Platelet Factor 4 (PF4) Release In Vitro From Unstimulated Human Platelets

1981 ◽  
Author(s):  
D S Cohen ◽  
J M Strohschein ◽  
R N Saunders ◽  
D I Cargill
Keyword(s):  
Platelet Rich Plasma ◽  
Venous Blood ◽  
Human Platelets ◽  
Platelet Factor 4 ◽  
Platelet Factor ◽  
Time Period ◽  
Time Periods ◽  
Endogenous Release ◽  
Two Populations

Human venous blood was collected (9.5:0.5) in an anticoagulant (provided in the PF4 RIA kit, Abbott Labs.) consisting of 2.5% EDTA, 0.025% adenosine and 7.0% procaine HCl. Platelet rich plasma (PRP) was stirred (900 RPM) at 37°C in a Payton aggregometer for intervals of 3, 6, 9 and 12 minutes. Unincubated aliquots of PRP were taken as the 0 time controls.A significant (p < 0.02) elevation in PF4 was observed with increasing intervals. Furthermore, from the data obtained, two populations of PF4 releasers were delineated: A high releasing (HR) group and a low releasing (LR) group. HR (n=10) displayed a baseline release of 10.3 ± 0.8 ng/ml and values of 36.1 ± 9.0 (p < 0.05 compared to baseline), 46.8 ± 8.2 (p < 0.01), 54.6 ± 7.9 (p < 0.01) and 87 ± 13.1 (p < 0.01) for the corresponding time periods. LR (n=7) exhibited significant but diminished release at each time period with a basal level of 10.1 ± 1.0 and 14.7 ± 1.4 (p < 0.05 compared to baseline), 15.3 ± 1.5 (p < 0.05), 19.7 ± 2.4 (p < 0.05) and 23.6 ± 2.0 (p < 0.01) respectively. Comparison of both populations revealed significant (p < 0.01) differences at the 6, 9 and 12 minute intervals. Total PF4 content from Triton lysed PRP displayed similar levels between groups: 360.0 ±22.6 (HR) and 380.2 ± 15.2 (LR).We conclude that an endogenous release of PF4 occurs in vitro in unstimulated human PRP even in a milieu designed to deter release. This should be a concern when PF4 values are to be assessed.

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.

The Effect of Lysed Platelets on Neutralization of Heparin In Vitro with Protamine as Measured by the Activated Coagulation Time (ACT)

1991 ◽  
Vol 66 (02) ◽  
pp. 213-217 ◽  
Author(s):  
Arthur P Bode ◽  
William J Castellani ◽  
Edna D Hodges ◽  
Susan Yelverton
Keyword(s):  
Platelet Rich Plasma ◽  
Coagulation Time ◽  
Platelet Factor ◽  
Platelet Suspension ◽  
Antiheparin Activity ◽  
Unit Increase ◽  
Heparin Anticoagulation ◽  
Heparin Activity ◽  
Activated Coagulation Time

SummaryThe effect of lysed platelets on the activated coagulation time (ACT) was studied in heparinized whole blood during titration with protamine. Frozen-thawed washed platelet suspension, or a chromatography fraction thereof, or autologous frozen-thawed platelet-rich plasma was added in various dilutions to freshly drawn blood anticoagulated with 3,000 USP units/1 heparin. After a 10 min incubation, the amount of protamine needed to restore the ACT to baseline ("protamine titration dose") was determined. We found that the protamine titration dose decreased in proportion to the amount of lysed platelet material added; expressed as a percentage of the total number of platelets present, each unit increase in lysed platelets produced a 1.7% ±0.8 (SD) reduction in the protamine dose needed to normalize the ACT. A heparin activity assay showed that this effect was not due to antiheparin activity of lysed platelets such as platelet factor 4 (PF4). Our data indicate that the procoagulant activity of platelet membranes reduced the sensitivity of the ACT to heparin. These findings suggest that membranous platelet microparticles may cause an inaccurate calculation, based on the ACT, of a protamine dose to reverse heparin anticoagulation in cardiopulmonary bypass procedures.

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.

Reaction of Acetaldehyde with Human Platelets

1992 ◽  
Vol 67 (01) ◽  
pp. 126-130 ◽  
Author(s):  
Olivier Spertini ◽  
Jacques Hauert ◽  
Fedor Bachmann
Keyword(s):  
Platelet Aggregation ◽  
Inhibitory Action ◽  
Ex Vivo ◽  
Platelet Rich Plasma ◽  
Shape Change ◽  
Reaction Medium ◽  
Human Platelets ◽  
Membrane Glycoproteins ◽  
Neutral Ph

SummaryPlatelet function defects observed in chronic alcoholics are not wholly explained by the inhibitory action of ethanol on platelet aggregation; they are not completely reproduced either in vivo by short-term ethanol perfusion into volunteers or in vitro by the addition of ethanol to platelet-rich plasma. As acetaldehyde (AcH) binds to many proteins and impairs cellular activities, we investigated the effect of this early degradation product of ethanol on platelets. AcH formed adducts with human platelets at neutral pH at 37° C which were stable to extensive washing, trichloracetic acid hydrolysis and heating at 100° C, and were not reduced by sodium borohydride. The amount of platelet adducts formed was a function of the incubation time and of the concentration of AcH in the reaction medium. At low AcH concentrations (<0.2 mM), platelet bound AcH was directly proportional to the concentration of AcH in the reaction medium. At higher concentrations (≥0.2 mM), AcH uptake by platelets tended to reach a plateau. The amount of adducts was also proportional to the number of exposures of platelets to pulses of 20 pM AcH.AcH adducts formation severely impaired platelet aggregation and shape change induced by ADP, collagen and thrombin. A positive correlation was established between platelet-bound AcH and inhibition of aggregation.SDS-PAGE analysis of AcH adducts at neutral pH demonstrated the binding of [14C]acetaldehyde to many platelet proteins. AcH adduct formation with membrane glycoproteins, cytoskeleton and enzymes might interfere with several steps of platelet activation and impair platelet aggregation.This in vitro study shows that AcH has a major inhibitory action on platelet aggregation and may account for the prolonged ex vivo inhibition of aggregation observed in chronic alcoholics even in the absence of alcoholemia.

Interactions of Staphylokinase with Human Platelets

1995 ◽  
Vol 73 (03) ◽  
pp. 472-477 ◽  
Author(s):  
H R Lijnen ◽  
B Van Hoef ◽  
D Collen
Keyword(s):  
Platelet Aggregation ◽  
Platelet Function ◽  
Specific Binding ◽  
Platelet Rich Plasma ◽  
Human Platelets ◽  
Normal Plasma ◽  
Atp Secretion ◽  
Platelet Disaggregation ◽  
Activation Rate

SummaryThe interactions of recombinant staphylokinase (SakSTAR) with human platelets were investigated in a buffer milieu, in a human plasma milieu in vitro, and in plasma from patients with acute myocardial infarction (AMI) treated with SakSTAR.In a buffer milieu, the activation rate of plasminogen by SakSTAR or streptokinase (SK) was not significantly altered by addition of platelets. Specific binding of SakSTAR or SK to either resting or thrombin- activated platelets was very low. ADP-induced or collagen-induced platelet aggregation in platelet-rich plasma (PRP) was 94 ± 2.7% or 101 ± 1.7% of control in the presence of 0.1 to 20 μM SakSTAR, with corresponding values of 95 ± 2.8% or 90 ± 4.6% of control in the presence of 0.1 to 4 μM SK. No effects were observed on platelet disaggregation. ATP secretion following collagen-induced platelet aggregation was 4.3 ± 0.26 μM for SakSTAR (at concentrations of 0.1 to 20 μM) and 4.4 ± 0.35 μM for SK (at concentrations of 0.1 to 4 μM), as compared to 3.4 ± 0.70 μM in the absence of plasminogen activator.Fifty % lysis in 2 h (C50) of 60 μl 125I-fibrin labeled platelet-poor plasma (PPP) clots prepared from normal plasma or from plasma of patients with Glanzmann thrombasthenia and immersed in 0.5 ml normal plasma, was obtained with 12 or 16 nM SakSTAR and with 49 or 40 nM SK, respectively. C50 values for lysis of 60 μl PRP clots prepared from normal or patient plasma were also comparable for SakSTAR (19 or 21 nM), whereas SK was 2-fold more potent toward PRP clots prepared from Glanzmann plasma as compared to normal plasma (C50 of 130 versus 270 nM).No significant effect of SakSTAR on platelet function was observed in plasma from patients with AMI treated with SakSTAR, as revealed by unaltered platelet count, platelet aggregation and ATP secretion.Thus, no effects of high SakSTAR concentrations were observed on human platelets in vitro, nor of therapeutic SakSTAR concentrations on platelet function in plasma.

scholarly journals Loss of 111Indium as indicator of platelet injury

Blood ◽  
1981 ◽  
Vol 58 (2) ◽  
pp. 350-353 ◽  
Author(s):  
JH Joist ◽  
RK Baker
Keyword(s):  
Small Molecules ◽  
Adenine Nucleotides ◽  
Human Platelets ◽  
Metabolic Energy ◽  
Platelet Factor ◽  
Platelet Protein ◽  
Threshold Rate ◽  
Immune Injury ◽  
Triton X

Abstract We previously demonstrated that platelets can be labeled with 111Inoxine with high labeling efficiency and that 111In is not liberated from labeled platelets during the platelet release reaction or prolonged in vitro storage. In view of these findings, we examined the potential usefulness of loss of 111In from labeled platelets as an indicator or platelet damage by comparing the loss of 111In with that of 51Cr and LDH (in some experiments also with platelet factor 3 availability) under different conditions of platelet injury. When washed human platelets labeled with either 51Cr-chromate or 111In-oxine were exposed to increasing concentrations of detergents (Triton X-100, lysolecithin), threshold, rate, and extent of loss of 111In, 51Cr and, LDH were similar. In contrast, when labeled platelets were depleted of metabolic energy by incubation in glucose-free Tyrode albumin solution or glucose-depleted plasma in the presence of antimycin A and 2-deoxy-D- glucose, loss of 51Cr (and PF3a) occurred earlier and progressed at a faster rate than that of 111In or LDH. Similar results were obtained when platelets were exposed to increasing concentrations of PlA1 antibody, causing complement-mediated immune injury. The findings indicate that with certain agents that cause rapid platelet disruption (lysis), different platelet constituents are lost at similar rates. However, under conditions of more subtle or slowly progressive platelet injury, small molecules such as adenine nucleotides (51Cr) may escape earlier and at faster rates than larger molecules such as LDH or 111In- binding platelet protein. Thus, neither 111In loss nor LDH loss appear to be suitable indicators for sublytic or prelytic platelet injury.

The procoagulant effects of factor V Leiden may be balanced against decreased levels of factor V and do not reflect in vivo thrombin formation in newborns

2006 ◽  
Vol 95 (03) ◽  
pp. 434-440 ◽  
Author(s):  
Satu Hyytiäinen ◽  
Ulla Wartiovaara-Kautto ◽  
Veli-Matti Ulander ◽  
Risto Kaaja ◽  
Markku Heikinheimo ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Platelet Rich Plasma ◽  
Venous Blood ◽  
Factor V Leiden ◽  
Factor V ◽  
Cord Plasma ◽  
Adult Plasma ◽  
Thrombin Formation

SummaryThrombin regulation in newborns remains incompletely understood.We studied tissue factor-initiated thrombin formation in cord plasma in vitro, and the effects of Factor VLeiden (FVL) heterozygosity on thrombin regulation both in vitro and in vivo in newborns. Pregnant women with known thrombophilia (n=27) were enrolled in the study. Cord blood and venous blood at the age of 14 days were collected from 11 FVL heterozygous newborns (FVL-positive) and from 16 FVL-negative newborns. Prothrombin fragment F1+2 and coagulation factors were measured. Tissue factor-initiated thrombin formation was studied in cord platelet-poor plasma (PPP) of FVL-negative and -positive newborns, and in both PPP and platelet-rich plasma (PRP) of healthy controls. The endogenous thrombin potential (ETP) in cord PPP or PRP was ∼60% of that in adult plasma, while thrombin formation started ∼55% and ∼40% earlier in cord PPP and PRP, respectively. Further, in FVL-positive newborns thrombin formation started significantly earlier than in FVL-negative newborns. Exogenous activated protein C (APC) decreased ETP significantly more in cord than in adult PRP. In FVL-negative cord plasma 5nM APC decreased ETP by 17.4±3.5% (mean±SEM) compared with only 3.5±3.8% in FVL-positive cord plasma (p=0.01). FVL-positive newborns showed similar levels of F1+2 but significantly decreased levels of factor V compared with FVL negative newborns both in cord plasma (FV 0.82±0.07 U/ml vs. 0.98±0.05 U/ml, p=0.03) and at the age of two weeks (FV 1.15±0.04 U/ml vs. 1.32±0.05 U/ml, p=0.03). In conclusion, newborn plasma showed more rapid thrombin formation and enhanced sensitivity to APC compared with adult plasma. FVL conveyed APC resistance and a procoagulant effect in newborn plasma. Lack of elevated F1+2 levels in FVL-positive infants, however, suggested the existence of balancing mechanisms; one could be the observed lower level of factor V in FVL heterozygous newborns.

scholarly journals Heparin-induced thrombocytopenia: in vitro studies on the interaction of dabigatran, rivaroxaban, and low-sulfated heparin, with platelet factor 4 and anti-PF4/heparin antibodies

Blood ◽  
2012 ◽  
Vol 119 (5) ◽  
pp. 1248-1255 ◽  
Author(s):  
Krystin Krauel ◽  
Christine Hackbarth ◽  
Birgitt Fürll ◽  
Andreas Greinacher
Keyword(s):  
Factor Xa ◽  
Platelet Factor 4 ◽  
Thrombin Inhibitors ◽  
Direct Thrombin Inhibitors ◽  
Platelet Factor ◽  
Heparin Induced Thrombocytopenia ◽  
Alternative Anticoagulation ◽  
Heparin Complexes ◽  
History Of

Abstract Heparin is a widely used anticoagulant. Because of its negative charge, it forms complexes with positively charged platelet factor 4 (PF4). This can induce anti-PF4/heparin IgG Abs. Resulting immune complexes activate platelets, leading to the prothrombotic adverse drug reaction heparin-induced thrombocytopenia (HIT). HIT requires treatment with alternative anticoagulants. Approved for HIT are 2 direct thrombin inhibitors (DTI; lepirudin, argatroban) and danaparoid. They are niche products with limitations. We assessed the effects of the DTI dabigatran, the direct factor Xa-inhibitor rivaroxaban, and of 2-O, 3-O desulfated heparin (ODSH; a partially desulfated heparin with minimal anticoagulant effects) on PF4/heparin complexes and the interaction of anti-PF4/heparin Abs with platelets. Neither dabigatran nor rivaroxaban had any effect on the interaction of PF4 or anti-PF4/heparin Abs with platelets. In contrast, ODSH inhibited PF4 binding to gel-filtered platelets, displaced PF4 from a PF4-transfected cell line, displaced PF4/heparin complexes from platelet surfaces, and inhibited anti-PF4/heparin Ab binding to PF4/heparin complexes and subsequent platelet activation. Dabigatran and rivaroxaban seem to be options for alternative anticoagulation in patients with a history of HIT. ODSH prevents formation of immunogenic PF4/heparin complexes, and, when given together with heparin, may have the potential to reduce the risk for HIT during treatment with heparin.

Characterisation of the conformational changes in platelet factor 4 induced by polyanions: towards in vitro prediction of antigenicity

2014 ◽  
Vol 112 (07) ◽  
pp. 53-64 ◽  
Author(s):  
Sven Brandt ◽  
Krystin Krauel ◽  
Kay E. Gottschalk ◽  
Thomas Renné ◽  
Christiane A. Helm ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Structural Changes ◽  
Cd Spectroscopy ◽  
Platelet Factor 4 ◽  
Drug Induced ◽  
Platelet Factor ◽  
Preclinical Drug Development ◽  
Varying Length ◽  
Endogenous Proteins

SummaryHeparin-induced thrombocytopenia (HIT) is the most frequent drug-induced immune reaction affecting blood cells. Its antigen is formed when the chemokine platelet factor 4 (PF4) complexes with polyanions. By assessing polyanions of varying length and degree of sulfation using immunoassay and circular dichroism (CD)-spectroscopy, we show that PF4 structural changes resulting in antiparallel β-sheet content >30% make PF4/polyanion complexes antigenic. Further, we found that polyphosphates (polyP-55) induce antigenic changes on PF4, whereas fondaparinux does not. We provide a model suggesting that conformational changes exposing antigens on PF4/polyanion complexes occur in the hairpin involving AA 32–38, which form together with C-terminal AA (66–70) of the adjacent PF4 monomer a continuous patch on the PF4 tetramer surface, explaining why only tetrameric PF4 molecules express “HIT antigens”. The correlation of antibody binding in immunoassays with PF4 structural changes provides the intriguing possibility that CD-spectroscopy could become the first antibody-independent, in vitro method to predict potential immunogenicity of drugs. CD-spectroscopy could identify compounds during preclinical drug development that induce PF4 structural changes correlated with antigenicity. The clinical relevance can then be specifically addressed during clinical trials. Whether these findings can be transferred to other endogenous proteins requires further studies.

scholarly journals The effect of resorcinol on bovine spermatozoa parameters in vitro

2020 ◽  
pp. 675-686
Author(s):  
M Massányi ◽  
M Halo ◽  
L Strapáková ◽  
T Slanina ◽  
P Ivanič ◽  
...  
Keyword(s):  
Cultivation Temperature ◽  
Negative Effects ◽  
Mtt Test ◽  
Progressive Motility ◽  
Time Period ◽  
Entire Duration ◽  
Bovine Spermatozoa ◽  
Time Periods ◽  
Motility Parameters

The goal of this study was to observe the effect of resorcinol on motility, viability and morphology of bovine spermatozoa. The semen was used from six randomly chosen breeding bulls. Ejaculate was diluted by different solutions of resorcinol in 1:40 ratio. Samples were divided into 7 groups with different concentrations of resorcinol (Control, RES1 – 4 mg/ml, RES2 – 2 mg/ml, RES3 – 1 mg/ml, RES4 – 0.5 mg/ml, RES5 – 0.25 mg/ml and RES6 – 0.125 mg/ml). Motility of spermatozoa was detected using CASA method at temperature of 37 °C in time periods 0, 1, 2, 3, 4 hours from the start of the experiment. Significant motility differences between all groups except control and RES6 with difference of 5.58 %, as well as between RES1 and RES2 groups with difference of 2.17 % were found. Progressive motility had the same significant differences. Spermatozoa viability (MTT test) decreased compared to control in all experimental groups during the entire duration of experiment. Observing morphologically changed spermatozoa, no significant changes were observed and a higher percentage of spermatozoa with separated flagellum in all experimental resorcinol groups compared to control were detected. Also, increased number of spermatozoa with broken flagellum, acrosomal changes and other morphological forms in the group with the highest concentration of resorcinol (RES1) were found. Results of our study clearly show negative effects on motility parameters of spermatozoa which depend on concentration, cultivation temperature and time period.

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