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

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.

Download Full-text

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

Blood ◽

10.1182/blood.v126.23.10.10 ◽

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.

Download Full-text

Comparison of effect of aloe Vera gel with aspirin and celecoxib on platelet aggregation.

The Professional Medical Journal ◽

10.29309/tpmj/2020.27.05.4007 ◽

2020 ◽

Vol 27 (05) ◽

pp. 973-978

Author(s):

Sidra Mushtaq ◽

Zobia Mushtaq ◽

Javeria Arif ◽

Mufakhara Fatima ◽

Sadida Bahawal ◽

...

Keyword(s):

Arachidonic Acid ◽

Platelet Aggregation ◽

Whole Blood ◽

Blood Count ◽

Complete Blood Count ◽

Platelet Rich Plasma ◽

Aloe Vera ◽

Medical Institute ◽

Aloe Vera Gel ◽

Study Setting

Objective: This study was designed to compare the effect of Aloe vera gel with aspirin and celecoxib on platelet aggregation. Study Design: Comparative Study. Setting: Post graduate Medical Institute Lahore, Children Hospital, Lahore. Period: September 2015 to September 2016. Material & Methods: Blood was withdrawn from anti-cubital vein, complete blood count was checked, platelet rich plasma was prepared by centrifuging citrated whole blood and then incubated with Aloe vera low (AVL), Aloe vera high (AVH), aspirin and celecoxib for 30 minutes at 37C. After adding the agonist arachidonic acid, reading was then taken for 3 minutes and percentage aggregation was recorded. Results: Platelet aggregation with aspirin, AVH and AVL was statistically significantly lower as compared to control and celecoxib groups. Conclusion: This study has demonstrateda dose dependentanti-platelet effect of Aloe vera gel which is comparable to aspirin.

Download Full-text

Fucosyltransferase VI Induces Platelet Activation: A Novel Property of a Plasma Glycosyltransferase.

Blood ◽

10.1182/blood.v114.22.4016.4016 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4016-4016

Author(s):

José-Tomás Navarro ◽

Shwan Tawfiq ◽

Roland Wohlgemuth ◽

Karin M. Hoffmeister ◽

Robert Sackstein

Keyword(s):

Flow Cytometry ◽

Platelet Aggregation ◽

Platelet Activation ◽

Light Transmission ◽

Conflicts Of Interest ◽

Platelet Rich Plasma ◽

Surface Protein ◽

Surface Flow ◽

Platelet Surface ◽

Biochemical Studies

Abstract Abstract 4016 Poster Board III-952 A number of glycosyltransferases are present in human plasma with the α(1→3) fucosyltransferase, Fucosyltransferase VI (FTVI), having the highest plasma concentration. Notably, elevated plasma levels of FTVI are associated with a variety of cancers and correlate with tumor load/progression. The well-known association of neoplasia with thromboembolic complications prompted us to examine whether FTVI has direct effect(s) on platelet function. We obtained human platelets from blood of healthy donors and separated from platelet-rich plasma by differential centrifugation. Freshly isolated platelets (x108/ml) were stirred and exposed at 37°C to varying concentrations (20, 40, 60 and 80 mU/mL) of glycosyltransferases FTVI, β-1-4-galactosyltransferase-I (βGalT-I), or α,2-3-N-sialyltransferase (α2,3-N-ST), or to 1 U/mL thrombin. Platelet aggregation and activation was assessed by aggregometry (light transmission) or by flow cytometry of FSC/SSC characteristics and of surface expression of P-Selectin, respectively. FT-VI reproducibly induced platelet aggregation and activation, whereas other glycosyltransferases (β4GalT-I and α2,3-N-ST) had no effect on platelets. FTVI activation of platelets was concentration-dependent, and the aggregation curve for FTVI was one wave, similar to that for thrombin. FTVI-induced platelet activation was independent of catalytic conversion of surface glycans, but was inhibited by FTVI denaturation, indicating that FTVI-induced platelet activation is a lectin-mediated process. To determine the membrane target(s) mediating FTVI-induced platelet activation, biochemical studies were performed after catalytic exofucosylation of the platelet surface. Flow cytometry after platelet exofucosylation showed formation of the carbohydrate structure sLex, detected by the mAb Heca452, but no formation of Lex (CD15). Western blot showed that enforced fucosylation induced sLex on a single platelet surface protein, and further biochemical studies revealed that this protein is GPIbα. These findings unveil a previously unrecognized property of FTVI as an activator of platelets, mediated via a specific lectin/carbohydrate interaction on GP1ba, and offer novel perspectives on the pathobiology of tumor-associated thrombogenesis. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

The active metabolite of prasugrel inhibits ADP-stimulated thrombo-inflammatory markers of platelet activation: Influence of other blood cells, calcium, and aspirin

Thrombosis and Haemostasis ◽

10.1160/th07-01-0010 ◽

2007 ◽

Vol 98 (07) ◽

pp. 192-200 ◽

Cited By ~ 33

Author(s):

Joseph Jakubowski ◽

You FuLi ◽

Marc Barnard ◽

Marsha Fox ◽

Matthew Linden ◽

...

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Whole Blood ◽

Blood Cells ◽

Inflammatory Markers ◽

Active Metabolite ◽

Platelet Rich Plasma ◽

Shape Change ◽

Flow Cytometric Analysis ◽

Platelet Surface

SummaryThe novel thienopyridine prodrug prasugrel, a platelet P2Y12 ADP receptor antagonist, requires in vivo metabolism for activity. Although pharmacological data have been collected on the effects of prasugrel on platelet aggregation,there are few data on the direct effects of the prasugrel’s active metabolite, R-138727, on other aspects of platelet function. Here we examined the effects of R-138727 on thrombo-inflammatory markers of platelet activation, and the possible modulatory effects of other blood cells, calcium, and aspirin. Blood (PPACK or citrate anticoagulated) from healthy donors pre- and post-aspirin was incubated with R-138727 and the response to ADP assessed in whole blood or platelet-rich plasma (PRP) by aggregometry and flow cytometric analysis of leukocyte-platelet aggregates,platelet surface P-selectin, and GPIIb-IIIa activation. Low-micromolar concentrations of R-138727 resulted in a rapid and consistent in-hibition of these ADP-stimulated thrombo-inflammatory markers.These rapid kinetics required physiological calcium levels, but were largely unaffected by aspirin. Lower IC50 values in whole blood relative to PRP suggested that other blood cells affect ADP-induced platelet activation and hence the net inhibition by R-138727. R-138727 did not inhibit P2Y12-mediated ADP-induced shape change, even at concentrations that completely inhibited platelet aggregation, confirming the specificity of R-138727 for P2Y12. In conclusion, R-138727, the active metabolite of prasugrel, results in rapid, potent, consistent, and selective inhibition of P2Y12-mediated up-regulation of thromboinflammatory markers of platelet activation.This inhibition is enhanced in the presence other blood cells and calcium,but not aspirin.

Download Full-text

Comparison of Rapid Platelet Function Assay with Whole Blood Platelet Impedance Aggregometry for the Definition of Aspirin Resistance.

Blood ◽

10.1182/blood.v106.11.4018.4018 ◽

2005 ◽

Vol 106 (11) ◽

pp. 4018-4018

Author(s):

Anna M. Dyszkiewicz-Korpanty ◽

Anne Kim ◽

James D. Burner ◽

Eugene P. Frenkel ◽

Ravindra Sarode

Keyword(s):

Arachidonic Acid ◽

Platelet Aggregation ◽

Platelet Function ◽

Whole Blood ◽

Platelet Rich Plasma ◽

Blood Platelet ◽

Aspirin Resistance ◽

Impedance Aggregometry ◽

Definition Of ◽

Induced Aggregation

Abstract The reported incidence of aspirin (ASA) resistance ranges from 5 to 30%. Various platelet function assays have been employed to detect aspirin resistance in patients with cardio- and cerebrovascular disease. Such a high proposed incidence of ASA resistance poses a critical need for a rapid point-of -care (POC) platelet function test. Unfortunately, no uniformly accepted definition of ASA resistance exists. Platelet aggregation studies that have been used to define ASA resistance are time consuming and require special technical expertise. The Ultegra Rapid Platelet Function -ASA (RPFA-ASA) has been developed as a POC test that is performed without sample processing. This optical method measures agglutination of fibrinogen-coated beads upon platelet activation with arachidonic acid. In the presence of aspirin effect, however, the agglutination of the beads is inhibited. The described cutoff of ≥ 550 Aspirin Reaction Units (ARU) is termed non-responsiveness to ASA based on a preclinical study and subsequent correlation with epinephrine-induced platelet aggregation in platelet rich plasma. Since RPFA-ASA uses whole blood, we validated its performance characteristics against a classic whole blood platelet aggregation assay (WBA). We studied 50 healthy volunteers, aged 25–75 (24 men, 26 women) with normal CBC, who had not ingested anti-platelet drugs for 14 days prior to the study. Baseline studies included WBA (dual channel aggregometer, Chrono-log Inc., Havertown, PA) using both arachidonic acid (AA -0.5; 0.25 mM) and collagen (1; 2 μg/mL) as well as an RPFA-ASA assay (Accumetrics Inc., San Diego, CA). These studies were repeated after 3 days of ASA (325 mg/d) intake. Based on a review of the literature, we defined an adequate ASA response as a completely inhibited AA-induced platelet aggregation and at least 30% inhibition of collagen-induced aggregation (both concentrations of the agonist). Thus, those with < 30% inhibition of aggregation response to collagen were considered ASA resistant. Eleven subjects were ASA resistant by WBA (20%; 8 females and 3 males (aged 25–63). In contrast, since all 50 subjects achieved ARU values of < 550 ARU, none were recognized as an ASA non-responder by the RPFA-ASA. While the current cutoff of < 550 ARU posed by the Ultegra RPFA-ASA does identify those who have taken ASA, the assay is unable to recognize ASA non-responders. Thus, based on these data, the appropriate cutoff for the recognition of ASA resistance by the RPFA-ASA should be re-adjusted to a significantly lower level to ensure appropriate assay results.

Download Full-text

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

Blood ◽

10.1182/blood.v118.21.2231.2231 ◽

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.

Download Full-text

The Effect of Tirofiban on Fibrinogen/Agonist-Induced Platelet Shape Change and Aggregation

Clinical and Applied Thrombosis/Hemostasis ◽

10.1177/1076029608316014 ◽

2008 ◽

Vol 14 (3) ◽

pp. 295-302 ◽

Cited By ~ 11

Author(s):

I. Anita Jagroop ◽

Dimitri P. Mikhailidis

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Whole Blood ◽

Platelet Rich Plasma ◽

Shape Change ◽

Adenosine Diphosphate ◽

Vascular Events ◽

Spontaneous Platelet Aggregation ◽

Induced Aggregation ◽

Platelet Shape

There is evidence linking raised plasma fibrinogen (fib) and platelet hyperactivity with vascular events. One way to inhibit platelets is to block the platelet membrane glycoprotein (GP) IIb/IIIa receptor, which binds circulating fib or von Willebrand factor and cross-links platelets at the final common pathway to platelet aggregation. Tirofiban is a potent and specific fib receptor antagonist, used in the treatment of unstable angina. The authors assessed the effect of tirofiban on spontaneous platelet aggregation (SPA), fib-induced, serotonin (5HT)-induced, and adenosine diphosphate (ADP)-induced aggregation in whole blood by calculating the percentage free platelet count. These various agonists were used alone and in combination. The authors also measured the effect of tirofiban on agonists-induced (ADP, 5HT) platelet shape change (PSC). The effect of fib on PSC was also evaluated in platelet-rich plasma using a high-resolution (0.07 fL) channelyzer. Tirofiban significantly inhibited SPA, fib (2, 4, 8 g/L), ADP, ADP + fib combination, and 5HT-induced aggregation. Tirofiban had no effect on agonist-induced PSC. There was no apparent change in platelet volume with fib. In conclusion, tirofiban does not appear to have an effect on PSC, an early phase of platelet activation. Tirofiban seems to be a nonspecific and an effective inhibitor of platelet aggregation (a later phase of platelet activation) in whole blood. The clinical significance of these findings remains to be established.

Download Full-text

Comparing a New Bovine Source Heparin to the Clinically Used Porcine Heparin for Platelet Function Effects and Heparin-Induced Thrombocytopenia Potential

Blood ◽

10.1182/blood-2018-99-118781 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2540-2540

Author(s):

Michelle Sung ◽

Jeanine Walenga ◽

Walter Jeske ◽

Omer Iqbal ◽

Mamdouh Bakhos

Keyword(s):

Arachidonic Acid ◽

Platelet Aggregation ◽

Platelet Activation ◽

Platelet Function ◽

Test Group ◽

The United States ◽

Platelet Factor ◽

Heparin Induced Thrombocytopenia ◽

Aggregation Response ◽

Significant Difference

Abstract Background Heparin is a sulfated polysaccharide obtained from intestinal mucosa with anticoagulant properties that is widely used as a standard clinical therapeutic agent to treat and prevent thrombosis. Heparin is known to affect platelet function, and among its side effects is heparin-induced thrombocytopenia (HIT) that can occur in about 1% of patients exposed to heparin. Presently, only porcine source heparin is approved for use in the United States. The aims of this study were to determine if platelet activation by physiological agonists and platelet aggregation induced by HIT antibodies would be equivalent in the presence of bovine source heparin and porcine source heparin. Materials and Methods Seven lots of bovine heparin from Eurofarma and 3 lots of commercial clinical grade porcine heparin (Pfizer/Hospira) were evaluated. The USP Reference Standard for porcine heparin was used to determine anti-Xa and anti-IIa potencies of the bovine heparins. For each study, blood was collected from healthy volunteers (n=5 per test group), anticoagulated with sodium citrate, and centrifuged to obtain platelet rich plasma (PRP). Platelet aggregation responses were assessed using the BioData PAP-8 platelet aggregometer. For the first aim to evaluate platelet function, PRP was combined with heparin at final concentrations of 10.0, 1.0, and 0.1 µg/mL, covering both therapeutic and prophylactic ranges. Platelet agonists included adenosine diphosphate (ADP), collagen, epinephrine, arachidonic acid, and thrombin receptor agonist peptide (TRAP). The aggregation response was quantitated in terms of primary slope (PS), area under the curve (AUC), maximum aggregation (MA), and final aggregation (FA). For the second aim to evaluate the HIT potential, antibodies to the complex of heparin-platelet factor 4 (H-PF4) from banked HIT patient apheresis fluid were combined with donor PRP and heparin. Heparins were tested at final concentrations of 0.1, 0.4, 0.8, 1, and 100 U/mL. PS and FA results were recorded. For all data, comparisons were analyzed with 2-Way ANOVA using SigmaPlot software. Results In the presence of either bovine (BMH) or porcine heparin (PMH), the normal platelet aggregation response of all donors was not altered from that obtained with saline (see representative aggregation tracing in the image below). All heparin concentrations produced the same response. There were no significant differences between the bovine and porcine heparins for each of the 4 platelet aggregation parameters for ADP, arachidonic acid, collagen, epinephrine, and TRAP. Variation in the PS for arachidonic acid and collagen need to be assessed in a larger pool of donors to assure the lack of significant difference. Platelet activation to H-PF4 antibodies was strong at 0.1 to 1 U/mL concentrations with the expected inhibition observed when using 100 U/mL heparin. The HIT potential between bovine heparin and porcine heparin demonstrated no significant difference between the heparins (see MA responses in the image below). There were no lot to lot differences for the bovine heparins or the porcine heparins in either the platelet aggregation studies or the assessment for HIT. Conclusion In these studies of platelet function, the bovine and porcine source heparins were comparable with regards to their effects on platelet aggregation induced by multiple different agonists and their HIT potential. Figure. Figure. Disclosures Walenga: Eurofarma: Research Funding.

Download Full-text

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

Blood ◽

10.1182/blood-2018-99-114450 ◽

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.

Download Full-text

Imaging Thromboembolism with 99mtc Labelled Chimeric Monoclonal Anti-Platelet Glycoprotein Iiia Antibody Fragment chSZ21F(ab)2: Evaluation In Beagle Canines

Blood ◽

10.1182/blood.v116.21.4387.4387 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4387-4387

Author(s):

Shundong Ji ◽

Wei Fang ◽

Ningzheng Dong ◽

Zuoxiang He ◽

Changgeng Ruan

Keyword(s):

Platelet Aggregation ◽

Conflicts Of Interest ◽

Platelet Rich Plasma ◽

Antibody Fragment ◽

Canine Model ◽

Platelet Glycoprotein ◽

Vein Thrombosis ◽

Focal Uptake ◽

Glycoprotein Iiia

Abstract Abstract 4387 Abstract Purpose A mouse/human chimeric monoclonal antibody F(ab)2 fragment, with high affinity for platelet glycoprotein IIIa was labelled with99mTc (99mTc-chSZ21F(ab)2), and evaluated in canine to image experimental venous thromboembolism (deep vein thrombosis [DVT]) and pulmonary embolism (PE). Methods ADP-stimulated platelet aggregation was performed to determine the affinity and specificity of chSZ21F(ab)2 to the GPIIb/IIIa receptor on plates of human or canine. ChSZ21F(ab)2 was modified with 2-iminotholane and incubated with 99mTc-glucoheptonate for imaging of 24-h-old DVT and PE in a canine model. Animals were imaged for up to 3 h after injection, heparinized, and sacrificed. Lungs and other tissues, including blood and normal lungs, were harvested and, concurrently, 99mTc was counted for determination of target to tissue ratios and the percentage of injected dose per gram of tissue. Results The concentration of chSZ21F(ab)2 required to inhibit 50% of platelet aggregation (IC50) in platelet-rich plasma was 11.6±7.9 nM for human and 24.9±18.8 nM for canine. In vivo, focal uptake was observed in planar images as early as 30 min (DVT), and 60 min (PE) after injection. Lesion uptake of 99mTc-chSZ21F(ab)2 at 3 h after injection was calculated in terms of percentage injected dose per gram (%ID/g) of tissue and averaged 0.076%ID/g PE and 0.083%ID/g DVT. Lesion-to-background ratios averaged 12.8 (PE-to-lung), 7.2 (DVT-to-blood), and 117.0(DVT-to-muscle). Conclusion 99mTc-chSZ21F(ab)2 with its modest affinity and high DVT and PE uptake is a promising agent for imaging vascular thrombosis. Disclosures: No relevant conflicts of interest to declare.

Download Full-text