Platelet inhibition assessed with VerifyNow, flow cytometry and PlateletMapping in patients undergoing heart surgery

Introduction

A substantial number of patients with coronary artery disease undergo cardiac surgery within five days of discontinuing anti-platelet treatment with aspirin and clopidogrel. The aims of this study were to describe the degree of platelet inhibition in patients with dual anti-platelet treatment scheduled for coronary artery bypass graft (CABG) surgery and to investigate whether the measured platelet inhibition correlated to intra- and postoperative risk for bleeding and transfusion requirements.

Material and Methods

Sixty patients were included. Platelet inhibition was analysed with flow cytometry including phosphorylation status of the vasodilator-stimulated phosphoprotein (VASP-assay) and two bed-side analyzers, VerifyNow-System and PlateletMapping, a modified thrombelastograph. All 60 patients were analysed with VerifyNow and PlateletMapping, and 48 were analysed with flow cytometry and VASP-assay.

Results

There was a correlation between the ADP-receptor inhibition as measured by VASP-assay and VerifyNowP2Y₁₂ (r = - 0.29, p < 0.05), and between VASP-assay and the expression of P-selectin (r = 0.29, p < 0.05) as measured by flow cytometry when platelets were stimulated with 5 µM ADP. VerifyNowP2Y₁₂ was the only measurement of platelet inhibition correlated to total blood loss (Spearman r = 0.29, p = 0.03) and red blood cell transfusion (Spearman r = 0.43, p < 0.01) requirements, although this might be confounded by aprotinin treatment.

Conclusion

We found a modest agreement between the methods for preoperative platelet inhibition, though not for PlateletMapping-MA_ADP. There was a correlation between preoperative platelet inhibition measured by VerifyNowP2Y₁₂ and surgical blood loss or transfusion requirements. However, for the individual patient, preoperative use of VerifyNowP2Y₁₂ as an instrument to decide bleeding and transfusion risk does not seem helpful.     

Platelet activation in patients with peripheral vascular disease: reproducibility and comparability of platelet markers

Background

Many markers of platelet activation have been described but their reproducibility and comparability in patient populations are poorly defined.

Objectives

We sought to compare markers of platelet and monocyte activation with platelet-monocyte aggregates, a proposed gold standard of in vivo platelet activation, and assess their reproducibility in patients with peripheral arterial disease: a population with substantial platelet activation, inflammation and risk of thrombotic events.

Patients/Methods

Thirty patients with peripheral vascular disease attended on two occasions to permit within-day and between-day comparisons. In vivo platelet and monocyte activation were determined by flow-cytometric quantification of platelet-monocyte aggregation, platelet surface expression of P-selectin and CD40L, platelet-derived microparticles, and monocyte surface expression of CD40 and CD11b. Plasma concentrations of platelet-derived microparticles, soluble P-selectin and CD40L were measured by enzyme-linked immunosorbant assays.

Results

Platelet-monocyte aggregation (36.7 ± 7.86%), and platelet surface expression of P-selectin (5.8 ± 1.65%) and CD40L (3.3 ± 1.45%) demonstrated comparable within-day (mean difference ± co-efficient of reproducibility; 0.9 ± 15.4%, 0.21 ± 1.65% and 0.2 ± 2.8% respectively) and between-day reproducibility (2.0 ± 12.4%, 0.10 ± 2.25% and 0.9 ± 6.4% respectively). Platelet-monocyte aggregates correlated well with other platelet (r = 0.30-0.50, P < 0.02) and monocyte (r = 0.27-0.47, P < 0.03) activation markers. Flow cytometric and assay quantified platelet-derived microparticles showed poorer reproducibility (co-efficient of reproducibility > 40).

Conclusions

In patients with peripheral arterial disease, measurements of platelet-monocyte aggregates have good reproducibility and consistently reflect other markers of platelet and monocyte activation.     

Platelet activation in psoriasis

Recent epidemiological studies have suggested that psoriasis represents a risk factor for thrombotic vascular diseases. In order to evaluate the possible role of hemostatic changes in the development of thrombotic episodes in psoriasis, some parameters of the hemostatic "balance" were investigated in 22 male psoriatic patients and compared to those of 22 male control subjects. Incidence of known risk factors for vascular diseases (diabetes, hypertension, smoking, dyslipidemia) was comparable in the two study groups. There were no statistically significant differences in platelet count, circulating platelet aggregates, platelet production of malondialdehyde (MDA), total plasma antithrombin and fibrinolytic activities. In patients with psoriasis the incidence of spontaneous platelet hyperaggregability and plasma levels of beta-thromboglobulin were significantly higher than in control subjects. Platelet regeneration time, measured as MDA recovery after aspirin ingestion, was significantly shorter in psoriatic patients. These data suggest that an in vivo platelet activation occurs in patients with psoriasis and could contribute to the development of thrombotic complications. The release of mitogenic and inflammatory substances by activated platelets may play a role in the histogenesis of psoriatic lesions.     

Platelet hyperreactivity in hemodialysis patients with frequently occluded vascular access

It is known that thrombosis is a leading cause of vascular access failure and that the formation of thrombus requires platelets. The activation of platelets induces the increase in intracellular Ca 2(+) levels ([Ca(2+)](i)) leading to aggregation and thrombosis. We compared the platelet [Ca(2+)](i) before and after stimulation between the patients with and without easily occluded vascular access. Our study included two groups of hemodialysis patients. Group 1 consisted of 21 patients who had received chronic hemodialysis therapy for more than 6 months. They had had more than three events (including three) of vascular access failures during the past year. Group 2 consisted of 21 hemodialysis patients with age, sex, and diabetes mellitus matched who had never suffered from any event of vascular access failure. We measured the basal and stimulated platelet [Ca(2+)](i) after stimulation with 1 U/ml thrombin, 1 micro M arachidonic acid, 1 micro M platelet activation factor (PAF), and 10 micro M adenosine diphosphate (ADP), respectively. Our results showed that in Ca 2(+)-containing media, there was no significant differences in the basal [Ca(2+)](i), but the maximal increases of [Ca(2+)](i) of platelets were higher (p <0.05) in group 1 than in group 2 after stimulating with PAF and ADP, but not with thrombin and arachidonic acid.We concluded that the causes for the susceptibility of some hemodialysis patients to vascular access occlusion were multifactorial. In addition to previously reported plasma factors, there was a sub-group of patients who showed greater elevations of agonists stimulated platelet intracellular calcium levels.     

Platelet adenine nucleotide levels in patients with Dacron vascular prostheses

Platelets adenine nucleotide levels were determined in 11 patients with Dacron bifemoral aortic prostheses using high performance liquid chromatography. Total platelet and dense granule adenine nucleotide levels were measured in neutralized perchloric acid extracts prepared from gel-filtered platelet suspensions and from platelet supernatants following thrombin-induced release of granule constituents respectively. Dense granule adenine nucleotide levels were significantly decreased in the patient group compared with age and sex-matched controls (p less than 0.01), whereas no differences in cytoplasmic adenine nucleotide levels were observed (p greater than 0.2). Platelet survival measurements were made on 3 patients; when grouped with control subjects, a positive correlation was observed between platelet survival and platelet dense granule ADP (r = 0.96; p less than 0.01). These results suggest that platelet dense granule adenine nucleotide content may be a clinically significant indicator of vascular graft thrombogenesis.     

Platelet activation in myeloproliferative disorders

Beta-thromboglobulin (beta-TG) and platelet factor four (PF4) are specific platelet proteins released when a process of platelet activation occurs. The present study was undertaken in order to measure beta-TG and PF4 both as absolute plasma value and ratio to platelet number in 69 patients with myeloproliferative disorders (MD). The aim was to establish whether the increase of the two proteins could depend on platelet number or indicated an "in vivo" platelet activation. In 74% of patients beta-TG was found elevated and PF4 was high in 68% of cases. However in 34.7% and in 31.9% of cases respectively, the elevation of the two platelet markers was correlated to platelet number and the ratio was normal. Only in about one third of cases an "in vivo" platelet activation could be admitted and this finding provides a more rational use of antiaggregating agents.     

Platelet activation in pregnancy-induced hypertension

BACKGROUND: Although excess platelet activation, as indicated by increased plasma beta thromboglobulin (beta-TG), has been shown in pregnancy-induced hypertension (PIH), platelet adhesion, platelet morphology and a comparison of platelet and soluble (plasma) levels of the adhesion molecules P-selectin (pPsel and sPsel, respectively) have not been studied. METHODS: We conducted a cross-sectional study of 35 consecutive women with PIH (age 31+/-6 years), 31 consecutive women with normotensive pregnancies (age 29+/-5 years) and 30 normotensive non pregnant women (age 30+/-5 years). Platelet adhesion was studied in vitro by binding to fibrinogen-coated microwells, platelet morphology [mass and volume by flow cytometry], whole-platelet P-selectin (pPsel) by ELISA of the lysate of 2 x 10(8) cells, and the plasma markers soluble P-selectin (sP-sel) and beta-TG, by ELISA. RESULTS: The women with PIH had significantly raised sPsel, pPsel and (as expected) beta-TG (all p<0.05), when compared to the normotensive pregnant women and controls. However, in PIH platelet adhesion was similar to that in the normotensive pregnancy, but still higher than the normal controls (p<0.001). There was no difference among the three groups with respect to platelet mass and volume. pPsel and platelet adhesion correlated with gestational age and with systolic and diastolic blood pressure (all p<0.05). CONCLUSIONS: Increased platelet activation and adhesion develop during normal pregnancy, with some indices being further altered in PIH.     

Platelet activation and platelet-erythrocyte aggregates in end-stage renal disease patients on hemodialysis

Activated platelets may engage in dynamic interplay with other blood cells. We examined the evidence for platelet activation and the formation of platelet-erythrocyte aggregates in chronic hemodialysis patients. Circulating activated platelets (P-selectin/CD63-positive platelets) were higher than normal controls (p < 0.001) and further increased during hemodialysis sessions, the increase being higher when patients were dialyzed with cellulosic than with synthetic membranes. We found direct evidence of uremic platelet-erythrocyte adherence in vitro and increased levels of circulating platelet-erythrocyte aggregates in dialysis patients, which represents a new observation in uremia. Platelet-erythrocyte aggregates were subject to further increase during hemodialysis, and again higher levels were found with cellulosic than synthetic membranes. This phenomenon was reproduced in vitro by both ADP and PAF, but not by either complement factor C3a or by heparin concentrations corresponding to those used for clinical hemodialysis. We conclude that platelet-erythrocyte aggregates occur in hemodialysis patients probably owing to a primary platelet activation mechanism.     

Platelet activation during treadmill exercise in patients with chronic peripheral arterial disease

β-Thromboglobulin (β-TG) and platelet factor 4 (PF-4) were measured in 59 patients with chronic peripheral arterial disease before and within 5 min. after treadmill exercising till occurrence of claudication. Plasma levels of β-TG before treadmill exercise ranged from 24 to 260 ng/ml with a geometric mean of 63.7 ng/ml, PF-4 levels ranged from 2 to 240 ng/ml with a mean of 18.5 ng/ml. These levels were significantly higher than those obtained in 28 normal individuals in which β-TG ranged from 7 to 39 ng/ml with a geometric mean value of 19.4 ng/ml and PF-4 from 1 to 19 ng/ml with a mean value of 4.6 ng/ml. No correlation between plasma β-TG or PF-4 and extent of arterial disease was found. β-TG levels, measured within 5 min. after treadmill exercise, showed a statistically significant increase to a mean value of 74.3 ng/ml but PF-4 did not rise significantly (mean value : 19.8 ng/ml) The supplementary increase of already elevated β-TG levels may be explained by enhanced in vivo platelet activation during treadmill exercising till occurrence of claudication. As the clearance of PF-4 from human plasma has been shown to be much faster than the clearance of β-TG, increases in PF-4 levels may be more difficult to detect during dynamic explorations of the vascular system.     

Platelet activation following cerebral angiography

Following cerebral angiography, the level of beta thromboglobulin rose in five of seven patients (P = less than 0.05). This would indicate that there had been generalised platelet activation. In view of the morbidity of cerebral angiography it is proposed that a trial of prophylactic anti-thrombotic therapy be instituted.     

Platelet activation and blood coagulation

Platelet activation and blood coagulation are complementary, mutually dependent processes in haemostasis and thrombosis. Platelets interact with several coagulation factors, while the coagulation product thrombin is a potent platelet-activating agonist. Activated platelets come in a procoagulant state after a prolonged elevation in cytosolic [Ca2+]i. Such platelets, e.g. when adhering to collagen via glycoprotein VI, expose phosphatidylserine (PS) at their outer surface and produce (PS-exposing) membrane blebs and microvesicles. Inhibition of aminophospholipid translocase and activation of phospholipid scramblase mediate the exposure of PS, whereas calpain-mediated protein cleavage leads to membrane blebbing and vesiculation. Surface-exposed PS strongly propagates the coagulation process by facilitating the assembly and activation of tenase and prothrombinase complexes. Factor IXa and platelet-bound factor Va support these activities. In addition, platelets can support the initiation phase of coagulation by providing binding sites for prothrombin and factor XI. They thereby take over the initiating role of tissue factor and factor VIIa in coagulation activation.     

Delayed awakening in dystonia patients undergoing deep brain stimulation surgery

We aimed to identify the incidence, duration and causes of delayed emergence from anesthesia in patients with dystonia undergoing surgery for deep brain stimulation (DBS) placement. A retrospective review of patients with dystonia who underwent DBS placement was conducted and the following characteristics were noted: age, gender, comorbid conditions, American Society of Anesthesiologists classification, anesthetic agents used, amount of initial dose, amount of infusion dose, duration of the infusion and the time needed for emergence. Twenty-four patients underwent 33 DBS procedures for dystonia. Propofol was administered to 21 patients, in 29 of the 33 procedures. Dexmedetomidine was administered to three patients, in four procedures. The average propofol loading dose was 0.7 mg/kg, and the infusion rate was 80 μg/kg per minute (min), for an average duration of 89 min. The average time of emergence was 36 min. Only 31% of patients emerged from propofol anesthesia during the expected time frame, 69% of patients had some degree of delayed emergence, and 24% had a significant delay in emergence. Delayed emergence was more common in younger patients due to the higher loading doses these patients received. This study shows a 69% incidence of delayed emergence in dystonia patients undergoing DBS surgery. It also suggests an association between delayed emergence and younger patients who receive higher loading doses. A possible cause of delayed emergence is excessive anesthetic potentiation of the low output pallidal state in dystonia which may depress the pallido-thalamo-cortical circuitry. Delayed emergence could also result from depression of the previously affected ventral pallidal inputs to the septo-hippocampal system that mediates general anesthesia and awareness. Complex neurotransmitter disturbances may also be involved.