scholarly journals Galectin-1 and platelet factor 4 (CXCL4) induce complementary platelet responses in vitro

PLoS ONE ◽  
2021 ◽  
Vol 16 (1) ◽  
pp. e0244736
Author(s):  
Annemiek Dickhout ◽  
Bibian M. E. Tullemans ◽  
Johan W. M. Heemskerk ◽  
Victor L. J. L. Thijssen ◽  
Marijke J. E. Kuijpers ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Platelet Aggregation ◽  
Light Transmission ◽  
Synergistic Effects ◽  
Platelet Factor 4 ◽  
Carbohydrate Binding ◽  
Platelet Factor ◽  
Neuraminidase Treatment ◽  
Platelet Surface

Galectin-1 (gal-1) is a carbohydrate-binding lectin with important functions in angiogenesis, immune response, hemostasis and inflammation. Comparable functions are exerted by platelet factor 4 (CXCL4), a chemokine stored in the α-granules of platelets. Previously, gal-1 was found to activate platelets through integrin αIIbβ3. Both gal-1 and CXCL4 have high affinities for polysaccharides, and thus may mutually influence their functions. The aim of this study was to investigate a possible synergism of gal-1 and CXCL4 in platelet activation. Platelets were treated with increasing concentrations of gal-1, CXCL4 or both, and aggregation, integrin activation, P-selectin and phosphatidyl serine (PS) exposure were determined by light transmission aggregometry and by flow cytometry. To investigate the influence of cell surface sialic acid, platelets were treated with neuraminidase prior to stimulation. Gal-1 and CXCL4 were found to colocalize on the platelet surface. Stimulation with gal-1 led to integrin αIIbβ3 activation and to robust platelet aggregation, while CXCL4 weakly triggered aggregation and primarily induced P-selectin expression. Co-incubation of gal-1 and CXCL4 potentiated platelet aggregation compared with gal-1 alone. Whereas neither gal-1 and CXCL4 induced PS-exposure on platelets, prior removal of surface sialic acid strongly potentiated PS exposure. In addition, neuraminidase treatment increased the binding of gal-1 to platelets and lowered the activation threshold for gal-1. However, CXCL4 did not affect binding of gal-1 to platelets. Taken together, stimulation of platelets with gal-1 and CXCL4 led to distinct and complementary activation profiles, with additive rather than synergistic effects.

scholarly journals Immunoglobulin G from patients with heparin-induced thrombocytopenia binds to a complex of heparin and platelet factor 4

Blood ◽  
1994 ◽  
Vol 83 (11) ◽  
pp. 3232-3239 ◽  
Author(s):  
JG Kelton ◽  
JW Smith ◽  
TE Warkentin ◽  
CP Hayward ◽  
GA Denomme ◽  
...  
Keyword(s):  
Minimum Degree ◽  
Equilibrium Dialysis ◽  
Fluid Phase ◽  
Platelet Factor 4 ◽  
Minimum Size ◽  
Sulfated Polysaccharides ◽  
Platelet Factor ◽  
Platelet Surface ◽  
Heparin Induced Thrombocytopenia

Abstract Heparin-induced thrombocytopenia (HIT) is an important complication of heparin therapy. Although there is general agreement that platelet activation in vitro by the HIT IgG is mediated by the platelet Fc receptor, the interaction among the antibody, heparin, and platelet membrane components is uncertain and debated. In this report, we describe studies designed to address these interactions. We found, as others have noted, that a variety of other sulfated polysaccharides could substitute for heparin in the reaction. Using polysaccharides selected for both size and charge, we found that reactivity depended on two independent factors: a certain minimum degree of sulfation per saccharide unit and a certain minimum size. Hence, highly sulfated but small (< 1,000 daltons) polysaccharides were not reactive nor were large but poorly sulfated polysaccharides. The ability of HIT IgG to recognize heparin by itself was tested by Ouchterlony gel diffusion, ammonium sulfate and polyethylene glycol precipitation, and equilibrium dialysis. No technique demonstrated reactivity. However, when platelet releasate was added to heparin and HIT IgG, a 50-fold increase in binding of radio-labeled heparin to HIT IgG was observed. The releasate was then depleted of proteins capable of binding to heparin by immunoaffinity chromatography. Only platelet factor 4-immunodepleted releasate lost its reactivity with HIT IgG and heparin. Finally, to determine whether the reaction occurred on the surface of platelets or in the fluid phase, washed platelets were incubated with HIT IgG or heparin and after a wash step, heparin or HIT IgG was added, respectively. Reactivity was only noted when platelets were preincubated with heparin. Consistent with these observations was the demonstration of the presence of PF4 on platelets using flow cytometry. These studies indicate that heparin and other large, highly sulfated polysaccharides bind to PF4 to form a reactive antigen on the platelet surface. HIT IgG then binds to this complex with activation of platelets through the platelet Fc receptors.

Comparison Of The Major Membrane Glycoproteins Of Human, Rabbit And Rat Platelets

1981 ◽  
Author(s):  
B Toor ◽  
J L McGregor ◽  
K J Clemetson ◽  
L McGregor ◽  
M Dechavanne ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Surface Composition ◽  
Sodium Dodecyl ◽  
Human Platelets ◽  
Membrane Glycoproteins ◽  
Neuraminidase Treatment ◽  
Platelet Surface ◽  
Rabbit Platelets ◽  
Rat Platelets

Rabbit and rat platelets have been extensively investigated under in vitro or in vivo conditions to try to understand the pathology of thrombosis in man. Here, surface-labelling techniques have been used to find out if the platelet surface has a similar composition in these two animals and in man or not. Human, rabbit and rat platelets were isolated, washed and surface-labelled by techniques specific for protein or for sugars (sialic acid or penultimate galactose/N-acetyl galactosamine residues). Labelled platelets were solubilized in sodium dodecyl sulphate and separated under reducing conditions on 7.5 % Laemmli polyacrylamide gels. Dried gels were exposed to film by fluorography or indirect autoradiography. Terminal Gal/Gal NAc residues (no neuraminidase treatment) were strongly labelled with rat and rabbit platelets compared to human platelets which labelled very poorly. Terminal sialic acid labelling with rat and rabbit platelets showed a weak labelling of a glycoprotein (GP) with the same M.Wt. as GPIb which is the most intensely labelled GP in man. However two GP (with rabbits) and one GP (in rats) were intensely labelled at a M.Wt. similar to that of GPIa in man. These GP had a different M.Wt. with terminal Gal/Gal NAc labelling. Bands with a similar M.Wt. to GPIIb and IIIa in man were strongly iodinated with rabbit platelets but with rat platelets only a single band at the position of GPIIb was strongly iodinated. These results strongly indicate that there are considerable differences in surface composition between rabbit, rat and human platelets.

scholarly journals Immunoglobulin G from patients with heparin-induced thrombocytopenia binds to a complex of heparin and platelet factor 4

Blood ◽  
1994 ◽  
Vol 83 (11) ◽  
pp. 3232-3239 ◽  
Author(s):  
JG Kelton ◽  
JW Smith ◽  
TE Warkentin ◽  
CP Hayward ◽  
GA Denomme ◽  
...  
Keyword(s):  
Minimum Degree ◽  
Equilibrium Dialysis ◽  
Fluid Phase ◽  
Platelet Factor 4 ◽  
Minimum Size ◽  
Sulfated Polysaccharides ◽  
Platelet Factor ◽  
Platelet Surface ◽  
Heparin Induced Thrombocytopenia

Heparin-induced thrombocytopenia (HIT) is an important complication of heparin therapy. Although there is general agreement that platelet activation in vitro by the HIT IgG is mediated by the platelet Fc receptor, the interaction among the antibody, heparin, and platelet membrane components is uncertain and debated. In this report, we describe studies designed to address these interactions. We found, as others have noted, that a variety of other sulfated polysaccharides could substitute for heparin in the reaction. Using polysaccharides selected for both size and charge, we found that reactivity depended on two independent factors: a certain minimum degree of sulfation per saccharide unit and a certain minimum size. Hence, highly sulfated but small (< 1,000 daltons) polysaccharides were not reactive nor were large but poorly sulfated polysaccharides. The ability of HIT IgG to recognize heparin by itself was tested by Ouchterlony gel diffusion, ammonium sulfate and polyethylene glycol precipitation, and equilibrium dialysis. No technique demonstrated reactivity. However, when platelet releasate was added to heparin and HIT IgG, a 50-fold increase in binding of radio-labeled heparin to HIT IgG was observed. The releasate was then depleted of proteins capable of binding to heparin by immunoaffinity chromatography. Only platelet factor 4-immunodepleted releasate lost its reactivity with HIT IgG and heparin. Finally, to determine whether the reaction occurred on the surface of platelets or in the fluid phase, washed platelets were incubated with HIT IgG or heparin and after a wash step, heparin or HIT IgG was added, respectively. Reactivity was only noted when platelets were preincubated with heparin. Consistent with these observations was the demonstration of the presence of PF4 on platelets using flow cytometry. These studies indicate that heparin and other large, highly sulfated polysaccharides bind to PF4 to form a reactive antigen on the platelet surface. HIT IgG then binds to this complex with activation of platelets through the platelet Fc receptors.

Pathogenic Role of Surface Platelet Factor 4 Complexes in Heparin-Induced Thrombocytopenia: Diagnostic and Therapeutic Implications.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 55-55 ◽  
Author(s):  
Lubica Rauova ◽  
Li Zhai ◽  
M. Anna Kowalska ◽  
Gowthami M. Arepally ◽  
Douglas B. Cines ◽  
...  
Keyword(s):  
Platelet Factor 4 ◽  
Thrombin Inhibitors ◽  
Current Therapy ◽  
Severe Thrombocytopenia ◽  
Pathogenic Role ◽  
Platelet Factor ◽  
Platelet Surface ◽  
Heparin Induced Thrombocytopenia

Abstract Heparin-induced thrombocytopenia (HIT) is caused by antibodies that recognize complexes between high molecular weight heparin and Platelet Factor 4 (PF4). Current therapy with direct thrombin inhibitors is not effective in all cases, likely because it acts downstream of antibody-induced platelet activation. More directed therapies to the underlying pathology in HIT may be more effective. Heparin and PF4 only bind HIT antibodies over a narrow molar ratio of reactants at which ultralarge soluble complexes are formed. We asked whether similar complexes form between PF4 and endogenous platelet glycosaminoglycans (GAG) and their pathogenic role in experimental HIT. Platelet surface GAG:PF4 complexes are indeed antigenic over a narrow molar range of reactants. Heparin is not required for either HIT-IgG or a HIT-like monoclonal antibody KKO to bind to PF4 on human or mouse platelet surfaces in vitro, but enhances antigenicity when very high levels of surface PF4 are present. Antigenicity is maximal at a PF4 concentration of 50 μg/mL (well within the range that can be achieved within a thrombus) and ~25 μg/mL heparin (~0.5 U/mL, which is within the therapeutic range) optimally enhances antigenicity when surface PF4 levels were increased 4-fold. Using transgenic mice lines each with platelets expressing a different level of hPF4, ranging from 0.5 – 6 X’s human platelet levels and all expressing FcRγIIA, were given KKO. The different lines developed thrombocytopenia proportional in severity and duration to hPF4 expression. A standard subcutaneous (sq) heparininzing dose (20 U/kg, sq daily) prolonged the duration of severe thrombocytopenia in high hPF4 expressing mice. We reasoned that altering the ratio of PF4 to GAG in either direction would alter antigenicity and could block the development of thrombocytopenia. In accordance with this concept, both high concentrations of anionic heparin (100 U/kg, sq daily) and cationic protamine sulfate (2 mg/kg, sq daily) decreased KKO binding in vitro and prevented KKO-induced thrombocytopenia in vivo as a demonstration of successful therapeutic intervention. These studies affirm a central role of surface GAG:PF4 complexes in the development of HIT, suggest ways to identify patients at high risk to develop HIT even prior to heparin exposure, and offers a new and rationale therapeutic paradigm based on disrupting surface antigen formation.

Increased Platelet Serotonin Content and Hypersecretion from Dense and A-Granules In Vitro In Tension-Type Headache

Cephalalgia ◽  
1993 ◽  
Vol 13 (5) ◽  
pp. 349-353 ◽  
Author(s):  
Giovanni D'Andrea ◽  
Lena Hasselmark ◽  
Michela Alecci ◽  
Francesco Perini ◽  
KMA Welch
Keyword(s):  
Platelet Aggregation ◽  
Tension Type Headache ◽  
Platelet Factor 4 ◽  
Healthy Controls ◽  
Platelet Factor ◽  
Serotonin Content ◽  
Platelet Serotonin ◽  
Serotonin Turnover ◽  
Type Headache

We investigated platelet aggregation and secretion from dense and a-granules in vitro in 28 tension-type headache (TH) patients and 26 healthy controls. We also measured basal platelet serotonin levels, Platelet aggregation was normal in TH, but the secretion of serotonin and platelet factor 4 (PF4) was significantly increased in response to 0.5 and 2.0 mg/ml collagen and to 1.0 mmol/l PAF. The basal platelet serotonin levels were also higher in patients than in controls. The mechanisms of platelet hypersecretion remain to be determined, but the increased secretion of serotonin is probably in part related to the increased basal levels. The increased platelet serotonin in TH patients may reflect an enhanced serotonin turnover.

In Vitro Efficacy of Platelet Glycoprotein IIb/IIIa Antagonist in Blocking Platelet Function in Plasma of Patients with Heparin-induced Thrombocytopenia

1998 ◽  
Vol 80 (12) ◽  
pp. 989-993 ◽  
Author(s):  
Koon-Hou Mak ◽  
Linda Brooks ◽  
Eric Topol ◽  
Kandice Kottke-Marchant
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Platelet Rich Plasma ◽  
Underlying Mechanism ◽  
Platelet Factor 4 ◽  
Platelet Glycoprotein ◽  
Platelet Factor ◽  
Heparin Induced Thrombocytopenia ◽  
Microparticle Formation

SummaryHeparin-induced thrombocytopenia (HIT) is an important complication following administration of heparin. Platelet activation and aggregation induced by heparin/platelet factor 4/immunoglobulin complexes are thought to be the underlying mechanism for this condition, so it was hypothesized that abciximab (a humanized murine monoclonal antibody directed against the glycoprotein IIb/IIIa receptor) would prevent heparin-induced platelet aggregation and activation in plasma from patients with HIT. Platelet aggregation was tested in vitro with platelet-poor plasma (obtained from 23 patients with HIT), platelet-rich plasma (from normal donors with known reactivity), heparin (0.5 U/ml), and ascending doses of abciximab (0.07-0.56 μg/ml). The ability of abciximab to prevent platelet activation was also evaluated using flow cytometry (P selectin expression, mepacrine release, microparticle formation) and platelet factor 4 immunoassay. In vitro, abciximab inhibited heparin-induced platelet aggregation in a dose-dependent fashion (IC50 0.103 μg/ml) and inhibited microparticle formation, the expression of P-selectin, release of mepacrine and platelet factor 4. These findings suggest that abciximab may be useful in treatment of patients with HIT and warrants further clinical evaluation.

Monocytes Are a Particularly Favorable Target for Surface Platelet Factor 4 (PF4) Antigenic Complex Formation in Heparin-Induced Thrombocytopenia: New Insights into the Thrombotic Risk in HIT

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 271-271
Author(s):  
Lubica Rauova ◽  
Gowthami M Arepally ◽  
Douglas B. Cines ◽  
Mortimer Poncz
Keyword(s):  
Complex Formation ◽  
Platelet Factor 4 ◽  
Drug Induced ◽  
Platelet Factor ◽  
Platelet Surface ◽  
Thrombotic Risk ◽  
Heparin Induced Thrombocytopenia ◽  
Antigenic Complex

Abstract Monocytes are a Favored Target for Surface Platelet Factor 4 (PF4) Antigenic Complex Formation in Heparin-Induced Thrombocytopenia: New Insights into the Thrombotic Risk in HIT Lubica Rauova, Gowthami Arepally, Douglas Cines and Mortimer Poncz HIT is a drug-induced autoimmune thrombocytopenia caused by antibodies to heparin/PF4 complexes that predispose to thrombotic complications. The studies described below examine how monocytes (Mo) may contribute to the thrombotic risk. We demonstrated previously that glycosaminoglycans (GAG) on the surface of platelets bind PF4, forming complexes that are recognized by HIT antibody, leading to platelet activation via the platelet FcγRIIA receptor in vitro and thrombocytopenia/thrombosis in vivo. However, heparin not only induces antibodies to develop against the PF4/GAG surface antigenic complexes, but also rapidly removes the same PF4/GAG complexes from the platelet surface, which may limit the likelihood of developing HIT and help limit its duration. This led us to study the involvement of Mo, which are a rich potential source of tissue factor and are known to be activated in HIT. Moreover, unlike platelets, which are coated with GAG composed almost entirely of chrondroitin sulfate (CS), Mo also express heparan sulfate, which has the capacity to bind PF4 with greater avidity and be resistant to the effect of plasma heparin. We found that Mo bind PF4 with greater avidity than platelets and higher concentrations of UFH are needed to remove PF4/GAG complexes and reduce the binding of a HIT monoclonal antibody KKO. In contrast to platelets, dissociation of PF4/GAG complexes from monocytes requires heparinases in addition to chondroitinases. In addition, macrophages GAG undergo hypersulfation during inflammation. Because clinical studies have shown inflammation predisposes to HIT, we examined the binding of KKO to unstimulated and bacterial lipopolysaccharide (LPS, E. coli serotype 011) stimulated cultured macrophages. Macrophages were derived from primary human Mo or murine bone marrow, cultured in the presence of M-CSF and stimulated with 0–500 ng/mL of LPS for 72 hrs. LPS increased KKO binding in the presence of PF4 2.7±0.7-fold compared to unstimulated cells (p&lt;0.002) and the stimulated cells required ~2-fold higher concentrations of heparin to remove surface PF4/GAG complexes. Addition of [35S]sulfate during the last 24 hrs of incubation lead to a 4.1±0.1-fold increase in the incorporation of 35S into surface GAG after LPS stimulation (p&lt;0.0001). These results provide important insights into the potential role of Mo in the prothrombotic sequelae of HIT. Compared to platelets, Mo are relatively resistant to “antigen down-regulation” by heparin and are more likely to bind anti-PF4/GAG HIT antibodies and become activated. The relative resistance of Mo to the dissociation PF4/GAG complexes from the cell surface also suggest a role in the development of Delayed-Onset HIT after heparin withdrawal.

Half-Life of Platelet Factor 4 (PF-4) in Plasma and Platelets from Macaca Mulatta

1977 ◽  
Vol 37 (01) ◽  
pp. 073-080 ◽  
Author(s):  
Knut Gjesdal ◽  
Duncan S. Pepper
Keyword(s):  
Macaca Mulatta ◽  
Half Life ◽  
Human Platelet ◽  
Gel Filtration ◽  
Platelet Factor 4 ◽  
Active Protein ◽  
Platelet Factor ◽  
Membrane Bound

SummaryHuman platelet factor 4 (PF-4) showed a reaction of complete identity with PF-4 from Macaca mulatta when tested against rabbit anti-human-PF-4. Such immunoglobulin was used for quantitative precipitation of in vivo labelled PF-4 in monkey serum. The results suggest that the active protein had an intra-platelet half-life of about 21 hours. In vitro 125I-labelled human PF-4 was injected intravenously into two monkeys and isolated by immuno-precipita-tion from platelet-poor plasma and from platelets disrupted after gel-filtration. Plasma PF-4 was found to have a half-life of 7 to 11 hours. Some of the labelled PF-4 was associated with platelets and this fraction had a rapid initial disappearance rate and a subsequent half-life close to that of plasma PF-4. The results are compatible with the hypothesis that granular PF-4 belongs to a separate compartment, whereas membrane-bound PF-4 and plasma PF-4 may interchange.

The Effect of Dimethyl Sulfoxide on In Vitro Platelet Function

1976 ◽  
Vol 36 (01) ◽  
pp. 221-229 ◽  
Author(s):  
Charles A. Schiffer ◽  
Caroline L. Whitaker ◽  
Morton Schmukler ◽  
Joseph Aisner ◽  
Steven L. Hilbert
Keyword(s):  
Platelet Count ◽  
Platelet Aggregation ◽  
Dimethyl Sulfoxide ◽  
Platelet Function ◽  
Platelet Volume ◽  
Short Term ◽  
Platelet Factor ◽  
Short Term Exposure ◽  
Structural Abnormalities

SummaryAlthough dimethyl sulfoxide (DMSO) has been used extensively as a cryopreservative for platelets there are few studies dealing with the effect of DMSO on platelet function. Using techniques similar to those employed in platelet cryopreservation platelets were incubated with final concentrations of 2-10% DMSO at 25° C. After exposure to 5 and 10% DMSO platelets remained discoid and electron micrographs revealed no structural abnormalities. There was no significant change in platelet count. In terms of injury to platelet membranes, there was no increased availability of platelet factor-3 or leakage of nucleotides, 5 hydroxytryptamine (5HT) or glycosidases with final DMSO concentrations of 2.5, 5 and 10% DMSO. Thrombin stimulated nucleotide and 5HT release was reduced by 10% DMSO. Impairment of thrombin induced glycosidase release was noted at lower DMSO concentrations and was dose related. Similarly, aggregation to ADP was progressively impaired at DMSO concentrations from 1-5% and was dose related. After the platelets exposed to DMSO were washed, however, aggregation and release returned to control values. Platelet aggregation by epinephrine was also inhibited by DMSO and this could not be corrected by washing the platelets. DMSO-plasma solutions are hypertonic but only minimal increases in platelet volume (at 10% DMSO) could be detected. Shrinkage of platelets was seen with hypertonic solutions of sodium chloride or sucrose suggesting that the rapid transmembrane passage of DMSO prevented significant shifts of water. These studies demonstrate that there are minimal irreversible alterations in in vitro platelet function after short-term exposure to DMSO.

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