Evaluation of platelet function in thrombocytopenia

Whole blood aggregometry is a functional assay for determination of platelet function. Until now, whole blood aggregometry has not been considered feasible at low platelet counts. Hence, the objectives of the present study were to explore platelet function in thrombocytopenia using a novel index of impedance aggregometry adjusted for platelet count and evaluate the association to platelet function assessed by flow cytometry. Hirudin anticoagulated blood was collected from 20 healthy volunteers, 20 patients with primary immune thrombocytopenia (ITP), and 17 hematological cancer patients. Platelet function was analyzed by impedance aggregometry and by flow cytometry. Collagen, adenosine diphosphate, thrombin receptor agonist peptide-6, and ristocetin were used as agonists for both analyses. Thrombocytopenia in healthy whole blood was induced in vitro employing a recently published method. Platelet aggregation of thrombocytopenic patients was evaluated relative to the aggregation of healthy volunteers at the same platelet count. In flow cytometry, platelet function was described as expression of the platelet surface glycoproteins: bound fibrinogen, CD63, and P-selectin. Similar platelet counts were obtained in the patient groups (p = 0.69) (range: 13–129 × 10⁹/l). Aggregation adjusted for platelet count was significantly increased in ITP patients compared to healthy platelets across all agonists. The platelet aggregation was high in the 95% prediction interval, with 18 ITP patients above the prediction interval in at least two agonists. In contrast, the platelet aggregation was low in the prediction interval in cancer patients, and three cancer patients with platelet aggregation below the prediction interval in at least one agonist. ITP patients displayed increased expression of bound fibrinogen and CD63 following activation, compared with particularly cancer patients, but also compared with healthy platelets. This study demonstrated the feasibility of a novel approach to perform platelet function analyses in thrombocytopenia using impedance aggregometry adjusted for platelet count.     

Does renal carcinoma affect the expression of P-selectin on platelets?

We studied changes in the expression of P-selectin on the blood platelet plasma membrane. The aim of the study was to determine the influence of renal carcinoma on P-selectin expression associated with changes in platelet morphology. Venous blood was collected from 30 patients with renal carcinoma and from 24 control subjects for cytometric analysis and to evaluate platelet morphology. P-selectin being the CD62P receptor on blood platelets was marked by anti-CD61/62P MoAb, and the results were presented as the percentage of CD62P-positive cells. Changes in the expression of the CD62P on the platelet plasma membrane during activation were investigated by flow cytometry in a comparative study of in vivo activation and in vitro platelet reactivity. Platelet activation reflected by P-selectin expression was higher in the group of patients (4.45 +/-1.96), compared to control (2.48 +/-1.66) (p < 0.05). However, adenosine diphosphate [ADP] -stimulated platelet reactivity in renal cancer patients increased only by 0.24% (p > 0.05), while following activation by thrombin by 0.54% (p < 0.05). Moreover, a higher (4.72 +/-2.02), statistically significant percentage of platelets with P-selectin expression was found in patients with disseminated neoplastic changes in renal parenchyma, compared to patients with a single localized neoplastic lesion (4.17 +/-1.89) (p < 0.05). A statistically significant difference was noted in the platelet count and anisocytosis in renal cancer patients. Renal cancer enhances P-selectin expression. It is due to the presence of intensified thrombinogenesis and other platelet agonists in the blood.     

Acoustophoretic purification of platelets: feasibility and impact on platelet activation and function

Purity, limited platelet activation, and preservation of platelet function are important stakes of preparation of platelet concentrates (PC) for clinical use. In fact, contaminating red blood cells and leukocytes, as well as activated and/or poorly functional platelets in PC, represents a risk of poor efficiency and adverse side effects during platelet transfusion. Therefore, optimization of preparation and storage of PC is still an active field of research. Shear-induced platelet activation is an unwanted side effect of the hard-spin (up to 5000g) step of centrifugation-based methods currently used in blood banks to prepare PC from whole blood samples. Here, we evaluated the effectiveness of an acoustic-based fractionation device for the isolation of human platelets from whole blood bags. The purity, activation status, and functionality of platelets isolated by acoustopheresis were compared with those of platelets isolated using a reference protocol known to produce limited platelet activation and consisting of two consecutive soft-spin centrifugations (120g and 1200g). Platelet concentration and purity were determined using an automated hematology analyzer. Platelet activation status and platelet reactivity to collagen and thrombin were assessed in flow cytometry by measurement of surface expression of P-selectin and activated integrin αIIbβ3. The ability of isolated platelets to incorporate into a thrombus when transfused to NOD/SCID mice was investigated by intravital microscopy using the ferric chloride-induced thrombosis model. Blood fractionation by acoustophoresis led to the elimination of more than 80% of red blood cells and leukocytes from the platelet fraction, whose mean purity was of 92.8 ± 12.8%. The activation status and reactivity to collagen and thrombin of acoustophoresis-isolated platelets were similar to those of platelets isolated by soft-spin centrifugation. Finally, acoustophoresis-isolated platelets were tethered, adhered to the vessel wall, and incorporated into a growing thrombus following ferric chloride-induced vascular injury. Together, our results indicate that acoustophoresis is a suitable method for the isolation of human platelets with minimal platelet activation and preservation of platelet function.     

Low-dose aspirin does not lower in vivo platelet activation in healthy smokers

Smoking causes atherosclerosis, and smokers have increased thromboxane (TXA₂) formation. As aspirin inhibits TXA₂ production we speculated that smokers would preferentially profit from inhibition of the TXA₂ pathway by aspirin. Increased expression of P-selectin, a constituent of the alpha-granules of platelets, and increased levels of circulating (c)P-selectin in plasma are markers for platelet activation. The aim of this study was to compare P-selectin expression on platelets between smokers and nonsmokers, and to compare with placebo the effect of 2 weeks administration of 100 mg/d aspirin on platelet activation in smokers. Smokers exhibited higher P-selectin expression on platelets than non-smokers (2.7 ± 1.8% v 1.6 ± 0.6%, P = 0.018), thus confirming increased platelet activation. Aspirin did not reduce platelet activation as demonstrated by unchanged P-selectin expression on platelets and cP-selectin plasma levels.     

Quality of harvested autologous platelets compared with stored donor platelets for use after cardiopulmonary bypass procedures

Platelet dysfunction has a major contribution in bleeding after cardiopulmonary bypass (CPB) and transfusion of platelets is frequently used to secure haemostasis. Allogeneic platelets prepared for transfusion are functionally impaired. Autologous platelets harvested preoperatively require a shorter storage time before transfusion and their use also avoids the risks associated with transfusion of allogeneic blood products. For the first time, we have compared the functional quality of autologous platelets with allogeneic platelets prepared by two methods, immediately before infusion. Platelet activation was assessed by P-selectin expression and fibrinogen binding using flow cytometry. We also monitored the effects of CPB surgery and re-infusion of autologous platelets on platelet function. Autologous platelet-rich plasma (PRP) contained a significantly lower (P < 0.05) percentage of P-selectin-positive and fibrinogen-positive platelets compared with allogeneic platelet preparations, and also contained a significantly higher (P < 0.05) percentage of responsive platelets. Allogeneic platelets prepared by donor apheresis were more activated and less responsive than those produced by centrifugation of whole blood. In patients' blood, the percentage of platelets expressing P-selectin or binding fibrinogen increased significantly after CPB (P < 0.05), while the percentage of platelets responsive to in vitro agonists was decreased (P < 0.05 in autologous transfusion patients), consistent with platelet activation during the procedure. The percentage of activated platelets decreased (statistically not significant) after re-infusion of autologous PRP. P-selectin expression had returned to pre-CPB levels 24 h post-operatively. Autologous platelet preparations display minimal activation, but remain responsive. Conservation of platelet function may contribute to the potential clinical benefits of autologous transfusion in cardiopulmonary bypass.     

Activation of calcium-dependent calmodulin by calcium(II)3(3,5-diisopropylsalicylate)6(H2O)6 decreases thrombin receptor activating peptide-induced P-selectin expression.

We examined the influence of 3,5-diisopropylsalicylic acid (3,5-DIPS) and calcium(II)3 (3,5-diisopropylsalicylate)6 (H2 O)6 [Ca(II)3 (3,5-DIPS)6 ], a new activator of calcium-dependent calmodulin-triggered nitric oxide synthase, on thrombin-induced platelet P-selectin expression. Citrated whole blood samples were incubated with either ethanol vehicle, 3,5-DIPS, or Ca(II)3 (3,5-DIPS)6. These whole blood samples were also co-incubated with thrombin receptor activating peptide (TRAP) or adenosine diphosphate (ADP), to up-regulate P-selectin (CD62P) on platelets. Both TRAP and ADP up-regulated P-selectin on platelets compared with platelets in whole blood samples that were not incubated with either platelet activator. Co-incubation of whole blood samples with TRAP, ADP together with 3,5-DIPS, or Ca(II)3 (3,5-DIPS)6 revealed that Ca(II)3 (3,5-DIPS)6 caused a decrease in platelet P-selectin expression for TRAP, ADP, and no-activator co-incubated samples of whole blood. Incubation of platelets with 3,5-DIPS also caused a decrease in ADP-induced up-regulation of P-selectin but failed to affect TRAP or no-activator-treated platelets. Incubation of whole blood with Ca(II)3 (3,5-DIPS)6 induced some hemolysis. We found that hemolysis increases basal P-selectin expression on platelets. We therefore conclude that Ca(II)3 (3,5-DIPS)6 decreased not only basal, but also hemolysis-induced P-selectin expression on platelets. In contrast, incubation of haemolysed whole blood with SIN-1 (standard nitric oxide-releasing drug) had no effect on P-selectin expression. In summary, Ca(II)3 (3,5-DIPS)6, a new calmodulin-dependent nitric oxide synthase activator, decreases P-selectin expression of human platelets in response to thrombin receptor activation. Improved calcium-dependent calmodulin activators may become useful drugs for the treatment of disorders associated with platelet activation, and P-selectin may decrease expression due to hemolysis.     

Lung damage may induce thrombocytopenia

Thrombocytopenia is common in patients with lung diseases. Lung injury is associated with deranged platelet homeostasis. The potential mechanisms underlying this association were investigated in this study. The number and morphology of circulating megakaryocytes (MK) were investigated in vivo during cardiopulmonary bypass (CPB) in patients undergoing elective cardiac surgery for heart disease. The association between lung injury and thrombocytopenia was further investigated in vitro in rats with lung injury. MK and platelet counts, and platelet activation between the prepulmonary (right atrial) and postpulmonary (left atrial) blood were also compared. In human, the total number of MK in central venous was higher than those of peripheral arteries during normal circulation. During CPB, the total MK and Type-4 MK of central venous and peripheral arteries were significant increased when compared with that in normal circulation. In healthy rats, the postpulmonary blood had lower MK count, higher platelet count, and similar P-selectin expression. However, the lung-damaged animals showed no such differences in either MK or platelet count, but P-selectin expression was greater in the postpulmonary blood. Peripheral platelets in the lung-injured rats were significantly lower than those in their respective controls. The lungs may play an active role in the regulation of MK levels and the lung damage may reduce circulating platelets.     

Evaluation of Random Donor Platelets Produced from Buffy Coat Stored for 24?h at Ambient Temperature: Should This be Implemented in India?

Whole blood derived platelets are made from platelet rich plasma (PRP) method or buffy coat (BC) method. In India majority of random donor platelets (RDPs) are prepared by PRP method. However, BC method offers the advantage of less platelet activation and fewer WBC contamination. Presently in India RDPs are prepared within 8 h of whole blood collection, whereas, in Europe this time limit is up to 24 h. Our aim was to evaluate the platelet count, WBC contamination, platelet CD62P expression, and biochemical parameters of RDPs prepared from BC within 8 h and within 24 h of collection. We prepared 40 units of RDP by the BC method from whole blood stored at room temperature within 8 h of collection (fresh BC), and another 40 units from BC stored at 22 °C for <24 h (stored BC). We analyzed the platelet counts, CD62P expression, WBC counts, glucose levels, pH, PO₂, PCO₂ in both the groups of RDPs, 24 h after respective preparation. The platelet counts from stored BC was higher in fresh BC. CD62P expression was low in stored BC compared to fresh BC. There were no differences of pH, pO₂, pCO₂ and glucose levels in fresh BC and stored BC. WBC contamination was more in fresh BC. Our study stored BC contained higher platelet counts, less WBC contamination and less platelet activation. We conclude that RDP prepared from stored BC is the better method for RDP production.     

Platelet adhesion changes during storage studied with a novel method using flow cytometry and protein-coated beads

The aim of the present study was to set up and evaluate a novel method for studies of platelet adhesion and activation in blood and platelet suspensions such as platelet concentrate (PC) samples using protein-coated polystyrene beads and flow cytometry. To demonstrate its usefulness, we studied PCs during storage. PCs were prepared by aphaeresis technique (n?=?7). Metabolic variables and platelet function was measured on day 1, 5, 7 and 12 of storage. Spontaneous and TRAP-6-induced adhesion to fibrinogen- and collagen-coated beads was analyzed by flow cytometry. P-selectin and phosphatidyl serine (PS) expression was assessed on platelets bound to beads as well as on non-adherent platelets. Platelet adhesion to fibrinogen beads had increased by day 12 and adhesion to collagen beads at day 7 of storage (p?<?0.05). TRAP-6 stimulation significantly increased the platelet adhesion to fibrinogen beads (p?<?0.05) as well as the P-selectin and PS exposure on platelets bound to beads (p?<?0.01) during the first 7 days of storage, but by day 12, significant changes were no longer induced by TRAP-6 stimulation. We demonstrate that our adhesion assay using protein-coated polystyrene beads can be used to assess the adhesion properties of platelets during storage without the addition of red blood cells. Therefore it may offer a useful tool for future studies of platelet adhesive capacity in transfusion medicine and other settings.     

Effects of erythropoietin on platelet reactivity and thrombopoiesis in humans

A recent study in dogs suggested that erythropoietin (EPO) not only promotes the synthesis of increased numbers of reticulated platelets but that these newly produced platelets are hyperreactive compared with controls. Because of the increasing use of EPO in the perioperative setting, we characterized the effects of EPO on platelet reactivity in healthy human volunteers. In a randomized, controlled trial, we studied the effects of EPO on platelet reactivity, thrombopoiesis, and endothelial activation in circumstances similar to those of autologous blood donation. Thirty healthy male volunteers received placebo or EPO (100 or 500 U/kg of body weight given intravenously) three times a week for 2 weeks and underwent phlebotomy on days 8 and 15. Thrombin receptor-activating peptide induced expression of P-selectin, and CD63 increased 2- to 3-fold during EPO treatment. The enhanced platelet reactivity was also reflected by a 50% increase in soluble P-selectin in plasma. Plasma E-selectin levels increased in a dose-dependent fashion by more than 100% during EPO treatment, indicating substantial activation of endothelial cells. A 10% to 20% increase in platelet counts was observed in both EPO groups on day 5. In the placebo group, platelets increased only several days after the first phlebotomy. The increase in platelet counts was not reflected by changes in the amounts of reticulated platelets or circulating progenitor cells. In summary, we found that EPO markedly enhances endothelial activation and platelet reactivity, which may adversely affect patients at cardiovascular risk. However, the increased platelet reactivity could be exploited in patients with platelet dysfunction. (Blood. 2000;95:2983-2989)     

Alteration of platelet function in dogs mediated by interleukin-6

To determine if interleukin-6 (IL-6) administration influences platelet function, platelet activation was analyzed sequentially in IL-6-treated (80 micrograms/kg/d) and control dogs. Platelet activation was determined in whole blood by flow cytometry by quantitating the binding of a monoclonal antibody to platelet surface P-selectin after stimulation with graded doses of thrombin. Administration of IL-6 resulted in a twofold decrease in the thrombin concentration required for induction of half-maximal P-selectin expression (ED50) compared with control animals. The ED50 returned to normal after cessation of IL- 6 administration. As measured by P-selectin expression, enhanced responsiveness to the strong agonist platelet activating factor (PAF) was also observed in the IL-6-treated dogs. IL-6 had no effect on the susceptibility of platelets to thrombin activation when incubated with anticoagulated dog blood. The data show that, in addition to augmenting the platelet count in normal dogs, IL-6 enhances the sensitivity of platelets to activation in response to thrombin and PAF.     

Platelet activation as a potential mechanism of GP IIb/IIIa inhibitor-induced thrombocytopenia.

The blockade of the platelet integrin glycoprotein (GP) IIb/IIIa has proved to be an effective antiplatelet therapy. Profound thrombocytopenia has repeatedly been described as an adverse effect in patients treated with GP IIb/IIIa inhibitors, but its mechanism has not been elucidated yet. With use of flow cytometry, the activation status of platelets was monitored in 26 patients presenting with acute myocardial infarction who were treated with the GP IIb/IIIa inhibitor abciximab alone or in combination with the fibrinolytic agent reteplase. Fibrinogen and PAC-1 (a GP IIb/IIIa activation-specific monoclonal antibody) binding, as well as P-selectin expression on unstimulated platelets were constant in 25 patients throughout a follow-up of 7 days. In 1 patient (D.F.), the percentage of platelet-binding fibrinogen increased from 2.2% to 17.8%, for PAC-1 from 2.8% to 13.2%, and for P-selectin expression from 10.2% to 58.3% 10 minutes after the start of treatment. Furthermore, D.F. had a decrease in single platelet count in ethylenediaminetetraacetic acid-, citrate-, and heparin-anticoagulated and native blood. Blood films revealed platelet aggregates. In vitro testing of D.F.'s blood 2 and 4 weeks after initial admission demonstrated a reinduction of fibrinogen and PAC-1 binding to platelets, an increase of P-selectin expression, and formation of platelet aggregates following exposition of platelets to abciximab in vitro. In summary, this report describes the induction of platelet activation by a GP IIb/IIIa inhibitor in vivo and reinduction in vitro in direct association with thrombocytopenia. Platelet activation by GP IIb/IIIa inhibitors may be one potential mechanism for GP IIb/IIIa inhibitor-induced thrombocytopenia.     

Activation of Blood Platelets after Percutaneous Transluminal Coronary Angioplasty and Coronary Artery Bypass Graft Surgery

We have evaluated the activation of platelets in blood samples taken from patients with stable angina undergoing balloon angioplasty (percutaneous transluminal coronary angioplasty [PTCA]) (n=11) or coronary artery bypass grafting (CABG) under hypothermic (n=11) or normothermic conditions (n=11). We have found that surface expression of P-selectin on platelets in whole blood from PTCA patients upon thrombin treatment was significantly reduced, as compared with control platelets from healthy subjects. This effect was partially reversed when platelets washed from the same blood sample were used, but even then P-selectin expression was significantly lower in PTCA patients than it was in control subjects. There was a significant increase in basal expression of P-selectin in blood platelets taken from patients who underwent CABG under normothermic conditions (warm blood cardioplegia) as opposed to hypothermic patients (cold crystalloid cardioplegia). These platelets retain the ability to respond to agonists, although to a much lower extent than do those from healthy control donors. The surface exposure of P-selectin on resting and thrombin-treated platelets isolated from CABG surgery patients was not different from that of the control platelets. The adhesion to fibrinogen of resting and thrombin-treated platelets from patients who underwent balloon angioplasty as well as CABG surgery under normothermic and hypothermic conditions was significantly reduced when compared with the fibrinogen of the control platelets. These results suggest that the function of platelet fibrinogen receptor is impaired in patients with stable angina pectoris and that PTCA and CABG surgery activates platelets.     

Agonist-inducible platelet activation in chronic idiopathic autoimmune thrombocytopenia

OBJECTIVE: There are only few studies on agonist-inducible platelet activation in chronic idiopathic autoimmune thrombocytopenia (cAITP). MATERIALS AND METHODS: We compared agonist (TRAP-6, ADP, Arachidonic acid, Epinephrine, and Ristocetin) -inducible P-selectin expression and PAC-1 binding in 40 patients with cAITP (f/m ratio 23/17) with those in 20 healthy controls. Results were correlated with platelet counts, detectable platelet antibodies, and reticulated platelets. RESULTS: The in vivo activation of platelets determined the in vitro inducible response to agonists. The stronger the in vivo activation the less the number of platelets responding to agonists, as illustrated by the inverse correlation of P-selectin expression ex vivo and the relative increase after the exogenous addition of agonists. The agonist-inducible platelet activation was not associated with the presence of detectable platelet antibodies to GPIb/IX or GPIIb/IIIa. Agonist-inducible platelet activation was also not correlated with counts of reticulated platelets. CONCLUSION: Agonist-inducible activation of platelets in cAITP is affected mainly by their in vivo activation.     

P-Selectin expression, platelet aggregates, and platelet-derived microparticle formation are increased in peripheral arterial disease.

Platelet volume has been reported to be increased in vascular disease. Therefore, we studied the relationship of mean platelet volume and platelet count as well as flow cytometrically measured platelet size and platelet function in 50 patients with peripheral arterial disease and 50 healthy volunteers. Platelet activation was measured by P-selectin expression analysis on resting and on stimulated platelets, and the determination of platelet aggregates and platelet-derived microparticles using flow cytometry. P-Selectin expression on platelets was significantly elevated in patients suffering from peripheral arterial disease (all P<0.0001). Platelet aggregates (P<0.0001) and platelet-derived microparticles (P<0.0001) were significantly higher in the patient group compared with controls, whereas mean platelet volume and platelet count showed no significant differences. Platelet count was inversely related to mean platelet volume in patients and controls (r = -0.43, P<0.001). The present study supports the hypothesis of platelet hyperreactivity and circulating activated platelets in peripheral arterial disease. Mean platelet volume, and platelet count cannot be used as predictive markers for platelet activation in peripheral arterial disease patients.     

Platelet function tests predict bleeding in patients with acute myeloid leukemia and thrombocytopenia

Bleeding is frequent among patients with thrombocytopenia. We studied whether in vitro platelet function predicts future bleeding in patients with acute myeloid leukemia (AML), and thrombocytopenia. Adult AML patients with platelet count <5.0 × 10¹⁰/L were included. Detailed bleeding history and blood samples were collected at inclusion and every 7 days. We analyzed hematology and coagulation parameters. With flow cytometry we evaluated platelet activation (activated GPIIb/IIIa, P-selectin, and CD63 expression), and platelet aggregation. Agonists were thrombin-receptor activating peptide (TRAP) and collagen-related peptide (CRP-XL). During 18 months, sixty participants were enrolled and followed for a total of 114 weeks. Bleeding occurred in 52 weeks, and was not associated with clinical, hematology or coagulation parameters. Predictors of bleeding were assessed using multivariate logistic regression, adjusted for platelet count, sex, and age. Bleeding history and receiver operating curves were compared using c-index. Reduced TRAP-induced platelet aggregation had Odds ratio 3.00 (95% confidence interval [CI] 1.38-6.60). Reduced CRP-XL-induced platelet aggregation had Odds ratio 4.00 (95%-CI 1.70-9.20) for bleeding. Overall, C-index was 0.71 for the models including platelet aggregation results, 0.72 for activated GPIIb/IIIa-positive platelets after stimulation with TRAP, 0.68 for percent P-selectin positive platelets with TRAP and 0.63 for the platelet count. Among patients receiving no platelet transfusion, C-index was 0.83–0.91 for bleeding; highest for models using platelet aggregation. Change in platelet aggregation did not correlate with the number of platelet concentrates administered. In conclusion, platelet aggregation and platelet activation results predict bleeding better than platelet count alone, among AML patients with thrombocytopenia.     

Platelet P-selectin expression in patients with sickle cell disease who undergo apheresis

Activated platelets have been identified in patients with sickle cell disease. However, the association of platelet P-selectin expression and automated red cell exchange procedures in these patients is not well known. We hypothesized that altered whole platelet P-selectin expression is associated with automated red cell exchange. Flow cytometric quantification of platelet P-selectin expression was carried out in 23 patients with sickle cell disease before and after automated red cell exchange. P-selectin expression was quantified as a binding index for platelet P-selectin (the percentage of positive platelets multiplied by the mean fluorescence of positive platelets). The patients were divided into two groups: individuals with painful vaso-occlusive crises (four women and five men; group 1) and those in a steady state (six women and eight men; group 2). The 33 exchange procedures were evaluated prospectively and used acid-citrate-dextrose A solution (whole blood to anticoagulant ratio = 14:1). Platelet P-selectin expression did not significantly change after automated red cell exchange. Clinical factors such as the volume of replacement fluid and the citrate infusion rate did not correlate with postapheresis platelet P-selectin expression. In addition, the association of platelet P-selectin expression and automated red cell exchange was independent of other laboratory factors (hematocrit level, hemoglobin S level, platelet count, and nitric oxide level). Finally, the difference between the study groups regarding platelet P-selectin expression before and after apheresis was insignificant. In conclusion, automated red cell exchange procedures do not induce platelet P-selectin expression in patients with sickle cell disease in the steady state or in vaso-occlusive crisis.     

In vivo effects of eltrombopag on platelet function in immune thrombocytopenia: no evidence of platelet activation

The effects of eltrombopag, a thrombopoietin-receptor agonist, on platelet function in immune thrombocytopenia (ITP) are not fully characterized. This study used whole blood flow cytometry to examine platelet function in 20 patients receiving eltrombopag treatment at days 0, 7, and 28. Platelet surface expression of activated GPIIb/IIIa, P-selectin, and GPIb was measured with and without low and high adenosine diphosphate (ADP) and thrombin receptor activating peptide (TRAP) concentrations. Before eltrombopag treatment with no ex vivo agonist, platelet activation was higher in ITP patients than controls. Platelet GPIb and activated GPIIb/IIIa expression without added agonist was unchanged following eltrombopag treatment, whereas a slight increase in P-selectin was observed. Expression of P-selectin and activated GPIIb/IIIa in response to high-dose ADP was lower during eltrombopag treatment than at baseline. Eltrombopag led to a slight increase in platelet reactivity to TRAP only in responders to eltrombopag but not to levels above those in controls; whole blood experiments demonstrated that this increase was probably because of higher platelet counts rather than higher platelet reactivity. In conclusion, although thrombocytopenic ITP patients have higher baseline platelet activation than controls, eltrombopag did not cause platelet activation or hyper-reactivity, irrespective of whether the platelet count increased.     

Left atrial platelet activity with rheumatic mitral stenosis: correlation study of severity and platelet P-selectin expression by flow cytometry

BACKGROUND: Previous studies have demonstrated that platelet activation, evaluated by measuring the secretory substances of platelets (ie, platelet factor 4 and beta-thromboglobulin), occurs in the peripheral blood of patients with rheumatic mitral stenosis (MS). However, the differences in platelet activation between peripheral and atrial blood, and the relationship between regional left atrial platelet P-selectin expression and the severity of MS have never been investigated. METHODS AND RESULTS: A total of 16 patients with symptomatic MS undergoing percutaneous transluminal mitral valvuloplasty were studied (group 1). The fractions of platelets expressing P selectin in the prevalvuloplasty left atrial, right atrial, peripheral venous, and arterial blood were determined by flow cytometry. The mitral valve area was calculated by means of the Doppler pressure half-time method. Peripheral venous platelet activity also was evaluated in 23 control patients (including 15 healthy volunteers who were in sinus rhythm [group 2] and 8 patients who had chronic lone atrial fibrillation [group 3]). The fraction of peripheral venous platelets expressing P selectin among group 1 patients was significantly higher than that of group 2 or 3 patients (p = 0.008). In group 1 patients, the fraction of platelets expressing P selectin in the left atrium was significantly higher than that in the right atrium, the femoral vein, or the femoral artery (p < 0.01). Correlation analysis demonstrated that there was a significantly direct relationship between the severity of MS and the fraction of left atrial platelets expressing P selectin (p = 0.01; r = -0.620). The fraction of peripheral venous platelets expressing P selectin among group 2 patients did not differ from that of group 3 patients CONCLUSIONS: In patients with rheumatic MS, increased regional left atrial platelet P-selectin expression had a significantly direct relationship with the severity of MS. The increased regional left atrial platelet P-selectin expression was not reflected in peripheral venous blood samples.     

Increased platelet activation in subjects chronically exposed to cadmium: A pilot study

Cadmium exposure has been reported to be associated with the risk of vascular disorders. Here, we investigated platelet activity in subjects with chronic cadmium exposure. Eighteen and 15 women participated in this study as chronically cadmium-exposed and control non-exposed subjects, respectively. Plasma P-selectin and CD40 ligand (CD40L), soluble markers of platelet activation, were measured. Platelet aggregation in whole blood, P-selectin and activated glycoprotein (aGP) IIb/IIIa expression on platelets and platelet–leukocyte aggregates were determined. The levels of plasma P-selectin and CD40L increased in subjects with chronic cadmium exposure compared with control subjects. Platelet aggregation induced by adenosine diphosphate (ADP) was higher in cadmium-exposed subjects than control subjects. Cadmium-exposed subjects had higher baseline and ADP-induced aGPIIb/IIIa expression on platelets than control subjects. Platelet–neutrophil aggregates also increased in cadmium-exposed subjects. Blood cadmium correlated with ADP-induced aggregation, aGPIIb/IIIa expression and platelet–neutrophil aggregates, while urinary cadmium correlated with soluble P-selectin. However, cadmium only at high concentration (15?µM) could potentiate ADP-induced platelet activation in vitro. In conclusion, our pilot data show that cadmium-exposed subjects have increased baseline platelet activation and reactivity.