Platelet-rich Plasma Releasate Research Articles

Platelet-rich plasma activation in combination with biphasic osteochondral scaffolds–conditions for maximal growth factor production

The combination of scaffolds and biological factors may enhance articular cartilage repair. Little is known regarding the activation and subsequent growth factor release of platelet-rich plasma (PRP) in contact with biosynthetic scaffolds. The purpose of this study was i) to identify whether the addition of thrombin was required to activate PRP in the presence of a collagen osteochondral scaffold and ii) to compare the activity of PRP when applied to both collagen- and polylactide-based osteochondral scaffolds. Equal combined volumes of test substances were used (n=3): 500μl PRP alone or on scaffolds; 375μl PRP+125μl autologous thrombin on scaffolds; 455μl PRP+45μl bovine thrombin on scaffolds. Scaffolds and/or PRP were cultured in vitro in DMEM/F12 medium for 10days. TGF-β1, PDGF-AB and bFGF were measured using ELISA. A similar cumulative release profile in all growth factors was found over the 10-day period i.e. a burst release and further physiological prolonged release. A significantly higher release of PDGF-AB was seen in the PRP+collagen scaffold groups at all time points, compared to scaffold+PRP+thrombins (P<0.001). A significantly increased cumulative volume of PDGF-AB was released from the collagen scaffold compared to the polylactide scaffold (P<0.001). This study shows that polylactide and particularly collagen osteochondral scaffolds activate PRP in vitro. If PRP is combined with these scaffolds clinically, no exogenous activation with thrombin is required to achieve growth factor release, which may be of benefit for articular cartilage repair applications.

Combined release of platelet-rich plasma and 3D-mesenchymal stem cell encapsulation in alginate hydrogels modified by the presence of silica

We report the modified release of platelet-rich plasma from alginate platelet-rich plasma hydrogels altered by the presence of silica. These PRP–alginate–silica compositions can be used as injectable carriers for viable mesenchymal stem cells.

Therapeutic Treatment with Sustained-Release Platelet-Rich Plasma Restores Blood Perfusion by Augmenting Ischemia-Induced Angiogenesis and Arteriogenesis in Diabetic Mice

Objective: The objective of this investigation was to establish the effectiveness of sustained-release platelet-rich plasma (PRP) on perfusion and neovascularization in diabetic murine hind limb ischemia. Methods: After surgery in streptozotocin-induced diabetic mice, the mice were randomly assigned to the following 4 experimental groups: control (C), 100 µl of the sustained-release form of platelet-poor plasma (PPP), 100 µl of the solution form of PRP (PRP-sol), and 100 µl of the sustained-release form of PRP (PRP-sr). Endpoint evaluations were: blood perfusion by laser Doppler perfusion imaging (LDPI), vascular density by anti-vWF, and mature vessel density by anti-smooth muscle actin antibody. Results: This study demonstrated that a sustained release of PRP increases the perfusion of ischemic tissue as measured by LDPI (57 ± 12; 56 ± 9; 72 ± 7, and 98 ± 4 for the C, PPP, PRP-sol, and PRP-sr groups, respectively; p < 0.05), capillary density (151 ± 16; 158 ± 12; 189 ± 39, and 276 ± 39 for groups C, PPP, PRP-sol, and PRP-sr, respectively; p < 0.05), and mature vessel density (28 ± 2; 31 ± 3; 52 ± 10, and 85 ± 13 for the C, PPP, PRP-sol, and PRP-sr groups, respectively; p < 0.05). Conclusion: A sustained release of PRP containing potent angiogenic growth factors restores blood perfusion by stimulating angiogenesis and arteriogenesis.

Platelet-Rich Plasma Releasate Promotes Differentiation of Tendon Stem Cells into Active Tenocytes

Background Platelet-rich plasma (PRP) has been used to enhance tendon healing in clinical settings. However, the cellular mechanisms underlying PRP treatment of injured tendons remain unclear. The aim of this study was to determine the effects of PRP, in the form of PRP-clot releasate (PRCR), on tendon stem cells (TSCs), a newly discovered cell population in tendons. Hypothesis The PRCR treatment promotes differentiation of TSCs into tenocytes that are activated to proliferate quickly and increase collagen production. Study Design Controlled laboratory study. Methods After PRCR treatment, cell morphology, expression of stem/progenitor cell marker nucleostemin, and population doubling time were examined. In addition, gene and protein analyses were performed using reverse transcription-polymerase chain reaction, immunocytochemistry, and Western blot to characterize the type of cells that had differentiated after PRCR treatment. Results The TSCs without PRCR treatment were small and exhibited an irregular shape, whereas with increasing PRCR dosage, TSCs became large, well spread, and highly elongated with downregulation of nucleostemin expression. The PRCR treatment also markedly enhanced TSC proliferation, tenocyte-related gene and protein expression, and total collagen production, all of which indicated that PRCR treatment induced differentiation of TSCs into activated tenocytes. Conclusion The PRCR treatment promotes differentiation of TSCs into active tenocytes exhibiting high proliferation rates and collagen production capability. Clinical Relevance The findings of this study suggest that PRP treatment of injured tendons is “safe” as it promotes TSC differentiation into tenocytes rather than nontenocytes, which would compromise the structure and function of healing tendons by formation of nontendinous tissues. Moreover, they suggest that PRP treatment can enhance tendon healing because tenocytes induced to differentiate by PRP are activated to proliferate quickly and produce abundant collagen to repair injured tendons that have lost cells and matrix.

Biochemical investigation of the effects of human platelet releasates on human articular chondrocytes

The aim of the present study was to demonstrate the mitogenic and differentiating properties of platelet-rich plasma releasates (PRPr) on human chondrocytes in mono- and three-dimensional cultures. In order to assess if PRPr supplementation could maintain the chondrocyte phenotype or at least inhibit the cell de-differentiation even after several days in culture, we performed a proteomic study on several cell cultures independently grown, for different periods of time, in culture medium with FCS, human serum (HS), and releasates obtained from PRP and platelet-poor plasma (PPP). We found that PRP treatment actually induced in chondrocytes the expression of proteins (some of which novel) involved in differentiation.

Angiogenic properties of sustained release platelet-rich plasma: Characterization in-vitro and in the ischemic hind limb of the mouse

While single growth factor has limitation to induce optimal neovascularization, platelet-rich plasma (PRP) is an autologous reserver of various growth factors. However, little is known about the mechanism of PRP-related neovascularization.The objective of this investigation was to characterize the angiogenic and growth factor content of PRP and to determine, in vitro, its effect on endothelial cell proliferation. Additionally, this experiment sought to determine the effectiveness of different compositions of PRP (solution versus sustained release) on perfusion and neovascularization in a murine model of hind limb ischemia. Different growth factors were measured by enzyme-linked immunosorbent assay (ELISA). In vivo study, we used gelatin hydrogel as a sustained release carrier for growth factors in PRP. We induced hind limb ischemia by excising right femoral artery in wild type C57BL6 mice. After surgery, mice were randomly assigned to four experimental groups; control (C), 100 muL of sustained release form of platelet-poor plasma (PPP), 100 muL of solution form of PRP (PRP-sol), 100 muL of sustained release form of PRP (PRP-sr); each formulation was administered via an intramuscular injection to the ischemic hind limb. Endpoint evaluations were blood perfusion by laser Doppler perfusion image, vascular density by anti Von Willebrand factor (vWF), and mature vessel density by anti smooth muscle actin (SMA) antibody. Green fluorescent protein (GFP+) transgenic mice were generated by transplantation of bone marrow derived mononuclear cells to wild type C57BL6 mice, and finally CD34+ cell in the ischemic site of transgenic mice was detected by staining with anti-CD34 antibody. In vitro study showed that PRP containing different growth factors induces endothelial cell proliferation and capillary tube formation. In vivo study demonstrated that sustained release of PRP increased perfusion of ischemic tissue as measured by laser Doppler perfusion imaging (LDPI) (57 +/- 12, 56 +/- 9, 72 +/- 7, 98 +/- 4 for groups C, PPP, PRP-sol, and PRP-sr, respectively; P < .05); capillary density (151 +/- 16, 158 +/- 12, 189 +/- 39, 276 +/- 39 for groups C, PPP, PRP-sol, and PRP-sr, respectively, P < .05) and mature vessel density (28 +/- 2, 31 +/- 3, 52 +/- 10, 85 +/- 13 for groups C, PPP, PRP-sol, and PRP-sr, respectively, P < .05) . Sustained release PRP also increases CD34+ cells in the ischemic site of transgenic mice (6 +/- 3 vs 18 +/- 5/mm(2) for groups control and PRP-sr respectively, P < .05). Sustained release of PRP containing potent angiogenic growth factors restores blood perfusion presumably by stimulating angiogenesis, arteriogenesis, as well as vasculogenesis in the mouse hind limb ischemia. PRP is a natural reserver of various growth factors that can be collected autologously and is costeffective. Thus for clinical use, no special considerations concerning antibody formation and infection risk are needed.Some clinical devices to automatically prepare PRP are available at present. PRP are consistently being used clinically inthe department of orthopedics and plastic surgery (oral, maxillary facial) for a long time. On the basis of researchevidence, some publications have reported positive results in either bone or soft tissue healing. However, some researchconcludes that there is no or little benefit from PRP. This is likely due to faster degradation of growth factors in PRP sincesome authors suggest using sustained release form of PRP to deliver optimal effect of PRP. Gelatin hydrogel is also beingused clinically as a slow, sustained release of carrier for growth factors in our center recently.

Residual platelet thromboxane A2 and prothrombotic effects of erythrocytes are important determinants of aspirin resistance in patients with vascular disease.

Permanent inactivation of cyclooxygenase-1 and inhibition of platelet thromboxane A(2) (TxA(2)) constitute the main mechanisms underlying the prevention of vascular disease by aspirin. We studied platelet TxA(2) synthesis and its impact on platelet reactivity and platelet-erythrocyte [platelet-rich plasma (PRP)-RBC] interactions in 533 aspirin-treated patients with vascular disease. Seventy aspirin-free and 16 aspirin-treated normal subjects were evaluated as controls. Collagen (1 mug mL(-1))-induced platelet activation ((14)C-5HT release) and recruitment (proaggregatory activity of cell-free releasates from activated platelets) were assessed in PRP, PRP + RBC, and whole blood (WB). TxA(2) was quantified in releasates from WB. Aspirin inhibited TxA(2) synthesis and platelet function in all patients, but to different degrees. Forty-two patients (8%) displayed partial (<95%) inhibition of TxA(2) relative to that of aspirin-free controls. They produced >3.5 ng mL(-1) TxA(2) and had higher platelet reactivity than 491 patients who had undetectable TxA(2) or produced residual TxA(2) (R-TxA(2); </=3.5 ng mL(-1)). Patients with R-TxA(2) were distributed into TxA(2) quartiles. Patients in the third and fourth quartiles had significantly elevated (14)C-5HT release in PRP, which was markedly amplified in PRP + RBC and WB. TxA(2) in the fourth quartile translated into increased platelet aggregation and recruitment. Significant correlations were found between R-TxA(2) and platelet hyperfunction. Biochemical markers (TxA(2) synthesis, (14)C-5HT release) and biological assays (platelet aggregation and recruitment) used to monitor the aspirin effect in a large population of patients presenting with vascular disease have evidenced the importance of R-TxA(2) and the prothrombotic effects of RBC in aspirin resistance.

The influence of intrinsic coagulation pathway on blood platelets activation by oxidized cellulose

Oxidized cellulose is an effective hemostat that works naturally to aid in blood coagulation. The mechanism of its action is not very well understood. Little effect on blood coagulation, but a pronounce decrease in platelet count has been reported upon the addition of the oxidized cellulose to the whole blood. As a marker of platelet activation and aggregation we used serotonin release reaction and turbidity changes in time. We found that oxidized cellulose did not activate washed platelets reconstituted in plasma-free medium or plasma-free medium with fibrinogen; no reduction of platelet count was observed. Serotonin release in platelet-rich plasma incubated with oxidized cellulose started in the range from 5 to 10 min. Serotonin release from platelets reconstituted in plasma deficient in either coagulation factor V, VIII, IX, or XII was delayed. Blood platelets activation by oxidized cellulose requires calcium ions present in dispersion of oxidized cellulose. Factor XIII deficiency had no influence on blood platelets activation by oxidized cellulose. Our results clearly indicate the significance of intrinsic coagulation pathway activation on blood platelets activation by oxidized cellulose and so indirectly on the hemostyptic effect of oxidized cellulose.

Release of platelet‐derived growth factors and proliferation of fibroblasts in the releasates from platelets stored in the liquid state at 22°C after stimulation with agonists

Fresh platelet (PLT)-rich plasma (PRP) treated with thrombin plus calcium chloride (CaCl(2)) is used to prepare a PLT gel to promote hemostasis and wound healing in a variety of surgical procedures. The effects of various agonists on stimulating the release of growth factors from liquid-preserved PLTs and the effects of the PLT releasate on the growth of fibroblasts in tissue culture were investigated. Plateletpheresis PLTs stored at 22 degrees C as high-yield PLTs for 3 to 6 days or outdated PLTs for 9 days were treated with agonists to assess release of platelet-derived growth factor (PDGF) AA, PDGF AB, PDGF BB, transforming growth factor-beta1 (TGF-beta1), and osteocalcin and the proliferation of fibroblasts treated with the PLT releasates in tissue culture. All treatments except for CaCl(2) alone and zeolite-CaCl(2) produced significant increases in PDGF AA compared to PRP. Thrombin-CaCl(2) produced significant increases in PDGF BB. Treatment by all the agonists produced similar increases in PDGF AB. TGF-beta1 and osteocalcin levels after treatment were similar to those in PRP. PRP releasate before and after stimulation with different agonists increased proliferation of fibroblasts in tissue culture. High-yield and outdated liquid-preserved PLTs released PDGF AA, AB, and BB but not TGF-beta1 or osteocalcin. The releasate from untreated PRP stimulated the proliferation of fibroblasts in tissue culture similar to the releasates from PRP treated with the different agonists. Further studies are needed to assess whether or not high-yield and outdated PLTs may be useful in wound healing.

4:48 Platelet rich plasma stimulates stem cell chemotaxis, proliferation and potentiates osteogenic differentiation

Purpose of study: Platelets are known to perform multiple functions during injury and tissue repair. As a repository of multiple growth factors such as PDGF, EGF, VEGF, and TGF-β, degranulation of platelets at wound sites serves to initiate or enhance the healing cascade. Armed with this knowledge, clinicians have used platelet rich plasma (PRP) in conjunction with bone graft materials to enhance osseous repair. As mesenchymal stem cells (MSDs) are known to be an essential component of the bone repair process, we sought to characterize their response to PRP. Because we have previously reported the profound mitogenic effect of PRP on MSCs, the purpose of this study was to extend our understanding of PRP's effect on MSCs during the chemotactic and cell differentiation phases of healing.Methods used: PRP was isolated from fresh human blood using a commercially available point-of-care system, designed to obtain PRP from a small amount of blood. Samples of the starting material and PRP were analyzed to determine the absolute concentrations of platelets. PRP, platelet poor plasma (PPP) and unfractionated blood were clotted with thrombin/CaCl2. PRP and PPP releasates were diluted in serum-free DMEM to create working stocks. Human NSCs (hMSCs) were isolated and culture expanded from bone marrow, as described previously. The ability of concentrated platelet releasate to stimulate the chemotactic migration of hMSCs was measured using a modified Boyden chamber. Cells were allowed to migrate for 4 hours, then stained and counted. The ability of PRP to support proliferation of hMSCs without loss of their osteogenic potential was demonstrated by first expanding hMSCs in media supplemented to 10% with PRP, or PPP, releasate. After 5 to 7 days in the test media, hMSCs were grown in media containing osteoinductive supplements (OS). Morphologic changes, alkaline phosphatase activity and matrix mineralization were used as differentiation markers.of findings: The efficiency of platelet recovery was approximately 70%, thus creating an approximately five-fold increase in platelet-derived growth factors. Exposure of hMSCs to PRP releasate caused a significant chemotactic effect in a dose-dependent manner. Control cultures with PPP or serum-free media exhibited significantly lower chemotactic rates. Human MSCs exposed to OS media after growth in PRP-supplemented media continued to proliferate more rapidly than the controls, and also displayed greater alkaline phosphatase activity and mineral deposition on a per cell basis, thus indicating an enhancement of differentiated function.Relationship between findings and existing knowledge: These studies demonstrate a direct effect of PRP releasate on purified human MSCs, which play a pivotal role in the process of musculoskeletal tissue repair. In view of the data presented, and clinical reports of enhanced bone healing, we suggest that local application of PRP causes migration of hMSCs to the wound site, followed by their substantial replication to form a repair blastema. As the bioactive factors diffuse away from the fibrin scaffold, now densely populated by hMSCs, the cells cease dividing and are primed to respond to the residual PRP releasate and endogenous inductive cues that stimulate differentiation.Overall significance of findings: The demonstration that autologous PRP is a source of concentrated growth factors that possesses chemotactic, mitogenic and osteopromotive activity lends further credence to its therapeutic role in clinical orthopedics.Disclosures: Device or drug: Symphony Platelet Concentrate System. Status: Approved.Conflict of interest: Stephen Haynesworth, grant research support; Stephen Haynesworth, consultant; Sudha Kadiyala, other support; Sudha Kadiyala, stockholder; Scott Bruder, employee; Scott Bruder, stockholder, JNJ, DePuy AcroMed.

Leukocytes can enhance platelet-mediated aggregation and thromboxane release via interaction of P-selectin glycoprotein ligand 1 with P-selectin.

Platelet--leukocyte conjugates have been observed in patients with unstable coronary syndromes and after cardiopulmonary bypass. In vitro, the binding of platelet P-selectin to leukocyte P-selectin glycoprotein ligand-1 (PSGL1) mediates conjugate formation; however, the hemostatic implications of these cell--cell interactions are unknown. The aims of this study were to determine the ability of leukocytes to modulate platelet agonist--induced aggregation and secretion in the blood milieu, and to investigate the role of P-selectin and PSGL-1 in mediating these responses. Blood was drawn from healthy volunteers for in vitro analysis of platelet agonist--induced aggregation, secretion (adenosine triphosphate, beta-thromboglobulin, and thromboxane), and platelet-leukocyte conjugate formation. Experiments were performed on live cells in whole blood or plasma to simulate physiologic conditions. Whole-blood impedance and optical aggregometry, flow cytometry, and enzyme-linked immunosorbent assays were performed in the presence and absence of blocking antibodies to P-selectin and PSGL1. The platelet-specific agonists, thrombin receptor activating peptide and adenosine diphosphate, were used to elicit platelet activation responses. Inhibition of platelet--leukocyte adherence by P- selectin and PSGL1 antibodies decreased agonist--induced aggregation in whole blood. The presence of leukocytes in platelet-rich plasma increased aggregation, and this increase was attenuated by P-selectin blocking antibodies. Data from flow cytometry confirmed that platelet-leukocyte conjugate formation contributed to aggregation responses. Blocking antibodies reduced platelet agonist--induced thromboxane release but had no impact on adenosine triphosphate and beta-thomboglobulin secretion. Leukocytes can enhance platelet agonist--induced aggregation and thromboxane release in whole blood and platelet-rich plasma under shear conditions in vitro. Interaction of platelet P-selectin with leukocyte PSGL1 contributes substantially to these effects.

Native macromolecular heparin proteoglycans exocytosed from stimulated rat serosal mast cells strongly inhibit platelet-collagen interactions.

Mast cells, the major source of tissue heparin, line the vascular system. On stimulation, rat serosal mast cells release soluble heparin proteoglycans (HEP-PGs) of very high molecular weight (7500(K)). We compared the effects of HEP-PGs and standard heparins (average molecular weights, 15,000 and 5,000) on platelet-collagen interactions in vitro. In contrast with the standard heparins, HEP-PGs completely inhibited collagen-induced platelet aggregation and serotonin release in platelet-rich plasma. The inhibition caused by HEP-PGs depended on its macromolecular structure. In flowing blood, HEP-PGs also inhibited platelet deposition on a collagen-coated surface both at low and high shear rates. Although HEP-PGs did not block glycoprotein (GP) Ia/IIa-mediated platelet adhesion, they attenuated subsequent platelet activation and aggregation, as well as fibrinogen binding to platelets after collagen stimulation. HEP-PGs did not bind to platelets but bound tightly to von Willebrand factor (vWf) and enhanced its binding to collagen. Although platelet adhesion at high shear rate and vWf binding to GP Ib after ristocetin stimulation were not markedly affected, HEP-PGs reduced thrombin-induced aggregation and vWf binding to GP IIb/IIIa. These findings imply that activation of vascular mast cells with ensuing secretion of HEP-PGs may locally attenuate the thrombogenicity of matrix collagen by inhibiting its platelet-activating capacity.

CHARACTERISTICS OF PROTEIN KINASE C-INDEPENDENT EXOCYTOSIS IN HUMAN PLATELETS

We evaluated the characteristics of the protein kinase C (PKC)-independent mechanism for ATP release in platelet-rich plasma. When ADP (10 μM) and U46619 (1 μM) were both added as agonists, a significant release was observed immediately after stimulation. The PKC inhibitor, Ro-31-7549 (10 μM), or a cyclooxygenase inhibitor, aspirin (400 μM) or indomethacin (20 μM), partially inhibited ATP release with little effect on platelet aggregation. The ATP release observed in the presence of Ro-31-7549 was abolished by a cyclooxygenase inhibitor or by preventing aggregation without stirring. In the nonstirred condition, thromboxane B 2 formation was reduced by 93%. When sodium arachidonate (1 mM) rather than U46619 was used with ADP, ATP release in the presence of Ro-31-7549 was abolished by stopping the stirring with no effect on thromboxane B 2 formation. In contrast, ADP/U46619-induced ATP release observed in the presence of aspirin was only partially inhibited when the stirring was stopped. This release was also inhibited dose-dependentl by Ro-31-7549 at concentrations between 1 and 10 μM. These results suggest that PKC-independent ATP-release in this system requires aggregation and is inhibited by a cyclooxygenase inhibitor, while PKC-dependent exocytosis is insensitive to aggregation and a cyclooxygenase inhibitor. © 1997 Elsevier Science Ltd

Evaluation of Whole-Blood Lumiaggregation

An evaluation of whole-blood lumiaggregation was conducted in a normal population. Platelet aggregation and adenosine triphosphate (ATP) secretion were monitored in a three-phase study that analyzed sample dilution, agonist dose response, and method comparison. In the first phase, the blood:saline ratio was varied; in the second phase, the concentration of the agonists was varied ; and in the last phase, a comparison of impedance aggregation and ATP release in whole blood to optical aggregation and ATP release in platelet-rich plasma (PRP) was performed. Five common platelet agonists— collagen, adenosine diphosphate (ADP), arachidonic acid, thrombin, and ristocetin—were used in this evaluation of the lumiaggregometer (Chrono-Log Corp., Havertown, PA, U.S.A.). The data revealed that the optimum blood:saline ratio for conducting platelet antigen studies is 1:1, although with some agonists other dilutions can be used. The agonist dose-response phase basically confirmed the manufacturer's concentration recommendations. Additionally, it was determined that platelet aggregation using the whole-blood impedance technique compared to the PRP optical method yielded similar information. Furthermore, the advantages of whole-blood impedance aggregation include its use in microsamples and more timely results due to minimal sample preparation. Key Words: Platelet aggregation—Lumiaggregation—Whole blood—Platelet-rich ptasma—ATP.

Reduced platelet serotonin content and release in familial hypercholesterolaemia

Plasma and platelet serotonin (5-HT) concentrations, and resting and collagen-induced 5-HT release in platelet-rich plasma were studied in normal and familial hypercholesterolaemic (FH) subjects. Platelet 5-HT concentrations were significantly reduced (−37%, P<0.01) in FH patients whilst mean plasma concentrations, although increased, were not significantly different from those in normal subjects. Platelet 5-HT correlated negatively with plasma cholesterol when the data for normal subjects and FH patients were combined ( r=−0.48, P=0.005). It also correlated negatively with low-density lipoprotein (LDL) (FH data, r=−0.59, P=0.03; normal and FH data, r=−0.49, P=0.004) but positively with high-density lipoprotein (HDL) (FH, r=0.79, P=0.001; normal and FH, r=0.37, P=0.03). Collagen (5–160 μg/ml) stimulated platelet 5-HT release occurred in a concentration-dependent manner. In FH patients stimulated 5-HT release was reduced (10 μg/ml collagen, −40%, P<0.05) and accompanied by increased collagen EC 50 values ( P<0.02). Resting 5-HT release was increased substantially in FH patients but not significantly. Our data provide evidence for a relationship between circulating cholesterol and platelet serotonergic mechanisms. It is proposed that abnormalities relating to platelet-plasma 5-HT dynamics, perhaps due to enhanced platelet activity or decreased platelet uptake, may contribute to the cardiovascular complications in FH.

2425 Central nitric oxide-prostaglandin system mediates the inhibition of rat gastric acid secretion through activation of central sympatho-adrenomedullary outflow

Three different NO donors, S-nitrosoglutathione (GSNO), sodium nitroprusside (SNP) and 3-morpholino-sydnonimine hydrochloride (SIN-1) were used in order to investigate mechanisms of platelet inhibition through cGMP-dependent and -independent pathways both in human and rat. To this purpose, we also evaluated to what extent cGMP-independent pathways were related with the entity of NO release from each drug.SNP, GSNO and SIN-1 (100 μM) effects on platelet aggregation, in the presence or absence of a soluble guanylate cyclase inhibitor (ODQ), on fibrinogen receptor (αIIbβ3) binding to specific antibody (PAC-1), and on the entity of NO release from NO donors in human and rat platelet rich plasma (PRP) were measured.Inhibition of platelet aggregation (induced by ADP) resulted to be greater in human than in rat. GSNO was the most powerful inhibitor (IC50 values, μM): (a) in human, GSNO = 0.52 ± 0.09, SNP = 2.83 ± 0.53, SIN-1 = 2.98 ± 1.06; (b) in rat, GSNO = 28.4 ± 6.9, SNP = 265 ± 73, SIN-1 = 108 ± 85. GSNO action in both species was mediated by cGMP-independent mechanisms and characterized by the highest NO release in PRP. SIN-1 and SNP displayed mixed mechanisms of inhibition of platelet aggregation (cGMP-dependent and independent), except for SIN-1 in rat (cGMP-dependent), and respectively lower or nearly absent NO delivery. Conversely, all NO-donors prevalently inhibited PAC-1 binding to αIIbβ3 through cGMP-dependent pathways.A modest relationship between NO release from NO donors and cGMP-independent responses was found. Interestingly, the species difference in NO release from GSNO and inhibition by cGMP-independent mechanism was respectively attributed to S-nitrosylation of non-essential and essential protein SH groups.

Serotonin content in human blood platelets is not stable

A large variation of platelet serotonin content over short time intervals has been shown. This oscillation of platelet serotonin does not result from the accumulation of random technical variations, but can be considered as an objectively existing phenomenon which might be due to alternation of serotonin release and uptake processes in platelet-rich plasma. Sex and age do not influence the variation of platelet serotonin.

Platelet-activating factor (PAF)-induced platelet aggregation modulation by plasma adenosine and methylxanthines

This study examined the role of plasma adenosine in the modulation of platelet-activating factor (PAF) activity on platelet aggregation and serotonin (5-HT) release in human platelet-rich plasma (PRP). In addition, the effects of methylxanthines (e.g. theophylline and caffeine) were studied on PAF-induced platelet aggregation in PRP isolated from blood samples from healthy subjects. Also, PAF-induced platelet aggregation was examined in PRP samples of patients receiving theophylline treatment. These studies demonstrate that plasma adenosine levels (0.1 to 0.3 μM) play a key role in negative modulation of PAF activity on platelet aggregation and 5-HT release. After depletion of plasma adenosine, the platelet-aggregating activity of PAF was increased greatly (>10-fold). PAF at concentrations of 0.1 to 12 μM caused no 5-HT release in PRP containing normal amounts of adenosine (blood collected in the presence of 2′-deoxycoformycin and dilazep), whereas PAF at 0.1 μM caused 5-HT release (45%) in adenosine-depleted PRP, demonstrating that plasma adenosine is much more inhibitory of 5-HT release than platelet aggregation. The adenosine antagonists theophylline (50 μM), caffeine (50 μM) and a xanthine derivative, 3,7-dimethyl-l-propargylxanthine (DMPX, 10 μM) (a more specific adenosine A 2 receptor antagonist), potentiated PAF activity on platelet aggregation in PRP samples containing adenosine. Also, patients receiving theophylline treatments showed significantly greater platelet aggregation induced by PAF in their PRP samples. PAF induced a rapid increase (80% in 15 sec) in intracellular Ca 2+ mobilization, which was strongly inhibited by adenosine (IC 50, 0.3 μM). Our studies suggest that agents that can increase plasma adenosine levels (e.g. inhibitors of adenosine uptake and adenosine metabolism) or methylxanthines may be useful in altering (inhibiting or enhancing, respectively) PAF actions on platelets and other tissues.

The effect of olive oil supplementation on human platelet function, serum cholesterol-related variables and plasma fibrinogen concentrations: A pilot study

Seven healthy volunteers ingested 21 g olive oil daily for 8 weeks. Platelet aggregation (in platelet rich plasma and in whole blood) and thromboxane A 2 release (in platelet rich plasma) were reduced after supplementation; some of these values achieved statistical significance. There were no significant changes in platelet count and median platelet volume or in the concentrations of plasma fibrinogen or serum cholesterol, low density lipoprotein and high density lipoprotein after supplementation. Platelet membrane oleic acid content increased significantly ( P<0.01), whereas arachidonic acid content decreased significantly ( P<0.05). There was a significant negative correlation between the content of these two fatty acids, whether all values were considered together or whether values after 4 and 8 weeks were considered separately. This correlation suggests the possibility that an excess of oleic acid impairs the incorporation of arachidonic acid into platelet phospholipids. This phenomenon could account for the observed inhibition of platelet aggregation and reduced thromboxane A 2 release, since arachidonic acid is the substrate for thromboxane A 2. It is of interest that populations consuming large amounts of olive oil have a low incidence of ischaemic heart disease. Olive oil should no longer be considered an inactive placebo in studies assessing platelet function.

Extracellular Na+, but not Na+/H+ exchange, is necessary for receptor-mediated arachidonate release in platelets

The effect of extracellular Na+ removal and replacement with other cations on receptor-mediated arachidonate release in platelets was studied to investigate the role of Na+/H+ exchange in this process. Replacement with choline+, K+, N-methylglucamine+ (which abolished the thrombin-induced pHi rise) or Li+ (which allowed a normal thrombin-induced pHi rise) significantly decreased arachidonate release in response to all concentrations (threshold to supra-maximal) of thrombin and collagen. This inhibition was not reversed by NH4Cl (10 mM) addition, which raised the pHi in the absence of Na+, but, on the contrary, NH4Cl addition further decreased the extent of thrombin- and collagen-induced arachidonate release, as well as decreasing 'weak'-agonist (ADP, adrenaline)-induced release and granule secretion in platelet-rich plasma. No detectable pHi rises were seen with collagen (1-20 micrograms/ml) and ADP (10 microM) in bis-(carboxyethyl)carboxyfluorescein-loaded platelets. Inhibition of thrombin-induced pHi rises was seen with 0.5-5 microM-5-NN-ethylisopropylamiloride (EIPA), but at these concentrations EIPA had little effect on thrombin-induced arachidonate release. At higher concentrations such as those used in previous studies (20-50 microM), EIPA inhibited aggregation/release induced by collagen and ADP in Na+ buffer as well as in choline+ buffer (where there was no detectable exchanger activity), suggesting that these concentrations of EIPA exert 'non-specific' effects at the membrane level. The results suggest that (i) Na+/H+ exchange and pHi elevations are not only necessary, but are probably inhibitory, to receptor-mediated arachidonate release in platelets, (ii) inhibition of receptor-mediated release in the absence of Na+ is most likely due to the absent Na+ ion itself, and (iii) caution should be exercised in the use of compounds such as EIPA, which, apart from inhibiting the Na+/H+ exchanger, have other undesirable and misleading effects in platelets.