scholarly journals Simultaneous Measurement of Platelet Factor 4 (PF4) and β-Thromboglobulin (βTG) Release and Fibrinopeptide A (FPA) Cleavage

1977 ◽  
Author(s):  
K. L. Kaplan ◽  
H. L. Nossel
Keyword(s):  
Platelet Activation ◽  
Simultaneous Measurement ◽  
Platelet Rich Plasma ◽  
Arterial Disease ◽  
Protein Release ◽  
Clinical Samples ◽  
Platelet Factor ◽  
Fibrin Formation ◽  
Platelet Protein ◽  
Disease Entities

Platelet activation and fibrin formation occur in thrombo-embolism, arterial disease, and intravascular coagulation. Selective involvement in certain disease entities and combined involvement in others has been suggested on the basis of turnover studies. The development in this laboratory of sensitive and specific radioimmunoassays for two released platelet proteins, PF4 and βTG, and the availability of the radioimmunoassay for FPA as an index of fibrin formation have allowed studies of the physiologic basis for differential involvement of platelets and fibrin formation. Simultaneous measurement of platelet activation, monitored by radioimmunoassay for PF4 and βTG as well as aggregometry and 14C-serotonin (5HT) release, and FPA cleavage were carried out in citrated platelet rich plasma, whole blood and gel-filtered platelets. Collagen and ADP aggregated platelets and released 5HT, PF4 and βTG without detectable FPA cleavage indicating that thrombin action on fibrinogen is not involved in aggregation or release induced by these agents. Thrombin cleaved FPA at concentrations 100-fold less than those required for platelet protein release, and platelet protein release could be detected at lower thrombin concentrations than 5HT release. This might be due to greater sensitivity of the PF4 and βTG assays in detecting release or to different mechanisms of release of the proteins and 5HT. These results suggest that, in clinical samples, elevated FPA with normal PF4 and βTG might be due to concentrations of circulating thrombin sufficient to cleave FPA but too low to induce platelet release, and that the converse situation, with elevated PF4 and βTG but normal FPA might imply platelet activation by exposed subendothelial collagen with no thrombin action.

scholarly journals Fibronectin modulates formation of PF4/heparin complexes and is a potential factor for reducing risk of developing HIT

Blood ◽  
2019 ◽  
Vol 133 (9) ◽  
pp. 978-989 ◽  
Author(s):  
Krystin Krauel ◽  
Patricia Preuße ◽  
Theodore E. Warkentin ◽  
Catja Trabhardt ◽  
Sven Brandt ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Platelet Rich Plasma ◽  
Enzyme Linked Immunosorbent Assay ◽  
Heparin Binding ◽  
Plasma Fibronectin ◽  
Platelet Factor ◽  
Washed Platelet ◽  
Plasma Factor ◽  
Heparin Complexes ◽  
Patient Groups

Abstract Heparin-induced thrombocytopenia (HIT) is caused by platelet-activating anti–platelet factor 4 (PF4)/heparin antibodies. Platelet activation assays that use “washed” platelets are more sensitive for detecting HIT antibodies than platelet-rich plasma (PRP)–based assays. Moreover, heparin-exposed patients vary considerably with respect to the risk of PF4/heparin immunization and, among antibody-positive patients, the risk of subsequent “breakthrough” of clinical HIT with manifestation of thrombocytopenia. We used washed platelets and PRP, standard laboratory HIT tests, and physicochemical methods to identify a plasma factor interfering with PF4/heparin complexes and anti-PF4/heparin antibody–platelet interaction, thus explaining differences in functional assays. To investigate a modulating risk for PF4/heparin immunization and breakthrough of HIT, we also tested 89 plasmas from 2 serosurveillance trials. Fibronectin levels were measured in 4 patient groups exhibiting different degrees of heparin-dependent immunization and expression of HIT. The heat-labile plasma protein, fibronectin, inhibited PF4 binding to platelets in a dose-dependent fashion, particularly in washed (vs PRP) systems. Fibronectin also inhibited PF4/heparin binding to platelets, anti-PF4/heparin antibody binding to PF4/heparin complexes, and anti-PF4/heparin antibody–induced platelet activation as a result of PF4/heparin complex disruption. In addition, plasma fibronectin levels increased progressively among the following 4 patient groups: enzyme-linked immunosorbent assay (ELISA)+/serotonin-release assay (SRA)+/HIT+ < ELISA+/SRA+/HIT− ∼ ELISA+/SRA−/HIT− < ELISA−/SRA−/HIT−. Altogether, these findings suggest that fibronectin interferes with PF4/heparin complex formation and anti-PF4/heparin antibody–induced platelet activation. Reduced fibronectin levels in washed platelet assays help to explain the greater sensitivity of washed platelet (vs PRP) assays for HIT. More importantly, lower plasma fibronectin levels could represent a risk factor for PF4/heparin immunization and clinical breakthrough of HIT.

ULTRASTRUCTURAL INVESTIGATIONS ON THE MECHANISM OF “SHEAR-INDUCED PLATELET ACTIVATION”

1987 ◽  
Author(s):  
L J Wurzinger ◽  
R Opitz ◽  
H Schmid-Schönbein
Keyword(s):  
Shear Stress ◽  
Platelet Activation ◽  
Platelet Rich Plasma ◽  
Adenine Nucleotides ◽  
Coagulation System ◽  
Laminar Shear Stress ◽  
Shear Forces ◽  
Platelet Factor ◽  
Membrane Defects ◽  
Biochemical Activation

High shear forces are suspected to play a triggering role in the initiation of arterial thrombosis, by activating platelets and the coagulation system. In an earlier study a shear stress of 170 N/m2 acting for only 7 milliseconds (ms) on platelet rich plasma (PRP) was found to induce a significant increase in platelet factor 3 availability (Thromb. Haemost. 54: 381-386; 1985). To clarify the question whether platelets can be activated directly by mechanical forces in analogy to smooth muscle cells, electron micrographs of platelets subjected to laminar shear stress were analysed with morphometric methods. The level of activation of platelet suspensions was quantified by assessing 1) the elongation of platelet profiles giving a measure for the “flatness” of the discoid resting platelets, and 2) the centralization of granules.Exposure to a shear stress of 170 N/m2 for 113 ms leaves ca. 15 % of the platelets irreversibly damaged, featuring degenerative ballooning, with break-down of internal structure and cell membrane defects. The remaining 85 % appear typically activated with rounded shape, extension of pseudopods and centralization of granules. Addition of “ADP-scavengers” to the suspension medium totally changes the appearance of sheared platelets: still a comparable proportion of them has undergone irreversible degenerative changes, but the “surviving” population lacks ultrastructural signs of platelet activation. This is reflected in values of the morphometric parameters which are close to the level of unsheared control samples.It is therefore concluded that “shear-induced platelet activation” cannot be ascribed to a direct stimulating effect of shear forces, but rather to secondary biochemical activation by adenine nucleotides leaking from a small percentage of shear destroyed platelets. The latter process, however, requires a well stirred though undiluted environment, as it is provided in vortices and eddies.

Functional Microparticles Are up Regulated in Patients with Anti-Heparin/Platelet Factor 4 Antibodies: A Potential Mechanism of Thrombogenesis in the Heparin-Induced Thrombocytopenia Syndrome.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2105-2105
Author(s):  
Josephine Cunanan ◽  
Michelle Kujawski ◽  
He Zhu ◽  
Margaret Prechel ◽  
Jeanine Walenga ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Medical Center ◽  
Annexin V ◽  
Platelet Factor 4 ◽  
Cellular Damage ◽  
Clinical Samples ◽  
Limiting Factor ◽  
Therapeutic Effects ◽  
Platelet Factor ◽  
Heparin Induced Thrombocytopenia

Abstract Heparin-induced thrombocytopenia (HIT) is one of the most catastrophic adverse effects of heparin therapy, representing a complex syndrome involving immunopathologic and hemostatic disorders. Vascular and blood cellular damage results in the generation of microparticles (MP). These MP are formed from stress conditions/cellular disruption and apoptosis. Cellular MP mediated pathophysiologic responses include platelet activation, up regulation of adhesion molecules, monocyte activation, up regulation of tissue factor and endothelial dysfunction. Several methods based on flow cytometric and other immunologic probes have been used to measure MP in the HIT syndrome. Recently, a functional method based on the complexation of MP with annexin V promoting the generation of factor Xa and thrombin has become available (Hyphen Biomedical, Neuville-Oise, France). To validate the hypothesis that functional MP are elevated in the HIT syndrome, this method was utilized for the quantitation of MP in sera ELISA positive for anti-heparin/platelet factor 4 (HIT) antibodies. Specimens (n = 53) were selected from archived samples that had been referred to Loyola University Medical Center for the laboratory diagnosis of HIT by quantitating anti-heparin/PF4 antibodies by ELISA and by evaluating HIT antibody induced platelet activation using the 14C Serotonin Release Assay (SRA). All selected specimens were positive for HIT antibodies in the GTI PF4 Enhanced ELISA with a broad range of antibody titers (absorbance range of 0.4 – 2.5). Eleven of these specimens were positive in the SRA. In addition, serial samples from HIT patients treated with argatroban (from the ARG-911 clinical study) were included (n = 23). The normal samples represented control sera obtained from healthy human volunteers (n = 25) and processed in the same manner as the clinical samples. Test samples were added to microtiter plates coated with streptavidin and biotinylated annexin V. MP present in the test sample bound to annexin V via exposed surface phospholipids. Following incubation and washing steps, a FXa – FVa mixture containing calcium and prothrombin was added. The assay was optimized so that MP associated phospholipid was the limiting factor for the generation of thrombin. In normal non-HIT sera, the MP levels ranged 5.6 – 10.1 nM (6.1 ± 2.8 nM). The pre-treatment, baseline levels of circulating MP in the suspected HIT patients ranged from 4.2 – 26.8 nM (15.8 ± 7.3 nM). Interestingly, SRA positive/ELISA positive samples had relatively higher levels of MP (19.9 ± 7.7 nM; range 11.5 – 29.8 nM) than SRA negative/ELISA positive samples (14.2± 4.6; range 6.8–21.2). In the ARG-911 study, sequential blood samples exhibited MP levels at the baseline ranging from 8.2 – 38.6 nM (21.8 ± 10.8 nM), whereas after 3 days of argatroban treatment were reduced to 5.1 – 19.2 nM (12.6 ± 6.3). The results of these studies suggest that circulating functional MP are increased in patients with ELISA positive HIT antibodies. Anticoagulation with such direct thrombin agents as argatroban effectively decreases the circulating functional MP levels. Since the elevated MP levels may mediate thrombin and FXa generation, the therapeutic effects of these drugs in HIT may be related to the decreased activation of coagulation and related thrombogenic processes.

scholarly journals Detection of Platelet-Activating Antibodies Associated with Heparin-Induced Thrombocytopenia

2020 ◽  
Vol 9 (4) ◽  
pp. 1226 ◽  
Author(s):  
Brigitte Tardy ◽  
Thomas Lecompte ◽  
François Mullier ◽  
Caroline Vayne ◽  
Claire Pouplard
Keyword(s):  
Platelet Activation ◽  
Platelet Rich Plasma ◽  
Serotonin Release ◽  
Membrane Expression ◽  
Platelet Factor ◽  
Strict Adherence ◽  
Heparin Induced Thrombocytopenia ◽  
Functional Assays ◽  
Healthy Donors ◽  
Sensitivity Specificity

Heparin-induced thrombocytopenia (HIT) is a prothrombotic immune drug reaction caused by platelet-activating antibodies that in most instances recognize platelet factor 4 (PF4)/polyanion complexes. Platelet activation assays (i.e., functional assays) are more specific than immunoassays, since they are able to discern clinically relevant heparin-induced antibodies. All functional assays used for HIT diagnosis share the same principle, as they assess the ability of serum/plasma from suspected HIT patients to activate fresh platelets from healthy donors in the presence of several concentrations of heparin. Depending on the assay, donors’ platelets are stimulated either in whole blood (WB), platelet-rich plasma (PRP), or in a buffer medium (washed platelets, WP). In addition, the activation endpoint studied varies from one assay to another: platelet aggregation, membrane expression of markers of platelet activation, release of platelet granules. Tests with WP are more sensitive and serotonin release assay (SRA) is considered to be the current gold standard, but functional assays suffer from certain limitations regarding their sensitivity, specificity, complexity, and/or accessibility. However, the strict adherence to adequate preanalytical conditions, the use of selected platelet donors and the inclusion of positive and negative controls in each run are key points that ensure their performances.

Fibrin Generation in Heparin-Induced Thrombocytopenia (HIT): Pathomechanistic Background for Novel Therapy and Prophylaxis

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 635-635
Author(s):  
Valerie Tutwiler ◽  
Hyun Sook Ahn ◽  
Rudy Fuentes ◽  
Vincent M. Hayes ◽  
Sergei V. Zaytsev ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Platelet Activation ◽  
Human Blood ◽  
Whole Blood ◽  
Platelet Adhesion ◽  
Thrombin Inhibitors ◽  
Single Chain ◽  
Monocyte Activation ◽  
Platelet Factor ◽  
Fibrin Formation

Abstract Abstract 635 HIT is an immune mediated prothrombotic disorder often associated with life- and limb-threatening thrombosis caused by antibodies to a complex between platelet factor 4 (PF4) and heparin. Platelet activation and clearance are considered key to the pathogenesis. Direct thrombin inhibitors, the most commonly used anticoagulant therapy in the treatment of HIT, provide incomplete prevention against development of new thrombi and little effect on the incidence of loss of limb and life. Thus, there is a need for a better understanding of pathogenesis of HIT and new approaches to therapy. We and others have shown that HIT not only is associated with platelet activation, but also involves activation of monocytes and endothelial cells, which together increase thrombin generation that may affect both the amount and structural properties of the resultant fibrin clot. However, this proposed increase in fibrin formation though suspected, has never been directly investigated. Previously, we have shown in the cremaster muscle laser-injury model of thrombosis induced by the HIT-like murine monoclonal anti-hPF4/heparin antibody KKO that transgenic mice expressing both human PF4 (hPF4+) and hFcγRIIA developed larger, more fibrin-rich occlusive thrombi than in control mice expressing hPF4 or hFcγRIIA alone. To quantify fibrin formation in a more controlled setting, we simulated HIT in a BioFlux microfluidic channel system coated with von Willebrand factor by perfusing whole blood at a venule shear stress of 20 dyne/cm2 at 37°C for 10 min. Platelets were labeled by adding Calcein-AM (3 μM) and fibrin was visualized by adding Alexa 647 labeled fibrinogen (1.5 μg/ml) to the whole blood prior to the perfusion. Using NaCitrate-anticoagulated human blood, we observed that recalcified human blood samples exposed to KKO plus hPF4 formed large platelet thrombi and an extensive fibrin network, with fibers radiating from the platelet aggregates and often organized along the direction of flow. In contrast, control samples exposed to a combination of the non-pathogenic anti-hPF4 monoclonal antibody RTO plus hPF4 showed little fibrin and less organization. Quantitative fluorescence analysis showed nine times more fibrin formed after stimulation with KKO plus PF4 than RTO plus PF4. Inhibition of KKO-mediated platelet activation by blocking FcgRIIA with Fab fragments of monoclonal antibody IV.3 in whole blood suppressed platelet adhesion by > 80%, but decreased fibrin formation by only ∼40%. On the other hand, addition of a selective inhibitor of the Syk tyrosine kinase PRT-060318 (Reilly et al., Blood 2011:117:2241–6; kindly provided by Dr. Uma Sinha, Portola Pharmaceuticals) to whole blood at a concentration of 3 μM suppressed both platelet adhesion and fibrin formation by 80% and 70%, respectively. IV.3 inhibits platelet activation alone, while we have shown that PRT-060318 inhibits both platelet activation and monocyte activation with the subsequent release of tissue factor-rich microparticles. These results provide a mechanistic basis for the use of novel therapies in HIT such as fibrinolytic agents. To do so, we studied a novel chimeric pro-fibrinolytic composed of a C-terminal thrombin-specific activatable low molecular weight urokinase (uPA-T) that has its plasmin-activation site replaced by a thrombin cleavage site and linked at its N-terminus to a single-chain variable region (scFv) that binds with high affinity to human platelet aIIb, designed to deliver the agent to sites of incipient thrombosis. Preliminary results show that uPA-T profoundly suppressed fibrin accumulation in both in vitro and in an in vivo model of HIT. This novel approach to therapy takes advantage of our growing understanding of the pathogenesis of the prothrombotic nature of HIT including monocyte activation and formation of fibrin-rich clots. Such therapeutics may be especially effective as targeted therapy in HIT. Disclosures: Cines: Amgen Inc.: Consultancy; GlaxoSmithKline: Consultancy; Eisai: Consultancy.

scholarly journals Platelet protein S limits venous but not arterial thrombosis propensity by controlling coagulation in the thrombus

Blood ◽  
2020 ◽  
Vol 135 (22) ◽  
pp. 1969-1982 ◽  
Author(s):  
Sara Calzavarini ◽  
Raja Prince-Eladnani ◽  
François Saller ◽  
Luca Bologna ◽  
Laurent Burnier ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Activated Protein C ◽  
Vena Cava ◽  
Protein S ◽  
Limiting Factor ◽  
Factor X ◽  
Platelet Factor ◽  
Shear Rates ◽  
Platelet Protein ◽  
Factor X Activation

Abstract Anticoagulant protein S (PS) in platelets (PSplt) resembles plasma PS and is released on platelet activation, but its role in thrombosis has not been elucidated. Here we report that inactivation of PSplt expression using the Platelet factor 4 (Pf4)-Cre transgene (Pros1lox/loxPf4-Cre+) in mice promotes thrombus propensity in the vena cava, where shear rates are low, but not in the carotid artery, where shear rates are high. At a low shear rate, PSplt functions as a cofactor for both activated protein C and tissue factor pathway inhibitor, thereby limiting factor X activation and thrombin generation within the growing thrombus and ensuring that highly activated platelets and fibrin remain localized at the injury site. In the presence of high thrombin concentrations, clots from Pros1lox/loxPf4-Cre− mice contract, but not clots from Pros1lox/loxPf4-Cre+ mice, because of highly dense fibrin networks. Thus, PSplt controls platelet activation as well as coagulation in thrombi in large veins, but not in large arteries.

scholarly journals Antibody Stabilization of Neutrophil Extracellular Trap-Platelet Factor 4 Complexes Is Therapeutic in a Murine Model of Endotoxemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 271-271
Author(s):  
Amrita Sarkar ◽  
Gowthami Arepally ◽  
M. Anna Kowalska ◽  
Douglas B. Cines ◽  
Lubica Rauova ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Platelet Activation ◽  
Degradation Products ◽  
Umbilical Vein ◽  
Dnase I ◽  
Platelet Factor 4 ◽  
Neutrophil Extracellular Trap ◽  
Clinical Samples ◽  
Platelet Factor ◽  
Positively Charged

Abstract Sepsis is characterized by life-threatening organ dysfunction caused by a dysregulated response to infection. Neutrophils play a crucial role in sepsis during which they release neutrophil extracellular traps (NETs), webs of negatively charged cell-free DNA (cfDNA) complexed with positively-charged histones that kill pathogens but also damage host tissue. While it has been proposed that NET digestion may be beneficial in the treatment of sepsis, it is also possible that this strategy leads to the release of harmful NET degradation products (NDPs), such as cfDNA, histones, and myeloperoxidase (MPO) that cause endothelial damage. An alternative approach would be treatment strategies that prevent NET lysis and the release of NDPs. We posited that NET-bound platelet factor 4 (PF4, CXCL4) may have this effect. PF4 is a highly-positively charged, platelet-specific chemokine released in high concentrations following platelet activation and aggregates polyanionic molecules like heparin, polyphosphates, and DNA. We have found that PF4 causes physical compaction of NETs without inducing histone release. We studied the effect of this compaction using an in vitro microfluidic assay in which NETs released from neutrophils stimulated by tissue-necrosis factor (TNF) α were infused over a layer of cultured human umbilical vein endothelial cells (HUVECs) ± PF4 (25 μg/ml) and incubated for 12 hours. Channels exposed to PF4 were "heathier" with significantly more residual attached endothelial cells (44±24 vs. 150±29 cells/hpf, n=6, p=0.0002). We have previously shown that mice that overexpress PF4 are protected from mortality in lipopolysaccharide (LPS) endotoxemia. Compared to wildtype animals, PF4 null mice have increased circulating levels of NET markers including cfDNA, citrullinated histones, and MPO. Treatment with exogenous PF4 leads to a decrease in plasma levels of NET components and a reduction in mortality. In a microfluidic assay in which channel-adherent NETs were treated with increasing concentrations of PF4 (0-25 µg/ml) and then infused with DNase I (100 U/ml), we found that compacted NET-PF4 complexes become resistant to DNase I digestion. Prior studies have revealed that when PF4 binds to polyanionic molecules, it exposes HIT-like antigenic sites. We confirmed that the NET-PF4 complexes similarly bind HIT IgG isolated from clinical samples and the HIT-like monoclonal antibody KKO. Interestingly, KKO binding further enhanced DNase I resistance, a phenomenon not seen with a polyclonal anti-PF4 antibody (abcam). We hypothesized that KKO which causes PF4 oligomerization unlike the polyclonal antibody, further stabilized the crosslinked NET-PF4 complexes, providing additional protection from DNase I digestion and preventing the release of NDPs. We then asked whether KKO may serve as a targeted therapy in the treatment of sepsis, enhancing NET-PF4 complex resistance to nuclease digestion and leading to the sequestration of NDPs. As KKO is an IgG2bk antibody, we were unable to use pepsin digestion to remove the Fc region that causes platelet activation. We therefore used an IgG-specific endoglycosidase to develop a deglycosylated version of KKO (DG-KKO) that retains the ability to bind to NET-PF4 complexes (data not shown), but has a reduced capacity to interact with hematopoietic cell Fc receptors. DG-KKO has a markedly decreased ability to activate human platelets compared to KKO in the presence of added PF4 as measured by P-selectin level (3710±140 control vs. 1580±300 treated MFI, n = 2). Compared to control, DG-KKO infusion in the murine endotoxemia model prevented thrombocytopenia (mean platelet count 286±16 control vs. 537±69 treated X103/µl, n=4-5, p=0.016), the release of cfDNA (8.5±0.3 vs. 3.7±0.7 µg/ml, n=4-5, p=0.016), and the emission of MPO-DNA complexes (234±14 vs. 99±5 % increase from non-LPS injected controls, n=4-5, p=0.016) As depicted in Figure 1, we propose that in sepsis, NETosis occurs (Step 1) causing the release of harmful NDPs (Step 2). PF4 expelled from activated platelets stabilizes PF4-NET complexes (Step 3). Infused DG-KKO enhances NET stability and decreases release of NDPs following DNase I digestion (Step 4). These studies provide mechanistic insights into the release of NDPs during endotoxemia and offer a targeted, novel therapeutic to prevent their contribution to inflammatory states such as sepsis. Disclosures Poncz: Incyte Corporation: Consultancy, Research Funding.

Defibrotide Does Not Cross-React with HIT Antibodies. Implications in the Management of HIT.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1054-1054
Author(s):  
Jawed Fareed ◽  
Debra A. Hoppensteadt ◽  
Massimo Iacobelli ◽  
Jeanine M. Walenga
Keyword(s):  
Platelet Activation ◽  
Platelet Rich Plasma ◽  
Serotonin Release ◽  
Antithrombotic Agent ◽  
Platelet Factor ◽  
Prolonged Incubation ◽  
Heparin Induced Thrombocytopenia ◽  
Orally Bioavailable ◽  
Microparticle Formation ◽  
Heparin Exposure

Abstract Defibrotide is a mammalian DNA derived antithrombotic and anti-ischemic agent that does not produce systemic anticoagulation. Heparin-induced thrombocytopenia (HIT) is an immune disease related to heparin exposure, in which patients are at risk of developing life- and limb-threatening thrombosis. Studies were performed to determine whether defibrotide cross-reacts with HIT antibodies. Sera from 141 clinically confirmed HIT patients were tested for platelet activation in the presence of defibrotide (1 – 100 μg/ml) and unfractionated heparin (1 – 100 μg/ml). 103 sera (73%) produced platelet aggregation and serotonin release activities with heparin. Only 4 samples (2%) showed a weak reactivity with defibrotide which was eliminated by heparinase treatment indicating heparin contamination in the patient sample. Further studies revealed that defibrotide does not complex with platelet factor 4 (PF4), and that prolonged incubation of defibrotide with platelet rich plasma does not mobilize PF4. Studies of patients treated with extended dosing of intravenous or oral defibrotide (n=270) demonstrated that HIT antibodies are not generated with defibrotide treatment. Studies carried out on the effect of purified IgG from HIT patients revealed that defibrotide blunts platelet activation and microparticle formation as measured by flow cytometry. These studies suggest that defibrotide may be a useful antithrombotic agent for the management of patients with HIT. Moreover, unlike heparin, defibrotide does not promote platelet activation, rather it is capable of suppressing the hypercoagulable state associated with HIT. Defibrotide is orally bioavailable and can be used for extended anticoagulant management of HIT patients.

Flow Cytometry Analysis of Intracellular VASP Phosphorylation for the Assessment of Activating and Inhibitory Signal Transduction Pathways in Human Platelets

1999 ◽  
Vol 82 (09) ◽  
pp. 1145-1152 ◽  
Author(s):  
Ulrike Schwarz ◽  
Jörg Geiger ◽  
Ulrich Walter ◽  
Martin Eigenthaler
Keyword(s):  
Flow Cytometry ◽  
Platelet Activation ◽  
Intracellular Signaling ◽  
Platelet Rich Plasma ◽  
Umbilical Vein ◽  
Cardiovascular Complications ◽  
Human Platelets ◽  
Mitogen Activated Protein Kinase ◽  
Therapeutic Interventions ◽  
Platelet Protein

SummaryIncreased platelet adhesion or aggregation are key events in the pathogenesis of cardiovascular diseases. Exact determination of the platelet activation state is essential to recognize, prevent, and treat cardiovascular complications due to platelet dysfunction. Initial phases of platelet activation and inhibition are characterized by phosphorylation of specific intracellular proteins. However, methodological problems often prevent analysis of platelet protein phosphorylation under clinical conditions. A novel flow cytometry-based method using a phosphorylation-specific antibody was developed for fast and easy quantification of the phosphorylation state of a specific intracellular platelet protein. This method was used to analyze various platelet receptors and their intracellular signaling which may be impaired in genetic or acquired disorders or altered due to therapeutic interventions. In a first clinical application, the inhibitory effects of ticlopidine and clopidogrel on the platelet P2YAC ADP receptor were monitored.Abbreviations: ADP: adenosine 5’-diphosphate; cAMP: cyclic adenosine-3’,5’-monophosphate; cGMP: cyclic guanosine-3’,5’-monophosphate; HUVECs: human umbilical vein endothelial cells; MAPK: mitogen-activated protein kinase; PG-E1: prostaglandin E1; PRP: platelet-rich plasma; SNP: sodium nitroprusside; VASP: vasodilator-stimulated phosphoprotein

In Vitro Efficacy of Platelet Glycoprotein IIb/IIIa Antagonist in Blocking Platelet Function in Plasma of Patients with Heparin-induced Thrombocytopenia

1998 ◽  
Vol 80 (12) ◽  
pp. 989-993 ◽  
Author(s):  
Koon-Hou Mak ◽  
Linda Brooks ◽  
Eric Topol ◽  
Kandice Kottke-Marchant
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Platelet Rich Plasma ◽  
Underlying Mechanism ◽  
Platelet Factor 4 ◽  
Platelet Glycoprotein ◽  
Platelet Factor ◽  
Heparin Induced Thrombocytopenia ◽  
Microparticle Formation

SummaryHeparin-induced thrombocytopenia (HIT) is an important complication following administration of heparin. Platelet activation and aggregation induced by heparin/platelet factor 4/immunoglobulin complexes are thought to be the underlying mechanism for this condition, so it was hypothesized that abciximab (a humanized murine monoclonal antibody directed against the glycoprotein IIb/IIIa receptor) would prevent heparin-induced platelet aggregation and activation in plasma from patients with HIT. Platelet aggregation was tested in vitro with platelet-poor plasma (obtained from 23 patients with HIT), platelet-rich plasma (from normal donors with known reactivity), heparin (0.5 U/ml), and ascending doses of abciximab (0.07-0.56 μg/ml). The ability of abciximab to prevent platelet activation was also evaluated using flow cytometry (P selectin expression, mepacrine release, microparticle formation) and platelet factor 4 immunoassay. In vitro, abciximab inhibited heparin-induced platelet aggregation in a dose-dependent fashion (IC50 0.103 μg/ml) and inhibited microparticle formation, the expression of P-selectin, release of mepacrine and platelet factor 4. These findings suggest that abciximab may be useful in treatment of patients with HIT and warrants further clinical evaluation.

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