Comparative Studies on the HIT Antibody Mediated Platelet Aggregation / Serotonin Release by Synthetic Pentasaccharide and Two Chemoenzymatically Synthesized Heptasaccharides

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2231-2231
Author(s):  
Jeanine M. Walenga ◽  
Chris Aranda ◽  
Robert Linhardt ◽  
Jian Liu ◽  
Mary Lewis ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Whole Blood ◽  
Conflicts Of Interest ◽  
Positive Control ◽  
Serotonin Release ◽  
Platelet Factor 4 ◽  
Aggregation Assay ◽  
Platelet Factor ◽  
Synthetic Oligosaccharides ◽  
Induced Aggregation

Abstract Abstract 2231 Synthetic oligosaccharides such as the Pentasaccharide (Arixtra) and its derivatives are antithrombotic agents which are clinically used in the management of thrombotic indications. These agents are claimed to be devoid of triggering the generation of HIT antibodies and therefore do not produce HIT syndrome. Several additional synthetic oligosaccharides are also developed for the management of thrombotic indications. More recently, two novel ultra low molecular weight heparins (ULMWHs) were synthesized chemoenzymatically. These ULMWHs are both heptasaccharides with AT pentasaccharide-binding sites within their structures which is comparable to the pentasaccharide. The IC50 of the anti-Xa effects of the agents are comparable to pentasaccharide, ranging from 0.7 to 1.0 ug/ml in comparison to pentasaccharide which is 0.8 ug/ml. All agents produced comparable anticoagulant effects in the Heptest clotting time. The purpose of this study is to compare the effects of the pentasaccharide and the two heptasaccharides namely ULMWH1 and ULMW2 in the HIT mediated platelet aggregation and serotonin release assays. In addition platelet factor 4 release in whole blood was also studied. The HIT mediated platelet aggregation studies were carried out utilizing a HIT antibody positive pool plasma preparation. PRP collected from 10 individual donors (250ul) was mixed with 200ul of HIT pool plasma and equilibrated at 37° C for 3 minutes. 50ul of 1, 10, and 100 ug/ml of each of these agents was added to trigger the platelet aggregation responses. Enoxaparin was used as a positive control in the same concentration ranges. The serotonin release assay was carried out using the standard method in the same concentration range monitoring the release of 14C serotonin with each of these agents. The PF4 release was also measured using an ELISA method for serotonin measurement in whole blood samples incubated with each of these agents at concentrations of 0, 10 and 100ug/ml. The pentasaccharide and the two heptasaccharides did not produce any aggregation of platelets in the HIT aggregation assay at all concentrations whereas Enoxaparin at concentrations of > 1ug/ml produces positive aggregation responses. In the 14C assay none of the agents produced any release of serotonin however Enoxaparin produced 14C release at all concentration studied. Similarly, the pentasachhardide and heptasaccharides did not produce any platelet factor 4 from the whole blood incubation studies, however Enoxaparin produced a measurable release of platelet factor 4. Interestingly, unlike Enoxaparin, the anti-Xa and heptest effects of these agents were not neutralized by platelet factor 4 or protamine sulfate. These results demonstrate that the pentasaccharide and chemoenzymatically synthesized ULMWH1 and ULMWH2 do not meditate HIT antibody induced aggregation and serotonin release. Therefore, these heptasaccharides may exhibit comparable safety profile to the pentasaccharide in heparin compromised patients. Disclosures: No relevant conflicts of interest to declare.

scholarly journals A Humanized Anti FcγRIIa Scfv Inhibits Platelet Activation, Aggregation and Thrombus Formation in Heparin-Induced Thrombocytopenia (HIT)

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 10-10
Author(s):  
Jose Perdomo ◽  
Jaa Yien New ◽  
Zohra Ahmadi ◽  
Xing-Mai Jiang ◽  
Beng H Chong
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Immune Complexes ◽  
Whole Blood ◽  
Conflicts Of Interest ◽  
Dependent Manner ◽  
Single Chain ◽  
Platelet Factor ◽  
Heparin Induced Thrombocytopenia ◽  
Micro Fluidics

Abstract Introduction. Heparin is widely used as an anticoagulant to prevent thrombosis and to treat venous thromboembolism and myocardial infarction. A complication of heparin use is the development of heparin-induced thrombocytopenia (HIT), which is a limb- and life-threatening disorder due to associated thrombotic events. HIT arises through the formation of immune complexes between heparin, platelet factor 4 and HIT autoantibodies. These immune complexes engage with FcγRIIa receptors on platelets, leading to platelet activation and aggregation and subsequent initiation of the coagulation pathway. Current HIT treatment consists of cessation of heparin administration and substitution with parenteral anticoagulants such as argatroban and danaparoid. While these anticoagulants are generally beneficial in reducing thrombocytopenia, they are only partially effective since the risk of thrombosis continues due to the underlying FcγRIIa-mediated platelet activation. Thus, alternative anticoagulants do not reduce morbidity and mortality rates, highlighting the need for more effective HIT interventions. Methods. IV.3 is a monoclonal antibody that recognizes and blocks the FcγRIIa receptor and is used in assays to confirm the presence of HIT antibodies. We derived the VH and VL sequences of IV.3 and constructed a single-chain variable fragment (scFv) antibody in the form of VH-linker-VL. Using a complementarity determining region grafting and point mutation approach the scFv was humanized with the aim of reducing potential immunogenicity for future clinical applications. The molecule was expressed in E. coli and purified by FPLC. We reconstituted the HIT condition in a micro-fluidics device on a Vena8 Fluoro+ biochip coated with vWf using whole blood flowing at 20 dyne/cm2 at 37oC. Whole blood was stained with DiOC6 and the formation of platelet aggregates was monitored by fluorescence microscopy. Video images were acquired at 1 frame every 2 sec for 460 sec. Results. The purified scFv interacts with FcγRIIa on platelets. Platelet aggregation and serotonin release assays show that the scFv effectively prevents aggregation and activation induced by HIT immune complexes. We demonstrate that in the HIT condition reconstituted in a micro-fluidics system the scFv precludes thrombus deposition in a dose-dependent manner as determined by thrombus coverage area and mean thrombus diameter (Figure 1). Conclusions. These data provide evidence that a humanized scFv binds and neutralizes FcγRIIa on platelets. This interaction prevents HIT immune complex-induced platelet aggregation and activation in vitro and stops thrombus deposition ex vivo. This molecule, therefore, inhibits a critical initiating event in HIT and may serve as a potential treatment for this condition. Disclosures No relevant conflicts of interest to declare.

EFFECT OF HEPARIN-PROTAMINE VERSUS HEPARIN-PLATELET FACTOR 4 COMPLEXES ON WHOLE BLOOD PLATELET AGGREGATION

1994 ◽  
Vol 81 (SUPPLEMENT) ◽  
pp. A635
Author(s):  
B. A. Ryan ◽  
M. N. DʼAmbra ◽  
R. M. LaRue ◽  
K. E. Lynch ◽  
T. E. Maione
Keyword(s):  
Platelet Aggregation ◽  
Whole Blood ◽  
Blood Platelet ◽  
Platelet Factor 4 ◽  
Platelet Factor ◽  
Blood Platelet Aggregation

scholarly journals Cleavage of Anti-PF4/H IgG Antibodies By Ides, and Potential Benefit in the Treatment of Heparin-Induced Thrombocytopenia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 518-518
Author(s):  
Claire Kizlik-Masson ◽  
Quentin Deveuve ◽  
Yuhang Zhou ◽  
Caroline Vayne ◽  
Gilles Thibault ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Whole Blood ◽  
Cell Activation ◽  
Conflicts Of Interest ◽  
Microfluidic Channels ◽  
Venous Flow ◽  
Platelet Factor ◽  
Cellular Activation ◽  
Heparin Induced Thrombocytopenia ◽  
Impedance Aggregometry

Abstract Heparin-induced thrombocytopenia (HIT) is a severe drug-adverse event due to platelet-activating antibodies (Abs) directed against platelet factor 4 (PF4)/heparin (H) complexes. In most patients, HIT Abs are IgG that directly activate platelets and monocytes in the presence of heparin via FcγRIIA receptors. The interaction between the Fc fragment of anti-PF4/H IgG and FcγRIIA is thus a key step for cellular activation in HIT. Several bacterial proteases such as IdeS (IgG-degrading enzyme of Streptococcus pyogenes) are cleaving IgG in the lower hinge region of heavy chain leading to the formation of single cleaved IgG (scIgG) and then of Fab'2. Importantly, cleavage of IgG by IdeS can abolish their ability to bind FcγR and suppress the cellular effects resulting from this interaction. The aim of this study was therefore to evaluate whether anti-PF4/H IgG cleavage by IdeS could inhibit cell activation induced by HIT antibodies, and their pathogenicity. To achieve this objective, we studied the effects of IdeS on platelet responses to 5B9, a monoclonal chimeric anti-PF4/H IgG1 recently developed in our laboratory, and which fully mimics the effects of human HIT antibodies (Kizlik-Masson et al, J Thromb Haemost, 2017). IdeS was demonstrated to quickly (6 minutes) cleave purified 5B9 IgG, leading to the formation of sc5B9, without any reduction in its binding ability to PF4/H complex. However, flow cytometry experiments showed that heparin-dependent binding of sc5B9 to platelets and FcγRIIA was dramatically reduced compared to those of uncleaved 5B9. In addition, functional assays (serotonin release assays and platelet aggregation tests) also confirmed that sc5B9 was unable to induce platelet activation and aggregation in the presence of heparin. Incubation of IdeS (0.02 U/µg of IgG; 6 minutes) in whole blood containing 5B9 IgG or HIT plasma samples also lead in every sample tested to the cleavage of anti-PF4/H Abs, which fully abolished their capacity to induce heparin-dependent platelet aggregation, as demonstrated by impedance aggregometry (Multiplate analyzer). As expected, no effect of IdeS was observed on platelet aggregation induced by collagen (1 µg/mL), or ADP (10 µM). Moreover, tissue factor (TF) gene expression induced in monocytes by 5B9 in the presence of heparin was also completely abolished after addition of IdeS (0.02 U/µg of IgG; 6 minutes) in whole blood, whereas no inhibitory effect of this protease on TF expression induced by LPS was evidenced. We also showed that platelet aggregation and fibrin formation induced by 5B9 with heparin was completely inhibited after IdeS treatment when whole blood was perfused in vWF-coated microfluidic channels with shear rates similar to those of venous flow (500s-1). Finally, IdeS was also showed to prevent efficiently thrombocytopenia and hypercoagulability (with no increase in thrombin/anti-thrombin plasma levels) induced by 5B9 in transgenic mice expressing human PF4 and FcγRIIA receptors, when previously treated by this protease (0.5 µg/g) before IV injection of heparin. In conclusion, the cleavage of anti-PF4/H IgG by IdeS prevents heparin-dependent cellular activation induced by HIT antibodies, thereby reducing their pathogenicity. Therefore, injection of IdeS could be considered as a potential treatment in patients with severe HIT, particularly in those who necessitate emergent cardiac surgery with cardiopulmonary bypass and thus anticoagulation with unfractionated heparin, which remains the safest and easiest anticoagulant to be used in this specific surgical procedure. Disclosures No relevant conflicts of interest to declare.

scholarly journals Dabigatran Neutralizing Antobody, Idarucizumab, Exhibits Procoagulant and Platelet Activation Responses in Whole Blood. Potential Clinical Implications

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2622-2622
Author(s):  
Zafar Siddiqui ◽  
Omer Iqbal ◽  
Debra Hoppensteadt ◽  
Mary Lewis ◽  
Rohan Rege ◽  
...  
Keyword(s):  
Arachidonic Acid ◽  
Platelet Aggregation ◽  
Blood Coagulation ◽  
Platelet Activation ◽  
Whole Blood ◽  
Conflicts Of Interest ◽  
Platelet Rich Plasma ◽  
Antibody Fragment ◽  
Platelet Factor ◽  
Activation Profile

Abstract Introduction: Idarucizumab is a humanized monoclonal antibody fragment which is capable of neutralizing Dabigatran and is currently clinically available for the control of bleeding associated with Dabigatran. Although this antibody is capable of neutralizing the anticoagulant effects of Dabigatran, its effect on the blood coagulation and platelet activation profile are not completely understood. There is limited data on the effect of Idarucizumab on the blood coagulation and platelet activation profile. The purpose of this study is to determine the effect of this antibody on blood coagulation and platelet activation profile. Materials: Idarucizumab was purchased from Brigham and Women's Hospital (Boston, MA) and was provided as a 50 mg/mL solution. Dabigatran was of synthetic origin and obtained from Sellec Chemical (Houston, TX). Whole blood from healthy volunteers was collected in plastic syringes using a sterile method for the TEG and ACT analysis. Citrated whole blood was used for the preparation of platelet rich plasma which was used in platelet aggregation studies. Such agonists as arachidonic acid, ADP, epinephrine, thrombin, and collagen were used. Methods: The TEG studies were carried on a Haemoscope 500 instrument. Native whole blood was supplemented with Idarucizumab in a concentration range of 0-10 mg/mL. Saline was used as a control. The TEG profile was measured for 15-30 minutes. Such parameters as R time, K time, angle, and max amplitude were recorded. The ACT studies were carried out in celite tubes in a concentration of 0-5 mg/mL. The agonist-induced platelet aggregation profile was studied by pre incubating platelet rich plasma with Idarucizumab at a fixed concentration of 1.0 mg/mL and studying its effect on the aggregation profile of such agonists as arachidonic acid, ADP, epinephrine, thrombin, and collagen. Both the slope and percent aggregation were measured. The effect of Idarucizumab on HIT antibodies mediated platelet aggregation was studied by pre incubating platelets with Idarucizumab and determining its effect on the HIT antibody mediated aggregation of platelets. Pooled plasma from symptomatic HIT patients was pre incubated with Idarucizumab at 1.0 mg/mL followed by the addition of HIT antibody pool in a 1:10 dilution. The aggregation profile was noted for up to an hour. Results: Idarucizumab produced a dose-dependent hypercoagulable effect in the TEG profile of native whole blood resulting in a reduction in R time and max amplitude at 35% and 45% respectively. At high concentrations, Idarucizumab produced a marked effect on the clot retraction. In the ACT studies, Idarucizumab produced a mild shortening of the ACT at a 5 mg/mL at 6%. Idarucizumab produced variable augmentation of different agonists mediated platelet aggregation. In the HIT mediated aggregation studies, Idarucizumab produced a strong augmentation of HIT antibody mediated platelet aggregation. At 1.0 ug/mL, Idarucizumab produced almost a 20% increase in platelet aggregation. Conclusions: These studies indicate that Idarucizumab produces mild procoagulant effects on whole blood coagulation process as studied by TEG and ACT. This agent also produces the augmentation of the platelet aggregation profile by various agonists including Anti-heparin platelet factor IV antibodies. These procoagulant effects of Idarucizumab may contribute to the potential hypercoagulable/prothrombotic events associated with its use. Disclosures No relevant conflicts of interest to declare.

Platelet Coagulant Activities and Retinal Vein Thrombosis

1977 ◽  
Vol 38 (02) ◽  
pp. 0399-0406 ◽  
Author(s):  
Peter N. Walsh ◽  
Richard E. Goldberg ◽  
Richard L. Tax ◽  
Larry E. Magargal
Keyword(s):  
Platelet Aggregation ◽  
Venous Thrombosis ◽  
Retinal Vein Occlusion ◽  
Serotonin Release ◽  
Platelet Factor ◽  
Trigger Mechanism ◽  
Local Conditions ◽  
Plasma Coagulation ◽  
Vein Thrombosis ◽  
Vein Occlusion

SummaryTo determine whether platelets play a role in the pathogenesis of retinal vein occlusion (RVO), platelets and coagulation were evaluated in 28 patients with RVO. Platelet coagulant activities concerned with the initiation and early stages of intrinsic coagulation were 2–4 fold increased in 9 patients with acute primary RVO but not in patients with acute secondary (10 patients) or chronic (9 patients) RVO. Platelet factor 3 activity, platelet aggregation, serotonin release by platelets and plasma coagulation were normal in all patients. Platelets may provide a trigger mechanism for venous thrombosis in the eye when local conditions permit.

Dipyridamole Inhibits Platelet Aggregation in Whole Blood

1983 ◽  
Vol 50 (04) ◽  
pp. 852-856 ◽  
Author(s):  
P Gresele ◽  
C Zoja ◽  
H Deckmyn ◽  
J Arnout ◽  
J Vermylen ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Whole Blood ◽  
Ex Vivo ◽  
Platelet Rich Plasma ◽  
Significant Negative Correlation ◽  
Significant Inhibition ◽  
Oral Drug ◽  
Human Blood Samples ◽  
Induced Aggregation

SummaryDipyridamole possesses antithrombotic properties in the animal and in man but it does not inhibit platelet aggregation in plasma. We evaluated the effect of dipyridamole ex vivo and in vitro on platelet aggregation induced by collagen and adenosine- 5’-diphosphate (ADP) in human whole blood with an impedance aggregometer. Two hundred mg dipyridamole induced a significant inhibition of both ADP- and collagen-induced aggregation in human blood samples taken 2 hr after oral drug intake. Administration of the drug for four days, 400 mg/day, further increased the antiplatelet effect. A significant negative correlation was found between collagen-induced platelet aggregation in whole blood and dipyridamole levels in plasma (p <0.001). A statistically significant inhibition of both collagen (p <0.0025) and ADP-induced (p <0.005) platelet aggregation was also obtained by incubating whole blood in vitro for 2 min at 37° C with dipyridamole (3.9 μM). No such effects were seen in platelet-rich plasma, even after enrichment with leukocytes. Low-dose adenosine enhanced in vitro inhibition in whole blood.Our results demonstrate that dipyridamole impedes platelet aggregation in whole blood by an interaction with red blood cells, probably involving adenosine.

Platelet Aggregation, β-Thromboglobulin and Platelet Factor 4 in Diabetes Mellitus and in Patients with Vasculopathy

1984 ◽  
Vol 52 (03) ◽  
pp. 236-239 ◽  
Author(s):  
J Fritschi ◽  
M Christe ◽  
B Lämmle ◽  
G A Marbet ◽  
W Berger ◽  
...  
Keyword(s):  
Diabetes Mellitus ◽  
Platelet Aggregation ◽  
Discriminant Analysis ◽  
Plasma Levels ◽  
Platelet Activating Factor ◽  
Platelet Factor 4 ◽  
Platelet Factor ◽  
Aggregation Response ◽  
The Difference

SummaryWe have studied 155 subjects, 48 normals, 36 diabetics without complications, 44 with complications and 27 patients with macroangiopathy. β-Thromboglobulin (β-TG) and platelet factor 4 (PF4) are elevated in the patients groups. There is no correlation between the plasma levels of β-TG and the stages of either retinopathy or macroangiopathy or nephropathy. The difference is more marked between normals and diabetics with neuropathy (p = 0.026). The aggregation response to ADP and platelet activating factor (PAF) is enhanced at lower stimulator concentration. Using the β-TG, PF4 and aggregation values the discriminant analysis allows a distinction of several subgroups especially with nephropathy and neuropathy (Table 6).

Serotonin‐release assay‐positive but platelet factor 4‐dependent enzyme‐immunoassay negative: HIT or not HIT ?

2020 ◽  
Author(s):  
Theodore E. Warkentin ◽  
Maureen A. Smythe ◽  
Mona A. Ali ◽  
Naveed Aslam ◽  
Jo‐Ann I. Sheppard ◽  
...  
Keyword(s):  
Enzyme Immunoassay ◽  
Serotonin Release ◽  
Platelet Factor 4 ◽  
Platelet Factor ◽  
Release Assay
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