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.

Abstract 13: Suppression of Atherosclerosis by CD39

2014 ◽  
Vol 34 (suppl_1) ◽  
Author(s):  
Yogendra Kanthi ◽  
Matthew Hyman ◽  
Hui Liao ◽  
Amy Baek ◽  
Scott Visovatti ◽  
...  
Keyword(s):  
Shear Stress ◽  
Platelet Activation ◽  
Receptor Expression ◽  
Plaque Burden ◽  
Laminar Shear Stress ◽  
Shear Forces ◽  
Lipoprotein Uptake ◽  
Laminar Shear ◽  
Deficient Mice

Atherosclerotic plaque rupture and thrombosis remains the leading cause of death in the United States. We investigated the role of CD39, a potent ecto-enzymatic regulator of platelet activation and leukocyte trafficking, in atherosclerosis. We generated mice deficient in CD39 on a hyperlipidemic, apoE -/- background and noted a two-fold higher plaque burden in when compared to apoE -/- controls ( P =0.003). We noted higher levels of circulating markers of platelet activation, soluble P-selectin (39%) and RANTES (60%), in the CD39-deficient mice ( P =0.003 and P =0.015, respectively, n=7-11). CD39-haploinsufficient mice had 1.8-fold greater enhanced platelet reactivity in response to ADP compared to controls ( P =0.03, n=3-8). Macrophages from CD39-deficient mice had significantly higher lipoprotein uptake in vitro . Correspondingly, CD39 overexpression in RAW cells inhibited scavenger receptor expression and lipoprotein uptake. Altered fluid mechanics contribute to atherosclerosis, with non-laminar shear stress enhancing regional plaque formation as seen in arterial bifurcations. We examined coronal sections of aortas from apoE −/− mice and observed that CD39 is poorly expressed in the endothelium in regions of turbulent blood flow, where plaque develops, supporting our hypothesis that endothelial CD39 can be induced by fluid phase shear forces. HUVEC treated with physiologic laminar shear stress (LS) (15 dynes/cm 2 ) had a 5.9-fold increase in CD39 protein ( P =0.004, N=3-7) and a concordant increase in nucleotidase activity ( P =0.03 N=3) compared to static controls (SS). We identified Krüppel like factor 2 (KLF2) as an upstream candidate for transcriptional regulation of CD39 induction by fluid shear forces. Silencing KLF2 in vitro led to a 55% decrease in CD39 mRNA induction with LS vs SS controls ( P =0.002, N=3-4). Chromatin immunoprecipitation revealed that KLF2 binds to the CD39 ( P =0.01, N=3) and this binding was further enhanced under laminar shear stress ( P =0.0007, N=3). These data show that CD39, an anti-thrombotic, anti-inflammatory enzyme is a critical regulator of atherosclerosis by modulating platelet, macrophage and endothelial function and mechanistically identify KLF2 as a direct, upstream regulator of CD39 expression.

Potentiation by red blood cells of shear-induced platelet aggregation: relative importance of chemical and physical mechanisms

Blood ◽  
1984 ◽  
Vol 64 (6) ◽  
pp. 1200-1206 ◽  
Author(s):  
RC Reimers ◽  
SP Sutera ◽  
JH Joist
Keyword(s):  
Shear Stress ◽  
Platelet Aggregation ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Platelet Rich Plasma ◽  
Adenine Nucleotides ◽  
Laminar Shear Stress ◽  
Platelet Surface ◽  
Surface Transport ◽  
Laminar Shear

Abstract Evidence has been reported to indicate that red blood cells (RBCs) may potentiate platelet adherence and platelet aggregation (PAG) in different flow systems in vitro as well as hemostatic platelet plug formation in response to vascular injury. In this study, we demonstrate that RBCs enhance PAG induced by well-defined, low-intensity, uniform, laminar shear stress. Potentiation by RBCs of shear-induced PAG was associated with appreciable loss of adenine nucleotides from 14C- adenine-labeled RBCs, the extent of which increased with increasing RBC concentration. The concentrations of RBC-derived ADP measured in the medium after shear, as determined by both high pressure liquid chromatography and the luciferin/luciferase system, were within the range of concentrations of ADP which may trigger PAG or potentiate PAG induced by low concentrations of other platelet agonists in the aggregometer. To assess the relative contribution of chemical (ADP) and physical (platelet surface transport) mechanisms in the RBC-mediated potentiation of shear-induced PAG, aliquots of citrated platelet-rich plasma (C-PRP) were exposed to shear stress in the presence of untreated RBCs or RBCs exposed to an antihemolytic concentration (5 mumol/L) of the membrane stabilizing agent, chlorpromazine (CPZ). Potentiation of shear-induced PAG in the RBC-CPZ system was significantly less than that in the untreated RBC system. However, CPZ- induced reduction of PAG potentiation was associated with an increase rather than a decrease in loss of adenine nucleotides from RBC. Furthermore, shear-induced PAG in C-PRP as well as ADP- and collagen- induced PAG in C-PRP in the aggregometer was significantly inhibited by 5 mumol/L CPZ, indicating that the observed reduced potentiation of shear-induced PAG by RBCs in the presence of CPZ was due to a direct inhibitory effect of the drug on platelets rather than a reduction of shear-induced liberation of ADP from RBCs. When aliquots of C-PRP were exposed to shear stress in the presence of RBCs completely depleted of ADP by fixation in 1% glutaraldehyde, potentiation of PAG was approximately half of that observed with intact RBCs. These findings indicate that both RBC-derived ADP and RBC-mediated platelet surface transport are involved in the potentiation by RBCs of PAG induced by laminar shear stress.

scholarly journals Potentiation by red blood cells of shear-induced platelet aggregation: relative importance of chemical and physical mechanisms

Blood ◽  
1984 ◽  
Vol 64 (6) ◽  
pp. 1200-1206 ◽  
Author(s):  
RC Reimers ◽  
SP Sutera ◽  
JH Joist
Keyword(s):  
Shear Stress ◽  
Platelet Aggregation ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Platelet Rich Plasma ◽  
Adenine Nucleotides ◽  
Laminar Shear Stress ◽  
Platelet Surface ◽  
Surface Transport ◽  
Laminar Shear

Evidence has been reported to indicate that red blood cells (RBCs) may potentiate platelet adherence and platelet aggregation (PAG) in different flow systems in vitro as well as hemostatic platelet plug formation in response to vascular injury. In this study, we demonstrate that RBCs enhance PAG induced by well-defined, low-intensity, uniform, laminar shear stress. Potentiation by RBCs of shear-induced PAG was associated with appreciable loss of adenine nucleotides from 14C- adenine-labeled RBCs, the extent of which increased with increasing RBC concentration. The concentrations of RBC-derived ADP measured in the medium after shear, as determined by both high pressure liquid chromatography and the luciferin/luciferase system, were within the range of concentrations of ADP which may trigger PAG or potentiate PAG induced by low concentrations of other platelet agonists in the aggregometer. To assess the relative contribution of chemical (ADP) and physical (platelet surface transport) mechanisms in the RBC-mediated potentiation of shear-induced PAG, aliquots of citrated platelet-rich plasma (C-PRP) were exposed to shear stress in the presence of untreated RBCs or RBCs exposed to an antihemolytic concentration (5 mumol/L) of the membrane stabilizing agent, chlorpromazine (CPZ). Potentiation of shear-induced PAG in the RBC-CPZ system was significantly less than that in the untreated RBC system. However, CPZ- induced reduction of PAG potentiation was associated with an increase rather than a decrease in loss of adenine nucleotides from RBC. Furthermore, shear-induced PAG in C-PRP as well as ADP- and collagen- induced PAG in C-PRP in the aggregometer was significantly inhibited by 5 mumol/L CPZ, indicating that the observed reduced potentiation of shear-induced PAG by RBCs in the presence of CPZ was due to a direct inhibitory effect of the drug on platelets rather than a reduction of shear-induced liberation of ADP from RBCs. When aliquots of C-PRP were exposed to shear stress in the presence of RBCs completely depleted of ADP by fixation in 1% glutaraldehyde, potentiation of PAG was approximately half of that observed with intact RBCs. These findings indicate that both RBC-derived ADP and RBC-mediated platelet surface transport are involved in the potentiation by RBCs of PAG induced by laminar shear stress.

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.

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 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.

PLATELET ALTERATIONS IN RESPONSE TO REPETITIVE, SHORT-DURATION LAMINAR SHEAR STRESS

1987 ◽  
Author(s):  
J H Joist ◽  
J E Bauman ◽  
S P Sutera
Keyword(s):  
Shear Stress ◽  
Pulse Duration ◽  
Platelet Rich Plasma ◽  
Pulse Amplitude ◽  
Pulse Number ◽  
Dense Granule ◽  
Laminar Shear Stress ◽  
Ramp Function ◽  
Laminar Shear ◽  
Granule Release

We examined platelet aggregation (PAG = loss of single platelets), platelet dense granule release, and platelet injury (LDH loss) in normal human citrated platelet-rich plasma subjected to biologically more relevant repetitive, laminar shear stress of 25 and 50 dyn/cm2 in a computer-controlled cone-plate viscometer. Shear pulse duration (1-3 sec), shear pulse ramp function (rate of shear stress increase and decrease per pulse, 0.6-4 sec), number of shear pulses (1-20) and pauses between shear pulses (0-5 sec) were varied in different combinations to assess the effects of each variable on platelet alterations. Maximum PAG (92±8%) was observed with three 1 sec shear pulses, 0.6 sec ramp function and 1 sec between shear pause. PAG decreased with increasing ramp function, increasing number of shear pulses (>10), and increasing pause duration. Rapid platelet deaggregation (starting at 5 sec) was observed after a single 1 sec shear exposure. The rate of deaggregation decreased with increasing shear pulse number, increasing shear pulse amplitude, and increasing shear pulse duration. In contrast to PAG, dense granule release increased progressively with increasing shear pulse number, duration, and amplitude. No appreciable platelet injury (LDH loss) was observed under the conditions used. The findings indicate that massive reversible PAG can be induced by a single 1 sec shear pulse and that the extent of PAG with more prolonged, repetitive shear exposure is largely a function of platelet deaggregation rather than PAG. Thus, data previously reported from our laboratory and other investigators using prolonged (>5 sec) exposure of platelets to shear stress may require reevaluation.

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.

Abstract 1946: Regulation OF Entpdase1 (CD39) Expression by Fluid-phase Shear Forces

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Matthew C Hyman ◽  
Chih-Wen Ni ◽  
Scott H Visovatti ◽  
Douglas Nam ◽  
Mithun K Neral ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Western Blotting ◽  
Umbilical Vein ◽  
Fluid Phase ◽  
Shear Stresses ◽  
Laminar Shear Stress ◽  
Shear Forces ◽  
Fluorescent Intensity ◽  
Fold Induction ◽  
Laminar Shear

Sudden death due to thrombotic occlusion of a ruptured plaque is the leading cause of mortality in patients with atherosclerosis. Little is known about how the apyrase ENTPDase1 (CD39), a primary regulator of platelet activation in vivo , is affected at the plaque surface. By examining coronal sections of atherosclerotic plaques from apoE −/− mice, we determined that CD39 is lost specifically in endothelium overlying the plaque surface, but not in adjacent endothelium. This led to the hypothesis that CD39 expression in the vascular wall may be driven by laminar, fluid-phase shear forces. To distinguish the effects of hyperlipidemia from that of shear forces, coronal aortic arch sections from wild type mice were examined at the greater curvature (an area defined by laminar shear stresses) and the ostia of the left subclavian artery (an area defined by non-laminar blood flow). Fluorescent intensity quantification revealed a near 6-fold induction of CD39 staining in areas exposed to laminar shear stresses. This finding was confirmed by confocal microscopy of en face aorta preparations as well. In vitro studies with human umbilical vein endothelial cells in a cone plate viscometer showed a 10.8-fold induction of CD39 mRNA by laminar shear stress (~15 dyne/cm 2 ) when compared to fluid stasis (p<0.001). By comparison oscillatory flow (0 ± 5 dyne/cm 2 , 1 Hz) did not induce a significant change in CD39 mRNA when compared to static control. Western blotting revealed that the CD39 mRNA induced by laminar shear manifested as a more than 4.3-fold induction of protein (p<0.01). Flow cytometry confirmed the western blotting results showing a 3.8-fold induction of CD39 protein on the surface of cells exposed to laminar shear. These are the first data to show that CD39 is regulated by shear, and that it is lost from endothelium overlying atherosclerotic plaque in regions of turbulent flow. These results could be of great clinical relevance as they demonstrate that CD39, the enzyme that prevents aberrant platelet activation, is lost on the plaque surface. These data support the hypothesis that regional vascular zones are athero- and thrombo-resistant due to local ectonucleotidase expression modulated by dynamic/shear forces in flowing blood. This research has received full or partial funding support from the American Heart Association, AHA Midwest Affiliate (Illinois, Indiana, Iowa, Kansas, Michigan, Minnesota, Missouri, Nebraska, North Dakota, South Dakota & Wisconsin).

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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