Platelet function testing in cardiovascular diseases.
Explore the source record for details and available documents.
Angina, Unstable↗
Biomedical subjects
Alan D Michelson
Publications and source records attributed to Alan D Michelson.
Release of soluble CD40L from platelets is regulated by glycoprotein IIb/IIIa and actin polymerization.
OBJECTIVES: The purpose of this study was to examine the effects of glycoprotein (GP) IIb/IIIa antagonists (abciximab, eptifibatide, and tirofiban) and other inhibitors on translocation of CD40L from intraplatelet stores to the platelet surface and on the release of soluble CD40L (sCD40L) from platelets. BACKGROUND: CD40L is a proinflammatory and prothrombotic ligand in the tumor necrosis factor family. METHODS: Platelet surface CD40L was measured by flow cytometry, and sCD40L was measured by enzyme-linked immunosorbent assay. RESULTS: Translocation of CD40L from intraplatelet stores to the platelet surface was not inhibited by GP IIb/IIIa antagonists. However, release of sCD40L from the surface of activated platelets was inhibited by GP IIb/IIIa antagonists in a dose-dependent manner, in concert with inhibition of PAC1 binding to platelets (a surrogate marker for fibrinogen binding). Release of sCD40L from activated platelets was also markedly reduced in Glanzmann platelets (deficient in GP IIb/IIIa). Ethylenediaminetetraacetic acid was an effective inhibitor of sCD40L release, but only when added before platelet activation. Both cytochalasin D (an inhibitor of actin polymerization) and GM6001 (an inhibitor of matrix metalloproteinases [MMPs]) inhibited the release of sCD40L from platelets when added before, as well as 3 min after, platelet activation. However, neither cytochalasin D nor GM6001 affected translocation of CD40L to the platelet surface. CONCLUSIONS: The GP IIb/IIIa antagonists inhibit release of sCD40L from activated platelets. Release of sCD40L from platelets is regulated, at least in part, by GP IIb/IIIa, actin polymerization, and an MMP inhibitor-sensitive pathway. In addition to their well-characterized inhibition of platelet aggregation, GP IIb/IIIa antagonists may obviate the proinflammatory and prothrombotic effects of sCD40L.
Actin Depolymerizing Factors↗
Application of flow cytometry to platelet disorders.
Flow cytometry is a powerful and versatile tool that can be used to yield definitive information regarding the phenotypic status of platelets. The method provides a quantitative assessment of the physical and antigenic properties of platelets (e.g., surface expression of receptors, bound ligands, components of granules, or interactions of platelets with other platelets, other blood cells, or components of the plasma coagulation system), thereby facilitating the diagnosis of inherited or acquired platelet disorders (e.g., Bernard-Soulier syndrome, Glanzmann thrombasthenia, storage pool disease), the pathological activation of platelets (e.g., in the setting of acute coronary syndromes, cerebrovascular ischemia, peripheral vascular disease, cardiopulmonary bypass), and changes in the ability of platelets to activate via specific stimuli (e.g., efficacy of antiplatelet therapies). Accordingly, this review summarizes the key technical and methodologic components of flow cytometric analysis of platelets, as well as specific examples of its application to diagnosis and patient care.
Blood Platelet Disorders↗
Antithrombotic therapy in children: the Seventh ACCP Conference on Antithrombotic and Thrombolytic Therapy.
This article about antithrombotic therapy in children is part of the 7th American College of Chest Physicians Conference on Antithrombotic and Thrombolytic Therapy: Evidence-Based Guidelines. Grade 1 recommendations are strong and indicate that the benefits do, or do not, outweigh the risks, burden, and costs. Grade 2 suggests that individual patients' values may lead to different choices (for a full understanding of the grading see Guyatt et al, CHEST 2004; 126:179S-187S). Among the key recommendations in this article are the following. In neonates with venous thromboembolism (VTE), we suggest treatment with either unfractionated heparin or low-molecular-weight heparin (LMWH), or radiographic monitoring and anticoagulation therapy if extension occurs (Grade 2C). We suggest that clinicians not use thrombolytic therapy for treating VTE in neonates, unless there is major vessel occlusion that is causing the critical compromise of organs or limbs (Grade 2C). For children (ie, > 2 months of age) with an initial VTE, we recommend treatment with i.v. heparin or LMWH (Grade 1C+). We suggest continuing anticoagulant therapy for idiopathic thromboembolic events (TEs) for at least 6 months using vitamin K antagonists (target international normalized ratio [INR], 2.5; INR range, 2.0 to 3.0) or alternatively LMWH (Grade 2C). We suggest that clinicians not use thrombolytic therapy routinely for VTE in children (Grade 2C). For neonates and children requiring cardiac catheterization (CC) via an artery, we recommend i.v. heparin prophylaxis (Grade 1A). We suggest the use of heparin doses of 100 to 150 U/kg as a bolus and that further doses may be required in prolonged procedures (both Grade 2 B). For prophylaxis for CC, we recommend against aspirin therapy (Grade 1B). For neonates and children with peripheral arterial catheters in situ, we recommend the administration of low-dose heparin through a catheter, preferably by continuous infusion to prolong the catheter patency (Grade 1A). For children with a peripheral arterial catheter-related TE, we suggest the immediate removal of the catheter (Grade 2C). For prevention of aortic thrombosis secondary to the use of umbilical artery catheters in neonates, we suggest low-dose heparin infusion (1 to 5 U/h) (Grade 2A). In children with Kawasaki disease, we recommend therapy with aspirin in high doses initially (80 to 100 mg/kg/d during the acute phase, for up to 14 days) and then in lower doses (3 to 5 mg/kg/d for > or = 7 weeks) [Grade 1C+], as well as therapy with i.v. gammaglobulin within 10 days of the onset of symptoms (Grade 1A).
Child↗
Combined effects of mild hypothermia and glycoprotein IIb/IIIa antagonists on platelet-platelet and leukocyte-platelet aggregation.
Endovascular cooling was assessed as a potential treatment for percutaneous coronary intervention in patients with acute myocardial infarction. Here we show that mild hypothermia: (1) inhibits platelet aggregation; (2) augments eptifibatide- and tirofiban- but not abciximab-induced inhibition of platelet aggregation; (3) increases the formation of adenosine diphosphate-induced leukocyte-platelet aggregates; and (4) diminishes the glycoprotein IIb/IIIa antagonist-induced decrease in leukocyte-platelet aggregates.
Abciximab↗
Spirochete-platelet attachment and thrombocytopenia in murine relapsing fever borreliosis.
Thrombocytopenia is common in persons infected with relapsing fever Borreliae. We previously showed that the relapsing fever spirochete Borrelia hermsii binds to and activates human platelets in vitro and that, after platelet activation, high-level spirochete-platelet attachment is mediated by integrin alpha IIb beta 3, a receptor that requires platelet activation for full function. Here we established that B hermsii infection of the mouse results in severe thrombocytopenia and a functional defect in hemostasis caused by accelerated platelet loss. Disseminated intravascular coagulation, immune thrombocytopenic purpura, or splenic sequestration did not play a discernible role in this model. Instead, spirochete-platelet complexes were detected in the blood of infected mice, suggesting that platelet attachment by bacteria might result in platelet clearance. Consistent with this, splenomegaly and thrombocytopenia temporally correlated with spirochetemia, and the severity of thrombocytopenia directly correlated with the degree of spirochetemia. Activation of platelets and integrin alpha IIb beta 3 were apparently not required for bacterium-platelet binding or platelet clearance because the bacterium-bound platelets in the circulation were not activated, and platelet binding and thrombocytopenia during infection of beta 3-deficient and wild-type mice were indistinguishable. These findings suggest that thrombocytopenia of relapsing fever is the result of platelet clearance after beta 3-independent bacterial attachment to circulating platelets.
Animals↗
Neonatal platelet function.
Healthy newborns maintain normal circulating platelet counts, with a platelet ultrastructure that does not differ from adults. In vitro assessments of intrinsic platelet function, however, have demonstrated transient hyporesponsiveness that is most marked in platelets from preterm infants. Decreased responses were originally considered to be the result of platelet activation and degranulation during labor and delivery, but more recent studies of platelet activation markers have not supported this theory. Decreased activation responses are due to relative deficiencies of phospholipid metabolism, calcium mobilization, granule secretion, and aggregation. These result in turn from differences in intrinsic signal transduction in the neonatal platelet compared with the adult. In contrast, there is enhanced platelet adhesion due to the presence in neonatal plasma of larger, more functionally potent von Willebrand factor multimers. These ultralarge multimers may result from decreased activity of von Willebrand factor-cleaving protease in neonatal plasma and are associated with shorter bleeding times and Platelet Function Analyser-100 closure times in neonates. In the immediate newborn period, this enhanced platelet adhesion may compensate for the decreased intrinsic platelet activation in healthy neonates, but may leave sick neonates at increased risk of bleeding.
Blood Platelet Disorders↗
Quantification of abciximab-induced platelet inhibition is assay dependent: a comparative study in patients undergoing percutaneous coronary intervention.
BACKGROUND: The best method for measuring the degree of platelet inhibition with glycoprotein (GP) IIb-IIIa antagonists during percutaneous coronary intervention (PCI) and the optimal degree of periprocedural inhibition is uncertain. Low molecular weight heparins have been reported to cause less platelet activation than unfractionated heparin. Therefore, compared with unfractionated heparin (UHF), a low molecular weight heparin could enhance measured platelet inhibition. In this study, we compared 3 methods of measuring platelet inhibition and investigated the effects of half doses of abciximab in combination with either UFH or the low molecular weight heparin dalteparin in patients undergoing PCI with planned abciximab administration. METHODS: Abciximab-induced platelet inhibition was measured serially by means of 3 assays: 1) GP IIb-IIIa receptor occupancy, 2) binding of the activated GP IIb-IIIa-specific monoclonal antibody PAC1, and 3) agglutination of platelets with fibrinogen-coated beads (RPFA). Forty patients were randomly allocated to receive either UFH (70 U/kg) or dalteparin (60 IU/kg), followed by a half dose of abciximab (0.125 mg/kg) administered twice at 10-minute intervals. Assays were obtained 10 minutes after each half dose of abciximab and 8 to 10 and 24 hours after abciximab administration. RESULTS: No differences between UFH and dalteparin were observed. At each time-point measured, the mean percent platelet inhibition as determined by means of the receptor occupancy assay and PAC1 binding assay was less than the degree of inhibition determined by means of the RPFA. CONCLUSIONS: The results of targeted levels of platelet inhibition cannot be extrapolated between different clinical trials of GP IIb-IIIa antagonists unless the same assay is used. Dalteparin, compared with UFH, does not enhance platelet inhibition or receptor occupancy by abciximab, as demonstrated by means of 3 separate assays.
Abciximab↗
The cleaved peptide of PAR1 is a more potent stimulant of platelet-endothelial cell adhesion than is thrombin.
PURPOSE: Platelet-endothelial cell adhesion is an important pathologic response to vessel injury or inflammation. On binding to its endothelial or platelet G protein-linked seven-transmembrane domain receptor, protease-activated receptor-1 (PAR1), thrombin releases a 41-amino acid peptide (TR(1-41)). We examined the effect of TR(1-41) on platelet activation and on platelet-endothelial cell adhesion. METHODS: A monolayer of confluent human saphenous vein endothelial cells was incubated with washed human platelets. Platelets were stimulated with either TR(1-41), TR(21-41), scrambled TR(1-41), adenosine diphosphate (ADP)-epinephrine (EPI), thrombin, or thrombin receptor activating peptide (TRAP). Platelet activation was identified with flow cytometry. The magnitude of platelet-endothelial cell adhesion was determined with a laser scanning cytometer that scanned the monolayer of endothelial cells and identified fluorescently bound platelets. RESULTS: Maximal thrombin stimulation (0.1 U/mL) induced a threefold increase in platelets bound to endothelial cells compared with buffer alone. Stimulation with TR(1-41) (20 mmol/L) tripled the number of platelets bound to endothelial cells compared with thrombin. Scrambled sequence of TR(1-41) (20 mmol/L) and TR(21-41) (20 mmol/L), neither of which induces platelet activation, had minimal effect on platelet adhesion. Both TRAP (20 mmol/L) and ADP-EPI (20 mmol/L) induced less platelet-endothelial cell adhesion than did thrombin. TR(1-41)-induced platelet-endothelial cell adhesion was partially blocked by glycoprotein (GP)IIb-IIIa-specific monoclonal antibody, 10E5 (10 mg/mL). CONCLUSIONS: TR(1-41), the cleaved peptide of PAR1, is a more potent stimulant of platelet-endothelial cell adhesion than is thrombin, TRAP, or ADP-EPI, and this adhesion is at least in part mediated by the platelet GPIIb-IIIa receptor.
Cell Adhesion↗
Two novel mutations in the alpha IIb calcium-binding domains identify hydrophobic regions essential for alpha IIbbeta 3 biogenesis.
The recently published crystal structure of the external domains of alphaVbeta3 confirms the prediction that the aminoterminal portion of alphaV, which shares 40% homology with alphaIIb, folds into a beta-propeller structure and that the 4 calcium-binding domains are positioned on the bottom of the propeller. To gain insight into the role of the calcium-binding domains in alphaIIb biogenesis, we characterized mutations in the second and third calcium-binding domains of alphaIIb in 2 patients with Glanzmann thrombasthenia. One patient inherited a Val298Phe mutation in the second domain, and the other patient inherited an Ile374Thr mutation in the third domain. Mammalian cell expression studies were performed with normal and mutant alphaIIb and beta3 cDNA constructs. By flow cytometry, expression of alphaIIb Val298Phe/beta3 in transfected cells was 28% of control, and expression of alphaIIbIle374Thr/beta3 was 11% of control. Pulse-chase analyses showed that both mutant pro-alphaIIb subunits are retained in the endoplasmic reticulum and degraded. Mutagenesis studies of the Val298 and Ile374 residues showed that these highly conserved, branch-chained hydrophobic residues are essential at these positions and that biogenesis and expression of alphaIIbbeta3 is dramatically affected by structural variations in these regions of the calcium-binding domains. Energy calculations derived from a new model of the alphaIIb beta-propeller indicate that these mutations interfere with calcium binding. These data suggest that the alphaIIb calcium-binding domains play a key structural role in the beta-propeller, and that the structural integrity of the calcium-binding domains is critical for integrin biogenesis.
Binding Sites↗
The Ultegra rapid platelet-function assay: comparison to standard platelet function assays in patients undergoing percutaneous coronary intervention with abciximab therapy.
BACKGROUND: Despite the proven benefit of glycoprotein IIb/IIIa inhibitors during percutaneous coronary intervention, significant interpatient variability exists in antiplatelet response. Furthermore, a diminished degree of platelet inhibition is an independent predictor of adverse cardiac events, highlighting the need for accurate and precise monitoring of platelet function. METHODS: Patients (n = 192) who underwent elective percutaneous coronary intervention at 4 centers were enrolled. The following 3 time points were studied: 1, baseline, before abciximab bolus administration; 2, during, within 1 hour of abciximab bolus administration; and 3, post, 24 hours after abciximab bolus administration or at the time of patient discharge, whichever occurred first. The following 3 assays were compared at all time points: Ultegra rapid platelet-function assay (Ultegra RPFA), conventional turbidometric platelet aggregometry, and receptor binding assay with [125I]-abciximab. Variability in Ultegra RPFA measurements between operators was determined with performance of the assays at the point of care and in the laboratory. A sub-study of 22 patients at 1 center was performed in which the laboratory scientist performed all 3 assays in duplicate at each time point. RESULTS: Comparison with the receptor binding assay and conventional platelet aggregometry in 120 patients showed that the Ultegra RPFA correlated with aggregometry (r = 0.89) and with the receptor binding assay (r = 0.89). There was good agreement (r = 0.80) between values obtained by intended users and those obtained by laboratory scientists. Furthermore, Ultegra RPFA values had equivalent precision to the standard assays. CONCLUSION: The Ultegra RPFA has equivalent accuracy and precision when compared with the 2 reference assays studied. Ultegra RPFA measurements are not operator-dependent and are not influenced by concomitant medications, hematologic parameters, or demographics.
Abciximab↗
An additional mechanism of action of abciximab: dispersal of newly formed platelet aggregates.
BACKGROUND: The ability of abciximab to prevent fibrinogen binding to activated platelets indicates it may also promote dissolution of platelet-rich thrombi. The present study examined the capacity of abciximab to reverse platelet aggregation in vitro. METHODS AND RESULTS: Experiments were performed on blood from healthy non-medicated donors. Platelet aggregate formation and disaggregation were monitored turbidimetrically. Platelet-bound fibrinogen was measured by flow cytometry. For disaggregation studies, platelets were first stimulated with either ADP or the 11-mer thrombin receptor activating peptide (TRAP), then varying amounts of abciximab were added at periodic intervals after agonist addition. Platelet disaggregation was detected by comparing the extent of light transmittance at 4 min after addition of either abciximab or saline to PRP. ATP release was simultaneously monitored by chemi-luminescence. When added 1 min after low concentrations of ADP, abciximab rapidly (< 1 min) dispersed platelet aggregates in a dose-dependent manner, with complete disaggregation observed with 6.25 microg/mL of the beta3 antagonist. In contrast, equivalent concentrations of abciximab did not induce appreciable disaggregation to platelets stimulated with TRAP (10 microM). Platelet counts of samples that had undergone complete disaggregation, as assessed by aggregometry, were equivalent to baseline, indicating dispersal of aggregates to single cells. Concentrations of abciximab that produced complete disaggregation induced partial displacement of platelet-bound fibrinogen (52 +/- 8% inhibition of fibrinogen binding at 12.5 microg/ml abciximab). The disaggregation effectiveness of abciximab decreased as the time between ADP and subsequent abciximab addition widened, and as the amount of both dense granule release and agonist stimulation increased. However, pre-treatment of platelets with acetylsalicylic acid (ASA) did not potentiate platelet disaggregation induced by abciximab. CONCLUSIONS: These data indicate that abciximab facilitates the dispersal of newly formed platelet aggregates in vitro, by partially displacing fibrinogen from activated GPIIb/IIIa receptors. In vivo, abciximab may destabilize coronary thrombi by preventing aggregate formation and dispersing mural thrombi.
Abciximab↗
Platelet-monocyte aggregates in patients with chronic venous insufficiency remain elevated following correction of reflux.
INTRODUCTION: An increased number of circulating platelet-monocyte aggregates (PMAs) is present in patients with all clinical classes of chronic venous insufficiency (CVI). The purpose of this study was to determine whether patients with CVI maintain elevated levels of PMAs following complete surgical correction of chronic venous insufficiency. METHODS: Patients with superficial venous insufficiency and a normal deep venous system documented by duplex scan were included in the study. Venous blood was drawn from a superficial vein in the leg and an antecubital vein prior to vein stripping and again six weeks postoperatively. Control subjects without evidence of venous disease had blood drawn from an antecubital vein. Whole blood flow cytometry was used to analyze the samples for the presence of platelet-monocyte aggregates following incubation with buffer or 0.5 microM adenosine diphosphate (ADP). RESULTS: Postoperative duplex scanning demonstrated elimination of venous reflux in the superficial venous system and normal deep vein physiology in all nine patients. Preoperatively, patients with CVI had significantly elevated levels of circulating PMAs in both arm and leg samples without stimulation by an agonist compared to controls (15.2+/-1.1 and 14.3+/-1.3 vs 7.4+/-0.3 for controls, p<0.02 for each), and after stimulation by 0.5 microM ADP (33.7+/-4.7 and 34.3+/-5.2 vs 12.5+/-3.8 for controls, p<0.04 for each). There was no significant change in the number of PMAs in either patient arm or leg blood samples six weeks following correction of venous reflux by removal of the diseased veins. CONCLUSIONS: Complete correction of chronic venous insufficiency did not diminish the elevated circulating levels of platelet-monocyte aggregates. We conclude that the presence of an increased number of PMAs identified in patients with CVI is not secondary to the presence of venous reflux, but may be involved with the primary etiology of chronic venous insufficiency. This finding also suggests that a stimulus other than venous hypertension may be important in triggering the leukocyte activation seen in patients with chronic venous disease.
Adult↗
How platelets work: platelet function and dysfunction.
This article briefly reviews (a) how platelets normally function and (b) the clinical approach to disorders of platelet numbers and function.
Animals↗
Platelet inhibitor therapy: mechanisms of action and clinical use.
This article briefly reviews the mechanisms of action and clinical uses of platelet inhibitor therapy.
Animals↗