Impaired clot retraction in factor XIII A subunit–deficient mice

Blood ◽  
2010 ◽  
Vol 115 (6) ◽  
pp. 1277-1279 ◽  
Author(s):  
Kohji Kasahara ◽  
Masayoshi Souri ◽  
Mizuho Kaneda ◽  
Toshiaki Miki ◽  
Naomasa Yamamoto ◽  
...  
Keyword(s):  
Knockout Mice ◽  
Factor Xiii ◽  
Platelet Rich Plasma ◽  
Tissue Transglutaminase ◽  
Adenosine Diphosphate ◽  
Wild Type ◽  
Clot Retraction ◽  
A Subunit ◽  
Fxiii Deficiency

Abstract Factor XIII (FXIII) is a plasma transglutaminase that cross-links fibrin monomers, α2-plasmin inhibitor, and so forth. Congenital FXIII deficiency causes lifelong bleeding symptoms. To understand the molecular pathology of FXIII deficiency in vivo, its knockout mice have been functionally analyzed. Because prolonged bleeding times, a sign of defective/abnormal primary hemostasis, were commonly observed in 2 separate lines of FXIII A subunit (FXIII-A) knockout mice, a possible role or roles of FXIII in platelet-related function was investigated in the present study. Although platelet aggregation induced by adenosine diphosphate or collagen was normal, clot retraction (CR) was lost in the platelet-rich plasma (PRP) of FXIII-A knockout mice. In contrast, there was no CR impairment in the PRP of tissue transglutaminase-knockout mice compared with that of wild-type mice. Furthermore, a transglutaminase inhibitor, cystamine, halted CR in the PRP of wild-type mice. These results indicate that the enzymatic activity of FXIII is necessary for CR, at least in mice.

Male-specific cardiac pathologies in mice lacking either the A or B subunit of factor XIII

2008 ◽  
Vol 99 (02) ◽  
pp. 401-408 ◽  
Author(s):  
Masayoshi Souri ◽  
Shiori Koseki-Kuno ◽  
Naoki Takeda ◽  
Mitsunori Yamakawa ◽  
Yasuchika Takeishi ◽  
...  
Keyword(s):  
Factor Xiii ◽  
Cardiac Fibrosis ◽  
Survival Rates ◽  
Heart Tissue ◽  
Wild Type ◽  
B Subunit ◽  
Reparative Process ◽  
Male Specific ◽  
Fxiii Deficiency

SummaryFactor XIII (FXIII) is a proenzyme of plasma transglutaminase consisting of enzymatic A subunits (FXIII-A) and non-catalytic B subunits (FXIII-B), and acts in haemostasis and wound healing. We generated mice lacking either FXIII-A or FXIII-B to investigate the physiological functions of FXIII in vivo. A longitudinal study was carried out using the gene-targeted mice to explore the possible effects of FXIII deficiency on aging. Survival rates of FXIII-A-/- males decreased to approximately 50% at 10 months after birth, although most FXIII-A-/- females and both genders of wild-type mice survived. Four FXIII-A-/- males died of severe intra-thoracic haemorrhage, and a large haematoma was found in their hearts. Haemorrhage, haemosiderin deposition and/or fibrosis were observed in the hearts of other dead FXIII-A-/- males. Fibrosis together with haemosiderin deposition was also found in the hearts of FXIII-A-/- males sacrificed. The in-vivo cardiac function was normal in FXIII-A-/- mice when compared with wild-type mice despite the presence of significant cardiac fibrosis. Although survival rates for both genders of the FXIII-B-/- and wild-type mice did not differ, mild fibrosis together with haemosiderin deposits were only found in the hearts of the sacrificed FXIII-B-/- males. Carditis and fibrosis in FXIII-deficient mice might be caused by a faulty or delayed reparative process that was initiated by abnormal haemorrhagic events within heart tissue. It is important therefore to examine possible cardiac involvement in human patients with congenital FXIII deficiency.

Factor XIII A subunit-deficient mice developed severe uterine bleeding events and subsequent spontaneous miscarriages

Blood ◽  
2003 ◽  
Vol 102 (13) ◽  
pp. 4410-4412 ◽  
Author(s):  
Shiori Koseki-Kuno ◽  
Mitsunori Yamakawa ◽  
Gerhard Dickneite ◽  
Akitada Ichinose
Keyword(s):  
Molecular Pathology ◽  
Factor Xiii ◽  
Vaginal Bleeding ◽  
Critical Role ◽  
Uterine Bleeding ◽  
Bleeding Events ◽  
A Subunit ◽  
Deficient Mice ◽  
Fxiii Deficiency

AbstractTo understand the molecular pathology of factor XIII (FXIII) deficiency in vivo, its A subunit (FXIIIA)-knockout (KO) mice were functionally analyzed. Although homozygous FXIIIA female KO mice were capable of becoming pregnant, most of them died due to excessive vaginal bleeding during gestation. Abdominal incisions revealed that the uteri of the dead mice were filled with blood and that some embryos were much smaller than others within a single uterus. A series of histologic examinations of the pregnant animals suggested that massive placental hemorrhage and subsequent necrosis developed in the uteri of the FXIIIA KO mice on day 10 of gestation. This was true regardless of the genotypes of fetuses. These results are reminiscent of spontaneous miscarriage in pregnant humans with FXIII deficiency and indicate that maternal FXIII plays a critical role in uterine hemostasis and maintenance of the placenta during gestation. (Blood. 2003;102:4410-4412)

scholarly journals Effects of factor XIII deficiency on thromboelastography. Thrombelastography with calcium and lysis by addition of streptokinase is more sensitive than solubility tests

2016 ◽  
Vol 8 ◽  
pp. e2016037 ◽  
Author(s):  
Marta E Martinuzzo
Keyword(s):  
Acetic Acid ◽  
Factor Xiii ◽  
Maximum Amplitude ◽  
Clot Lysis ◽  
Thrombin Time ◽  
Spontaneous Bleeding ◽  
A Subunit ◽  
One Year ◽  
Fxiii Deficiency

Background:Homozygous or double heterozygous factor XIII (FXIII) deficiency is characterized by soft tissue hematomas, intracranial and delated spontaneous bleeding. Alterations of thromboelastographic (TEG) parameters in these patients have been reported. The aim of the study was to show results of TEG, TEG Lysis (Lys 60) induced by subthreshold concentrations of streptokinase (SK), and to compare them to the clot solubility studies results in samples of a1 year old girl with homozygous or double heterozygous FXIII deficiency.Materials and Methods:Case: A one year girl with history of bleeding from umbilical cord. During her first year of live several hematomas in soft upper limb tissue after punctures for vaccination and a gluteal hematoma appeared. One additional sample of a heterozygous and three of acquired FXIII deficiency were also evaluated.Materials and Methods: clotting tests, von Willebrand factor (vWF) antigen and activity,  plasma FXIII-A (pFXIII-A) subunit were measured by an immunoturbidimetric assays in a foto-optical coagulometer. Solubility tests were performed with Ca2+-5 M urea and thrombin-2% acetic acid. Basal and post FXIII concentrate infusion samples were studied. TEG was performed with CaCl2 or CaCl2 + SK (3.2 U/mL) in a Thromboelastograph. Results: Prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time, fibrinogen, factor VIIIc, vWF and platelet aggregation were normal. Antigenic pFXIII-A subunit was < 2%. TEG presented a normal reaction time (R), 8 min, prolonged k (14 and11min), low Maximum amplitude (MA), 39 and 52 mm,  and Clot Lysis (Lys60) slightly increased (23 and 30%) at diagnosis and post FXIII concentrate infusion (pFXIII-A= 37%), respectively. In Sample at diagnosis clot solubility was abnormal, 50 and 45 min with Ca-Urea and thrombin-acetic acid, respectively, but normal (>16 hours) 1 day post FXIII infusion. Analysis of FXIII deficient and normal plasma mixtures (< 2 -102% of pFXIII-A), showed that Ca-urea solubility was abnormal at pFXIII-A < 9%, thrombin-acetic acid at pFXIII-A<18%, but TEG MA and elasticity at 23% and Lys60 with SK at pFXIII-A< 40%.Conclusions: TEG parameters MA and elasticity, and Lys 60 in TEG with Ca2+ and in TEG Ca2+ and SK are more sensitive to low levels of pFXIII than solubility tests. The increased Lys60 induced by subthreshold concentration of SK could probably reflect the clot characteristics “in vivo” in many patients with pFXIII levels between 5-40% and could be potentially considered as screening test.

scholarly journals Preoperative administration of the 5-HT4 receptor agonist prucalopride reduces intestinal inflammation and shortens postoperative ileus via cholinergic enteric neurons

Gut ◽  
2018 ◽  
Vol 68 (8) ◽  
pp. 1406-1416 ◽  
Author(s):  
Nathalie Stakenborg ◽  
Evelien Labeeuw ◽  
Pedro J Gomez-Pinilla ◽  
Sebastiaan De Schepper ◽  
Raymond Aerts ◽  
...  
Keyword(s):  
Knockout Mice ◽  
Postoperative Ileus ◽  
Intestinal Inflammation ◽  
Electrical Field Stimulation ◽  
Enteric Neurons ◽  
Postoperative Treatment ◽  
Wild Type ◽  
Clinical Recovery ◽  
Preoperative Administration

ObjectivesVagus nerve stimulation (VNS), most likely via enteric neurons, prevents postoperative ileus (POI) by reducing activation of alpha7 nicotinic receptor (α7nAChR) positive muscularis macrophages (mMφ) and dampening surgery-induced intestinal inflammation. Here, we evaluated if 5-HT4 receptor (5-HT4R) agonist prucalopride can mimic this effect in mice and human.DesignUsing Ca2+ imaging, the effect of electrical field stimulation (EFS) and prucalopride was evaluated in situ on mMφ activation evoked by ATP in jejunal muscularis tissue. Next, preoperative and postoperative administration of prucalopride (1–5 mg/kg) was compared with that of preoperative VNS in a model of POI in wild-type and α7nAChR knockout mice. Finally, in a pilot study, patients undergoing a Whipple procedure were preoperatively treated with prucalopride (n=10), abdominal VNS (n=10) or sham/placebo (n=10) to evaluate the effect on intestinal inflammation and clinical recovery of POI.ResultsEFS reduced the ATP-induced Ca2+ response of mMφ, an effect that was dampened by neurotoxins tetrodotoxin and ω-conotoxin and mimicked by prucalopride. In vivo, prucalopride administered before, but not after abdominal surgery reduced intestinal inflammation and prevented POI in wild-type, but not in α7nAChR knockout mice. In humans, preoperative administration of prucalopride, but not of VNS, decreased Il6 and Il8 expression in the muscularis externa and improved clinical recovery.ConclusionEnteric neurons dampen mMφ activation, an effect mimicked by prucalopride. Preoperative, but not postoperative treatment with prucalopride prevents intestinal inflammation and shortens POI in both mice and human, indicating that preoperative administration of 5-HT4R agonists should be further evaluated as a treatment of POI.Trial registration numberNCT02425774.

FUNDC2 regulates platelet activation through AKT/GSK-3β/cGMP axis

2018 ◽  
Vol 115 (11) ◽  
pp. 1672-1679 ◽  
Author(s):  
Qi Ma ◽  
Weilin Zhang ◽  
Chongzhuo Zhu ◽  
Junling Liu ◽  
Quan Chen
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Platelet Rich Plasma ◽  
Thrombus Formation ◽  
Mitochondrial Protein ◽  
Dependent Manner ◽  
Clot Retraction ◽  
Akt Phosphorylation ◽  
Gsk 3Β

Abstract Aims AKT kinase is vital for regulating signal transduction in platelet aggregation. We previously found that mitochondrial protein FUNDC2 mediates phosphoinositide 3-kinase (PI3K)/phosphatidylinositol-3,4,5-trisphosphate (PIP3)-dependent AKT phosphorylation and regulates platelet apoptosis. The aim of this study was to evaluate the role of FUNDC2 in platelet activation and aggregation. Methods and results We demonstrated that FUNDC2 deficiency diminished platelet aggregation in response to a variety of agonists, including adenosine 5′-diphosphate (ADP), collagen, ristocetin/VWF, and thrombin. Consistently, in vivo assays of tail bleeding and thrombus formation showed that FUNDC2-knockout mice displayed deficiency in haemostasis and thrombosis. Mechanistically, FUNDC2 deficiency impairs the phosphorylation of AKT and downstream GSK-3β in a PI3K-dependent manner. Moreover, cGMP also plays an important role in FUNDC2/AKT-mediated platelet activation. This FUNDC2/AKT/GSK-3β/cGMP axis also regulates clot retraction of platelet-rich plasma. Conclusion FUNDC2 positively regulates platelet functions via AKT/GSK-3β/cGMP signalling pathways, which provides new insight for platelet-related diseases.

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.

scholarly journals Zinc-induced platelet aggregation is mediated by the fibrinogen receptor and is not accompanied by release or by thromboxane synthesis

Blood ◽  
1985 ◽  
Vol 66 (1) ◽  
pp. 213-219 ◽  
Author(s):  
P Heyns A du ◽  
A Eldor ◽  
R Yarom ◽  
G Marx
Keyword(s):  
Platelet Aggregation ◽  
Dense Body ◽  
Platelet Rich Plasma ◽  
Adenosine Monophosphate ◽  
Adenosine Diphosphate ◽  
Creatine Phosphate ◽  
Calcium Flux ◽  
Fibrinogen Receptor ◽  
Induced Aggregation

Abstract We demonstrate that zinc (0.1 to 0.3 mmol/L) induces aggregation of washed platelet suspensions. Higher concentrations (1 to 3 mmol/L) of zinc were needed to aggregate platelets in platelet-rich plasma obtained from blood anticoagulated with low-molecular-weight heparin, probably due to the binding of zinc to the plasma proteins. Zinc- induced aggregation of normal washed platelets required added fibrinogen and no aggregation occurred with thrombasthenic platelets or with normal platelets pretreated with a monoclonal antibody (10E5) that blocks the platelet fibrinogen receptor. These data indicate that the platelet membrane fibrinogen receptor-glycoproteins IIb and IIIa mediate the effect of zinc. Zinc-induced aggregation was blocked by the agent TMB-8, which interferes with the internal calcium flux, and by prostacyclin, which elevates platelet cyclic adenosine monophosphate levels. Zinc-induced aggregation was not accompanied by thromboxane synthesis or by the secretion of dense-body serotonin and was not affected by preexposure of platelets to acetylsalicylic acid. Experiments with creatine phosphate/creatine phosphokinase showed that the zinc effect on platelets was independent of extracellular adenosine diphosphate (ADP). Zinc had an additive effect when platelet aggregation was stimulated with subthreshhold concentrations of collagen or ADP. Together with the known effects of nutritional zinc on in vivo bleeding, on platelet aggregation, and on lipid metabolism, the results suggest that zinc may have an important bearing on normal hemostasis, thrombosis, and atherosclerosis.

scholarly journals Effect of Targeted Deletion of the Heme Oxygenase-2 Gene on Hemoglobin Toxicity in the Striatum

2005 ◽  
Vol 25 (11) ◽  
pp. 1466-1475 ◽  
Author(s):  
Yan Qu ◽  
Jing Chen ◽  
Luna Benvenisti-Zarom ◽  
Xin Ma ◽  
Raymond F Regan
Keyword(s):  
Heme Oxygenase ◽  
Knockout Mice ◽  
Protein Oxidation ◽  
Gene Deletion ◽  
Wild Type ◽  
Cell Culture Study ◽  
Targeted Deletion ◽  
Rate Limiting Step ◽  
Diphenyltetrazolium Bromide

The heme oxygenase (HO) enzymes catalyze the rate-limiting step in the breakdown of heme to iron, carbon monoxide, and biliverdin. A prior cell culture study demonstrated that deletion of HO-2, the isoform constitutively expressed in neurons, attenuated hemoglobin (Hb) neurotoxicity. The present study tested the hypothesis that HO-2 gene deletion is cytoprotective in a model of Hb toxicity in vivo. Stereotactic injection of 6 μL stroma-free Hb (SFHb) into the striatum significantly increased protein oxidation in wild-type mice at 24 to 72 h, as detected by an assay for carbonyl groups. At 72 h, carbonylation was increased 2.5-fold compared with that in the contralateral striatum. In HO-2 knockout mice, protein oxidation was not increased at 24 h, and was increased by only 1.7-fold at 72 h. Similarly, striatal lipid peroxidation, as detected by the malondialdehyde assay, was significantly greater in the SFHb-injected striata of wild-type mice than in knockout mice. Striatal cell viability, determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, was 45.0%±6.3% of that in contralateral striata in wild-type mice at 72 h; it was increased to 85%±8% in knockouts. Heme oxygenase-2 gene deletion did not alter weight loss or mortality after SFHb injection. Baseline striatal HO-1 expression was similar in knockout and wild-type mice; induction after SFHb injection occurred more rapidly in the latter. These results suggest that HO-2 gene deletion protects striatal cells from the oxidative toxicity of Hb in vivo. Pharmacologic or genetic strategies that target HO-2 may be beneficial after central nervous system hemorrhage, and warrant further investigation.

Quantification of the adhesion of platelets in hamster venules in vivo

1982 ◽  
Vol 215 (1199) ◽  
pp. 135-145 ◽  
Keyword(s):  
Platelet Rich Plasma ◽  
Fluorescein Isothiocyanate ◽  
Adenosine Diphosphate ◽  
Cheek Pouch ◽  
Video Tape ◽  
Flow Through ◽  
Platelet Aggregates ◽  
The Mean ◽  
Adhesion Of Platelets

(i) Citrated platelet-rich plasma freshly prepared from golden hamsters was mixed with fluorescein isothiocyanate (FITC) which made the platelets fluorescent. These platelets were injected intravenously into anaesthetized hamsters with exteriorized cheek pouch preparations superfused at 37 °C with Krebs-bicarbonate solution. The exposed microcirculation was observed microscopically by bright field or fluores­cent illumination. The flowing and sticking of fluorescent platelets was recorded on video tape for quantitative analysis. (ii) In four experiments 22–36%, mean 28%, of fluorescent platelets were circulating 2-3 h after their injection. In seven experiments the fluorescent platelets accounted for 0.6–3.3 %, mean 1.7 %, of circulating platelets. (iii) In venules 20–60 μm in diameter small proportions, mean 5.4%, of circulating fluorescent platelets stopped moving by sticking to the vessel walls. About 80 % of these platelets stuck for up to 1 s, a further 10-15% for up to 5 s, and only about 2% for longer than 2 min. There was an inverse relation between size of venule and proportion of platelets sticking in them. (iv) There was a direct relation between the mean velocities at which platelets flowed through the venules and the sizes of the venules. In the smaller venules the velocity distribution of the platelets had a clear maximum which was not as evident in larger venules. (v) In a few observations on arterioles, flowing platelets could not be seen, and arrested platelets only in a dilatation and at a capillary branch. (vi) Ethylenediamine tetraacetate in the superfusing fluid decreased platelet sticking in venules but did not abolish it. (vii) Adenosine diphosphate in the superfusing fluid caused the appear­ance of platelet aggregates in venules and of sticking platelets in arterioles during progressive diminution in blood flow through both types of vessel. (viii) The observations make it improbable that the release of platelet constituents affects normal venules or arterioles except, possibly, where haemodynamic conditions are affected by wall irregularities such as dilations or branching.

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