut-off levels to segregate non-responders from responders to clopidogrel therapy.

Use of native or platelet count adjusted platelet rich plasma for platelet aggregation measurements

Background: It is still not clear whether native or platelet count adjusted platelet rich plasma (PRP) should be used for platelet aggregation measurements. Aim: To evaluate the necessity of using...

The Effects of Platelet Count on Clot Retraction and Tissue Plasminogen Activator-Induced Fibrinolysis on Thrombelastography

Clot retraction and fibrinolysis may present as a decrease in amplitude on thrombelastography (TEG). The former represents normal or hyperactive platelet function, and the latter represents a fibrinolytic state. It is important to distinguish clot retraction from fibrinolysis because the treatment of each condition is different. To distinguish between these phenomena, we performed TEG with platelet-poor plasma (PPP) and platelet-rich plasma (PRP) with an increasing platelet count (range, 50-1200 x 10(9)/L) with or without abciximab. Maximum amplitude (MA) and the percentage decrease of amplitude at 30 and 60 min after MA were examined for each sample. Blood samples to which tissue plasminogen activator (tPA) was added served as positive controls for fibrinolysis. Morphological changes of clots and D-dimer levels were also examined. With higher platelet counts, the percentage decrease of amplitude after MA increased significantly at 30 and 60 min, but not in the abciximab samples. Morphological changes of clots have shown clot retraction in PRP, but not in PPP or PRP pretreated with abciximab. D-dimer levels increased only in samples to which tPA was added, but not in native PPP or PRP samples. In conclusion, we have shown that the decrease in amplitude at 30 and 60 min can be due to platelet-mediated clot retraction and can be attenuated by sample pretreatment with abciximab, which interrupts platelet-fibrin(ogen) binding.

Optimally functional fluorescein isothiocyanate‐labelled fibrinogen for quantitative studies of binding to activated platelets and platelet aggregation

Dynamic and quantitative studies of the binding of fibrinogen (Fg) to its receptor, GPIIb-IIIa, on activated platelets, leading to platelet aggregation, are best studied with fluorescently-labelled Fg by flow cytometry. Due to conflicting reports on the functionality of FITC-labelled Fg, we have developed a reproducible and 'mild' labelling of fibrinogen with FITC-celite at pH 7.4-8.5 for direct and dynamic studies of specific Fg binding to activated platelets evaluated for native platelet-rich plasma, for washed platelets, and for activated, fixed platelets. We have demonstrated the equivalence of FITC-labelled and unlabelled Fg for binding to activated GPIIb-IIIa receptors, and in the rate and extent of mediating platelet aggregation. We found that FITC-Fg labelled at pH > or = 9 had reduced to absent specific binding to activated platelets, whether using soluble FITC or FITC-celite. The FITC-labelled Fg must be diluted 3-fold with unlabelled Fg when evaluating maximal Fg binding to activated platelets in order to prevent autoquenching of the FITC-Fg which leads to underestimation of Fg levels. The dissociation constant (KD) of Fg on stable preparations of activated, fixed platelets, determined with FITC-Fg binding to platelets by flow cytometry, was in the range reported for 125I-labelled Fg, 70-255 nm with Bmax = 10000-25000 Fg per platelet (n = 20). The FITC-Fg was used to monitor Fg binding to activated platelets directly by plasma, as well as to evaluate platelet subpopulations which maximally bind Fg according to the concentration of ADP used as activator. It is expected that this 'mildly' labelled FITC-Fg will stimulate further studies of platelet activation directly in native anticoagulated blood/plasma, for both basic and clinical research.

Activated human platelets in plasma excite nociceptors in rat skin, in vitro

Extravascular activation of thrombocytes may contribute to nociceptor excitation and pain, since platelets store and, upon stimulation, release potential algogenic substances such as serotonin, histamine and precursor molecules of bradykinin. To test this hypothesis, a skin-nerve preparation of rat hairy skin, in vitro, was used that allows to record and characterize single afferent nerve fibers. In a first protocol, receptive fields of nociceptive C-fibers, at the corium side of the skin patch, were exposed to adenosine diphosphate (ADP), to heparinized human platelet-rich plasma (PRP) and to PRP activated by ADP. Such activated platelets excited 9 11 units characterized as mechano-heat responsive C-nociceptors (CMH); peak discharges of more than 10 spikes/s were observed. After application of activated PRP, 4 5 high threshold mechanosensitive C-units and 4 5 mechano-cold sensitive C -units became responsive to heat stimulation but only few of these fibers were excited ( 1 5 in each group). In a second series of experiments the exposure to native PRP was prolonged to test for the effect of spontaneous platelet activation resulting from cutaneous collagen. Prolonged exposure did, but not significantly, enhance fiber discharge. During subsequent exposure to activated PRP, the discharge commenced, on average, after a significant delay of about three minutes. With this protocol 5 7 CMH units were driven by activated platelets. Following both protocols, mechanical (v.Frey) and thermal thresholds of the CMH units were not significantly altered. The findings demonstrate that nociceptors can indeed be driven and sensitized by activated platelets. This pain inducing mechanism may be relevant to certain clinical conditions, and it appears promising to scrutinize the chemical factors involved.

Increased lipid binding to thrombi in coronary artery disease. Findings in patients without premedication in native (not anticoagulated) test systems.

Thrombi and clots were produced from native (i.e., not anticoagulated) platelet-rich and platelet-poor plasma from patients with coronary artery disease and control subjects who had not taken any medication known to influence plasma lipids, coagulation, or platelet aggregation. The clotting times were recorded, and the lipid content of clots, thrombi, platelets, plasma, and high density lipoprotein was analyzed. Thrombi produced from native platelet-rich plasma were 46% heavier in coronary artery disease patients and contained about 20% more phospholipids and free cholesterol and about twice the amount of triglycerides and esterified cholesterol in both absolute and relative amounts with respect to the corresponding lipids of plasma plus platelets. The elevated content of lipids not only increases the size of the thrombi but also changes their quality because of an increased content in plasmatic lipids, as platelets contain only trace amounts of triglycerides and cholesterol esters. In agreement herewith, fibrinogen and maximal amplitude on the thrombelastogram were increased in coronary artery disease patients, whereas the thrombus-forming time and clotting times of platelet-poor and platelet-rich plasma were shortened, indicating accelerated coagulation and activation of platelets. Analysis of these results suggests a disturbed interrelation in coronary artery disease between lipids and hemostasis, in which platelets, high density lipoprotein, and lipoproteins rich in triglycerides and cholesterol esters may play a role.

Influence of oral contraceptives on coagulation tests in native blood and plasma

Routine coagulation laboratory tests, clotting times in native (not anticoagulated) whole blood, platelet-rich and platelet-poor plasma, and recalcification times in citrated whole blood, platelet-rich and platelet-poor plasma were performed in 14 healthy premenopausal women. Blood was taken before and after one or two cycles of low-dose oral contraceptives. After oral contraceptives a reduction in clotting time in native platelet-rich plasma and activated partial thromboplastin time were observed. Recalcification times in whole blood and platelet-rich plasma were shorter than clotting times in their native counterparts. The observed changes are compatible with a procoagulant effect seen soon after the start of oral contraceptive use. The absence of these changes in the recalcification times in citrate systems suggests a masking effect of citrate. The reduction in clotting times in native platelet-rich but not in platelet-poor plasma indicates that the hypercoagulability in oral contraceptives users is mainly related to platelets. (Am J Obstet Gynecol 1990;163:417-20.)

A species difference in platelet aggregation induced by tumour cells

Ehrlich ascites tumour cells (EATC) induced the aggregation of human platelets but not of sheep or rabbit platelets in native platelet-rich plasma. Aggregation was initiated by the interaction of EATC with a component(s) of human plasma, possibly related to the complement system, which led to the release of cellular ADP, a potent platelet aggregating agent. EATC previously incubated with human platelet-poor plasma induced immediate aggregation in platelet-rich plasma from all three species. The species difference in platelet aggregation by EATC is therefore related to the activity or availability of plasma component(s) responsible for release of cellular ADP rather than to intrinsic differences in platelet responsiveness to the tumour cells.

Effect of contact factor (factor XII+factor XI) on aggregation of platelets.

Inhibition of platelet aggregation by contact factor eluate (CFE) is demonstrated. A similar inhibition of platelet aggregation is found following incubation of ellagic acid with native platelet rich plasma. Inhibition of platelet aggregation by CFE or ellagic acid was not found when PRP from a patient with Hageman’ disease was used. The electrophoretic mobility of platelets was increased by the action of CFE. The possible role of the contact factor as a regulator of the platelet plug in the early stages of haemostasis is discussed.

The influence of salicylate on platelets and whole blood adenine nucleotides.

1. The addition of sodium salicylate to freshly withdrawn human blood or native platelet-rich plasma significantly delays platelet aggregation in vitro.2. The administration of acetylsalicylic acid to human subjects also significantly delays platelet aggregation in their whole blood and there is a short delay in the formation of fibrin but this is not statistically significant.3. Salicylate, whether added to human blood in vitro as sodium salicylate or given by mouth as acetylsalicylic acid, significantly reduces the platelet clumping activity of adenosine diphosphate (ADP) added to whole blood in vitro.4. The administration of aspirin in high doses for several days produces a marked increase in the total adenine nucleotide content of whole blood. The percentage of adenosine triphosphate (ATP) was increased, that of ADP decreased while there is an obvious increase in the ATP: ADP ratio.5. There is little correlation between the plasma salicylate level and the delay aspirin produces in platelet aggregation in vitro or the changes that occur in the levels of ADP or ATP in whole blood during administration of aspirin.6. Significant correlations do occur, however, between the delay in platelet aggregation in vitro and (i) the percentage increase in the ATP concentration, (ii) the percentage decrease in ADP concentration, (iii) the percentage change in the ATP: ADP ratio observed during aspirin administration.

The Ultrastructural Localization and Release of Platelet Lipids

AbstractInvestigation of the ultrastructure of platelets of carefully prepared, native platelet rich plasma fixed in gluteraldehyde-osmium has revealed a process not apparent by other technics. Platelet granules undergo transformation into lameller particles with the configuration of lipid micelles. The platelet micelles are released through the platelet cell membrane prior to aggregation. Comparison of the platelet micelles with micelles formed by aqueous dispersion of extracted platelet lipids and brain cephalin revealed similarities of particle size and miceller configuration. The origin of platelet micelles from platelet granules, their release from the cell in early clot development, and their similarity to other lipids active in coagulation are provocative. Platelet micelles may be the active lipid substance contributed by platelets to coagulation, and the lipoprotein complexes of platelet granules the "hidden form" of the platelet lipid in normal circulating cells.