Heparin-Associated Thrombocytopenia: The Antibody Is Not Heparin Specific

1992 ◽  
Vol 67 (05) ◽  
pp. 545-549 ◽  
Author(s):  
A Greinacher ◽  
I Michels ◽  
C Mueller-Eckhardt
Keyword(s):  
Platelet Activation ◽  
Dextran Sulfate ◽  
Dermatan Sulfate ◽  
Solid Phase ◽  
Binding Capacity ◽  
Low Grade ◽  
Type Ii ◽  
Pentosan Polysulfate ◽  
Heparin Administration

SummaryIn this study the hypothesis was assessed whether heparin-associated thrombocytopenia (HAT) may be caused by an antibody dependent on polysulfated oligosaccharide epitopes, present not only on heparin but also on different polysulfated substances such as dextran sulfate and pentosan polysulfate. We found that the major factor for eliciting platelet activation with sera of HAT type II patients is neither the structure nor the AT III binding capacity of an oligosaccharide, but rather its grade of sulfation. This was shown by in vitro crossreactivity studies with 40 sera of HAT type II patients using unfractionated heparins, LMW heparins (Fragmin, Fraxiparin), enoxaparin, LMW heparinoid (Org 10172 and its subfractions), de-N-sulfated heparin, dermatan sulfate, dextran sulfate, pentosan polysulfate and dextran. Platelet activation was measured by the heparin induced platelet activation (HIPA) assay and the serotonin release assay (SRA). The platelet activating factor was isolated with the IgG fraction, but did not bind to heparin and dextran sulfate fixed to a solid phase. By isoimmune fixation electrophoresis a monoclonal gammopathy was ruled out in the three sera assessed. The in vivo effect of different LMW heparins and the heparinoid Org 10172 was observed in 10 patients with HAT type II. In a prospective study, a compatible heparin-like anticoagulant was selected for 10 HAT patients for whom further parenteral anticoagulation was required. The only substance that showed no crossreactivity in vitro was the LMW heparinoid Org 10172, which differs from heparin and LMW heparins by its low-grade sulfation. Upon treatment with the heparinoid, all 10 patients had a good clinical outcome, even if they had previously developed thromboembolic complications under LMW heparin administration. As Org 10172 contains a small amount of a LMW heparin-like substance (3%) this heparinoid should not be used in HAT patients without prior in vitro testing. We conclude that heparin-associated thrombocytopenia is not caused by a heparin-specific antibody and that a major factor contributing to the pathomechanism is the high grade of sulfation present in a variety of polysulfated oligosaccharides.

A solid-phase radioimmunoassay for human corticosteroid binding globulin

1985 ◽  
Vol 104 (2) ◽  
pp. 259-267 ◽  
Author(s):  
P. A. Robinson ◽  
M. S. Langley ◽  
G. L. Hammond
Keyword(s):  
Solid Phase ◽  
Binding Capacity ◽  
High Serum ◽  
Protein L ◽  
Parallel Displacement ◽  
Standard Curve ◽  
Corticosteroid Binding Globulin ◽  
Assay Tube ◽  
The Mean

ABSTRACT A radioimmunoassay (RIA) for human corticosteroid binding globulin (CBG) has been developed using 125I-labelled CBG and a monospecific solid-phase CBG-antiserum (CBG-Ab-cellulose). In an RIA of serum CBG concentrations, pure CBG standards (1–100 ng protein) or samples (1 : 200) were incubated (16 h at 20 °C) with 125I-labelled CBG and CBG-Ab-cellulose. After addition of 2 ml 0·9% NaCl, the tubes were centrifuged, supernatants were aspirated and the 125I-labelled CBG bound to the CBG-Ab-cellulose pellet was counted. The specificity of the RIA was confirmed by parallel displacement curves for serial dilutions of male, female and pregnancy sera, as well as pure CBG standards. The mean ± s.d. recovery (99±8%) of pure CBG (1·6–25·0 ng) added to a diluted serum sample verified the accuracy of the method, and a good correlation (r = 0·97; n = 43) existed between serum CBG cortisol binding capacity (nmol/l) measurements and CBG concentrations (mg protein/l) measured by RIA. Intra- and interassay precisions (C.V.) at low to high serum CBG concentrations were <5% and <9% respectively. The mean ± s.d. serum CBG concentrations (mg protein/l) measured by the RIA were: 21·8±4·6 in boys (n = 12), 20·0±4·2 in girls (n = 9), 20·7±2·7 in men (n = 6), 20·5±2·9 in women (n = 6) and 47·1 ±10·5 in pregnant women (n = 5). The sensitivity of the standard curve used in the routine RIA of serum CBG was 1·0 ng CBG/assay tube, but this could be increased to 0·2 ng/assay tube by reducing the amount of CBG-Ab-cellulose used. The RIA is suitable for both clinical and research purposes, and will aid the identification of abnormal forms of CBG and facilitate studies of the regulation of CBG production in vitro. J. Endocr. (1985) 104, 259–267

Inhibition of rat glomerular visceral epithelial cell growth by heparin

1988 ◽  
Vol 255 (4) ◽  
pp. F781-F786 ◽  
Author(s):  
S. Adler
Keyword(s):  
Growth Inhibition ◽  
Dextran Sulfate ◽  
Anticoagulant Activity ◽  
Sulfated Polysaccharides ◽  
Heparin Binding ◽  
Pentosan Polysulfate ◽  
The Media ◽  
Heparan Sulfates ◽  
Visceral Epithelial Cell

The effect of several glycosaminoglycans and sulfated polysaccharides on the growth of cultured rat glomerular visceral epithelial cells (GEC) was studied in vitro. Heparin, one preparation of heparan sulfate proteoglycan, dextran sulfate, and pentosan polysulfate significantly inhibited the growth of several GEC clones studied (36.0-77.1% inhibition at 100 micrograms/ml). Other glycosaminoglycans studied did not affect GEC growth. Growth inhibition by heparin was dose related and did not appear to reflect cytotoxicity. Heparins with high or low affinity for antithrombin inhibited growth to similar degrees. When heparin was fractionated into high- and low-anticoagulant activity fractions by physicochemical means the high activity fraction displayed significantly greater growth inhibition. The degree of growth inhibition significantly correlated with serum concentration in the media (r = 0.64; P less than 0.001). Removal of heparin binding factors from serum resulted in a loss of this correlation as well as less overall growth inhibition. These experiments suggest that interactions of GEC with heparan sulfates and other heparin-like molecules in the extracellular matrix may be important in the control of GEC growth.

The Effect of Human Serum on the Binding Activity of Radiolabelled Monoclonal Antibodies

1987 ◽  
Vol 73 (6) ◽  
pp. 547-554
Author(s):  
Silvia Camagni ◽  
Silvana Canevari ◽  
Marina Ripamonti ◽  
Delia Mezzanzanica ◽  
Rosaria Orlandi ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Human Blood ◽  
Solid Phase ◽  
Binding Capacity ◽  
Binding Activity ◽  
Ovarian Carcinomas ◽  
Radiolabelled Monoclonal Antibodies ◽  
In Vitro Stability ◽  
Murine Monoclonal Antibodies

Three murine monoclonal antibodies (MoAbs), MBrl and MOv2 of IgM isotype and MOv8 of IgG isotype, with restricted reactivity for breast or ovarian carcinomas, were labelled with 125I in the perspective of obtaining specific and stable radioimmunopharmaceutical reagents. The radiolabeled MoAbs were analyzed for their « in vitro » stability in human blood. They were incubated at 37 °C for various lengths of time in human or, as a control, in murine blood and their binding capacity was evaluated by solid-phase RIA and compared with that obtained after incubation with buffer. In human blood, serum and plasma, but not with other components such as erythrocytes, leukocytes, HSA and IgG, the MoAbs revealed a loss of binding reactivity which was marked and constant for the IgM MoAbs, and only occasional for the IgG MoAb. In murine serum the decrease was not so rapid. The same change in the binding capacity was observed when the MoAbs were labelled with 3H or 35S, excluding the involvement of dehalogenating mechanisms. In the perspective of using MoAbs for intracavity therapy the effect of ascitic or pleural fluids on their binding activity was also evaluated. The inhibition of the binding reactivity was not as evident and was not related to the MoAb isotype.

Adipose-on-a-chip: a dynamic microphysiological in vitro model of the human adipose for immune-metabolic analysis in type II diabetes

Lab on a Chip ◽  
2019 ◽  
Vol 19 (2) ◽  
pp. 241-253 ◽  
Author(s):  
Yunxiao Liu ◽  
Patthara Kongsuphol ◽  
Su Yin Chiam ◽  
Qing Xin Zhang ◽  
Sajay Bhuvanendran Nair Gourikutty ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
Immune Cells ◽  
Type Ii Diabetes ◽  
In Vitro Model ◽  
Low Grade ◽  
Type Ii ◽  
Metabolic Analysis ◽  
Vitro Model ◽  
Low Grade Inflammation

Infiltration of immune cells into adipose tissue is associated with chronic low-grade inflammation in obese individuals.

Polyphenols: Anti-Platelet Nutraceutical?

2018 ◽  
Vol 24 (2) ◽  
pp. 146-157 ◽  
Author(s):  
Valeria Ludovici ◽  
Jens Barthelmes ◽  
Matthias P. Nagele ◽  
Andreas J. Flammer ◽  
Isabella Sudano
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Therapeutic Potential ◽  
Critical Role ◽  
Organ Damage ◽  
Low Grade ◽  
Protective Effects ◽  
Dietary Polyphenols

Background: Coronary artery disease (CAD) is a disease progressing over many years. Genetic factors, as well as the exposure to risk factors, are continuously leading to endothelial dysfunction, vascular alterations and, eventually, organ damage, major cardiovascular events and deaths. Oxidative stress, platelet hyperactivity and low-grade inflammation are important modulators in this context, contributing to plaque formation. Since platelet activation plays a critical role in the development and progression of atherothrombotic events, the inhibition of platelet hyperactivity may contribute to decreased atherothrombotic risk. The consumption of bioactive foods, and plant-derived polyphenols in particular, might impart anti-thrombotic and cardiovascular protective effects. Methods: Aim of this work is to focus on the potential of dietary derived polyphenols to reduce platelet hyperactivity or hypercoagulability in addition to discussing their possible complementary anti-platelet therapeutic potential. All the relevant publications on this topic were systematically reviewed. Results: Various studies demonstrated that polyphenol supplementation affects platelet aggregation and function in vitro and in vivo, mainly neutralizing free radicals, inhibiting platelet activation and related signal transduction pathways, blocking thromboxane A2 receptors and enhancing nitric oxide production. Experimental data concerning the effect of dietary polyphenols on platelet aggregation in vivo are poor, and results are often conflicting. Only flavanols clearly mirrored in vivo showed the efficacy in vitro in modulating platelet function. Conclusion: Dietary polyphenols, and above all flavanols contained in cocoa and berries, reduce platelet activation and aggregation via multiple pathways. However, more controlled interventional studies are required to establish which doses are required as well as what circulating concentrations are sufficient to induce functional antiplatelet effects.

scholarly journals Effects of Hyperhomocysteinemia on the Platelet-Driven Contraction of Blood Clots

Metabolites ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 354
Author(s):  
Rustem I. Litvinov ◽  
Alina D. Peshkova ◽  
Giang Le Minh ◽  
Nail N. Khaertdinov ◽  
Natalia G. Evtugina ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Platelet Activation ◽  
Rat Model ◽  
Binding Capacity ◽  
Blood Clot ◽  
Fibrinogen Binding ◽  
Blood Clots ◽  
Clot Contraction ◽  
Dose Dependent

Hyperhomocysteinemia (HHcy) is associated with thrombosis, but the mechanistic links between them are not understood. We studied effects of homocysteine (Hcy) on clot contraction in vitro and in a rat model of HHcy. Incubation of blood with exogenous Hcy for 1 min enhanced clot contraction, while 15-min incubation led to a dose-dependent suppression of contraction. These effects were likely due to direct Hcy-induced platelet activation followed by exhaustion, as revealed by an increase in fibrinogen-binding capacity and P-selectin expression determined by flow cytometry. In the blood of rats with HHcy, clot contraction was enhanced at moderately elevated Hcy levels (10–50 μM), while at higher Hcy levels (>50 μM), the onset of clot contraction was delayed. HHcy was associated with thrombocytosis combined with a reduced erythrocyte count and hypofibrinogenemia. These data suggest that in HHcy, platelets get activated directly and indirectly, leading to enhanced clot contraction that is facilitated by the reduced content and resilience of fibrin and erythrocytes in the clot. The excessive platelet activation can lead to exhaustion and impaired contractility, which makes clots larger and more obstructive. In conclusion, HHcy modulates blood clot contraction, which may comprise an underappreciated pro- or antithrombotic mechanism.

Immobilized Glycosaminoglycans Reduce Thrombopoietin-Induced Apoptosis during In Vitro Expansion of Megakaryocyte Precursors from Cord Blood CD34+ Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1127-1127
Author(s):  
Vipuil Kishore ◽  
James F. Eliason ◽  
Howard W.T. Matthew
Keyword(s):  
Cell Expansion ◽  
Dextran Sulfate ◽  
Dermatan Sulfate ◽  
Annexin V ◽  
Hematopoietic Stem ◽  
Platelet Recovery ◽  
Cd34 Cells ◽  
Chitosan Membranes ◽  
Carboxy Methyl

Abstract Umbilical cord blood (UCB) provides a rich source of hematopoietic stem cells for transplantation after high dose chemotherapy. However, delayed platelet recovery is a serious limitation of UCB transplantation in adults. A suggested potential solution is the transfusion of expanded megakaryocyte progenitors derived from hematopoietic stem cells (HSCs). Previous work has shown that HSC proliferation and differentiation can be influenced by the use of glycosaminoglycans (GAGs). Specifically, GAGs are known to bind and modulate the activity of many cytokines and growth factors [Gupta et al. 1998, Madihally et al. 1999]. Direct GAG-receptor interactions are also believed to play a role in this modulation. Thrombopoietin (TPO), a relatively specific megakaryocyte/platelet cytokine, plays an important role in early hematopoiesis and megakaryopoiesis. However, it has been reported that TPO induces apoptosis in cells belonging to the megakaryocyte lineage [Ryu et al. 2001]. In this study, we examined the effects of various immobilized GAGs on the expansion and apoptosis of CD41+ megakaryocyte progenitors in vitro. GAG-derivatized, chitosan membranes were prepared in 24 well culture plates by first casting chitosan membranes from acetic acid solution, and then covalently binding the GAG component using carbodiimide chemistry. Saturating GAG surface densities were employed in all studies. The GAGs studied were heparin, hyaluronic acid, chondroitin-4-sulfate, dermatan sulfate, heparan sulfate and the GAG analogue carboxy-methyl dextran sulfate. Freshly isolated CD34+ cells from UCB were cultured in 24 well plates at a density of 25,000 cells/well using serum free media supplemented with bovine serum albumin, rh-insulin, human transferrin, FL (50 ng/ml), TPO (50 ng/ml) and SCF (10 ng/ml). Half medium changes were done twice per week. Wells were demidepopulated at days 7, 11, and 14 and simultaneous assays of viability, CD41 antigen expression, and apoptosis (via Annexin V expression) were conducted by flow cytometry. A rapid CD41+ cell expansion was observed on all GAG surfaces except chondroitin-4-sulfate from day 7 to day 11. Plastic and chondroitin-4-sulfate showed delayed CD41+ cell expansion but by day 14 the number of viable CD41+ cells on all surfaces were comparable. An initial high expansion of CD41+ cells on the carboxy-methyl dextran sulfate surface appeared to plateau between days 11 and 14. The Annexin V analysis revealed that the GAG surfaces had a substantially lower proportion of apoptotic megakaryocytes compared to the plastic and chitosan controls. In particular, heparin, chondroitin-4-sulfate and dermatan sulfate showed two fold lower levels (p&lt;0.05) of apoptotic megakaryocytes. These results suggest that GAGs have a substantial potential to reduce the TPO induced megakaryocyte apoptosis. The use of GAGs along with an optimal cytokine combination may accelerate the application of ex vivo expanded megakaryocyte transfusion, thereby shortening the time of platelet recovery in the thrombocytopenia induced by radiotherapy and chemotherapy. Figure Figure

A Proposed Role of Surfactant in Platelet Function and Treatment of Pulmonary Hemorrhage in Preterm and Term Infants

2018 ◽  
Vol 140 (4) ◽  
pp. 215-220
Author(s):  
Tal  Sadeh-Vered ◽  
Nurit  Rosenberg ◽  
Iris  Morag ◽  
Asaf A. Berg ◽  
Gili Kenet ◽  
...  
Keyword(s):  
Preterm Infants ◽  
Platelet Activation ◽  
Platelet Function ◽  
Complete Blood Count ◽  
Binding Capacity ◽  
Pulmonary Hemorrhage ◽  
Adenosine Diphosphate ◽  
Term Infants ◽  
Average Size

Background: We evaluated the effect of surfactant on platelet function as a potential contributing mechanism to the pathogenesis of pulmonary hemorrhage (PHEM) in term and preterm infants. Methods: Cord blood samples were collected from neonates following delivery. Complete blood count and platelet function were measured using a cone and platelet analyzer (CPA). Increasing surfactant concentrations were added to platelets in vitro, and the adhesion molecule P-selectin and the monoclonal antibody PAC-1 were evaluated following platelet activation by flow cytometry. Results: Forty-one infants (11 preterm and 30 term) were studied. CPA revealed a significant decrease in the average size of the aggregates and in platelet adhesion when surfactant was added. In term infants, the addition of surfactant to native platelets yielded an increased binding capacity of PAC-1 but did not affect P-selectin expression. In preterm infants, platelet activation with adenosine diphosphate in the presence of a high surfactant concentration (0.5 mg/mL) resulted in increased PAC-1 binding and no change in P-selectin expression. Conclusions: The platelets of preterm infants are less active (hyporesponsive) than those of term infants, both in their native state as well as after stimulation with various agonists. Surfactant may play an important role in treating PHEM in preterm infants.

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.

Transient heparin-induced platelet activation linked to generation of platelet 12-lipoxygenase

2013 ◽  
Vol 109 (06) ◽  
pp. 1099-1107 ◽  
Author(s):  
Greg S. McMahon ◽  
Chris I. Jones ◽  
Paul D. Hayes ◽  
A. Ross Naylor ◽  
Alison H. Goodall
Keyword(s):  
Molecular Weight ◽  
Platelet Aggregation ◽  
Platelet Activation ◽  
Adenosine Diphosphate ◽  
Aspirin Resistance ◽  
Low Molecular Weight ◽  
Heparin Administration ◽  
12 Lipoxygenase ◽  
Cox 1

SummaryPreviously we demonstrated that heparin administration during carotid endarterectomy (CEA) caused a marked, but transient increase in platelet aggregation to arachidonic acid (AA) and adenosine diphosphate (ADP), despite effective platelet cyclo-oxygenase-1 (COX-1) inhibition with aspirin. Here we investigated the metabolism of AA via platelet 12-lipoxygenase (12-LOX) as a possible mediator of the observed transient aspirin resistance, and compared the effects of unfractionated (UFH) and low-molecular-weight (LMWH) heparin. A total of 43 aspirinated patients undergoing CEA were randomised in the trial to 5,000 IU UFH (n=22) or 2,500 IU LMWH (dalteparin, n=21). Platelet aggregation to AA (4×10–3) and ADP (3×10–6) was determined, and the products of the COX-1 and 12-LOX pathways; thromboxane B2 (TXB2) and 12-hydroxyeicosatretraenoic acid (12-HETE) were measured in plasma, and in material released from aggregating platelets. Aggregation to AA increased significantly (∼10-fold) following heparinisation (p<0.0001), irrespective of heparin type (p=0.33). Significant, but smaller (∼2-fold) increases in aggregation to ADP were also seen, which were significantly lower in the platelets of patients randomised to LMWH (p<0.0001). Plasma levels of TxB2 did not rise following heparinisation (p=0.93), but 12-HETE increased significantly in the patients’ plasma, and released from platelets stimulated in vitro with ADP, with both heparin types (p<0.0001). The magnitude of aggregation to ADP correlated with 12-HETE generation (p=0.03). Heparin administration during CEA generates AA that is metabolised to 12-HETE via the 12-LOX pathway, possibly explaining the phenomenon of transient heparin-induced platelet activation. LMWH has less effect on aggregation and 12-HETE generation than UFH when the platelets are stimulated with ADP.

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