Effect of Pre-Analytical Conditions on the Thrombodynamics Assay

Introduction

Standardization of pre-analytical conditions is the obligatory step for all potential diagnostic tests. Spatial clot growth (Thrombodynamics) is a new global hemostasis assay that considers spatial organization of coagulation. The principal parameter is rate of fibrin clot growth from the tissue-factor coated surface. In this work we studied the pre-analytical variables of Thrombodynamics assay that include conditions of blood collection, sample preparation and storage.

Materials and Methods

Blood of apparently healthy volunteers was used. Eight types of citrate blood collection tubes were tested, centrifugation conditions for plasma preparation were evaluated and impact of plasma freezing/thawing was tested.

Results

Among the blood collection tubes tested, BD Vacutainer glass tubes showed a significantly higher clot growth rate compared to plastic tubes. There was no difference between 3.2% and 3.8% of sodium citrate. For plasma preparation, a single 15 min centrifugation at 1 600 g shows significantly increased clot growth rate compared to plasma obtained by two sequential centrifugations (15 min 1 600 g, 5 min 10 000 g). There was no significant difference between 1 600 g and 2 100 g if the second centrifugation was performed. For the second centrifugation there was no difference between 20 min at 1 600 g and 5 min at 10 000 g. Frozen-thawed plasma showed increased clot growth rate compared to fresh plasma.

Conclusion

The data represent the necessary steps for the standardization of Thrombodynamics assay and for the formulation of the operating guide.     

Pre-analytical and analytical variables affecting the measurement of plasma-derived microparticle tissue factor activity

Introduction

Elevated levels of tissue factor positive (TF⁺) microparticles (MPs) are observed in plasma from a variety of patients with an increased risk of thrombosis. We and others have described the measurement of TF activity in MPs isolated from plasma. The aim of this study was to investigate the effects of pre-analytical and analytical variables on TF activity of MPs isolated from blood of healthy volunteers either untreated or treated ex vivo with bacterial lipopolysaccharide.

Materials and methods

We evaluated the following parameters: use of different centrifugation speeds to isolate the MPs; comparison of TF activity of MPs isolated from platelet poor plasma versus platelet free plasma; effect of freeze/thaw on MP TF activity; and comparison of the MP TF activity assay with the measurement of TF protein by ELISA or flow cytometry.

Results

MPs prepared from platelet poor plasma by centrifugation at 20,000 × g or 100,000 × g for 15 minutes had similar levels of TF activity. However, significantly less TF activity was found in MPs isolated from platelet free plasma compared with platelet poor plasma. Interestingly, freeze/thawing of the plasma showed donor to donor variation in MP TF activity, with a moderate increase in some individuals.

Conclusion

TF⁺ MPs can be quantitatively isolated from platelet poor or platelet free plasma by centrifugation at 20,000 × g for 15 minutes. Measurement of MP TF activity in plasma may be used to detect a prothrombotic state in patients with various diseases.     

Use of the platelet function analyzer to minimize bleeding complications after renal biopsy

Background

The bleeding time is frequently used to screen primary haemostasis before surgical procedures, although it poorly predicts the risk of hemorrhage. The platelet function analyzer (PFA), which is also used to screen primary haemostasis, has a higher sensitivity and other advantages, like patient friendliness, higher degree of objectivity and analytical reliability, but needs more extensive clinical validation.

Methods

We compared the predictive values of the PFA-CTs (closure times) and bleeding time for bleeding events after renal biopsy. We prospectively evaluated the complications in patients that underwent a renal biopsy and were screened with PFA in advance (n = 170). For comparison we used a historical cohort of patients screened with the bleeding time (n = 132).

Results

When the PFA-CTs were normal, 26.0% of the patients had a mild bleeding event after the biopsy, which did not differ from the event rate with a normal bleeding time (29.4%). When one or both PFA-CTs were prolonged, 51.3% of the patients had post-biopsy bleeding events independently of the measures to correct the closure time(s), significantly more than with either a prolonged bleeding time (26.7%) or normal PFA-CTs (26.0%).

Conclusion

For bleeding events, the PFA has a higher positive and similar negative predictive value compared to the bleeding time. Taken into account the additional advantages of the PFA like patient friendliness and better analytical qualities, we prefer the PFA over the bleeding time as a screening tool for primary haemostasis before performing a renal biopsy.     

DDAVP: Does the drug have a direct effect on the vessel wall?

Evidence is presented that 1-deamino-8-d-arginine vasopressin (DDAVP), a vasopressin analog, has a direct effect on isolated vessel segments. The most significant finding is increased platelet adhesion and spreading at injury sites. An isologous human umbilical vein perfusion model was used to compare effects of DDAVP with those of epinephrine or zero drug controls. Scanning electron microscopy, in conjunction with morphometry, permitted quantification of platelet adhesion to subendothelium exposed by minimal injury in the model. In addition, umbilical vein effluents were tested for levels of factor VIII moieties (F VIII:C, F VIII:Rag, F VIII:RCof) and the prostanoids, 6 keto PGF1 alpha (stable metabolite of prostacyclin) and TXB2 (stable metabolite of thromboxane A2. Only F VIII:C from DDAVP treated segments was significantly (p less than 0.01) changed from controls.     

Platelet reactivity evaluated with the VASP assay following ticagrelor loading dose in acute coronary syndrome patients undergoing percutaneous coronary intervention

Background

The level of platelet reactivity (PR) inhibition obtained after P2Y12-ADP receptor antagonist loading dose (LD) is associated with the ischemic and bleeding risk following percutaneous coronary intervention (PCI) in acute coronary syndromes (ACS).

Objective

We aimed to evaluate the level of PR inhibition achieved by a 180 mg LD of ticagrelor and the rate of high on-treatment platelet reactivity (HTPR) in ACS patients undergoing PCI.

Methods

We performed a multicentre prospective observational study enrolling ACS patients undergoing PCI. Patients were included if they were admitted for ST-elevation myocardial infarction or non ST-elevation ACS. To assess PR, a VASP index was measured at least 6 and within 24 hours following a 180 mg LD of ticagrelor. HTPR was defined as a VASP index ≥ 50%.

Results

One hundred and fifteen patients were included: 31.3% of STEMI, 49.6% of NSTEMI and 19.1% of unstable angina. Following ticagrelor LD the mean VASP index was 17 ± 14%. However the response to ticagrelor was not uniform with a small inter-individual variability: inter quartile range: 7.6–22.8% and a rate of HTPR of 3.5%. A high number of patients, 65.6%, had a VASP index < 16%. None of the baseline characteristics of the study population was associated with PR. In addition, PR was similar in STEMI, NSTEMI and unstable angina (p = 0.9).

Conclusion

In ACS patients the level of PR inhibition achieved by a 180 mg loading dose of ticagrelor is not uniform and the rate of HTPR is 3.5%. A high proportion of patients exhibited a VASP index < 16%.     

Both antiplatelet and anticoagulant therapy may favorably affect outcome in patients with advanced heart failure. A retrospective analysis of the PRIME-II trial

INTRODUCTION: Current guidelines of chronic heart failure (CHF) do not recommend the use of oral anticoagulants (OAC) or antiplatelet therapy (APT). We performed a post-hoc analysis to evaluate the effect of the use of anti-thrombotic therapy with APT and OAC. PATIENTS AND METHODS: We examined 427 patients with advanced CHF, and assessed the effects of the use of APT or OAC at baseline on mortality. We employed a Cox-proportional hazard model to value the effects of APT or OAC use. RESULTS: After a mean follow-up of 3.4 years (range 2.0-5.4), 214 patients died (51%). Forty-one (41) percent (95%CI: 29-53%) of the patients on APT died, and 52% (47-57%) of the patients not on APT (P=0.07). Forty-eight (48) percent (42-54%) of the patients on OAC died, and 55% (46-63%) of the patients not on OAC (P=0.20). This effect of OAC was seen both in patients in sinus rhythm and in atrial fibrillation. After adjusting for important prognostic variables, such as age, LVEF, renal function, and NYHA class, both the use of APT (hazard ratio (HR) 0.62, 95% confidence interval (CI) 0.40-0.97; P=0.04) and the use of OAC (HR 0.60, 95%-CI 0.43-0.83; P<0.01) were related to an improved prognosis. CONCLUSION: This post-hoc analysis suggests that in CHF patients the use of APT or OAC is associated with a higher survival.     

Usefulness of the INNOVANCE® PFA P2Y test cartridge for the detection of patients with congenital defects of the platelet P2Y12 receptor for adenosine diphosphate

Introduction

The platelet function analyzer (PFA)-100® is used in clinical practice to screen patients with bleeding diathesis and suspected defects of primary hemostasis. A new cartridge, INNOVANCE® PFA P2Y, has been specifically developed to monitor patients’ response to drugs inhibiting the platelet P2Y₁₂ receptor for ADP. In this study, we compared the ability of INNOVANCE® PFA P2Y to detect congenital defects of the platelet P2Y₁₂ receptor to that of standard cartridge formulations currently in clinical use.

Materials and Methods

We studied two patients with severe P2Y₁₂ deficiency, one patient with heterozygous P2Y₁₂ deficiency and one with dysfunctional P2Y₁₂ receptor. Closure times were measured using 3 cartridges: collagen/ADP, collagen/epinephrine, and INNOVANCE® PFA P2Y. The results obtained in the four patients with P2Y₁₂ defects were compared to those obtained for 20 healthy controls.

Results

In 2 patients with severe P2Y₁₂ deficiency, closure times of INNOVANCE® PFA P2Y and collagen/ADP cartridges were > 300 s, while those of collagen/epinephrine cartridge were variable (186 s and > 300 s). In the patient with dysfunctional P2Y₁₂, closure time of INNOVANCE® PFA P2Y was > 300 s, while closure times of collagen/ADP and collagen/epinephrine were normal. Closure times of all cartridges were normal in the patient with heterozygous P2Y₁₂ deficiency.

Conclusion

Our study provides the first evidence that INNOVANCE® PFA P2Y cartridge is sensitive to congenital severe and moderate defects of the platelet P2Y₁₂ receptors.     

Mean platelet volume as marker of restenosis after percutaneous transluminal coronary angioplasty in patients with stable and unstable angina pectoris

INTRODUCTION: Several experimental and clinical studies have demonstrated that platelet size and function correlate since large platelets are hemostatically more reactive than platelets of normal size. Since platelets play a crucial role in vascular remodeling after percutaneous transluminal coronary angioplasty (PTCA), we investigated the influence of the mean platelet volume (MPV), a parameter of platelet size, on restenosis after PTCA. METHODS: The retrospective study comprised 174 patients who underwent elective PTCA and follow-up angiography within 6 months thereafter. According to the follow-up angiograms, the patients were assigned to group A ("restenosis", n=74) or group B ("no restenosis", n=100). Both groups were compared in regard to pre-procedural hematological routine parameters including MPV, platelet count, hematocrit, white blood cell count and fibrinogen. RESULTS: MPV was significantly increased in group A, compared with that in group B (8.75+/-0.99 fl vs. 8.04+/-0.74 fl, p<0.001). This difference in MPV was evident in patients with stable and unstable angina pectoris. In addition, MPV had an impact on the time-related incidence of angiographic restenosis, as early restenosis was associated with higher pre-procedural MPV values. Platelet count correlated inversely with MPV (r=-0.36, p<0.01) and was significantly lower in group A than in group B. The remaining hematological parameters were not different in both groups. CONCLUSIONS: The MPV seems to be a marker of coronary restenosis in patients undergoing PTCA. Patients with high pre-procedural MPV values might benefit from an intensified antiplatelet therapy after coronary interventions.     

A population-based study of an Italian genetic isolate reveals that mean platelet volume is not a risk factor for thrombosis

Introduction

Several studies revealed that mean platelet volume (MPV) was larger in the acute phase of arterial and venous thrombosis and predicted a poor clinical outcome. It has been suggested that MPV is a risk factor for thrombosis. However, it is unclear whether increased platelet size is a cause or a consequence of thrombosis.It was the objective of this study to verify whether MPV is a risk factor for arterial and venous thrombosis.

Methods

We search for associations between platelet parameters and thrombosis by a population-based study in 11,084 inhabitants of an Italian genetic isolate characterized by wide variability of platelet parameters. To validate this methodology of investigation, we also evaluated whether it was able to identify several well known thrombotic risk factors in the study population.

Results

Statistical analysis confirmed that male gender, ageing, hypertension, high total cholesterol, low HDL cholesterol, diabetes, obesity and smoking were risk factors for arterial thrombosis, while alcohol consumption had a protective effect. Female gender, ageing, pregnancy, estroprogestinic treatment, obesity, varicose veins were associated with venous thrombosis. At variance, MPV and platelet count were unrelated to previous thrombotic events. However, MPV was negatively correlated with the time since the last thrombotic event.

Conclusions

This study indicated that an epidemiologic study of a population isolate is appropriate for the identification of thrombotic risk factors, but it failed to identify such a role for MPV. Thus, we suggest that the increased MPV previously described in subjects with acute thrombosis was a consequence instead of a cause of thrombosis.     

Deficiency of PAR4 attenuates cerebral ischemia/reperfusion injury in mice

Stroke is the third leading cause of death in the USA. Antithrombotic therapy targeting platelet activation is one of the treatments for ischemic stroke. Here we investigate the role of one of the thrombin receptors, protease-activated receptor 4 (PAR4), in a mouse transient middle cerebral artery occlusion (MCAO) model. After a 60 min MCAO and 23 h reperfusion, leukocyte and platelet rolling and adhesion on cerebral venules, blood–brain barrier (BBB) permeability, and cerebral edema were compared in PAR4-deficient mice and wild-type mice. Cerebral infarction volume and neuronal death were also measured. PAR4−/− mice had more than an 80% reduction of infarct volume and significantly improved neurologic and motor function compared with wild-type mice after MCAO. Furthermore, deficiency of PAR4 significantly inhibits the rolling and adhesion of both platelets and leukocytes after MCAO. BBB disruption and cerebral edema were also attenuated in PAR4−/− mice compared with wild-type animals. The results of this investigation indicate that deficiency of PAR4 protects mice from cerebral ischemia/reperfusion (I/R) injury, partially through inhibition of platelet activation and attenuation of microvascular inflammation.     

Predictive value of platelet activation for the rate of cognitive decline in Alzheimer's disease patients

Vascular risk factors contribute to the progression of dementia in Alzheimer''s disease (AD) and influence platelet activation. However, the degree of platelet activation as a possible underlying mechanism of this progression has not been studied till now. Significantly higher baseline expression of both platelet activation biomarkers, activated glycoprotein IIb–IIIa complex and P-selectin, was observed in patients with AD with fast cognitive decline compared with AD patients with slow cognitive decline during a 1-year follow-up period. These results suggest that platelet activation could be a putative prognostic biomarker for the rate of cognitive decline and a potential new treatment target in AD patients.     

Influence of renal function and platelet turnover on the antiplatelet effect of aspirin

Introduction

Kidney disease predisposes to cardiovascular events. This study investigated the influence of renal function and platelet turnover on the antiplatelet effect of aspirin in patients with coronary artery disease.

Materials and Methods

We included 124 aspirin-treated patients with coronary artery disease and normal to moderately reduced renal function. All tests were performed one hour after aspirin ingestion. Renal function was assessed using creatinine, estimated glomerular filtration rate (eGFR), and cystatin C. The antiplatelet effect of aspirin was evaluated using the VerifyNow® Aspirin assay and multiple electrode aggregometry (MEA, Multiplate®) induced by collagen (1.0 μg/mL) and arachidonic acid (1.0 mmol/L). Von Willebrand factor was measured as a marker of endothelial dysfunction. Platelet turnover was evaluated by measurements of immature, reticulated platelets.

Results

Renal function did not influence the antiplatelet effect of aspirin evaluated by MEA (r = − 0.2-0.09, p = 0.03-0.77) or the VerifyNow® (r = − 0.12-0.11, all p-values > 0.1). In contrast, renal function correlated inversely with von Willebrand factor levels (r_creatinine = 0.48, p < 0.0001; r_eGFR = − 0.46, p < 0.001; r_{cystatin C} = 0.54, p < 0.0001). The number of immature platelets correlated with platelet aggregation according to MEA (r = 0.20-0.39, all p-values < 0.03), but not according to VerifyNow® (r = − 0.07, p = 0.50).

Conclusions

A reduced antiplatelet effect of aspirin may be explained by an increased number of immature platelets. Moderately impaired renal function was associated with high levels of von Willebrand factor, but not with a reduced antiplatelet effect of aspirin.     

The Cone-and-Plate(let) analyzer is not suitable to monitor clopidogrel therapy: A comparison with the flowcytometric VASP assay and optical aggregometry

Introduction

High on-clopidogrel platelet reactivity has been associated with an increased risk for atherothrombotic events. A new player on the horizon is the IMPACT-R ADP-test using ADP pre-stimulation. We here report the results of a thorough evaluation of this new device.

Materials and methods

The IMPACT-R ADP-test was evaluated in different categories of subjects. First, normal range values were determined in healthy subjects (n = 46). Second, the effect of 600 mg of clopidogrel was evaluated with the IMPACT-R ADP-test and two other well-validated methods (flowcytometric VASP-analysis and optical aggregometry) in 21 patients. Third, a head-to-head comparison between the IMPACT-R ADP-test and optical aggregometry was performed in a large cohort of patients on dual antiplatelet therapy.

Results

The results of the IMPACT-R ADP-test were highly variable throughout healthy subjects. The administration of a high clopidogrel loading dose resulted in a small but significant increase in surface coverage but 61.9% of the patients were still identified as clopidogrel nonresponder. In contrast, optical aggregometry and VASP-analysis identified 24% and 33% of these patients as a clopidogrel nonresponder, respectively. Head-to-head comparison with optical aggregometry in 451 patients showed only a modest correlation between both methods (r ∼ 0.20, p < 0.0001).

Conclusions

The IMPACT-R ADP-test is relatively insensitive to the effects of clopidogrel and cannot substitute for methods such as flowcytometric VASP-analysis and optical aggregometry. Further studies are required to establish the clinical usefulness of IMPACT-R ADP-test to accurately predict the occurrence of major adverse cardiovascular events in patients with high on-clopidogrel platelet reactivity before it can be implemented in clinical practice.     

Synergistic effect of cilostazol and dipyridamole mediated by adenosine on shear-induced platelet aggregation

INTRODUCTION: The Cilostazol Stroke Prevention Study found that cilostazol, a phosphodiesterase 3 inhibitor, can reduce the risk of subsequent stroke in Japanese patients with cerebral infarction. Here, we measured the effects of cilostazol in vitro on shear-induced platelet aggregation, an important mechanism of thrombosis at arterial bifurcations or stenotic lesions. We also evaluated the influences of intrinsic adenosine on the ability of cilostazol to inhibit shear-induced platelet aggregation by investigating the effect of dipyridamole, an inhibitor of cellular adenosine reuptake, in combination with cilostazol in vitro. MATERIALS AND METHODS: We measured platelet aggregation induced by a shear rate of 10,800 s(-1) in whole blood and in platelet-rich plasma from healthy volunteers using a cone-plate streaming chamber. RESULTS: Both cilostazol and adenosine dose-dependently inhibited shear-induced platelet aggregation in platelet-rich plasma samples. Adding a low concentration of adenosine (0.3 microM) did not inhibit shear-induced platelet aggregation, but significantly enhanced the inhibitory effect of cilostazol in platelet-rich plasma. Dipyridamole dose-dependently inhibited shear-induced platelet aggregation in whole blood and significantly enhanced the inhibitory effect of cilostazol on shear-induced platelet aggregation, but did not affect shear-induced platelet aggregation in platelet-rich plasma. The inhibitory effects of cilostazol combined with dipyridamole in whole blood were almost completely reversed by adenosine deaminase. CONCLUSIONS: Dipyridamole appears to synergistically enhance the inhibitory effect of cilostazol on shear-induced platelet aggregation by maintaining high plasma levels of adenosine.     

Evidence for an alternative mechanism of human platelet secretion involving peripheralization of secretory granules and formation of membrane-associated multivesicular structures

Stimulated platelets release the content of their granules to the environment by a process known as platelet secretion. The precise mechanism of platelet secretion is poorly understood. The most widely-held theory suggests that, during platelet activation, secretory granules centralize and their content is released into the surface-connected canalicular system. Using fixation techniques directed at preserving membrane structures, morphological evidence is provided that human platelet activation is associated with secretory granules migrating to the periphery of platelets, where they undergo transition to participate in the formation of membrane-associated multivesicular structures. These multivesicular structures then undergo dissolution resulting in granule content secretion to the environment. The formation of the membrane-associated multivesicular structures occurs in platelets in which some of the secretory granules have also become centralized. This suggests that during the activation of human platelets, membrane-associated multivesicular structure release may occur concurrently with other mechanisms of secretory granule content release. The present observations are consistent, however, with the hypothesis that during human platelet activation the formation of membrane-associated multivesicular structures by platelet secretory granules contributes importantly to the mechanism of platelet secretion.     

ADP‐induced platelet aggregation in acute ischemic stroke patients on aspirin therapy

Background and purpose: Aspirin is an important therapeutic regimen to prevent the recurrent ischemic events or death after acute ischemic stroke. In this study, we evaluated the relationship between the extent of adenosine diphosphate (ADP) ‐induced platelet aggregation and outcome in acute ischemic stroke patients on aspirin therapy. Methods: We selected 107 acute ischemic stroke patients who had been prescribed aspirin and evaluated platelet function test by using optic platelet aggregometer test after 5 days of taking it and investigated the prognosis 90 days after ischemic events. Kaplan–Meyer curve was used for survival analysis. Results: After stratification of the subjected patients by tertiles of ADP‐induced platelet aggregation, the events rates were 7.4%, 9.3% and 30.8% (P = 0.023). In multiple logistic regression analysis, old age over 70 years (OR, 13.7; 95% CI, 2.14–88.07; P = 0.001) and the increased ADP‐induced platelet aggregation had independent significance to the risk of primary end‐points after acute ischemic stroke (OR, 1.1; 95% CI 1.01 to 1.20; P = 0.026). Conclusions: This study showed that the increased ADP‐induced platelet aggregation under using aspirin is associated with poor outcome after acute ischemic stroke.     

Synergistic effect of a factor Xa inhibitor, TAK-442, and antiplatelet agents on whole blood coagulation and arterial thrombosis in rats

Introduction

Activated platelets facilitate blood coagulation by providing factor V and a procoagulant surface for prothrombinase. Here, we investigated the potential synergy of a potent factor Xa/prothrombinase inhibitor, TAK-442, plus aspirin or clopidogrel in preventing arterial thrombosis and whole blood coagulation.

Methods

Thrombus formation was initiated by FeCl₃-induced rat carotid injury. Bleeding time was evaluated with the rat tail transection model. Whole blood coagulation was assessed by thromboelastographic examination (TEG) for which blood obtained from control, aspirin-, or clopidogrel-treated rats was transferred to a TEG analyzer containing, collagen or adenosine diphosphate (ADP), and TAK-442 or vehicle.

Results

TAK-442 (3 mg/kg, po), aspirin (100 mg/kg, po) or clopidogrel (3 mg/kg, po) alone had no significant effect on thrombus formation, whereas the combination of TAK-442 with aspirin and clopidogrel remarkably prolonged the time to thrombus formation without additional significant prolongation of bleeding time. TEG demonstrated that the onset of collagen-induced blood coagulation were slightly longer in aspirin-treated rats than control; however, when the blood from aspirin-treated rats was subsequently treated in vitro with 100 nM TAK-442, the onset of clotting was significantly prolonged. In contrast, only marginal prolongation was observed with TAK-442 treatment of blood from control animals. The onset time of ADP-induced blood coagulation was slightly longer in clopidogrel-treated rats compared with control, and it was further extended by TAK-442 treatment.

Conclusion

These results demonstrate that blood coagulation can be markedly delayed by the addition of TAK-442 to antiplatelets treatment which could contribute to synergistic antithrombotic efficacy in these settings.     

Post interventional cardiology urinary thromboxane correlates with PlateletMapping® detected aspirin resistance

Introduction

We have previously defined aspirin resistance detected by TEG PlateletMapping using arachidonic acid (AA). This aspirin resistance is observed as platelet activation (> 20%) by AA in whole blood, even though the isolated platelets are inhibited by aspirin. This platelet activation in whole blood is due to a transcellular pathway mediated by platelets and leukocytes.

Methods

To determine if this PlateletMapping assay of aspirin resistance on pre-procedure blood samples correlated with an in vivo response we assayed the first voided urine samples collected 2-8 hours post interventional cardiology procedures for 11-dehydro thromboxane B2.

Results and Conclusions

We detected 27 aspirin resistant patients out of a total of 81 (33%), in agreement with our previous study. All of these patients were on aspirin therapy, confirmed by a < 20% aggregation response to AA by light transmission platelet aggregometry using isolated platelet rich plasma. Aspirin resistant patients urine samples (14 out of a total of 60 patients analyzed) contained significantly (P = 0.008) higher 11-dehydro thromboxane B2 levels than the other 46 aspirin sensitive patients urine samples. Since our previous study implicated 12- and 15-lipoxygenases in this pathway, we also assayed for polymorphisms to determine any correlation with aspirin resistance. A correlation was found in a polymorphism affecting the lipoxygenase domain of platelet 12-lipoxygenase. This result indicates that aspirin resistance detected in whole blood by the TEG PlateletMapping assay correlates with a physiological consequence in terms of thromboxane formation. This is the first report of such a correlation.     

Baseline platelet size is increased in patients with acute coronary syndromes developing early stent thrombosis and predicts future residual platelet reactivity. A case-control study

Introduction

Pre-procedural predictors of early stent thrombosis (ST) and future response to platelet inhibitors are in demand. We sought to evaluate the impact of baseline platelet indices on the occurrence of early ST and future residual platelet reactivity.

Materials and methods

Hundred and eight patients with acute coronary syndromes (ACS) in whom stents were implanted were included: 36 consecutive ST cases and 72 matched controls. Platelet indices assessed with flow cytometry before stent implantation were retrieved from the department's data base. Residual platelet reactivity specific to aspirin (aspirin reaction units-ARU) and clopidogrel (P2Y12 reaction units-PRU) was assessed prospectively with VerifyNow^® under dual antiplatelet treatment.

Results

Platelet size reported as mean platelet volume (MPV) or proportion of large platelets (LPLT) was significantly higher in ST cases compared with controls (10.4, 95% confidence intervals [CI], 10.1-10.8 vs. 9.7, CI, 9.5-9.9, P = 0.0004 and 35.8, CI, 34.2-37.3 vs. 33.3, CI, 32.2-34.3, P = 0.007, respectively). Dual aspirin and clopidogrel poor-responsiveness was diagnosed significantly more often in ST cases than in controls (19.6% vs. 1.4%, P = 0.004), whereas no difference was observed for single aspirin or clopidogrel poor-responsiveness. A strong correlation was found between MPV and both, ARU (r = 0.66, P < 0.0001) and PRU (r = 0.55, P < 0.0001). Similarly, higher LPLT was associated with higher ARU (r = 0.47, P < 0.0001) and PRU (r = 0.38, P = 0.0001).

Conclusions

Baseline platelet size is increased in patients with ACS developing early ST and correlates with future residual platelet reactivity under aspirin and clopidogrel therapy. Dual but not isolated aspirin or clopidogrel poor-responsiveness appears to be associated with early ST.     

Low Concentration Detergent Sclerosants Induce Platelet Activation but Inhibit Aggregation due to Suppression of GPIIb/IIIa Activation in vitro

Introduction

Sclerotherapy is associated with thromboembolic and ischemic neurological adverse events but the effects of sclerosants on platelet function are unknown. The aim of this study was to investigate the in vitro effects of detergent sclerosants Sodium Tetradecyl Sulphate (STS) and Polidocanol (POL) on platelet activation and aggregation.

Materials and Methods

Whole blood and platelet rich plasma samples were incubated with sclerosants. Platelet and platelet microparticle (PMP) counts were measured by flow cytometry. Platelet activation was examined by ELISA for soluble factors (sP-selectin, von Willebrand factor, sCD40L and serotonin) and by flow cytometry for membrane-bound markers (CD62p, CD63) and cytoplasmic calcium. Platelet aggregation was assessed by PFA-100®, light transmission and impedance (Multiplate®) aggregometry, and by flow cytometry for glycoprotein (GP) Ib and GPIIb/IIIa subunits, heterodimer expression and activation (PAC-1 binding).

Results

Both agents lysed platelets at high concentrations (≥ 0.1%) but induced platelet activation at lower concentrations as evident by a rise in membrane-bound and soluble markers, cytoplasmic calcium and release of phosphatidylserine + PMP. Agonist-stimulated platelet aggregation was inhibited by both sclerosants. Membrane expression of GPIb and GPIIb/IIIa individual subunits or heterodimer was not affected by sclerosants but the activation of GPIIb/IIIa was suppressed.

Conclusion

Low concentration sclerosants activated platelets and released microparticles but inhibited platelet aggregation due to suppression of GPIIb/IIIa activation.