scholarly journals The production of anti-PF4 antibodies in anti-phospholipid antibody-positive patients is not affected by COVID-19 vaccination

2021 ◽  
Author(s):  
Paola Lonati ◽  
Caterina Bodio ◽  
Mariangela Scavone ◽  
Giuliana Martini ◽  
Elisa Pesce ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Lupus Erythematosus ◽  
Solid Phase ◽  
Clinical Manifestations ◽  
Platelet Factor ◽  
Heparin Treatment ◽  
Before And After ◽  
Solid Phase Assay ◽  
Vitro Effect

Antibodies against cationic platelet chemokine, platelet factor 4 (PF4/CXCL4) have been described in heparin-induced thrombocytopenia (HIT) but also in patients positive for anti-phospholipid antibodies (aPL) even in the absence of heparin treatment and HIT-related clinical manifestations. Anti-PF4 antibodies have been recently described also in subjects who developed thrombosis with thrombocytopenia syndrome (TTS) in association with adenoviral vector-based, but not with mRNA-based COVID-19 vaccines. We investigated whether COVID-19 vaccination affects the production of anti-PF4 immunoglobulins detectable by solid-phase assay in aPL-positive patients and their ability to induce in vitro platelet activation. Anti-PF4 were found in 9/126 aPL-positive patients, 4/50 COVID-19, 9/49 other infections, and 1/50 aPL-negative systemic lupus erythematosus patients. Clinical manifestations of TTS were not observed in any aPL patient positive for anti-PF4, whose sera failed to cause platelet aggregations. The administration of COVID-19 vaccines did not affect the production of anti-PF4 immunoglobulins or their ability to cause platelet aggregation in 44 aPL-positive patients tested before and after vaccination. In conclusion, heparin treatment-independent anti-PF4 antibodies can be found in aPL-positive patients and asymptomatic carriers, but their presence, titer as well as in vitro effect on platelet activation are not affected by COVID-19 vaccination.

Partially desulfated heparin modulates the interaction between anti-protamine/heparin antibodies and platelets

2016 ◽  
Vol 115 (02) ◽  
pp. 324-332 ◽  
Author(s):  
Rabie Jouni ◽  
Heike Zöllner ◽  
Ahmad Khadour ◽  
Jan Wesche ◽  
Anne Grotevendt ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Platelet Factor ◽  
Standard Drug ◽  
Platelet Surface ◽  
Minimal Impact ◽  
Platelet Survival ◽  
Heparin Complexes ◽  
The Impact

SummaryProtamine (PRT) is the standard drug to neutralise heparin. PRT/heparin complexes induce an immune response similar to that observed in heparin-induced thrombocytopenia (HIT). Partially desulfated heparin (ODSH) was shown to interfere with anti-platelet factor 4/heparin antibodies (Abs), which are responsible for HIT. In this study, we analyse the impact of ODSH on the interaction between anti-PRT/heparin Abs and platelets. The ability of ODSH to prevent anti-PRT/heparin Ab-induced platelet destruction in vivo was investigated using the NOD/ SCID mouse model. ODSH improved platelet survival in the presence of PRT, heparin and anti-PRT/heparin Abs (median platelet survival after 300 minutes (min) with 20 μg/ml ODSH: 75 %, range 70–81 % vs without ODSH: 49%, range 44–59%, p=0.006). Furthermore, when ODSH was applied 60 min after Ab injection platelet survival was improved (median platelet survival after 300 min with ODSH: 83 %, range 77–93 % vs without ODSH: 59 %, range 29–61 %, p=0.02). In in vitro experiments ODSH inhibited platelet activation at concentrations > 16 μg/mL (p< 0.001), as well as PRT/heparin complex binding to platelets (mean fluorescence intensity [MFI] without ODSH: 85 ± 14 vs with ODSH: 15 ± 0.6, p=0.013). ODSH also displaced pre-bound complexes from the platelet surface (MFI without ODSH: 324 ± 43 vs with 32 μg/ml ODSH: 53 ± 9, p< 0.001). While interfering with platelet activation by anti-PRT/heparin Abs, up to a concentration of 16 μg/ml, ODSH had only minimal impact on neutralisation of heparin by PRT. In conclusion, our study shows that ODSH is able to inhibit platelet activation and destruction suggesting a potential clinical use to reduce anti-PRT/heparin Ab-mediated adverse effects.

scholarly journals The Effect of Procoagulative Phospholipids on the Synthetic Substrate Determined Thrombin Generation in Vitro

1977 ◽  
Author(s):  
F. Schulte ◽  
O. Klug ◽  
Ursula Roth
Keyword(s):  
Thrombin Generation ◽  
Chromogenic Substrate ◽  
Chromogenic Substrates ◽  
Platelet Factor ◽  
Synthetic Substrate ◽  
Thrombin Activity ◽  
Vitro Effect ◽  
Hydrolysis Of

The effect of procoagulative phospholipids (Procops) with platelet factor-3 like activity on the generation of thrombin in plasma can be determined in vitro with the aid of synthetic chromogenic substrates. Standard citrated plasma of two manufacturers and from different batches incubated with amounts of 2-10 meg Procops as 1:100 - 1:500 diluted Tachostyptan (micellar Procops in form of a pharmaceutical speciality) was activated. The generated amount of thrombin activity catalyses the hydrolysis of chromogenic substrate to a tripeptide and to p-nitroaniline which was measured kinetically with a spectrophotometer at 405 nm. At optimum concentrations of Procops, activities adequate to 80 (SD ± 7, VK 8. 8%) - 115 i. u. (SD ± 2. 5, VK 2.2%) thrombin per ml plasma were measured depending on the conditions of incubation and activation. Having made the basis of appropriate procedures for incubation and activation the in vitro effect of Procops on the thrombin generation in plasma is reproducibly measurable with the aid of chromogenic substrate.

Clinical evaluation of total and high-avidity anti-dsDNA antibody assays for the diagnosis of systemic lupus erythematosus

Lupus ◽  
2019 ◽  
Vol 28 (12) ◽  
pp. 1387-1396 ◽  
Author(s):  
W Yuan ◽  
H Cao ◽  
P Wan ◽  
R Shi ◽  
S Zhou ◽  
...  
Keyword(s):  
Systemic Lupus Erythematosus ◽  
Disease Activity ◽  
Lupus Erythematosus ◽  
Clinical Manifestations ◽  
Superior Performance ◽  
High Avidity ◽  
Systemic Lupus ◽  
Dsdna Antibody ◽  
In Vitro Diagnostic

Background This study evaluated the diagnostic performances of total and high-avidity (HA) anti-dsDNA enzyme immunoassays (EIA) in Chinese systemic lupus erythematosus (SLE) patients. Methods A total of 410 serum samples from 217 SLE patients, 54 patients with other systemic autoimmune diseases, and 139 healthy subjects were tested on total and HA anti-dsDNA EIA, as well as three commercial in vitro diagnostic kits: BioPlex 2200 ANA Screen, Kallestad anti-dsDNA EIA, and Crithidia Lucilae IFA. The disease activities of SLE patients were assessed using the modified SLE Disease Activity Index. The diagnostic performances of each assay were analyzed using Analyse-it software. Results The diagnostic performances of the total and HA anti-dsDNA EIA kits were comparable to other commercially available in vitro diagnostic assays. Receiver operating characteristic curve analysis demonstrated an area under the curve ranging from 0.85 to 0.89, with the total anti-dsDNA kit demonstrating the highest sensitivity and the HA kit showing higher specificity. An overall agreement of >90% was observed between the total and HA anti-dsDNA EIA kits and commercially available quantitative anti-dsDNA kits. The ratio of HA to total anti-dsDNA antibody was significantly higher among SLE patients with active disease status and/or kidney damage. All assays exhibited a significant correlation with disease activity and multiple clinical manifestations. Conclusions While the clinical performances of various anti-dsDNA assays showed adequate agreements, the BioPlex 2200 anti-dsDNA assay demonstrated the highest positive likelihood ratio and odds ratio. The HA anti-dsDNA EIA kit in association with the total anti-dsDNA kit provided superior performance in SLE diagnosis and monitoring disease activity.

scholarly journals Vesiculation of platelets during in vitro aging

Blood ◽  
1991 ◽  
Vol 77 (4) ◽  
pp. 887-895
Author(s):  
AP Bode ◽  
SM Orton ◽  
MJ Frye ◽  
BJ Udis
Keyword(s):  
Surface Area ◽  
Platelet Activation ◽  
Lactic Dehydrogenase ◽  
Prostaglandin E ◽  
Platelet Factor ◽  
Storage Container ◽  
Fluorescent Beads ◽  
In Vitro Aging ◽  
Two Populations

Membranous microparticles (MP) appearing in the supernatant plasma of stored platelet concentrates (PC) were analyzed by flow cytometry. Two populations of MP were arbitrarily delineated by light scatter as larger or smaller than 0.5 micron fluorescent beads. An estimate of MP concentration was obtained by adding a known amount of fluorescent beads to each sample before analysis of a set number of counts on the flow cytometer. The addition of platelet activation inhibitors (prostaglandin E-1, theophylline, and aprotinin) to the anticoagulant during preparation of PC combined with a reduction in surface area of the storage container caused approximately a 40% reduction in the number of MP appearing during storage relative to donor-matched controls. In addition, the inhibited concentrates had 84% less platelet factor 3 (PF3) activity in the supernatant and 61% less released lactic dehydrogenase. A reduction in surface area of the container in the controls partially offset these differences. A significant correlation was found (rs = .748) between PF3 levels and the concentration of larger MP. The inhibitors did not reduce the small number of MP found in stored platelet-poor plasma. Surface antigen analysis showed that the majority of MP in PC were platelet-derived; most were positive for glycoprotein (GP) IIbIIIa (73%) and/or for GPIb (43% to 46%). We conclude that procoagulant MP are released from platelets during storage as a result of platelet activation augmented by interaction of platelets with the bag wall.

STUDY OF THE IN VITRO EFFECT ON GLOMERULAR ALBUMIN PERMSELECTIVITY OF SERUM BEFORE AND AFTER RENAL TRANSPLANTATION IN FOCAL SEGMENTAL GLOMERULOSCLEROSIS1,2

1997 ◽  
Vol 64 (12) ◽  
pp. 1711-1715 ◽  
Author(s):  
Yann Godfrin ◽  
Jacques Dantal ◽  
Sabine Perretto ◽  
Dan Hristea ◽  
Christophe Legendre ◽  
...  
Keyword(s):  
Renal Transplantation ◽  
Before And After ◽  
Vitro Effect

SELECTIVE INHIBITION OF HUMAN MEGAKARYOCYTOPOIESIS IN VITRO BY HIGHLY PURIFIED PLATELET FACTOR 4

1987 ◽  
Author(s):  
A Gewirtz ◽  
W Y Xu ◽  
B Rucinski ◽  
S Niewiarowski
Keyword(s):  
T Cells ◽  
T Cell ◽  
Colony Formation ◽  
Solid Phase ◽  
Specific Inhibitor ◽  
Platelet Glycoprotein ◽  
Lymphocyte Function ◽  
Erythroid Progenitor ◽  
Platelet Factor

Platelet (plt) factor 4 (PF4) is an alpha granule protein which can modulate T lymphocyte function. T cells may help regulate megakaryocytopoiesis. Therefore, we hypothesized that T cell-PF4 interactions might play a role in autoregulating marrow megakaryocyte (MEG) production. To test this idea, we studied MEG colony formation in plasma clot cultures containing human serum derived solely from pit poor normal AB plasma, enriched hematopoietic progenitor cells (HPC), autologous T cells, and exogenous PF4. Highly purified PF4 (single band on SDS gel) was prepared from outdated human pits by a combination of heparin-agarose, Sephacryl G-200, and Sephadex G-50 column chromatography. HPC were prepared by depleting normal light density marrow mononuclear cells of adherent monocytes, and T cells. T cells were further fractionated into helper (Leu 3+) and suppressor (Leu 2+) subtypes by solid phase immunoabsorption ("panning"). MEG colonies were enumerated by indirect immunofluorescence with an anti-human platelet glycoprotein antiserum. HPC(5×105/ml) were co-cultured with Leu 3+, or Leu 2+ T cells at target;T cell ratios of 2:1 (n=3; n=4 respectively) and l:l(n=4; n=4 respectively) in the presence of 2.5 μg/ml PF4. Under these growth conditions, MEG colony formation was unchanged (p>0.5) when compared to colonies formed by HPC in the absence of PF4. When the above experiments were repeated (n=2-3/condition) at a higher PF4 concentration [25 μg/ml], MEG colony formation was markedly (>60%) inhibited. To determine if PF4 directly inhibited MEG or erythroid progenitor cell growth (CFU-Meg; CFU-E) in vitro, HPC were cloned in PF4 (25μg/ml) without added T cells. Mean ± SEM of MEG and CFU-E derived colonies formed without vs. with PF4 was as follows:These results suggest that: 1) PF4 may be a non-T cell dependent, lineage specific inhibitor of CFU-MEG, and 2) PF4 may play a role in autoregulating human megakaryocytopoiesis.

scholarly journals Furosemide and Platelet Functions

1977 ◽  
Author(s):  
I.S. Chohan ◽  
I. Singh ◽  
J. Vermvlen ◽  
M. Verstraete
Keyword(s):  
Platelet Aggregation ◽  
Ex Vivo ◽  
Metabolic Effects ◽  
Platelet Factor ◽  
Metabolic Products ◽  
Vitro Effect ◽  
Primary Platelet ◽  
Platelet Functions

Furosemide inhibits primary platelet aggregation by adenosine-5'-diphosphate and prolongs the latent period before Thrombofax- or collagen-induced platelet aggregation, both in vitro and ex vivo. Furosemide also inhibits the release of platelet factor 4 and 14C-serotonin. The inhibitory concentrations of furosemide in vitro range between 0.5 and 2.5 mM. The ex vivo effects were obtained after an intravenous injection of 40 mg furosemide.The furosemide concentrations required for ex vivo inhibition are hundred fold lower than those required for in vitro effect. This suggests in vivo metabolic effects of this drug, such as, calcium ions shifts, or action of other metabolic products of furosemide. In support of this concept platelet aggregation was found more disturbed six hours than 10 minutes after injection of the drug.

Single Center Experience with the Nanoparticle-Based Flow Immunoassay for Diagnosis of Heparin-Induced Thrombocytopenia (HIT).

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2189-2189
Author(s):  
Susanne Macher ◽  
Nazanin Sareban ◽  
Camilla Drexler ◽  
Gerhard Lanzer ◽  
Katharina Schallmoser
Keyword(s):  
Platelet Activation ◽  
Conflicts Of Interest ◽  
Laboratory Diagnosis ◽  
Heparin Therapy ◽  
Enzyme Linked Immunosorbent Assay ◽  
Platelet Factor ◽  
Heparin Induced Thrombocytopenia ◽  
Score System ◽  
Single Center Experience

Abstract Abstract 2189 Heparin-induced thrombocytopenia (HIT), caused by antibodies against heparin/platelet factor 4 (HPF4) complex, is a rare but potentially serious side effect of heparin therapy where due to high mortality, rapid diagnosis is crucial. For the detection of HPF4 antibodies we compared the new nanoparticle-based lateral-flow immunoassay (LFI-HIT, Milenia Biotec, Germany) and a particle gel immunoassay (PaGIA, BioRad, Germany) with an IgG-specific-PF4/polyanion enzyme-linked immunosorbent assay (IgG-ELISA, GTI Diagnostics, USA). Sera from 121 patients (54/67 f/m, median 73 years, range 14–94) with suspected HIT were prospectively tested. The LFI-HIT and the PaGIA were evaluated visually, the IgG-ELISA was positive at an optical density (OD) cutoff > 0.4. For most of the positive samples, the functional heparin-induced platelet activation (HIPA) assay was additionally performed to detect false positive serological results and to confirm a clinically relevant HIT by in vitro platelet-activation. Regarding HIT as a clinico-pathological syndrome, characteristics for HIT were evaluated for each patient by the 4Ts scoring system and divided into high, intermediate or low risk. Results of serological analyses and OD values are summarized in the table. Ten of 121 samples were positive in the LFI-HIT, 10/10 positive in the PaGIA and 8/10 positive in the IgG-ELISA. The HIPA was tested in 9/10 samples and was positive in 8/9 samples. Of the 2 samples positive for LFI-HIT and PaGIA but negative in the ELISA, 1 was HIPA positive, 1 HIPA negative, resulting in a specificity of 88.9% for the LFI-HIT assay correlated to the HIPA. From 111/121 LFI-HIT-negative samples, 2 were positive in the PaGIA, the IgG-ELISA (OD 1.318 and 2,019) and in the HIPA. Seven of the 111 LFI-HIT negative samples were positive only in the IgG-ELISA. Due to marginal positive reactions of 5/7 samples in the ELISA with OD values between 0.4 to 0.5, only 2 LIF-HIT negative IgG-ELISA positive samples were tested by HIPA and 1/2 was positive. Based on the ELISA, the sensitivity of the LFI-HIT was 91.9% (102/111 negative samples also negative in the ELISA) in contrast to 93.1% of the PaGIA. The specificity of the LFI-HIT was 80% (LFI-HIT and IgG-ELISA positive), compared to 57.9% of the PaGIA. Notably, the clinical risk estimated by the 4Ts score system (received from 92/121 patients) did not correlate with laboratory diagnosis of HIT, probably due to inadequate evaluation. Concluding our data, a reliable exclusion of HIT by rapid testing with the LFI-HIT only seems possible with additional analysis of HPF4 antibodies by IgG-ELISA and/or HIPA assay. LFI-HIT PaGIA IgG-ELISA OD IgG-ELISA HIPA assay Median (range) Samples n=121 Pos 10 Pos 10 Pos 8 2.366 (0.902-3.000) 7/7 pos Neg 2 0.199 and 0.170 1/2 pos, 1/2 neg Neg 0 - - - - Neg 111 Pos 9 Pos 2 1.318 and 2.019 2/2 pos Neg 7 0.110 (0.054-0.139) 6/6 neg Neg 102 Pos 7 0.436 (0.404-1.463) 1/2 pos, 1/2 neg Neg 95 0.082 (0.013-0.376) Disclosures: No relevant conflicts of interest to declare.

Oxidative stress and thromboxane-dependent platelet activation in inflammatory bowel disease: effects of anti-TNF-α treatment

2016 ◽  
Vol 116 (09) ◽  
pp. 486-495 ◽  
Author(s):  
Marco Guerci ◽  
Paola Simeone ◽  
Sandro Ardizzone ◽  
Alessandro Massari ◽  
Paolo Giuffrida ◽  
...  
Keyword(s):  
Inflammatory Bowel Disease ◽  
Platelet Activation ◽  
Bowel Disease ◽  
Infliximab Treatment ◽  
Soluble Cd40 Ligand ◽  
Tnf Α ◽  
Inflammatory Bowel ◽  
Vitro Effect

SummaryPatients with inflammatory bowel disease (IBD) are at higher risk of venous thromboembolism and coronary artery disease despite having a lower burden of traditional risk factors. Platelets from IBD patients release more soluble CD40 ligand (CD40L), and this has been implicated in IBD platelet hyper-activation. We here measured the urinary F2-isoprostane 8-iso-prostaglandin (PG)2α (8-iso-PGF2α), urinary 11–dehydro–thromboxane (TX) B2 (11-dehydro–TXB2) and plasma CD40L in IBD patients, and explored the in vitro action of anti-tumour necrosis factor (TNF)–α antibody infliximab on IBD differentiating megakaryocytes. Urinary and blood samples were collected from 124 IBD patients and 37 healthy subjects. Thirteen IBD patients were also evaluated before and after 6–week infliximab treatment. The in vitro effect of infliximab on patient-derived megakaryocytes was evaluated by immunoflorescence microscopy and by flow cytometry. IBD patients had significantly (p<0.0001) higher urinary 8–iso–PGF2α and 11–dehydro–TXB2 as well as plasma CD40L levels than controls, with active IBD patients displaying higher urinary and plasma values when compared to inactive patients in remission. A 6-week treatment with infliximab was associated with a significant reduction of the urinary excretion of 8–iso–PGF2α and 11–dehydro–TXB2 (p=0.008) and plasma CD40L (p=0.001). Infliximab induced significantly rescued pro-platelet formation by megakaryocytes derived from IBD patients but not from healthy controls. Our findings provide evidence for enhanced in vivo TX–dependent platelet activation and lipid peroxidation in IBD patients. Anti-TNF–α therapy with infliximab down-regulates in vivo isoprostane generation and TX biosynthesis in responder IBD patients. Further studies are needed to clarify the implication of infliximab induced-proplatelet formation from IBD megakaryocytes.Supplementary Material to this article is available online at www.thrombosis-online.com.

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