Binding of plasma factor XIII to thrombin-receptor activated human platelets

2009 ◽  
Vol 102 (07) ◽  
pp. 83-89 ◽  
Author(s):  
Béla Nagy ◽  
Zsuzsa Simon ◽  
Zsuzsa Bagoly ◽  
László Muszbek ◽  
János Kappelmayer
Keyword(s):  
Whole Blood ◽  
Factor Xiii ◽  
Coagulation Factor ◽  
Human Platelets ◽  
Thrombin Receptor ◽  
Type I ◽  
Fibrinogen Receptor ◽  
Activated Platelets ◽  
Fibrinogen Binding ◽  
Plasma Factor

SummaryPlatelet-bound coagulation factor XIII (FXIII) is targeted and concentrated at the site where platelet-rich thrombi are formed. Previous studies were in disagreement about the nature of FXIII binding to platelets. In this study, thrombin-receptor activating peptide (TRAP)-stimulated human whole blood and washed platelets were analysed by flow cytometry for the binding of FXIII using a monoclonal antibody against the A subunit of FXIII (FXIII-A). Here, we demonstrate that FXIII-A positivity significantly increased on activated platelets in whole blood compared to unstimulated sample, but not in washed platelets. GPIIb/IIIa receptor plays an essential role in FXIII binding, as fibrinogen receptor antagonist eptifibatide and fibrinogen binding inhibitor RGDS tetrapeptide significantly prevented the binding of FXIII. Furthermore, stimulated platelets from a patient with severe type I Glanzmann thrombasthenia showed insignificant FXIII-A positivity versus healthy controls. In addition, basal negligible amount of FXIII on washed platelets was only slightly increased when highly purified plasma FXIII (FXIII-A2B2) was added upon platelet activation by TRAP. Similarly, no remarkable FXIII-A positivity was observed when we used FXIII-A2B2 with γA/γA fibrinogen. However, γA/γ' fibrinogen significantly augmented FXIII binding on TRAP-stimulated platelets in the presence of non-activated FXIII-A2B2. We conclude that FXIII-A2B2 of plasma origin binds to thrombin-receptor activated platelets via GPIIb/IIIa receptor-bound fibrinogen with γ’-chain and is not capable of direct platelet binding.

Platelet Aggregation and Fibrinogen Binding in Human, Rhesus Monkey, Guinea-Pig, Hamster and Rat Blood: Activation by ADP and a Thrombin Receptor Peptide and Inhibition by Glycoprotein IIb/IIIa Antagonists

1993 ◽  
Vol 70 (03) ◽  
pp. 531-539 ◽  
Author(s):  
Nigel S Cook ◽  
Hans-Günter Zerwes ◽  
Carlo Tapparelli ◽  
Max Powling ◽  
Jagjit Singh ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Guinea Pig ◽  
Rhesus Monkey ◽  
Whole Blood ◽  
Flow Cytometric Analysis ◽  
Human Platelets ◽  
Thrombin Receptor ◽  
Important Species ◽  
Rat Blood ◽  
Fibrinogen Binding

SummaryPlatelet aggregation and fibrinogen binding in whole blood, induced either by ADP or by a 14 amino acid peptide mimicking an N-terminal region of the platelet thrombin receptor (TRP, thrombin receptor activating peptide), have been studied with blood from different species. Aggregation was assessed by counting the number of single platelets in blood before und after addition of the agonist with an automated cell counter. Both ADP (0.02-0.5 μM) and TRP (1-10 μM) were found to be potent agonists of platelet aggregation in human, rhesus monkey and guinea-pig blood, causing a near-maximal aggregatory response within 2 min of agonist addition. In contrast, hamster and rat platelets were much less responsive to both ADP and TRP in terms of the whole blood aggregation.Echistatin, RGDW and a synthetic glycoprotein (GP) IIb/IIIa antagonist, Ro 43-8857, inhibited fibrinogen binding to purified immobilized human GP-IIb/IIIa with IC50’s of 1.6, 88 and 11.4 nM, respectively. In whole human blood, the respective IC50’s (as determined by flow cytometric analysis of platelet fibrinogen binding) were 4.4, 1700 and 29.5 nM. The affinities of these three compounds for inhibiting fibrinogen binding in whole blood from rhesus monkeys and guinea-pigs were similar to the affinities for human platelets, but with rat blood echistatin, RGDW and Ro 43-8857 were all around 100-fold less potent. Ro 43-8857 was a potent inhibitor of ADP- or TRP-induced platelet aggregation in human, rhesus monkey and guinea-pig whole blood (IC50 of 69-320 nM) but was much less active in hamster blood.These results highlight important species differences in the response of platelets to activation by two different agonists and also in their inhibition by GP-IIb/IIIa antagonists. In particular, platelets from the rat and hamster were insensitive to agonists and antagonists, whereas guinea-pig and rhesus monkey platelets responded with an affinity similar to human platelets. Since these studies were performed in whole blood, the results should be representative of those expected in animal experiments. These recently developed methods for studying platelet responses in small aliquots of whole blood are simple to perform and provide important information concerning the optimal choice of species for subsequent in vivo studies with these compounds.

scholarly journals An investigation into the role of coagulation factor XIII in ADP- induced aggregation and fibrinogen binding with rabbit platelets

Blood ◽  
1982 ◽  
Vol 60 (4) ◽  
pp. 905-911
Author(s):  
EJ Harfenist ◽  
G Raychaudhuri ◽  
MA Packham ◽  
JF Mustard
Keyword(s):  
Factor Xiii ◽  
Specific Binding ◽  
Coagulation Factor ◽  
Factor Xiiia ◽  
Platelet Protein ◽  
Fibrinogen Binding ◽  
Plasma Factor ◽  
Rabbit Platelets ◽  
Platelet Aggregate ◽  
Induced Aggregation

Because there was a possibility that activated factor XIII (factor XIIIa) might stabilize a platelet-fibrinogen aggregate through its crosslinking action, we have isolated plasma factor XIII, activated it, and studied the effect of factor XIIIa at a concentration of 3.3 micrograms/ml on aggregation and 125I-fibrinogen binding of rabbit platelets stimulated with 9 microM ADP. Factor XIIIa did not cause aggregation in the absence of ADP, nor did it enhance ADP-induced aggregation or substantially stabilize the platelet aggregate. The presence of factor XIIIa did not affect the amount of fibrinogen bound to platelets immediately after stimulation with ADP, but it appeared to cause a slow specific binding of 125I-fibrinogen to platelets whether or not they were stimulated with ADP. This binding, which was not inhibited by prostaglandin E1, did not lead to aggregation and was accompanied by crosslinking of fibrinogen through its A alpha and gamma chains, either to other fibrinogen molecules or to a platelet protein or proteins.

scholarly journals An investigation into the role of coagulation factor XIII in ADP- induced aggregation and fibrinogen binding with rabbit platelets

Blood ◽  
1982 ◽  
Vol 60 (4) ◽  
pp. 905-911 ◽  
Author(s):  
EJ Harfenist ◽  
G Raychaudhuri ◽  
MA Packham ◽  
JF Mustard
Keyword(s):  
Factor Xiii ◽  
Specific Binding ◽  
Coagulation Factor ◽  
Factor Xiiia ◽  
Platelet Protein ◽  
Fibrinogen Binding ◽  
Plasma Factor ◽  
Rabbit Platelets ◽  
Platelet Aggregate ◽  
Induced Aggregation

Abstract Because there was a possibility that activated factor XIII (factor XIIIa) might stabilize a platelet-fibrinogen aggregate through its crosslinking action, we have isolated plasma factor XIII, activated it, and studied the effect of factor XIIIa at a concentration of 3.3 micrograms/ml on aggregation and 125I-fibrinogen binding of rabbit platelets stimulated with 9 microM ADP. Factor XIIIa did not cause aggregation in the absence of ADP, nor did it enhance ADP-induced aggregation or substantially stabilize the platelet aggregate. The presence of factor XIIIa did not affect the amount of fibrinogen bound to platelets immediately after stimulation with ADP, but it appeared to cause a slow specific binding of 125I-fibrinogen to platelets whether or not they were stimulated with ADP. This binding, which was not inhibited by prostaglandin E1, did not lead to aggregation and was accompanied by crosslinking of fibrinogen through its A alpha and gamma chains, either to other fibrinogen molecules or to a platelet protein or proteins.

Specific Cross-reaction of IgG Anti-phospholipid Antibody with Platelet Glycoprotein IIIa

1996 ◽  
Vol 75 (01) ◽  
pp. 168-174 ◽  
Author(s):  
Shigeru Tokita ◽  
Morio Arai ◽  
Naomasa Yamamoto ◽  
Yasuhiro Katagiri ◽  
Kenjiro Tanoue ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Heparan Sulfate ◽  
Disulfide Bonds ◽  
Dextran Sulfate ◽  
Human Platelets ◽  
Platelet Glycoprotein ◽  
Activated Platelets ◽  
Fibrinogen Binding ◽  
Dose Dependent Fashion ◽  
Glycoprotein Iiia

SummaryTo study the pathological functions of anti-phospholipid (anti-PL) antibodies, we have analyzed their effect on platelet function. We identified an IgG anti-PL mAb, designated PSG3, which cross-reacted specifically with glycoprotein (GP) IIIa in human platelets and inhibited platelet aggregation. PSG3 bound also to certain polyanionic substances, such as double-stranded DNA, heparan sulfate, dextran sulfate and acetylated-LDL, but not to other polyanionic substances. The binding of PSG3 to GPIIIa was completely inhibited by heparan sulfate and dextran sulfate, indicating that PSG3 recognizes a particular array of negative charges expressed on both GPIIIa and the specified polyanionic substances. Since neither neuraminidase- nor endoglycopeptidase F-treatment of GPIIIa had any significant effect on the binding of PSG3, this array must be located within the amino acid sequence of GPIIIa but not in the carbohydrate moiety. Reduction of the disulfide bonds in GPIIIa greatly reduced its reactivity, suggesting that the negative charges in the epitope are arranged in a particular conformation. PSG3 inhibited platelet aggregation induced by either ADP or collagen, it also inhibited fibrinogen binding to activated platelets in a dose-dependent fashion. PSG3, however, did not inhibit the binding of GRGDSP peptide to activated platelets. These results suggest that the PSG3 epitope on GPIIIa contains a particular array of negative charges, and possibly affects the fibrinogen binding to GPIIb/IIIa complex necessary for platelet aggregation.

Pretreatment of Human Platelets with Plasmin Inhibits Responses toThrombin, but Potentiates Responses to Low Concentrations of Aggregating Agents, Including the Thrombin Receptor Activating Peptide, SFLLRN

1997 ◽  
Vol 77 (04) ◽  
pp. 741-747 ◽  
Author(s):  
R L Kinlough-Rathbone ◽  
D W Perry ◽  
M L Rand ◽  
M A Packham
Keyword(s):  
Arachidonic Acid ◽  
Platelet Activating Factor ◽  
Human Platelets ◽  
Thrombin Receptor ◽  
Fibrinolytic Agents ◽  
Albumin Solution ◽  
Fibrinogen Receptor ◽  
Low Concentrations ◽  
Induced Aggregation

SummaryEffects of plasmin on platelets, that influence subsequent responses to aggregating agents, are relevant to attempts to prevent rethrombosis following administration of fibrinolytic agents. We describe plasmin-induced inhibition of platelet responses to thrombin, but potentiation of responses to other aggregating agents. Washed human platelets were labeled with 14C-serotonin, treated for 30 min at 37° C with 0, 0.1 or 0.2 CU/ml of plasmin, followed by aprotinin, washed and resuspended in a Tyrode-albumin solution with apyrase. Incubation with 0.2 CU/ml of plasmin almost completely inhibited thrombin-induced (0.1 U/ml) aggregation, release of 14C-serotonin, and increase in cytosolic [Ca2+]. In contrast, with plasmin-pretreated platelets, aggregation and release of 14C-serotonin were strongly potentiated in response to low concentrations of the thrombin receptor-activating peptide SFLLRN, ADP, platelet-activating factor, collagen, arachidonic acid, the thromboxane mimetic U46619, and the calcium ionophores A23187 and ionomycin. Aspirin or RGDS partially inhibited potentiation. Plasmin-pretreated platelets resuspended in plasma anticoagulated with FPRCH2C1 (PPACK) also showed enhanced responses to aggregating agents other than thrombin. The contrasting effects on responses to thrombin and SFLLRN are noteworthy. Plasmin cleaves GPIIb/IIIa so that it becomes a competent fibrinogen receptor, and binding of 125I-fibrinogen during ADP-induced aggregation was greatly potentiated within 10 s. Potentiation of aggregation by other agonists may be due to increased binding of released fibrinogen. Thus, platelets freed from a thrombus may have increased responsiveness to low concentrations of aggregating agents other than thrombin. These results provide further support for the use of inhibitors of platelet reactions in conjunction with administration of fibrinolytic agents.

Mobilization of Fibrinogen Receptor Sites on Human Platelets Stimulated with ADP is Prevented by Prostacyclin

1979 ◽  
Author(s):  
J. Hawiger ◽  
S. Parkinson ◽  
S. Timmons
Keyword(s):  
Inhibitory Effect ◽  
Human Platelets ◽  
Affinity Constant ◽  
Human Fibrinogen ◽  
Fibrinogen Receptor ◽  
Receptor Sites ◽  
Plasma Factor ◽  
Potent Inhibitory Effect

Fibrinogen is a plasma factor required for aggregation of human platelets by ADP. The mechanism of platelet-ADP-fibrinogen interaction was studied by measuring the equilibrium binding of 125I-fibrinogen to human platelets separated from plasma proteins. Binding of 125I-fibrinogen to platelets not stimulated with ADP was low and unaffected by an excess of unlabel led fibrinogen. However, when platelets were stimulated with 4μM of ADP, there was an eightfold increase In the number of available binding sites for human fibrinogen, with affinity constant of 1.9 x 109M-1. This striking increase in fibrinogen receptor sites on human platelets was specific for ADP as contrasted to ATP, AMP, and adenosine. Prostacyclin (Prostaglandin I2, PGI2), a novel prostaglandin produced by the blood vessel wall, completely blocked this ADP-induced increase in fibrinogen receptor sites on human platelets. The effect of PGI2 was prompt and concentration dependent, reaching maximum at 10-9M. 6-keto PGF2 a stable derivative ot PGI2, did not have such an effect. Thus movement of fibrinogen receptor sites on human platelet membrane stimulated with ADP is prevented by PGI2. This represents a new biologic property of this vascular hormone and contributes to better understanding of its potent inhibitory effect in vitro and in vivo on ADP-induced platelet aggregation requiring mobilization of fibrinogen receptor.

scholarly journals Val34Leu polymorphism of plasma factor XIII: biochemistry and epidemiology in familial thrombophilia

Blood ◽  
2000 ◽  
Vol 96 (7) ◽  
pp. 2479-2486 ◽  
Author(s):  
István Balogh ◽  
Gabriella Szôke ◽  
Levente Kárpáti ◽  
Ulla Wartiovaara ◽  
Éva Katona ◽  
...  
Keyword(s):  
Protective Effect ◽  
Venous Thrombosis ◽  
Factor Xiii ◽  
Coagulation Factor ◽  
Wild Type ◽  
Thrombin Cleavage ◽  
Plasma Factor ◽  
Thrombin Activation ◽  
A Subunit ◽  
The Mean

Abstract Val34Leu polymorphism of the A subunit of coagulation factor XIII (FXIII-A) is located in the activation peptide (AP) just 3 amino acids away from the thrombin cleavage site. This mutation has been associated with a protective effect against occlusive arterial diseases and venous thrombosis; however, its biochemical consequences have not been explored. In the current study it was demonstrated that the intracellular stability and the plasma concentration of FXIII of different Val34Leu genotypes are identical, which suggests that there is no difference in the rate of synthesis and externalization of wild-type and mutant FXIII-A. In contrast, the release of AP by thrombin from the Leu34 allele proceeded significantly faster than from its wild-type Val34 counterpart. By molecular modeling larger interaction energy was calculated between the Leu34 variant and the respective domains of thrombin than between the Val34 variant and thrombin. In agreement with these findings, the activation of mutant plasma FXIII by thrombin was faster and required less thrombin than that of the wild-type variant. Full thrombin activation of purified plasma FXIII of different genotypes, however, resulted in identical specific transglutaminase activities. Similarly, the mean specific FXIII activity in the plasma was the same in the groups with wild-type, heterozygous, and homozygous variants. Faster activation of the Leu34 allele hardly could be associated with its presumed protective effect against venous thrombosis. No such protective effect was observed in a large group of patients with familial thrombophilia.

The Regulatory Role Of Camp In Induction Of Human Platelet Receptor For Fibrinogen

1981 ◽  
Author(s):  
S E Graber ◽  
J Hawiger
Keyword(s):  
Human Platelets ◽  
Membrane Receptor ◽  
Fibrinogen Receptor ◽  
Fibrinogen Binding ◽  
Platelet Receptor ◽  
The Relationship ◽  
Camp Levels ◽  
Dose Dependent ◽  
Stimulation Of

Membrane receptor for fibrinogen plays an essential role in adhesion and aggregation of human platelets by allowing fibrinogen to bridge two or more platelets together. Whereas in normal, unstimulated platelets fibrinogen receptor is not available, it becomes mobilized upon stimulation of platelets with thrombin, ADP, and other stimuli. The mechanism(s) regulating availability of membrane receptor for fibrinogen remains unknown. Following our recent demonstration that prostacyclin (PGI2) prevents mobilization of fibrinogen receptor by thrombin and ADP (Nature 1980, 283,195), we investigated the relationship between cAMP levels and fibrinogen receptor availability. Platelets separated from plasma proteins were briefly exposed to a low thrombin concentration (0.05 U/ml) followed by hirudin to inactivate free thrombin. Binding of 125I-fi- brinogen and cAMP levels were determined in parallel samples. A dose-dependent rise in platelet cAMP levels from 3.3 pM to 10.3 pM/108 platelets in response to PGI2 (3×10-9M - 3×108M) was accompanied by a corresponding inhibition of 125I-fibrinogen binding. The degree of the cAMP increment correlated with binding inhibition (r=0.96). The inhibition of 125I-fibrinogen binding by PGI2 was sustained up to 120 min and was paralleled by a persistent rise in cAMP level. Stimulation of platelet cAMP synthesis “from within” by a ribosylation of the nucleotide regulatory component with subunit A1 of cholera toxin also increased cAMP levels and inhibited fibrinogen receptor mobilization.These results provide evidence that “up and down” regulation of fibrinogen receptor in platelets is linked to changes in cAMP levels induced by different types of adenyl cyclase antagonists and agonists.

scholarly journals Normalization of Blood Clotting Characteristics Using Prothrombin Complex Concentrate, Fibrinogen and Fxiii In an Albumin Based Fluid: Experimental Studies In Thromboelastometry.

2020 ◽  
Author(s):  
Tobias Koller ◽  
Nadia Kinast ◽  
Andres Guilarte Castellanos ◽  
Sergio Perez Garcia ◽  
Pilar Paniagua Iglesias ◽  
...  
Keyword(s):  
Whole Blood ◽  
Factor Xiii ◽  
Prothrombin Complex Concentrate ◽  
Massive Transfusion ◽  
Experimental Studies ◽  
Coagulation Factor ◽  
Fibrin Clot ◽  
Fibrinogen Concentrate ◽  
Coagulation Factor Concentrates

Abstract Background: Colloid fluids supplemented with adequate combinations of coagulation factor concentrates with capability to restore coagulation could be a desirable future treatment component in massive transfusion.Methods: Starting from a coagulation factor and blood cell free albumin solution we added Prothrombin Complex Concentrate, Fibrinogen Concentrate and Factor XIII in different combinations and concentrations to analyze their properties to restore thromboelastometry parameters without the use of plasma. Further analysis under presence of platelets was performed for comparability to whole blood conditions.Results: Albumin solutions enriched with Fibrinogen Concentrate, Factor XIII and Prothrombin Complex Concentrate at optimized concentrations show restoring coagulation potential. Prothrombin Complex Concentrate showed sufficient thrombin formation for inducing fibrinogen polymerization. The combination of Prothrombin Complex Concentrate and Fibrinogen Concentrate led to the formation of a stable in vitro fibrin clot. Fibrinogen and Factor XIII showed excellent capacity to improve fibrin clot firmness expressed as Amplitude at 10 minutes and Maximal Clot Firmness. Fibrinogen alone, or in combination with Factor XIII, was able to restore normal Amplitude at 10 minutes and Maximal Clot Firmness values. In the presence of platelets, the thromboelastometry surrogate parameter for thrombin generation (Clotting Time) improves and normalizes when compared to whole blood.Conclusions: Combinations of coagulation factor concentrates suspended in albumin solutions have the capacity to restore thromboelastometry parameters in the absence of plasma. This kind of artificial colloid fluids with coagulation-restoring characteristics might offer new treatment alternatives for massive transfusion.Trial registration: Study registered at the institutional ethic committee “Institut de Recerca, Hospital Santa Creu i Sant Pau, with protocol number IIBSP-CFC-2013-165.

Sign in / Sign up

Export Citation Format

Share Document