SEARCH · Search PubMed
Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.
Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
Several 'capture' assays are currently employed to identify specific platelet antibodies, but all require the use of murine monoclonal antibodies (MoAbs) against the antigen of interest. We have developed a new antigen capture assay for the detection of platelet reactive antibodies, based on platelet surface sialoglycoprotein labelling with biotin hydrazide, and a following immobilization of the biotinylated platelet proteins to microtiter wells that had been coated with streptavidin. The resulting solid phase can then be used in a simple ELISA to detect serum and platelet associated antibodies. We describe here two versions of this biotin-avidin immobilization of platelet glycoproteins (BAIPG) assay. In BAIPG assay type I, the test sera are directly incubated in microtiter wells previously coated with streptavidin plus biotinylated platelet proteins. The BAIPG type II procedure involves the incubation of sera with biotinylated platelets before platelet solubilization, and, after platelet lysis, the immobilization of the immune complexes to streptavidin-coated wells. In both cases, the bound antibodies are determined by alkaline phosphatase conjugated anti-human IgG. Using BAIPG type I, positive results were obtained in 7/33 patients with idiopathic thrombocytopenic purpura (ITP), 1/10 patients with secondary immune thrombocytopenia (SIT) and 4/17 with non-immune thrombocytopenia (NIT). The BAIPG type II test was positive in 13 out of 33 patients with ITP, in six out of ten patients with SIT, and in three out of the 17 patients with NIT. A comparison between BAIPG and monoclonal antibody immobilization of platelet antigens (MAIPA) assays showed a high degree of correlation between the two methods. These results suggest that the BAIPG assay is a valuable new tool for the detection of anti-platelet antibodies.
Blood platelets are the smallest cellular elements in mammalian blood. Because of their small size, platelets have an unusually large surface area: volume ratio and are exquisitely sensitive to a multitude of physiological and environmental stimuli. Platelets lack nuclei, but most possess functional mitochondria and remain capable of both anaerobic and aerobic energy metabolism, for which they utilise a variety of substrates including many which are cytotoxic and genotoxic for other (nucleated) cells. Nucleic acid precursors are amongst the potentially genotoxic compounds for which platelets have an apparently insatiable appetite. In particular platelets actively scavenge adenine and adenosine, which they convert to nucleotides and use in energy metabolism, but they also rapidly phosphorylase thymidine and liberate thymine into the extracellular medium. In addition, platelets contain non-metabolisable membrane-bound pools of adenine nucleotides which they secrete in response to strong agonists. Taken together, these observations suggest that blood platelets play an important role in nucleic acid precursor metabolism. In the previous paper we have shown that most thymidine phosphorylase activity present in normal human blood resides in the cytoplasm of platelets. Here we demonstrate that this enzyme activity can be modulated in a dose-dependent fashion, not only by substances recognised as platelet agonists and antagonists, but also by some compounds which are considered to be toxic, mutagenic and/or carcinogenic. The data which we present provide additional support for our previous suggestion that platelets regulate thymidine homeostasis and further imply that this is the normal, physiological, platelet function. Preliminary results suggest that assays of blood platelet thymidine metabolism may provide data with a wide variety of applications.
Indium-111-labeled autologous platelets, injected 48 hours after operation, were used to evaluate the thrombogenicity of prosthetic material and the effect of platelet inhibitor therapy in vivo. Dacron double-velour (Microvel) aortofemoral artery bifurcation grafts were placed in 16 patients and unilateral polytetrafluoroethylene femoropopliteal grafts were placed in 10 patients. Half the patients in each group received platelet inhibitors before operation (dipyridamole, 100 mg 4 times a day) and after operation (dipyridamole, 75 mg, and acetylsalicylic acid, 325 mg 3 times a day); the rest of the patients served as control subjects. Five-minute scintigrams of the graft region were taken with a gamma camera interfaced with a computer 48, 72, and 96 hours after injection of the labeled platelets. Platelet deposition was estimated from the radioactivities of the grafts and expressed as counts per 100 pixels per microcurie injected. Dipyridamole and aspirin therapy significantly reduced the number of platelets deposited on Dacron grafts and prevented platelet accumulation over 3 days. With the small amount of platelet deposition on polytetrafluoroethylene femoropopliteal artery grafts even in control patients, platelet inhibitor therapy had no demonstrable effect on platelet deposition on these grafts. It is concluded that (1) platelet deposition on vascular grafts in vivo can be quantitated by noninvasive methods, and (2) dipyridamole and aspirin therapy reduced platelet deposition on Dacron aortofemoral artery grafts.
An appreciable effort is directed toward designing strategies to minimize platelet interactions with artificial surfaces, because their reactivity is thought to promote thrombus formation and lead to materials failure. Although platelet glycoprotein Ib/IX (GPIb/IX) and glycoprotein IIb/IIa (GPIIb/IIIa) receptors are thought to mediate adhesion, whether GPIIb/IIIa receptors are activated and how this might occur are largely unknown and are the focus of this article. There are a few ways, other than thrombin generation, that blood contact with artificial surfaces can lead to GPIIb/IIIa activation. Complement activation can lead to products capable of activating platelets (C1q, C5b-9), and contact between platelet CD32 (FcgammaRII) receptors and immobilized immunoglobulin G could also activate platelets. In this article the potential role of these processes was evaluated by using various inhibitors in a microsphere-based platelet adhesion immunoassay. Polystyrene microspheres (10 microm) were incubated in platelet-rich plasma before flow cytometric analysis of beads for adherent platelets. The data eliminated occupancy of the FcgammaRII receptor (by use of IV.3 blocking antibody), C5b-9 production (by use of sCR1), and the indirect action of factor XIIa on complement components (by use of corn trypsin inhibitor) as playing roles in supporting platelet adhesion. Agents directed against the first complement component (benzamidine, pentamidine, pyridoxal-5-phosphate) were effective inhibitors of platelet adhesion and were also demonstrated to inhibit SC5b-9 and C3d levels on the bead surface after serum incubations. Because these agents are not highly specific, it can not be concluded that C1q is a mediator of adhesion. These agents were also demonstrated to inhibit fluorescein isothiocyanate-fibrinogen binding to activated washed platelets, therefore indicating that fibrinogen receptor expression is a requirement for platelet adhesion.
The detection of platelet-specific antibodies is of high clinical interest in diseases with immune thrombocytopenia. The glycoprotein-specific platelet-antigen capture (PAC)-assay developed in this study is especially suited to the differentiation of platelet-specific immunoglobulin (Ig) G subclasses and the determination of platelet-specific IgM in serum or on platelets. The problems with unspecific signals or low sensitivity usually seen with the detector antibodies available are effectively overcome, as unbound detector antibodies are removed at an early stage in the assay. We investigated 14 maternal alloantisera from cases of neonatal alloimmune thrombocytopenia (NAITP) and six sera from patients with autoimmune thrombocytopenic purpura (AITP). In NAITP sera, we found IgG1 alone in 57%, IgG1 + IgG3 in 21% and IgG1 + IgG2 in 14% of cases. One serum contained IgG1 + IgG2 + IgG3. In AITP, three out of the six sera contained IgG1 alone. One serum contained IgG1 + IgG2. One patient, with highly refractory AITP, had platelet-specific IgG1 + IgG2 + IgG3 in his serum. A patient with AITP in remission and normal platelet counts only showed platelet-bound IgG2. The detection of platelet-specific 'whole IgG' is possible too. However, at this time the commonly used monoclonal antibody-specific immobilization of platelet antigens (MAIPA) method should not be replaced for this purpose, as it is well standardized and used with similar results in many laboratories. The PAC assay sensitively detects the subclasses of platelet-specific IgG and human leucocyte antigen (HLA)-antibodies independently. It is easy to perform and takes less time than other platelet glycoprotein-specific methods.
A paired prospective study was performed to compare the in vitro storage characteristics and in vivo kinetics of platelets stored in granulocyte-platelet concentrates prepared by apheresis with platelets prepared from whole blood. Platelet and granulocyte-platelet concentrates were collected from five healthy volunteer autologous donors and stored for 16 to 18 hours at 20 to 24 degrees C with and without agitation, respectively. After storage, pH, platelet count, percent release of beta-thromboglobulin, morphologic score, and percent osmotic recovery were measured. In addition, the granulocyte-platelet concentrates were assayed for total leukocyte count, release of lysozyme, and by several in vitro tests of granulocyte function. The platelets in both products were labeled with 111In oxine and infused into the donors. The pH of both products was above 6.0 at the end of storage. The units stored as platelet concentrates compared with those stored as granulocyte-platelet concentrates showed a higher percent release of beta-thromboglobulin, 18.4 +/- 4.0 percent versus 5.9 +/- 3.2 percent (mean +/- SD), but significantly better morphologic scores, 676 +/- 21 versus 525 +/- 56, and better osmotic recovery scores, 72 +/- 10 percent versus 40 +/- 7 percent, respectively (all p less than 0.05). The platelet concentrates (compared with the granulocyte-platelet product) had significantly better in vivo recovery, 49.5 +/- 15.8 percent versus 38.9 +/- 11.5 percent, and survival, 6.1 +/- 1.3 days versus 2.4 +/- 0.4 days, respectively (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)
Current platelet crossmatch procedures to select compatible donors for alloimmunized thrombocytopenic patients are hampered by the lack of a convenient platelet storage method. This study examined the feasibility of using washed apheresis donor platelets stored for up to 1 year in a modified Hank's buffer solution at 4 degrees C as crossmatch reagents in an indirect IgG-enzyme immunoassay. Pooled and monospecific HLA and PlA1 antisera were used to determine the antigenic reactivity of donor platelets in relation to duration of storage. There were no significant differences between mean HLA and PlA1 antigen expression in fresh and stored platelets. HLA reactivity was detected on 12 of 13 donor platelet samples stored for 3 to 9 months and on 14 of 17 platelets stored for 12 to 14 months. PlA1 reactivity was maintained at 12 to 14 months for all 12 donor platelet samples tested. In addition, incompatibility remained in 23 of 24 paired fresh and stored platelet crossmatches using individual alloimmunized patient plasmas. These data indicate that both HLA and platelet-specific PlA1 antigen reactivity can be maintained adequately in liquid storage at 4 degrees C for up to 1 year. The availability of a convenient platelet storage method should facilitate the general application of platelet crossmatching procedures for alloimmunized patients.
BACKGROUND: Donor exposure risk and cost in platelet transfusion practice can be limited by increasing the recovery of platelets from donor units. STUDY DESIGN AND METHODS: This study presents results of continuous quality improvement efforts in platelet production and compares the in vivo therapeutic efficacy of currently produced platelet concentrates (PCs) with that of apheresis platelets. Production quality improvement measures included optimization of instrument performance (rotor speed trials), process (massaging whole-blood units, using cup liners, limiting spin-expression time, and refining plasma expression technique), and staff (intensive training with observation and ongoing quality control data feedback). Corrected count increments and increments per kg were calculated for transfusions of 4 pooled PCs and apheresis platelets over a 30-day period. RESULTS: The mean number of platelets per PC increased from 5.5 x 10(10) in 1975 to 9.69 x 10(10) in 1994. The mean platelet dose was 3.78 x 10(11) for 4 PCs and 4.17 x 10(11) for apheresis platelets. A total of 34 pooled PCs and 17 apheresis platelets was transfused to 21 patients. The mean increment, the increment per kg, and the corrected count increment were, respectively, 31 x 10(3) per microL, 4.8 x 10(2) per microL, and 14,700 for 4 PCs and 35.4 x 10(3) per microL, 5.4 x 10(2) per microL, and 14,700 for apheresis platelets. Differences were not significant. CONCLUSION: Therapeutic efficacy comparable to that of apheresis platelets can be obtained with 4 high-yield PCs.
The platelet high affinity binding site for thrombin appears to be described by a classical receptor-ligand interaction that is distinct from the platelet thrombin receptor/substrate, PAR-1. However, the identification and function of the high affinity binding site with respect to its physiological importance have continued to elude investigators. Prior studies using two mutant thrombins suggested that thrombin interaction with the platelet high affinity binding site is mediated through an extensive portion of the thrombin molecule involving residues within the substrate binding pocket and the anion binding exosite (Leong, L., Henriksen, R. A., Kermode, J. C., Rittenhouse, S. E., and Tracy, P. B. (1992) Biochemistry 31, 2567-2575) and may mimic a thrombin-hirudin interaction. To test this hypothesis, an anti-hirudin peptide antibody (anti-hirpeptide Ab) was raised against a peptide mimicking the COOH terminus of hirudin. The Ab recognized adherent platelets and those in suspension as determined by enzyme-linked immunosorbent assay and immunofluorescence microscopy, respectively. 125I-Thrombin binding to platelets was inhibited in the presence of the anti-hirpeptide Ab in a dose-dependent manner with maximal inhibition >90%. Analyses of data from binding studies of 125I-thrombin to platelets at a fixed Ab concentration indicated that the anti-hirpeptide Ab inhibited the high affinity binding interaction exclusively. In addition, thrombin-induced increases in platelet [Ca2+]i were enhanced by blocking the high affinity binding site with the Ab due to redistribution of the agonist to PAR-1. Thrombin Quick I-induced platelet calcium mobilization was unaffected by the presence of the Ab, consistent with the inability of thrombin Quick I to bind to the high affinity site. Even though glycoprotein (GP) Ib contains a hirudin-like region within the alpha subunit, the postulated high affinity binding site, direct binding of 125I-thrombin could not be demonstrated to transfected Chinese hamster ovary and L cells expressing the GP Ib-IX-V complex. Furthermore, an anti-GP Ib Ab, raised to the peptide region proposed as the thrombin high affinity site, did not enhance thrombin-induced platelet calcium mobilization. The anti-hirpeptide Ab recognized a population of platelet membrane proteins distinct from PAR-1 and GP Ib by three-color immunofluorescence using confocal microscopy. These combined studies demonstrate that the high affinity binding site for thrombin is a unique platelet protein distinct from GP Ib which modulates the effective thrombin concentration localized at the human platelet surface.
OBJECTIVE: The Cephotest is an activated partial thromboplastin time (APTT) test used to measure the activity of the intrinsic pathway of coagulation. To perform this test, blood is usually centrifuged to obtain plasma that is almost without erythrocytes and leucocytes and with only a minimal amount of platelets. MATERIAL AND METHODS: In the present experiments blood was centrifuged at different speeds to produce either platelet-poor plasma (PPP) or platelet-rich plasma (PRP). PPP and PRP obtained from the same whole blood samples from each of several persons were tested in pairs using the standard Cephotest reagent to observe the consequences of Cephotest being performed on plasma containing platelets. The same procedure was used with a Cephotest reagent with a reduced concentration of phosphatidylserine. In succeeding experiments the PRP was preincubated with SFLLRN or Ca-ionophore to activate the platelets, a procedure also known to produce platelet-derived microparticles. PPP and PRP were compared by thrombin time and reptilase time tests to find out at which stage platelets might influence the Cephotest. RESULTS: The results showed that the platelets did influence Cephotest when using both the regular reagent and the phospholipid-reduced agent. When using the regular reagent, PRP showed a tendency towards a longer APTT than PPP. It is suggested that this was caused by platelets consuming some of the first traces of thrombin generated. CONCLUSIONS: When using the phospholipid-reduced reagent, PRP showed a shorter APTT than PPP, probably because the platelets contributed phosphatidylserine to the system. When the platelets were activated before testing, their effects on the tests were increased. Microparticles that formed during platelet activation may have contributed to these effects.
BACKGROUND: Cancer is a complex multi-factorial disorder that may commonly show abnormal angiogenesis in such patients. Recently, platelets have been postulated to have a major role in both these processes, suggesting that antiplatelet strategies may be useful in cancer treatment. MATERIALS AND METHODS: To further investigate the role of platelets in angiogenesis, we used a novel platelet lysate assay to analyse platelet contents in breast cancer (n = 30) and prostate cancer (n = 30) patients and age- and sex-matched controls (n = 60). Markers of angiogenesis (vascular endothelial growth factor (VEGF), angiopoietin-1 and-2 (Ang-1, -2), and their respective receptors (Flt-1 and Tie-2) plus a marker of platelet activation (P-selectin (P-sel)), were all measured in platelet lysate by enzyme-linked immunsorbent assay. RESULTS: Platelet lysate from breast cancer patients contained higher levels of VEGF (P < 0.0001). Ang-1 (P = 0.0186) and P-sel (P = 0.0002), compared to healthy controls. Platelet lysate from prostate cancer patients had elevated VEGF (P = 0.008) but not Ang-1 or P-sel. There were no significant differences between levels of Fit-1 between patients and controls, and both Ang-2 and Tie-2 were undetectable in both patient groups and control platelet lysate. CONCLUSION: We have shown that our previously developed platelet lysate technique could be used to measure indices of angiogenesis, and their respective receptors, and that this assay can be applied to patients with cancer. Our study also provides further evidence that platelets may influence angiogenic abnormalities in human cancer. The platelet may be a useful target in anti-cancer strategies.