Ovulation is accompanied by a marked infiltration of leukocytes into thecal layers after the gonadotropin surge. P-selectin is known to play a critical role in the initial steps of leukocyte recruitment from the bloodstream during inflammation. Thus, the objective was to investigate the potential regulation of P-selectin by gonadotropins in equine preovulatory follicles. The full-length equine P-selectin cDNA was cloned by a combination of reverse transcription-polymerase chain reaction (RT-PCR) and 5'- and 3'-rapid amplification of cDNA ends. Results showed that equine P-selectin cDNA encodes an 829-amino acid protein that is highly conserved when compared to the human protein (80% identity). Semiquantitative RT-PCR/Southern blot analyses were performed to study the regulation of P-selectin transcript in preovulatory follicles isolated during estrus at 0, 12, 24, 30, 33, 36, and 39 h after an ovulatory dose of hCG (ovulation occurs between 39 and 42 h post-hCG in this model). Results showed that levels of P-selectin mRNA remained very low or undetectable throughout the ovulatory process in extracts prepared from the granulosa cell layer. In contrast, a significant increase in P-selectin transcript was observed between 30 and 39 h post-hCG in extracts obtained from thecal layers (P < 0.05). Likewise, immunohistochemistry revealed an increase of immunoreactive P-selectin protein in the vascular endothelium present in thecal layers of follicles isolated 36 and 39 h post-hCG. Thus, the present study describes, to our knowledge for the first time, the primary structure of equine P-selectin and the regulation of P-selectin transcript and protein in follicular thecal endothelial cells before ovulation.

Acute occlusion and subacute restenosis of the coronary artery are still the limiting factors of the otherwise successful interventional cardiology. Platelets and especially activated platelet populations play a key role concerning these typical and sometimes fatal complications. In this study we used flow-cytometry to determine the influence of the modern interventional technique of rotablation on platelet antigens and their possible alteration. A PTCA control group was included. We analyzed the fluorescence expression of structural antigens CD41a (GPII-IIIa) and CD42b (GPIb-V-IX), and of the activation-dependent antigens CD62p (P-selectin, PADGEM, GMP-140) and CD63 (GP53). Furthermore we analyzed the binding of fibrinogen to the platelet flow-cytometrically. CD41a and CD42b did not show significant alternations in fluorescence before, directly after and thirty minutes after finishing PTCA and rotablation (PTCA: CD41a p=0.8 and 0.9; CD42b p=0.5 and 0.2; rotablation: CD41a p=0.2 and 0.2; CD42b p=0.4 and 0.1). But platelet activation could be detected directly after PTCA and rotablation measuring the mean channel fluorescence intensity (MCFI) of CD62p, CD63 and fibrinogen binding (all p<0.05). Thirty minutes after finishing the procedures there were again significant changes in MCFI in PTCA (CD62p, CD63, fibrinogen binding; all p<0.05), but not in rotablation (CD62p p=0.1; CD63 p=0. 9; fibrinogen binding p=0.5). But MCFI for CD62p and fibrinogen binding in rotablation was higher than in PTCA. The results of our study show that rotablation also induces significant platelet activation that is higher than in PTCA alone. Flow cytometry is a sensitive and specific, multiparametric tool in establishing platelet activation. The individual platelet activation process is part of a complex cascade of events happening in the rotablated coronary segment leading to a vascular-molecular inflammatory process and consecutive clinical problems in some patients.

BACKGROUND

Formation of platelet-leukocyte aggregates (PLA) via the CD62p-ligand PSGL-1 represents an important mechanism by which leukocytes contribute to thrombotic and inflammatory events. Deficient variants (namely the Thr715Pro-SNP for CD62p and a VNTR-polymorphism for PSGL-1) might affect PLA formation and probably the response to clopidogrel (which is known to reduce PLA-formation).

METHODS

CD62p-expression, PLA-formation and the up-regulation of CD11b before (V1) and 24 hours after (V2) a loading dose of clopidogrel 225 mg were investigated in ten wild-type controls, ten heterozygote carriers of the Thr715Pro-allele and five carriers of the rare PSGL-1 B-allele (2 A/B and 3 B/B).

RESULTS

CD62p-expression before application of clopidogrel and under clopidogrel treatment in Pro715-haplotype samples did not differ from that in wild-type subjects. The response to clopidogrel was similar in all subjects. Pro715-carriers exhibited a significantly lower percentage of monocytes with platelets attached prior to clopidogrel treatment (ADP: median 22 (1st-3rd quartile 20-23), TRAP: 27 (25 - 38)) compared to the wild-type (ADP: 37 (31-44), TRAP: 55 (37-63)). These differences were not present under clopidogrel, and CD11b-expression was significantly reduced in both groups (controls: median 150 (quartile range 121 - 230) to 113 (121 - 230), Pro715-carriers: 147 (139 - 221) to 126 (109 - 170); all values refer to mean fluorescence intensity). Statistical analysis was not done in the case of PSGL-1 B-allele carriers, but PLA-formation before and under clopidogrel was always at the bottom end of the range seen in the control group and the Pro715-carriers or even below this range.

CONCLUSIONS

Minor phenotypic differences in the CD62p-PSGL-1 axis could be demonstrated in this study. Carriers of these polymorphisms showed a full response to clopidogrel comparable to that in control subjects.

BACKGROUND

Acute normovolemic hemodilution (ANH) has been widely used to prevent the massive blood loss during hepatic carcinoma. The influences of ANH on coagulation function are still controversy, especially in elderly patients. The study observed ANH effects on coagulation function and fibrinolysis in elderly patients undergoing the disease.

METHODS

Thirty elderly patients (aged 60-70 yr) with liver cancer (ASA I or II) taken hepatic carcinectomy from February 2007 to February 2008 were randomly divided into ANH group (n=15) and control group (n=15). After tracheal intubation, patients in ANH group and control group were infused with 6% hydroxyethyl starch (130/0.4) and Ringer's solution, respectively. Blood samples were drawn from patients in both groups at five different time points: before anesthesia induction (T1), 30 min after ANH (T2), 1 h after start of operation (T3), immediately after operation (T4), and 24 h after operation (T5). Then coagulation function, soluble fibrin monomer complex (SFMC), prothrombin fragment (F1+2), and platelet membrane glycoprotein (CD62P and activated GP IIb/GP IIIa) were measured.

RESULTS

The perioperative blood loss and allogeneic blood transfusion were recorded during the surgery. The perioperative blood loss was not significantly different between two groups (p>0.05), but the volume of allogeneic blood transfusion in ANH group was significantly less than in control group (350.0±70.7) mL vs. (457.0±181.3) mL (p<0.01). Compared with the data of T1, the prothrombin time (PT) and activated partial thromboplastin time (APTT) measured after T3 were significantly longer (p<0.05) in both groups, but within normal range. There were no significant changes of thrombin time (TT) and D-dimer between two groups at different time points (p>0.05). SFMC and F1+2 increased in both groups, but were not statistically significant. PAC-1-positive cells and CD62P expressions in patients of ANH group were significantly lower than those at T1 (p<0.05) and T2-T5 (p>0.05).

CONCLUSIONS

ANH has no obvious impact on fibrinolysis and coagulation function in elderly patients undergoing resection of liver cancer. The study suggested that ANH is safe to use in elderly patients and it could reduce allogeneic blood transfusion.

Swine platalets are very similar to those of humans and are therefore relevant to cardiovascular research. The swine coronary circulation mimics the human circulation and is large enough to obtain multiple blood samples in survival experiments. In swine regional ischemia similar to the human condition is easily obtainable, which makes the porcine model an ideal choice to study coronary artery disease. However, little is known about the similarity between swine and human platelet surface antigens. We tested the hypothesis that certain swine platelet antigens could crossreact with antihuman antibodies. Using FITC-conjugated monoclonal murine antihuman platelet antibodies, surface antigen expression was determined for human and Yorkshire swine platelets. Expression of CD9 (p24), CD42B (Ib), CD41b, (Ilb), CD61 (IIIa), CD41a (Ilb/IlIa), CD49b (VLA-2), CD62p, (P selectin), CD31 (PECAM-1D, and CD51/CD61 (vitronectin) was measured by flow cytometry. Significant crossreactivity with human platelets was observed consistently for swine platelet GP 1b and GP IIIa. Crossreactivity of the swine GPIb, and GP IIIa with the human receptors is evidence of receptor similarity between human and swine platelets. The implications of significant crossreactivity of these antigens and the lack of recognition of IIb/IIIa needs to be understood in cardiovascular research. Determining commercially available antihuman GP Ib and GP IIIa, rather than GP IIb/IIIa, would contribute to better elucidation of the effect of von Willebrand factor and the booming family of platelet inhibitors in the swine model of ischemia-reperfusion.

BACKGROUND

It is unknown whether the use of volumetric infusion pumps for the transfusion of red blood cells (RBCs) or platelet (PLT) concentrates (PCs) affects the quality of the blood components. We therefore investigated the in vitro quality of these components after use of infusion pumps.

METHODS

Ten different volumetric infusion pumps were used to simulate transfusion with RBCs and PCs. To prevent donor-dependent differences multiple units were pooled and divided into equal portions. The storage time of RBCs was 30 to 35 days (n=10 experiments), and for PCs, either 2 (n=5) or 7 days (n=5). For RBCs an infusion rate of 100 or 300mL/hr was used, and for PCs, 600mL/hr. Transfusions without an infusion pump served as a reference.

RESULTS

None of the infusion pumps induced an increase of free hemoglobin, annexin A5 binding, or formation of echinocytes in RBCs compared to reference units. In 2- and 7-day-old PCs no effect was shown on PLT concentration, annexin A5 binding, mean PLT volume, and morphology score compared to the reference. The CD62P expression of 2-day-old PCs was significantly lower after transfusion compared to the reference, that is, 11.7±2.1% versus 8.1±1.3% (p<0.01).

CONCLUSIONS

There was no adverse effect on the in vitro quality of RBCs or PCs after simulated transfusion using volumetric infusion pumps. A decrease in PLT activation was observed, which can probably be explained by capturing of activated or damaged PLTs in the 200-µm filter present in the infusion system.

BACKGROUND

For logistic reasons, possibilities to produce both platelet (PLT) concentrates prepared from fresh or overnight-stored whole blood (fresh and o/n PCs, respectively) are convenient. The consequences of both possibilities are not well described. The PLT pooling system used might also influence the condition of PCs. Our aim was to compare fresh and o/n PCs with different PLT pooling systems.

METHODS

Fresh and o/n PCs were prepared from buffy coats and plasma in PLT pooling systems of Baxter, Fresenius, Terumo, or Pall (n = 5). PCs were stored for 9 days. The in vitro quality was determined by the PLT count, pH, glucose, lactate, pO(2), pCO(2), CD62P expression, and annexin V binding.

RESULTS

The o/n PCs showed higher PLT count (approx. 460 x 10(9)/PC vs. approx. 310 x 10(9)/PC), pCO(2), and lactate concentration and lower pH, pO(2), glucose concentration, CD62P expression (until Day 5), and annexin V binding (until Day 7) compared with fresh PCs (p < 0.05). Only for o/n PCs in the Baxter and Fresenius systems did the pH and glucose concentration remain higher, and the lactate concentration and CD62P expression remained lower than that of o/n PCs in the Pall and Terumo systems. The pH for fresh PCs in the Baxter and Fresenius systems was more often greater than 7.4 than for fresh PCs in the Terumo or Pall systems.

CONCLUSIONS

The quality of PCs depended on whether PCs were prepared from fresh or overnight-stored whole blood and on the used PLT pooling system. The main difference between fresh and o/n PCs was the PLT count.

BACKGROUND

Muromonab-CD3 is a murine monoclonal antibody (MoAb) that is used in the prophylaxis and treatment of acute graft rejection. Activation of coagulation and fibrinolysis following anti-CD3 administration have been reported in some patients to lead to irreversible intragraft thrombosis.

METHODS

We have studied the effect of muromonab-CD3 infusion on platelets using flow cytometry in six patients who received three daily doses of muromonab-CD3 as prophylaxis of rejection before receiving a living donor renal transplant. Samples were collected before, 15 and 60 min after muromonab-CD3 infusion. Immunolabeling of platelets was performed in whole blood using dual-color analysis. The following conjugated MoAb were used: anti-CD41a, -CD36, -CD42b, -CD62P, -CD63, -factor V/Va and nonspecific Ig. Samples were analyzed with a FACScan flow cytometer (Becton Dickinson, Mountain View, CA, USA).

RESULTS

After muromonab-CD3 infusion, an increase in the binding of MoAb anti-factor V/Va to platelets was seen, which was only statistically significant (2.2% vs. 12.8%, P=.04) after 15 min of the second dose. No significant changes were seen in the other MoAbs studied. No thrombotic complications were observed after transplantation.

CONCLUSIONS

In uremic patients receiving muromonab-CD3 infusion as prophylaxis of graft rejection, an increase in the binding of anti-factor V/Va, denoting an increased exposure of anionic phospholipids in platelets, was seen. This increase in platelet procoagulant activity might contribute to the appearance of thromboses within renal graft seen in some patients who received muromonab-CD3.

OBJECTIVE

To compare the effects of one minimum alveolar concentration (MAC) desflurane and sevoflurane on the expression of CD42b (glycoprotein [GP] Ib), CD41 (GPIIb), CD61 (GPIIIa), CD62P (P-selectin), and CD63 in both unstimulated and adenosine diphosphate (ADP)-stimulated platelets in vitro.

METHODS

University laboratory.

METHODS

15 healthy volunteers.

METHODS

Platelet-rich plasma was obtained and divided into three groups: platelet-rich plasma exposed to air (group 1); air plus one MAC desflurane (6% vol; group 2), and air plus one MAC sevoflurane (2% vol; group 3), for 40 minutes. Percentage of antigen-positive cells (%(+)) mean channel fluorescence (MCF(Sigma)), and index of platelet activation for positive platelets (IPA(+)) as expression markers for GPIb, GPIIb, GPIIIa, P-selectin, and CD63, were measured.

RESULTS

In unstimulated platelets, expression markers for GPIIb and GPIIIa were significantly lower in groups 2 and 3 than group 1 (P < 0.001). P-selectin expression markers were significantly higher in group 2 than in group 1 or group 3 (P < 0.016). CD63 expression markers were significantly lower in group 3 than group 1 (P < 0.016). In ADP-stimulated platelets, expression markers for all glycoproteins were significantly higher in all groups.

CONCLUSIONS

Neither one MAC desflurane nor sevoflurane showed any significant change in ADP-stimulated platelets compared with the control group.

In spite of the frequent need of platelet transfusions, there is limited information on the association of platelet activation markers, in transfused patients with hematology/oncology disorders, with platelet function using flow cytometry. The goal of this study was to evaluate the changes of PAC-1 binding and CD62P expression, with or without agonists in patients after transfusions. Twenty-eight whole blood samples were obtained from 24 patients admitted to the department of Hematology & Oncology and transfused with platelets; these samples were compared to 30 healthy controls. Whole blood samples, either with or without agonists, such as 20 microM adenosine diphosphate (ADP) or 100 microM thrombin receptor activating peptide (TRAP), were stained with the fluorescein conjugated monoclonal antibodies PAC-1 or CD62P. Then, the percent expression for each marker was analysed using flow cytometry. ADP and TRAP induced an increased percentage of CD62P expression and PAC-1 binding after platelet transfusions compared to the samples studied before transfusion, and these findings were lower than those of the healthy controls. However, the expression of platelets without the agonists was not significantly changed, despite the transfusions. Therefore, agonist-induced platelet activation markers, studied by flow cytometry, appear to be more useful for the evaluation of platelet function after transfusions than platelet activation markers without agonists.

A series of events, such as increase of cytoplasmic free calcium (Ca2+) and expression of P-selectin (CD62P), an adhesion molecule, on the platelet surface, are significant indicators of platelet activation. We have used flow cytometry to examine Ca2+ mobilization and CD62P expression in platelets in whole blood obtained in women prior to, and after, different forms of hormone replacement therapy. Thirty-two women completed a protocol consisting of two consecutive 1-month periods under oestradiol (E2), administered orally (2 mg/day) or transdermally (50 microg/day) in random order, followed by a 4-week transdermal sequential regime, in which, during the last 14 days, either progesterone (300 mg/day) or medroxyprogesterone acetate (5 mg/day) was added to the 50 microg/day E2, administered orally in random order. None of the hormonal combinations determined significant changes in Ca2+ mobilization or CD62P expression in non-stimulated platelets. However, stimulation of platelets with adenosine diphosphate, but not with thrombin, caused a significant increase in cytoplasmic Ca2+ concentration during treatment with transdermal E2 plus progesterone. Also when stimulating with thrombin, transdermal E2 was more active than oral E2 in increasing CD62P expression, a difference that was not reduced by the addition of progestogens. In conclusion, hormone replacement therapy only increased Ca2+ mobilization or CD62P expression in stimulated platelets, and then followed a varied pattern that was dependent on the stimulant and on the specific hormonal formulation.

Low cardiorespiratory fitness (CRF) represents a major risk factor for atherosclerosis, and platelets play a key role in the development of this chronic inflammatory disease. Therefore, the purpose of this study was to assess the relationship between CRF and platelet function.

CRF and different aspects of platelet function were assessed in healthy, young, nonsmoking women. Results were compared between groups of low (LF), medium (MF) and high CRF (HF). Measurements were repeated in group LF after a supervised endurance training program lasting two menstrual cycles and obtained results were compared with groups MF and HF. CRF was quantified by maximal oxygen consumption (V˙O2max) determined by an incremental treadmill exercise test. V˙O2max criteria for groups were (mL·min·kg bodyweight): LF < 45, MF 45-55, HF>> 55. Platelet activation state and platelet reactivity were assessed by basal and agonist-induced surface expression of CD62P and CD40L as well as the intraplatelet amount of reactive oxygen species.

In group LF, basal platelet activation as well as agonist-induced platelet reactivity were increased compared with groups MF and HF. Between groups MF and HF parameters of platelet function were roughly equal despite a pronounced difference regarding CRF. Exercise training improved CRF in group LF and aligned platelet function to levels observed in groups MF and HF, although CRF still markedly differed.

Low levels of CRF favor a proinflammatory platelet phenotype. A relatively low dose of exercise is sufficient to normalize platelet function, whereas superior levels of physical activity and CRF do not provide any further substantial benefit, but also no appreciable adverse effects.

The effects of varying concentrations of platelet-activating factor (PAF), arachidonic acid (AA) and collagen on the expression of the platelet activation markers CD63 and CD62P were assessed in 10 normal subjects using flow cytometry. CD63 expression was significantly greater than CD62P expression, with PAF (80 nM) inducing mean maximum CD63 expression of 32.9 ± 6.4% and mean maximum CD62P expression of 5.5 ± 1.8%. AA (1 mM) induced maximum CD63 expression of 37.7 ± 7% and maximum CD62P expression of 9.3 ± 1%. Collagen (2-80 pg/ml) induced minimal expression but 800 pg/ml induced mean CD63 expression of 33.1 ± 4.1% and mean CD62P expression of 6.1 ± 0.8%. Greater CD63 and CD62P expression were induced by phorbol myristate acetate (1.6 pM, 70.9 ± 11% and 69.4 ± 9.9%, respectively) and thrombin (0.1 U/ml, 70.7 ± 9.3% and 73.5 ± 5.4%, respectively). With PAF and collagen only one platelet population was detected whereas with 1 mM AA two populations were observed. These results indicate that expression of platelet adhesion receptors depends on the nature and concentration of agonist and that subpopulations of platelets may exist. Importantly, PAF concentrations inducing moderate CD63 and CD62P expression did not induce platelet aggregation, suggesting that platelets can be activated independently of aggregation.

BACKGROUND

Platelet activation leads to signal transduction mechanisms, in which phosphotyrosine proteins play a relevant role.

METHODS

Platelet suspensions were independently activated by collagen and thrombin in the absence and in the presence of two tyrosine kinase inhibitors, tyrphostin 47 and genistein. Samples were processed to visualize morphological changes by electron microscopy, to evaluate changes in cytoskeletal assembly, to analyze modifications in the expression of activation dependent antigens, and the procoagulant activity at the surface level by flow cytometry. Additional experiments applying flow conditions were performed to assess the effect of inhibiting tyrosine phosphorylation on primary platelet adhesion and fibrin formation.

RESULTS

Inhibition of tyrosine phosphorylation blocked shape change and cytoskeletal assembly induced by collagen, and inhibited, though partially, those effects due to thrombin. Both activating agents induced the expression of the intraplatelet antigens CD62P and CD63 at the surface, although only collagen promoted expression of anionic phospholipids. Both tyrphostin 47 and genistein prevented those effects. The extent of platelet adhesion on both collagen-coated and subendothelial surfaces was significantly diminished by the presence of the tyrosine kinase inhibitors assayed. Fibrin formation was also significantly reduced.

CONCLUSIONS

Platelet shape change and secretion during platelet activation depends on tyrosine phosphorylation. In addition, primary adhesion of platelets induces signaling through tyrosine kinases to achieve full spreading, and results in the exposure of a procoagulant surface on platelets.

This study was purposed to investigate the effects of 25 Gy gamma-ray irradiation on the CD62p expression, platelet count and the mean platelet volume (MPV) of manually enriched platelet suspension in different time of shelf life at 22 degrees C. Each of 16 bags with plasma-rich platelet was divided into two bags, one of which was exposed to 25 Gy gamma-ray of 137Cs and the other ones was not exposed. 16 bags then were preserved for 72 hours according to AABB standards. The irradiated platelets were regarded as the observation group, and the other ones were regarded as the control group, the expression of p-selectin (CD62p) in the above 2 groups was detected by flow cytometry before irradiation and at 24, 72 hours after irradiation respectively; at the same time, the platelet count and MPV were assayed by using blood cell counter. The results showed that the expression level of CD62p on platelet in irradiated and control groups increased along with the prolonging of preservation time, the expression rate of CD62p on the platelets preserved for 24 hours was higher than that on fresh platelets with significant difference (p<0.05); the expression rate of CD62p on the platelets preserved for 72 hours obviously was enhanced as compared with platelets preserved for 24 hours (p<0.01). There were no significant differences in CD62p expression rate, platelet count and MPV between irradiated and control groups preserved for 24 and 72 hours (p>0.05), however the MPV of irradiated and control groups preserved for 72 hours was higher than that of fresh platelets (p<0.05). It is concluded that the gamma-ray irradiation does not affect the quantity and quality of platelets, but the preservation time for manually enriched platelet suspension should be shortened as far as possible.

An 11-year-old spayed-female German Shepherd dog was presented to the Veterinary Medical Teaching Hospital at Kansas State University with a history of weight loss, anorexia, depression, and lethargy for 2-3 weeks. Radiographic examination revealed a mass in the spleen and several round radiodense foci in the liver. CBC results included normocytic normochromic anemia, marked thrombocytopenia, and low numbers of neoplastic cells that frequently had cytoplasmic projections or blebs. A bone marrow aspirate contained about 80% neoplastic megakaryoblasts with the same microscopic features as those observed in peripheral blood. Using flow cytometry, cells of large size were identified in peripheral blood that expressed CD41/61, CD45, CD61, and CD62P (P-selectin) and were negative for markers of T cells, B cells, monocyte/macrophages, and dendritic cells. Because of the poor prognosis, euthanasia and subsequently necropsy were performed. On histopathologic examination, neoplastic megakaryoblasts were identified in spleen, liver, mesenteric lymph node, and the pulmonary vasculature. Using immunohistochemistry, the neoplastic megakaryoblasts weakly expressed von Willebrand factor. Based on microscopic and immunophenotypic findings, a diagnosis of acute megakaryoblastic leukemia (AMegL) was made. To our knowledge, this is the first report of AMegL in a domestic animal in which immunophenotyping by flow cytometry and a panel of antibodies against CD41/61, CD61, and CD62P were used to support the diagnosis.

OBJECTIVE

Platelet function abnormalities have been reported in blood donors who have not consumed aspirin. Our objective was to identify factors other than aspirin that may contribute to impaired platelet function in qualified volunteer blood donors.

METHODS

Blood samples were obtained from 24 donors following routine blood donation. Donors completed a study questionnaire that included questions about recent food consumption, medication and medical history. Platelet activation was measured using monoclonal antibodies and flow cytometry. CD62P expression and PAC-1 binding on platelets were used as indicators of platelet activation. Platelet function was measured on a platelet function analyser (PFA-100) using both collagen/epinephrine (cEPI) and collagen/ADP (cADP) cartridges.

RESULTS

Fifty-four per cent of donors (13 of 24) had normal platelet function. Thirty-eight per cent (nine of 24) had prolonged cEPI closure times, of whom four (17%) had no cEPI closure >> 300 seconds). No closure was associated with aspirin use (two donors) or chocolate consumption (two donors) before donation. Two donors (8%) had either a shortened cEPI or cADP closure time.

CONCLUSIONS

Platelet dysfunction in qualified blood donors is underestimated. Platelet function screening can identify donors with diet-related platelet dysfunction or with poor recollection of aspirin use.

SUMMARY: BACKGROUND: The Mirasol® pathogen reduction technology (PRT) for platelet concentrates (PC) uses riboflavin and UV light (270-360 nm). We evaluated the impact of PRT on platelets in comparison to standard single-donor PC. MATERIAL AND METHODS: Platelets were resuspended in autologous plasma. After 2 h rest without agitation, PC were split into an untreated control unit (C-PC) and an immediately treated unit (T-PC) (series I). In series IV, split PC were stored under agitation over night before PRT was carried out. Platelet quality was assessed by pH, glucose consumption, lactate production rate, LDH, soluble sCD62p and CD62p expression with and without TRAP (thrombin receptor-activating peptide) over 7 days. RESULTS: SERIES I: On day 5, pH values were lower for T-PC (6.8 ± 0.2 vs. 7.4 ± 0.1, C-PC), accompanied by a higher glucose consumption rate of 0.069 ± 0.016 vs. 0.035 ± 0.006 mmol/10(12) platelets/h and lactate production rate of 0.126 ± 0.031 vs. 0.063 ± 0.011 mmol/10(12) platelets/h. CD62p using TRAP was lower for T-PC (50 ± 11 vs. 62 ± 14%). Baseline activation was higher in T-PC (35 ± 12 vs. 28 ± 15%). Longer initial rest time had no impact on these results (series II/III/IV). CONCLUSION: PRT leads to an increase of platelet metabolism and activation independent of the length of the initial rest times. PC resuspended in autologous plasma should be stored at maximum up to day 5.

BACKGROUND

In Japan, no platelet (PLT) additive solutions (PASs) are officially approved for clinical use although blood centers often receive requests for washed PLTs to reduce adverse reactions. Recently, we developed a novel PAS called BRS-A based on clinically available bicarbonated Ringer's solution (BRS), Bicanate and acid-citrate-dextrose formula A (ACD-A), which has been shown to maintain the in vitro properties of PLTs in the condition of <5% residual plasma during 7-day storage. The aim of this study was to evaluate whether another clinically available BRS, Bicarbon with different electrolyte concentrations can be used as a PAS.

METHODS

Two types of BRS-As were prepared by adding 25 mL of ACD-A to 500 mL of Bicanate or Bicarbon BRSs. Bicanate-based BRS-A and Bicarbon-based BRS-A contain 0.9 or 0.5 mmol/L of magnesium chloride, 95.2 or 100.1 mmol/L of sodium chloride, 4.2 or 5.1 mmol/L of trisodium citrate, and 26.6 or 23.8 mmol/L of sodium bicarbonate, respectively; the other components were identical. Apheresis PLTs stored in these solutions with less than 5% plasma for 7-day storage were compared with regard to their in vitro properties.

RESULTS

The pH levels of all units were above 7 throughout storage. The mean PLT volume, hypotonic shock response, glucose consumption, lactate production, swirling, and CD62P and CD42b expression were similar during 7-day storage. The bicarbonate levels in Bicarbon-based BRS-A were lower than those in Bicanate-based BRS-A.

CONCLUSIONS

Differences in concentrations of electrolytes such as magnesium, sodium, citrate, and bicarbonate salts in BRS-A do not affect the in vitro properties of PLTs during 7-day storage. These results indicate that the use of another type of BRS-A based on Bicarbon as a PAS is feasible. Thus, BRS-A can be used in hospitals that do not stock Bicanate but have Bicarbon.

Platelet recovery, and viability, and function is strongly dependent on the method of the preparation of platelet concentrate (PC). The glucose consumption, decrease of pH, release of alpha granules during storage in platelet concentrate impair their clinical effectiveness.

OBJECTIVE

To compare of the quality of buffy-coat-derieved platelet concentrates prepared using automatic system terumo automated centrifuge and separator integration (TACSI) and stored over 7 days.

METHODS

PCs were prepared from buffy coats using manual method (group I), or automatic system TACSI (group II). Fifteen PCs prepared from the 5 buffy coats each were stored over 7 days in 22-24 degrees C and tested. Samples were taken from the PCs container on days 1 and 7. The following laboratory tests were performed: number of platelets, platelets derived microparticles, CD62P expression, platelet adhesion, pH, glucose, lactate dehydrogenase activity.

RESULTS

We have observed higher expression of CD62P in PCs prepared using manual method compared to the PCs produced automatically Platelet recovery was significantly higher in PCs prepared using automatic systems compare to manual method.

CONCLUSIONS

Compared to manual methods, automatic system for preparation of buffy coats, is more efficient and enable production of platelets concentrates of higher quality.

The purpose of this investigation was to obtain information on platelet-leukocyte conjugate formation in whole blood and on factors that affect it. We also measured platelet and leukocyte activation by quantitating the expression of CD62P and CD11b. In both cases a flow cytometric approach was used. The results show that platelet-monocyte and platelet-polymorphonuclear leukocyte (PMNL) conjugate formation is enhanced by simply stirring blood, with optimum conjugate formation occurring after 10 min. In the case of monocytes,conjugate formation was enhanced by adenosine diphosphate (ADP). Both monocyte and PMNL conjugate formation was enhanced by phorbol myristate acetate (PMA), but L-formyl methionyl lysyl proline (FMLP) was either without effect (monocytes) or inhibitory (PMNL). EDTA also inhibited conjugate formation (implying involvement of divalent cations), as did dextran sulphate (implying involvement of P-selectin = CD62P). Interestingly GR144053F, which acts at GpIIb-IIIa on platelets to interfere with fibrinogen binding, and also glycyl prolyl arginyl proline (GPRP), a peptide that interferes with the interaction between CD11c on leukocytes and fibrinogen, did not inhibit platelet-monocyte conjugate formation, but did inhibit the platelet-PMNL interaction; this indicates that GpIIb-IIIa on platelets and CD11c on leukocytes and fibrinogen are involved in mediating the interaction between platelets and PMNL but not platelets and monocytes. Surprisingly arginyl-glycyl aspartyl serine (RGDS) inhibited the formation of both types of conjugate but this may be because it also inhibited both platelet and leukocyte activation as measured by CD62P and CD11b exposure and/or interferes with the binding of adhesion molecules other than fibrinogen. The results show that a flow cytometric procedure can be effective in obtaining rapid information on platelet-leukocyte conjugate formation in whole blood and on factors that are involved in its regulation. It is suggested that the technique may be applicable to the study of platelet-leukocyte conjugate formation in whole blood in disease, and also to study the effects of drugs interfering with conjugate formation.

BACKGROUND

To investigate the changes of platelet microparticle (PMPs), monocyte-platelet aggregation (MPAs), and the platelet membrane glycoprotein GPIIb/IIIa ligands (PAC-1) and P-hormone (CD62P) activation ratio changes in acute coronary syndrome (ACS) patients.

METHODS

92 patients were divided into ACS group (54 cases) and coronary angiography negative group (38 cases). 30 cases of age/gender matched healthy control group were recruited. The flow cytometry analysis in each group of PMPs, the MPAs expression of CD62P, GPIIb/IIIa activation ratio, and ROC curve were performed to evaluate the sensitivity and specificity of each parameter.

RESULTS

The healthy control group showed MPAs 5.94 +/- 1.93%, PMPs 1.89 +/- 0.53%, and PAC-1 2.86 +/- 0.93%, the coronary angiography-negative group showed MPAs 11.97 +/- 4.92%, PMPs 3.08 +/- 1.38%, and PAC-1 3.38 +/- 0.92%, and the ACS group showed MPAs 46.27 +/- 17.74%, PMPs 5.28 +/- 2.44%, and PAC-1 5.34 +/- 2.44%. In the ACS group, the area under the ROC curve of each indicator for identifying suspected ACS patients were MPAs (0.952), PMPs (0.807), PAC-1 (0.770), and CD62p (0.656). MPAs showed the highest sensitivity (94.4%) and specificity (84.2%) for the diagnosis of ACS.

CONCLUSIONS

acute coronary syndrome, platelet microparticle, monocyte-platelet aggregation, CD62P, GPIIb/IIIa.

Tethering of PMNL by platelets via CD62P has been shown to cause PMNL activation. Co-incubation of purified PMNL with platelets that were activated with thrombin and then fixed and washed, resulted in the formation of platelet-PMNL conjugates as well as in a generation of reactive oxygen species that were measured as luminol-enhanced chemiluminescence. When platelets were thrombin activated in the presence of RGDS to prevent binding of fibrinogen to membrane receptors, they had a reduced capacity to adhere to PMNL, but ROS generation was enhanced. In samples of citrated whole blood RGDS as well as the more specific platelet fibrinogen receptor antagonist GR144053F or a dissociation of the platelet glycoprotein IIb/IIIa complex markedly enhanced ROS generation that was induced by stirring the samples for 10 min at 1000 rpm, by 175%, 95% and 138%, respectively. Removal of platelets from the whole blood samples also resulted in an enhancement of stirring-induced ROS generation, which was inversely correlated to the platelet count. These data provide some evidence that platelets are capable of inhibiting ROS generation in PMNL by a mechanism that involves platelet-bound fibrinogen and probably depends on fibrinogen-mediated platelet-PMNL contact.