Effects of glycosaminoglycan from Urechis unicinctus on the P2Y1 receptor pathway and expression of related factors in rat platelets.

The aim of this study was to examine the effects of glycosaminoglycan (GAG) from Urechis unicinctus on the P2Y1 receptor pathway and expression of related factors in rat platelets. The concentration of calcium ion (Ca2+) in rat platelets was determined by double wavelength Fura-2 fluorescence spectrophotometry, and the concentrations of inositol trisphosphate (IP3) and glycoprotein IIb/IIIa (GPIIb/IIIa) in rat platelets were measured using the enzymatic immunoassay method. The phosphorylation levels of phospholipase C (PLC), phospholipase A2 (PLA2), protein kinase C (PKC), and p38 mitogen-activated protein kinase (p38MAPK) were also detected by Western blot. It was found that the GAG from U. unicinctus significantly reduced the Ca2+ and IP3 levels in rat platelets (p<0.05, p<0.01). Moreover, medium and high concentrations of GAG significantly reduced the concentration of the platelet membrane GPIIb/IIIa in rats (p<0.05, p<0.01). The phosphorylation levels of PLC, PLA)2), PKC and p38MAPK in rat platelets were also inhibited by GAG and P)2)Y)1) receptor blocker MRS2179 (p<0.05, p<0.01). However, the degree of inhibition of GAG was lower than that of MRS2179. The results laid a foundation for further utilization of the glycosaminoglycan.

Comparison of platelet function and viscoelastic test results between healthy dogs and dogs with naturally occurring chronic kidney disease.

OBJECTIVE To compare platelet function and viscoelastic test results between healthy dogs and dogs with chronic kidney disease (CKD) to assess whether dogs with CKD have platelet dysfunction and altered blood coagulation. ANIMALS 10 healthy control dogs and 11 dogs with naturally occurring CKD. PROCEDURES Blood and urine were collected once from each dog for a CBC, serum biochemical analysis, urinalysis, and determination of the urine protein-to-creatinine ratio, prothrombin time, activated partial thromboplastin time, plasma fibrinogen concentration, and antithrombin activity. Closure time was determined by use of a platelet function analyzer and a collagen-ADP platelet agonist. Thromboelastography (TEG) variables (reaction time, clotting time, α angle, maximum amplitude, and global clot strength [G value]) were determined by use of recalcified nonactivated TEG. Platelet expression of glycoprotein Ib (GPIb; receptor for von Willebrand factor), integrin αIIbß3 (αIIbß3; receptor for fibrinogen), and P-selectin (marker for platelet activation) was assessed by flow cytometry. RESULTS Compared with healthy control dogs, the median closure time was prolonged, the median maximum amplitude and G value were increased, and the median clotting time was decreased for dogs with CKD. Platelet expression of both αIIbß3 and P-selectin was also significantly increased for dogs with CKD, compared with that for control dogs. Platelet expression of GPIb, αIIbß3, and P-selectin was not correlated with closure time or any TEG variable. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that dogs with CKD frequently had evidence of platelet dysfunction and hypercoagulability that were not totally attributable to alterations in platelet surface expression of GPIb, αIIbß3, and P-selectin.

Calcium-Channel Blockers Attenuate the Antiplatelet Effect of Clopidogrel.

AIMS: Dihydropyridine calcium-channel blockers (CCBs) inhibit cytochrome 3A4 and could therefore interfere with the conversion of clopidogrel to its active form. The impact of CCBs on the antiplatelet effect of clopidogrel has not been studied with assays directly capturing platelet activation to adenosine diphosphate (ADP), so far. We therefore sought to investigate platelet activation in response to ADP by flow cytometry in clopidogrel-treated patients without and with CCBs. METHODS: Platelet surface P-selectin expression and activated glycoprotein (GP) IIb/IIIa in response to ADP were determined by flow cytometry in 302 patients on dual antiplatelet therapy with aspirin and clopidogrel after successful angioplasty with stent implantation. RESULTS: Ninety-two patients (30.5%) received CCBs. Patients with concomitant CCB therapy showed significantly higher platelet surface expressions of P-selectin and activated GPIIb/IIIa in response to ADP than patients without CCBs (both P ≤ 0.03). Moreover, the fold increase of P-selectin and activated GPIIb/IIIa in response to ADP was significantly more pronounced in patients taking CCBs (both P ≤ 0.03). The associations of ADP-inducible activated GPIIb/IIIa and fold increase of activated GPIIb/IIIa after the addition of ADP with CCB therapy remained significant after adjustment for differences in patient characteristics and factors that were previously associated with clopidogrel response by multivariate regression analyses (both P < 0.05). High levels of ADP-inducible P-selectin and activated GPIIb/IIIa were seen significantly more frequent in patients with CCBs than in patients without CCB therapy (both P ≤ 0.01). CONCLUSION: Dihydropyridine CCBs attenuate the effect of clopidogrel on ADP-inducible platelet activation in patients undergoing angioplasty and stenting for cardiovascular disease.

Case-control study of platelet glycoprotein receptor Ib and IIb/IIIa expression in patients with acute and chronic cerebrovascular disease.

BACKGROUND: Animal models have been instrumental in defining thrombus formation, including the role of platelet surface glycoprotein (GP) receptors, in acute ischemic stroke (AIS). However, the involvement of GP receptors in human ischemic stroke pathophysiology and their utility as biomarkers for ischemic stroke risk and severity requires elucidation. AIMS: To determine whether platelet GPIb and GPIIb/IIIa receptors are differentially expressed in patients with AIS and chronic cerebrovascular disease (CCD) compared with healthy volunteers (HV) and to identify predictors of GPIb and GPIIb/IIIa expression. METHODS: This was a case-control study of 116 patients with AIS or transient ischemic attack (TIA), 117 patients with CCD, and 104 HV who were enrolled at our University hospital from 2010 to 2013. Blood sampling was performed once in the CCD and HV groups, and at several time points in patients with AIS or TIA. Linear regression and analysis of variance were used to analyze correlations between platelet GPIb and GPIIb/IIIa receptor numbers and demographic and clinical parameters. RESULTS: GPIb and GPIIb/IIIa receptor numbers did not significantly differ between the AIS, CCD, and HV groups. GPIb receptor expression level correlated significantly with the magnitude of GPIIb/IIIa receptor expression and the neutrophil count. In contrast, GPIIb/IIIa receptor numbers were not associated with peripheral immune-cell sub-population counts. C-reactive protein was an independent predictor of GPIIb/IIIa (not GPIb) receptor numbers. CONCLUSIONS: Platelet GPIb and GPIIb/IIIa receptor numbers did not distinguish between patient or control groups in this study, negating their potential use as a biomarker for predicting stroke risk.

Laropiprant attenuates EP3 and TP prostanoid receptor-mediated thrombus formation.

The use of the lipid lowering agent niacin is hampered by a frequent flush response which is largely mediated by prostaglandin (PG) D(2). Therefore, concomitant administration of the D-type prostanoid (DP) receptor antagonist laropiprant has been proposed to be a useful approach in preventing niacin-induced flush. However, antagonizing PGD(2), which is a potent inhibitor of platelet aggregation, might pose the risk of atherothrombotic events in cardiovascular disease. In fact, we found that in vitro treatment of platelets with laropiprant prevented the inhibitory effects of PGD(2) on platelet function, i.e. platelet aggregation, Ca(2+) flux, P-selectin expression, activation of glycoprotein IIb/IIIa and thrombus formation. In contrast, laropiprant did not prevent the inhibitory effects of acetylsalicylic acid or niacin on thrombus formation. At higher concentrations, laropiprant by itself attenuated platelet activation induced by thromboxane (TP) and E-type prostanoid (EP)-3 receptor stimulation, as demonstrated in assays of platelet aggregation, Ca(2+) flux, P-selectin expression, and activation of glycoprotein IIb/IIIa. Inhibition of platelet function exerted by EP4 or I-type prostanoid (IP) receptors was not affected by laropiprant. These in vitro data suggest that niacin/laropiprant for the treatment of dyslipidemias might have a beneficial profile with respect to platelet function and thrombotic events in vascular disease.

Classification of Iranian patients with Glanzmann's Thrombasthenia using a flow cytometric method.

Glanzmann's Thrombasthenia (GT) is a rare inherited autosomal recessive platelet disorder caused by a deficiency or dysfunction of the GPIIb-IIIa receptor on platelets, which is characterized by a lack of platelet aggregation in response to multiple physiologic agonists and a life-long bleeding disorder. Flow cytometry is a rapid and highly sensitive method that can detect reduced levels of receptors, as well as absolute deficiency. The aim of this study was to classify Iranian GT patients by a flow cytometric method, and to correlate these findings with the severity of clinical bleeding. The expression of GPIIb-IIIa on the platelet surface was assessed in 123 GT patients using quantitative flow cytometry to determine the most common subtype among these patients. We used a panel of antibodies to detect the expression of glycoproteins GPIb, GPIIb, GPIIIa, as well as Integrin αv. Patients were also interviewed with regard to the severity and frequency of bleeding, according to history and gender, in order to evaluate the nature of their bleeding phenotype, and classify them as mild, moderate or severe bleeders, in accordance with the Glanzmann's Thrombasthenia Italian Team (GLATIT) protocol. In the detailed analysis of the results of our investigation, 95 out of 123 (77.5%) were classified as type I; 20 (16%) as type II with residual GPIIb-IIIa, and eight (6.5%) as GT variants. The variant type was diagnosed by the inability of GPIIb-IIIa to bind fibrinogen, as evidenced by the absence of platelet aggregation in response to physiologic agonists. There was no significant correlation between bleeding severity and different subtypes of GT. This study demonstrates that GT type I is the most common subtype among Iranian patients. There was no correlation between severity of symptoms and cytometric phenotype of the disease. The identification of families at risk may significantly decrease the incidence of the severe form of the disorder if genetic counseling is provided.

Inhibition of platelet aggregation and immunomodulation of NK lymphocytes by administration of ascorbic acid.

Platelets aggregation around migrating tumor cells offers protection against the cytotoxic activity of the natural killers cells (NKC). The ascorbic acid in 3 x 10(-3) M concentration completely inhibited platelet aggregation, decreased thromboxane B2 levels, and inhibited the expression of platelet membranic receptor GpIIb/IIIa in non stimulated platelets, and increased the NKC cytotoxicity in an average rate of 105, 61, and 285% in the NKC/targets cells ratios 12.5:1, 25:1 and 50:1 respectively. The results suggest the role of ascorbic acid in increasing the susceptibility of tumor cells to NKC; the ascorbic acid could be used as part of a multidrug therapy to treat diseases which up to now have been treated only through chemotherapy.

Supernates from stored red blood cells inhibit platelet aggregation.

BACKGROUND: Previously, we reported that red blood cells (RBCs) stored in AS-5 accumulated proinflammatory substances during storage. We observed in those studies that supernates from nonleukoreduced (NLR) RBCs reduced mean anti-CD41a-fluorescein isothiocyanate (FITC) fluorescence on platelets (PLTs), indicative of decreased expression of glycoprotein (GP)IIb/IIIa on the PLT membrane. The objective of this study was to determine if supernates from stored RBCs impaired PLT aggregation as a consequence of reduction in GPIIb/IIIa expression. STUDY DESIGN AND METHODS: Leukoreduced (LR) and NLR RBC units were prepared in AS-5 and stored at 1 to 6 degrees C for 6 weeks. Supernates from RBC samples collected every 2 weeks were mixed with freshly collected type-matched blood and incubated for 30 minutes at 37 degrees C. PLTs in each incubated blood sample were evaluated for GPIIb/IIIa expression by flow cytometry and for aggregation response to collagen by whole blood aggregometry. RESULTS: Supernates from stored NLR RBCs reduced CD41a-FITC fluorescence on PLTs by 15% to 31%. A reduction in fluorescence was induced by supernates of RBCs stored for 14 days and increased as storage time increased. Supernates from Day 42 NLR RBCs reduced the mean amplitude of PLT aggregation by 31% compared to Day 0 supernates and lengthened the time before onset of aggregation by 21%. In addition, amplitude correlated directly and lag time correlated inversely with CD41a-FITC fluorescence in all samples. Supernates from prestorage LR RBCs did not affect PLT CD41a-FITC fluorescence or aggregation response. CONCLUSIONS: Substances that decrease expression of GPIIb/IIIa and inhibit PLT aggregation accumulate in NLR RBCs. Accumulation of this material is prevented by leukoreduction.

Effect of alphaIIb Ala477Pro(A446P) mutation of the platelet glycoprotein on the expression of alphaIIbbeta3 complex.

OBJECTIVE: To explore the effect of glycoprotein (GP) alphaIIb Ala477Pro(A446P) mutation on the expression of integrin alphaIIbbeta3. METHODS: The alphaIIbA477P (A446P) eukaryotic expression plasmid alphaIIbA477P (A446P)-pcDNA3.1(+) was constructed by site-directed mutagenesis. Cos-7 cells were transfected with the mutational plasmid or the normal plasmid alphaIIb-pcDNA3.1(+) with beta3-pcDNA3.1(+). The expression of alphaIIbA477P (A446P)was tested with the RT-PCR, Western blot, and flow cytometry. RESULTS: RT-PCR revealed the transcriptional script of alphaIIbA477P (A446P). Western blot showed the expression of alphaIIbA477P (A446P) protein. Flow cytometry demonstrated that the expression of alphaIIbA477P (A446P)beta3 on the membrane was only 12.95% of the normal alphaIIbbeta3 complex. CONCLUSION: alphaIIbA477P (A446P) mutation distinctly reduces the expression of alphaIIbbeta3 complex on the membrane. This mutation may interfere the formation of alphaIIbbeta3 complex or impair the proper conformation of alphaIIb subunit.

Dependence of platelet function on underlying liver disease in orthotopic liver transplantation.

OBJECTIVE: The purpose of the present study was to explore the platelet function during the perioperative period of orthotopic liver transplantation (OLT) due to the underlying liver disease. METHODS: The blood coagulation parameters, platelet surface markers and the determination of platelet aggregation were analyzed in 34 patients who underwent OLT. Blood samples were drawn preoperatively, anhepatic, 10 min and 1 hour after reperfusion, 1 day, 3 and 7 days postoperatively. Conventional coagulation screens, thrombopoietin (TPO) serum levels, P-selectin, GPIIb/IIIa and GPIb binding sites on the surface of platelets as evaluated by flow cytometry and platelet aggregation response were measured. RESULTS: Coagulation factors, maximum aggregation and rate of aggregation were significantly different before transplantation due to the underlying liver disease. Further we found a markedly depressed GPIIb/IIIa and P-selectin expression and a reduced rate of aggregation in all patients throughout the study. In contrast maximum aggregation of platelets was restored on the third day after reperfusion without intergroup differences and almost comparable to healthy controls. An inverse correlation was found between peripheral platelet count pre-transplantation and peak TPO concentrations one weak post-transplantation. CONCLUSIONS: In the entire process of OLT, coagulation factors, maximum aggregation and rate of platelet aggregation depend on the surgical phases during transplantation and on the underlying liver disease. The data obtained in this study might contribute to a better understanding of the pathophysiology and assessment of bleeding risk in OLT.

No evidence for systemic platelet activation during or after orthotopic liver transplantation.

Platelet function is thought to deteriorate during liver transplantation as a result of platelet activation and proteolysis of platelet receptors by plasmin following reperfusion. However, this hypothesis has never been formally tested. Twenty patients undergoing a first or second liver transplant were included in the study. Blood samples were taken at standardized time points during transplantation and up to 10 days after transplantation. Platelet activation was assessed by detection of the activation markers P-selectin and activated integrin alphaIIbbeta3 with flow cytometry. Proteolytic cleavage of platelet receptors was assessed by flow cytometry measurement of the constitutively expressed platelet receptors glycoprotein Ibalpha and integrin alphaIIbbeta3. In addition, using enzyme-linked immunosorbent assay techniques, we measured plasma levels of platelet activation products beta-thromboglobulin and platelet factor 4 and plasma levels of cleaved fragments of glycoproteins Ibalpha and V. Flow cytometry analyses provided no evidence of substantial platelet activation during transplantation. In fact, the expression of activated integrin alphaIIbbeta3 decreased postoperatively; this indicated that platelets were in a slightly activated state prior to surgery. Plasma levels of beta-thromboglobulin and platelet factor 4 also substantially decreased after transplantation. In addition, no changes were observed in the constitutively expressed platelet receptors or in the plasma levels of platelet receptor fragments, and this indicated a lack of substantial receptor proteolysis. In conclusion, no evidence was found for significant activation of circulating blood platelets or the proteolysis of key platelet receptors during liver transplantation. These findings suggest that the platelet functional capacity does not decrease during liver transplantation. Liver Transpl 15:956-962, 2009. (c) 2009 AASLD.

Identification of five novel 14-3-3 isoforms interacting with the GPIb-IX complex in platelets.

BACKGROUND: Binding of von Willebrand factor to the platelet glycoprotein (GP)Ib-IX complex initiates a signaling cascade leading to integrin alpha(IIb)beta(3) activation, a key process in hemostasis and thrombosis. Interaction of 14-3-3zeta with the intracytoplasmic domain of GPIb appears to be a major effector of this activation pathway. OBJECTIVE: The aim of our study was to determine whether other members of the 14-3-3 family bind to GPIb-IX. RESULTS: In this study, western blot analyses showed that platelets also contain the 14-3-3beta, 14-3-3gamma, 14-3-3epsilon, 14-3-3eta and 14-3-3theta isoforms, but lack 14-3-3sigma. Coimmunoprecipitation studies in platelets and CHO transfectants demonstrated that all six 14-3-3 isoforms expressed in platelets, including, as previously reported, 14-3-3zeta, bind to GPIb-IX. In addition, their interaction was found to critically require the same GPIbalpha domains (580-590 and 605-610) already identified as essential for 14-3-3zeta binding, in agreement with the conservation of the sequence of the I-helix among these different isoforms. Pull-down experiments indicated that all six 14-3-3 isoforms present in platelets bind to GPIbbeta. In contrast, deletion or mutation of the GPIbbeta intracytoplasmic tail did not affect the interaction of GPIb-IX with the 14-3-3 isoforms, questioning the importance of this domain. CONCLUSIONS: Our study suggests that, to inhibit GPIb-induced integrin alpha(IIb)beta(3) activation, a more appropriate strategy than inhibiting individual 14-3-3 isoforms would be to target the 14-3-3-binding motif on GPIb or, alternatively, the conserved 14-3-3 I-helix.

Inhibition of in vivo tumour growth by the blocking of host alpha(v)beta3 and alphaII(b)beta3 integrins.

BACKGROUND: The alpha(v)beta3 integrin in the endothelial cell membrane is important for the growth and migration of capillaries into tumour tissue and is also a survival factor for these cells. The alphaII(b)beta3 (GPIIb/IIIa) integrin is responsible for platelet activation and, with concomitant release of different stored proangiogenic factors, and tumour cell-platelet interactions. MATERIALS AND METHODS: An immunodeficient nude rat model was used to study tumour growth in tibial bone, with tumour cells negative for the target alpha(v)beta3 and alphaII(b)beta3 integrins. RESULTS: Daily intraperitoneal injections of m7E3 F(ab')2 antibody fragment, blocking human and rat alpha(v)beta3 and alphaII(b)beta3 integrins, reduced the measured size of the tumours growing in the tibial bone by 35% (p = 0.012), and also the microvessel density in these tumours. The concentration of the important proangiogenic factor bFGF was significantly reduced by 41% in the treated tumours. The treatment slightly increased the time to the appearance of the tumour from 22.2 to 24.9 days, indicating a small but significant effect on the early stages of tumour growth and invasion through the bone tissue. CONCLUSION: Integrin-targeted treatment reduced tumour growth, solely targeting the host angiogenesis. This treatment strategy should be further exploited for use in combination with conventional treatment strategies, or the combined targeting of alternative antiangiogenic pathways.

Hyperbaric oxygen treatment induces platelet aggregation and protein release, without altering expression of activation molecules.

OBJECTIVES: To investigate the effect of hyperbaric oxygen (HBO) on platelet physiology. DESIGN AND METHODS: Human platelets were exposed to HBO (97.7% O(2), balance CO(2) at 2.2 ata) or control (CON; 5% CO(2), balance air at 1 ata) for 90 min, and analyzed for aggregation, protein release, ()NO production, and activation. RESULTS: HBO induced 29.8+/-3.0% of platelets to aggregate compared with CON (5.5+/-0.9%). Proteins observed to be released in greater abundance from HBO- compared with CON-treated platelets included 14-3-3 zeta and alpha-2-macroglobulin. Release of ()NO by platelets was unaffected following exposure to HBO, as was platelet activation as measured by surface expression of PECAM-1, CD62P and the activated form of alpha(IIB)beta(IIIa). CONCLUSIONS: Exposure to HBO induces both platelet aggregation and protein release. Further study will better define the precise mechanisms and effects of HBO on platelet activation.

The complement factor properdin induces formation of platelet-leukocyte aggregates via leukocyte activation.

Both the complement system and platelet-leukocyte aggregates are involved in chronic and acute stages of atherosclerosis. Properdin, a positive regulator of the complement system, is secreted by leukocytes and endothelial cells. In the present study, the role of properdin in the formation of platelet-leukocyte aggregates was investigated. Incubation of human whole blood with properdin (25-200 microg/ml) resulted in a dose-dependent formation of platelet-leukocyte aggregates, with an increase of up to 2.2-fold compared to controls (p < 0.05), as analysed by flow cytometry. In addition, properdin significantly amplified ADP-induced aggregation of platelets with leukocytes by 53% (p < 0.05), while it had no effect on ADP-induced aggregation of platelets alone. Consistent with these results, properdin did not activate platelets as shown by the expression of activated GPIIb/IIIa (PAC-1 epitope) and P-selectin (CD62P) on the platelet surface. However, properdin significantly induced expression of CD11b (MAC-1) on leukocytes by 12-fold (p < 0.05) as a measure of leukocyte activation. In conclusion, the complement system component properdin induces the formation of platelet-leukocyte aggregates via leukocyte activation. The data establish a link between the complement system and platelet-leukocyte aggregates with potential significance in atherosclerotic vascular disease.

Effect of curcumin on the adhesion of platelets to brain microvascular endothelial cells in vitro.

AIM: To determine whether curcumin prevents the adhesion of platelets to brain microvascular endothelial cells (BMECs) cultured in vitro. METHODS: [3H]Adenine- labeled platelets were incubated with BMECs to investigate the role of curcumin in the adhesion of platelets to BMECs. The number of platelets adhering to the BMECs monolayer was determined by liquid scintillation spectroscopy. The thrombin-induced expression of platelets P-selectin, glycoprotein IIb (GPIIb), and glycoprotein IIIa (GPIIIa) on the cell surface, was measured by flow cytometry. P-selectin mRNA levels of BMECs were determined by RT-PCR. The TNF-alpha- induced expressions of P-selectin and E-selectin on the surface of BMECs were determined by Western blotting. RESULTS: The adhesion between thrombin-activated platelets and normal BMECs, and that of TNF-alpha-activated BMECs and normal platelets were significantly increased, and this increase could be inhibited by curcumin (30-240 micromol/L) in a concentration-dependant manner. The platelets activated with thrombin and BMECs stimulated by TNF-alpha demonstrated an upregulated expressions of P-selectin and E-selectin, and this increase, when pretreated with curcumin for 30 min, could be restrained dose dependently. Curcumin also inhibited the increase of the GPIIb/GPIIIa expression of thrombinactivated platelets in a concentration-dependent manner. CONCLUSION: Curcumin can inhibit the platelets to BMECs. This effect may be related to the decreased expressions of P-selectin, E-selectin, and GPIIb/GPIIIa on platelets and BMECs.

Expression of platelet surface receptors and early changes in platelet function in patients with STEMI treated with abciximab and clopidogrel versus clopidogrel alone.

BACKGROUND: Platelets play a crucial role in the pathogenesis of acute coronary syndromes (ACS). The efficacy of antiplatelet treatment is pivotal in the success of percutaneous coronary intervention (PCI) performed in patients with ACS. OBJECTIVE: The aim of the study was to investigate the effects of clopidogrel with or without abciximab on the expression of platelet surface receptors and platelet function in patients with ST-segment elevation myocardial infarction (STEMI) undergoing PCI. MATERIALS AND METHODS: Thirty patients with STEMI were included in the study. During acute primary coronary intervention, patients received aspirin (acetylsalicylic acid) and clopidogrel in a loading dose of 300mg. Clopidogrel was the only antiplatelet therapy used by nine patients (group B). Twenty-one patients (group A) received additional abciximab. Blood samples were collected and analyzed twice: before and up to 22 hours after administration of antiplatelet therapy. The platelet aggregation was established as primary platelet-related hemostasis (closure time [CT] assessed using the PFA100 system). The absolute number of platelet surface antigens as CD41a, CD42a, CD42b, CD61, and CD62P were determined by flow cytometry analysis. RESULTS: The study revealed a statistically significant increase in CT induced by adenosine diphosphate and adrenaline (epinephrine) +130 seconds (p < 0.0001) and +94 seconds (p < 0.0001), respectively, in group A patients post-therapy. While in group B the parameters of CT did not change after treatment. In addition, the absolute number of CD41a antigens (glycoprotein [GP] IIb/IIIa) increased significantly after treatment in group A. No significant changes were observed after treatment in the expression of CD62P (P-selectin) antigens in either treatment group. There was a significant reduction in the percentage of CD62P-positive platelets in group B after antiplatelet therapy. CONCLUSIONS: The absolute number of GP IIb/IIIa receptors increases and platelets are not activated up to 12 hours after cessation of abciximab therapy. Treatment of STEMI patients undergoing PCI with a loading dose of clopidogrel reduces the percentage of active platelets but does not influence the CT.

The association between platelet autoantibody specificity and response to intravenous immunoglobulin G in the treatment of patients with immune thrombocytopenia.

We retrospectively investigated the association between platelet autoantibody specificity and response to intravenous immunoglobulin G (IVIG) in 17 patients with immune thrombocytopenia (ITP). Platelet-associated antibodies against glycoprotein (GP) IIb/IIIa, GPIb/IX, and GPIa/IIa were detected in 13, 10, and 8 patients, respectively. A response occurred in 7 of 7 patients without anti-GPIb/IX, but in only 3 of 10 patients with anti-GPIb/IX (p<0.01). There was no difference in the response rates in patients with or without anti-GPIIb/IIIa or anti-GPIa/IIa. We conclude that ITP patients with anti-GPIb/IX may be less responsive to IVIG.

Reticulated platelets and uninhibited COX-1 and COX-2 decrease the antiplatelet effects of aspirin.

BACKGROUND: The mechanisms for the variability in antiplatelet effects of aspirin are unclear. Immature (reticulated) platelets may modulate the antiplatelet effects of aspirin through uninhibited cyclooxygenase (COX)-1 and COX-2. OBJECTIVES: To evaluate the role of reticulated platelets in the antiplatelet effects of aspirin. METHODS: Sixty healthy volunteers had platelet studies performed before and 24 h after a single 325-mg dose of aspirin. Platelet studies included light transmission aggregometry; P-selectin and integrin alpha(IIb)beta(3) expression, and serum thromboxane B(2) (TxB(2)) levels. Reticulated platelets and platelet COX-2 expression were measured using flow cytometry. RESULTS: Subjects were divided into tertiles based on the percentage of reticulated platelets in whole blood. Baseline platelet aggregation to 1 microg mL(-1) collagen, and postaspirin aggregations to 5 microm and 20 microm ADP and collagen, were greater in the upper than in the lower tertile of reticulated platelets. Stimulated P-selectin and integrin alpha(IIb)beta(3) expression were also higher in the upper tertile both before and after aspirin. Platelet COX-2 expression was detected in 12 +/- 7% (n = 10) of platelets in the upper tertile, and in 7 +/- 3% (n = 12) of platelets in the lower two tertiles (P = 0.03). Postaspirin serum TxB(2) levels were higher in the upper (5.5 +/- 4 ng mL(-1)) than in the lower tertile (3.2 +/- 2.5 ng mL(-1), P = 0.03), and decreased even further with ex vivo additional COX-1 and COX-2 inhibition. The incidence of aspirin resistance (>or= 70% platelet aggregation to 5 microm ADP) was significantly higher in the upper tertile (45%) than in the lower tertile (5%, P < 0.0001). CONCLUSIONS: Reticulated platelets are associated with diminished antiplatelet effects of aspirin and increased aspirin resistance, possibly because of increased reactivity, and uninhibited COX-1 and COX-2 activity.