Thrombosis-associated hypofibrinogenemia: novel abnormal fibrinogen variant FGG c.8G>A with oxidative posttranslational modifications.

Here, we present the first case of fibrinogen variant FGG c.8G>A. We investigated the behaviour of this mutated fibrinogen in blood coagulation using fibrin polymerization, fibrinolysis, fibrinopeptides release measurement, mass spectrometry (MS), and scanning electron microscopy (SEM). The case was identified by routine coagulation testing of a 34-year-old man diagnosed with thrombosis. Initial genetic analysis revealed a heterozygous mutation in exon 1 of the FGG gene encoding gamma chain signal peptide. Fibrin polymerization by thrombin and reptilase showed the normal formation of the fibrin clot. However, maximal absorbance within polymerization was lower and fibrinolysis had a longer degradation phase than healthy control. SEM revealed a significant difference in clot structure of the patient, and interestingly, MS detected several posttranslational oxidations of fibrinogen. The data suggest that the mutation FGG c.8G>A with the combination of the effect of posttranslational modifications causes a novel case of hypofibrinogenemia associated with thrombosis.

Fibrin Clot Formation under Oxidative Stress Conditions.

During coagulation, the soluble fibrinogen is converted into insoluble fibrin. Fibrinogen is a multifunctional plasma protein, which is essential for hemostasis. Various oxidative posttranslational modifications influence fibrinogen structure as well as interactions between various partners in the coagulation process. The aim was to examine the effects of oxidative stress conditions on fibrin clot formation in arterial atherothrombotic disorders. We studied the changes in in vitro fibrin network formation in three groups of patients-with acute coronary syndrome (ACS), with significant carotid artery stenosis (SCAS), and with acute ischemic stroke (AIS), as well as a control group. The level of oxidative stress marker malondialdehyde measured by LC-MS/MS was higher in SCAS and AIS patients compared with controls. Turbidic methods revealed a higher final optical density and a prolonged lysis time in the clots of these patients. Electron microscopy was used to visualize changes in the in vitro-formed fibrin network. Fibers from patients with AIS were significantly thicker in comparison with control and ACS fibers. The number of fibrin fibers in patients with AIS was significantly lower in comparison with ACS and control groups. Thus, oxidative stress-mediated changes in fibrin clot formation, structure and dissolution may affect the effectiveness of thrombolytic therapy.

Targeting Human Thrombus by Liposomes Modified with Anti-Fibrin Protein Binders.

Development of tools for direct thrombus imaging represents a key step for diagnosis and treatment of stroke. Nanoliposomal carriers of contrast agents and thrombolytics can be functionalized to target blood thrombi by small protein binders with selectivity for fibrin domains uniquely formed on insoluble fibrin. We employed a highly complex combinatorial library derived from scaffold of 46 amino acid albumin-binding domain (ABD) of streptococcal protein G, and ribosome display, to identify variants recognizing fibrin cloth in human thrombus. We constructed a recombinant target as a stretch of three identical fibrin fragments of 16 amino acid peptide of the Bß chain fused to TolA protein. Ribosome display selection followed by large-scale Enzyme-Linked ImmunoSorbent Assay (ELISA) screening provided four protein variants preferentially binding to insoluble form of human fibrin. The most specific binder variant D7 was further modified by C-terminal FLAG/His-Tag or double His-tag for the attachment onto the surface of nanoliposomes via metallochelating bond. D7-His-nanoliposomes were tested using in vitro flow model of coronary artery and their binding to fibrin fibers was demonstrated by confocal and electron microscopy. Thus, we present here the concept of fibrin-targeted binders as a platform for functionalization of nanoliposomes in the development of advanced imaging tools and future theranostics.

Enhanced plasma protein carbonylation in patients with myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) represent a heterogeneous group of pre-leukemic disorders, characterized by ineffective hematopoiesis and the abnormal blood cell development of one or more lineages. Oxidative stress, as an important factor in the carcinogenesis of onco-hematological diseases, is also one of the known factors involved in the pathogenesis of MDS. An increase of reactive oxygen species (ROS) may lead to the oxidation of DNA, lipids, and proteins, thereby causing cell damage. Protein carbonylation caused by ROS is defined as an irreversible post-translational oxidative modification of amino acid side chains, and could play an important role in signaling processes. The detection of protein carbonyl groups is a specific useful marker of oxidative stress. In this study, we examined 32 patients divided into three different subtypes of MDS according to the World Health Organization (WHO) classification criteria as refractory anemia with ringed sideroblasts (RARS), refractory cytopenia with multilineage dysplasia (RCMD), refractory anemia with excess blasts-1,2 (RAEB-1,2). We found significant differences in protein carbonylation between the group of all MDS patients and healthy controls (P=0.0078). Furthermore, carbonylated protein levels were significantly elevated in RARS patients compared to healthy donors (P=0.0013) and to RCMD patients (P=0.0277). We also found a significant difference in the total iron binding capacity (TIBC) between individual subgroups of MDS patients (P=0.0263). Moreover, TIBC was decreased in RARS patients compared to RCMD patients (P=0.0203). TIBC moderately negatively correlated with carbonyl levels (r=-0.5978, P=0.0054) in the MDS patients as a whole. Additionally we observed changes in the carbonylated proteins of RARS patients in comparison with healthy controls and their negative controls. Using tandem mass spectrometry (LC-MS/MS) we identified 27 uniquely carbonylated proteins of RARS patients, which were generated by ROS and could influence the pathophysiology of low-risk MDS. These data indicate that increased protein carbonylation is related with RARS as low-risk MDS subgroup. We suggest that this type of post-translational modification in MDS disease is not "only" a consequence of oxidative stress, but also plays an active role in the pathophysiology and iron metabolism within the RARS subgroup of MDS. Measurement of plasma carbonyl levels and the isolation of carbonylated plasma proteins, followed by their identification, could serve as a potential diagnostic and prognostic tool in MDS.

Plasma levels of aminothiols, nitrite, nitrate, and malondialdehyde in myelodysplastic syndromes in the context of clinical outcomes and as a consequence of iron overload.

The role of oxidative stress in the initiation and progression of myelodysplastic syndromes (MDS) as a consequence of iron overload remains unclear. In this study we have simultaneously quantified plasma low-molecular-weight aminothiols, malondialdehyde, nitrite, and nitrate and have studied their correlation with serum iron/ferritin levels, patient treatment (chelation therapy), and clinical outcomes. We found significantly elevated plasma levels of total, oxidized, and reduced forms of cysteine (P < 0.001), homocysteine (P < 0.001), and cysteinylglycine (P < 0.006) and significantly depressed levels of total and oxidized forms of glutathione (P < 0.03) and nitrite (P < 0.001) in MDS patients compared to healthy donors. Moreover, total (P < 0.032) and oxidized cysteinylglycine (P = 0.029) and nitrite (P = 0.021) differed significantly between the analyzed MDS subgroups with different clinical classifications. Malondialdehyde levels in plasma correlated moderately with both serum ferritin levels (r = 0.78, P = 0.001) and serum free iron levels (r = 0.60, P = 0.001) and were significantly higher in patients with iron overload. The other analyzed compounds lacked correlation with iron overload (represented by serum iron/ferritin levels). For the first time our results have revealed significant differences in the concentrations of plasma aminothiols in MDS patients, when compared to healthy donors. We found no correlation of these parameters with iron overload and suggest the role of oxidative stress in the development of MDS disease.

A novel natural mutation AαPhe98Ile in the fibrinogen coiled-coil affects fibrinogen function.

The aim of this study was to investigate the structure and function of fibrinogen obtained from a patient with normal coagulation times and idiopathic thrombophilia. This was done by SDS-PAGE and DNA sequence analyses, scanning electron microscopy, fibrinopeptide release, fibrin polymerisation initiated by thrombin and reptilase, fibrinolysis, and platelet aggregometry. A novel heterozygous point mutation in the fibrinogen Aα chain, Phe98 to Ile, was found and designated as fibrinogen Vizovice. The mutation, which is located in the RGDF sequence (Aα 95-98) of the fibrinogen coiled-coil region, significantly affected fibrin clot morphology. Namely, the clot formed by fibrinogen Vizovice contained thinner and curled fibrin fibers with reduced length. Lysis of the clots prepared from Vizovice plasma and isolated fibrinogen were found to be impaired. The lysis rate of Vizovice clots was almost four times slower than the lysis rate of control clots. In the presence of platelets agonists the mutant fibrinogen caused increased platelet aggregation. The data obtained show that natural mutation of Phe98 to Ile in the fibrinogen Aα chain influences lateral aggregation of fibrin protofibrils, fibrinolysis, and platelet aggregation. They also suggest that delayed fibrinolysis, together with the abnormal fibrin network morphology and increased platelet aggregation, may be the direct cause of thrombotic complications in the patient associated with pregnancy loss.

Fibrinogen Sumperk II: dysfibrinogenemia in an individual with two coding mutations.

Fibrinogen­a 340-kDa glycoprotein­plays a crucial role in blood coagulation, platelet aggregation, wound healing, and other physiological processes. A mutation in fibrinogen may lead to congenital dysfibrinogenemia,a rare disease characterized by the functional deficiency of fibrinogen. About 580 cases of abnormal fibrinogens have been reported worldwide; thereof 335 cases in the fibrinogen Aa chain[1]. To our knowledge, only five cases of abnormal fibrinogens with two mutations [2­6] and one case of two different mutations in the same family [7] have been described earlier. A 52-year-old female was examined for bleeding. Routine hemostasis screening resulted in a diagnosis of dysfibrinogenemia. Functional testing revealed prolonged fibrin polymerization, prolonged lysis of the clot, abnormal fibrin morphology,and fibrinopeptides release. Genetic analysis showed two heterozygous nonsense mutations­previously described mutation AaGly13Glu and a novel mutation Aa Ser314Cys. The mutation Aa Gly13-Glu was found in her brother and niece, but there was no evidence in either of the mutation Aa Ser314Cys. While mutation Aa Gly13Glu is responsible for abnormal fibrinopeptide release and prolonged thrombin time, the novel mutation Aa Ser314Cys seems to affect fibrin morphology and fibrinolysis.

Two novel fibrinogen variants in the C-terminus of the Bß-chain: fibrinogen Rokycany and fibrinogen Znojmo.

Hereditary dysfibrinogenemia is a rare disorder wherein an inherited abnormality in fibrinogen structure may result in defective fibrin function and/or structure. Congenital hypofibrinogenemia is a rare autosomal bleeding disorder, either recessive or dominant, characterized by a low fibrinogen plasma level. A 28-year-old asymptomatic woman (fibrinogen Rokycany) and a 54-year-old man with thrombosis and pulmonary embolism (fibrinogen Znojmo) were investigated for a suspected fibrinogen mutation after abnormal coagulation tests results were obtained. DNA sequencing showed the heterozygous point mutation Bß Asn351Lys in fibrinogen Rokycany and the heterozygous point mutation Bß Arg237Ser in fibrinogen Znojmo, respectively. The kinetics of fibrinopeptide release was found to be normal in both cases. Fibrinolysis was impaired in the Znojmo variant. The average fibril diameters of Znojmo fibrin was slightly increased, but not differing significantly from normal; formed by less fibrils with abrupt fibril terminations. Rheological studies revealed a softer clot. Rokycany fibrin was formed by significantly narrower fibrils than normal fibrin; and the clot was denser than the control clot. Rheological studies revealed a stiffer clot. Impaired fibrinolysis and abnormal clot morphology may be the cause of thrombotic episodes in the patient with Znojmo mutation. New cases of hypofibrinogenemia and dysfibrinogenemia, found by routine coagulation testing, were genetically identified as a novel fibrinogen variants Bß Asn351Lys (fibrinogen Rokycany) and Bß Arg237Ser (fibrinogen Znojmo), respectively.

A novel fibrinogen variant--Liberec: dysfibrinogenaemia associated with gamma Tyr262Cys substitution.

OBJECTIVE: A 22-yr-old woman had abnormal preoperative coagulation test results and congenital dysfibrinogenaemia was suspected. PATIENTS AND METHODS: The patient from Liberec (Czech Republic) had a low fibrinogen plasma level as determined by Clauss method, normal fibrinogen level as determined by immunoturbidimetrical method, and prolonged thrombin time. To identify the genetic mutation responsible for this dysfibrinogen, genomic DNA extracted from the blood was analysed. Fibrin polymerisation measurement, kinetics of fibrinopeptide release, fibrinogen clottability measurement and scanning electron microscopy were performed. RESULTS: DNA sequencing showed the heterozygous fibrinogen gamma Y262C mutation. Kinetics of fibrinopeptide release was normal, however fibrin polymerisation was impaired. Fibrinogen clottability measurement showed that only about 45% molecules of fibrinogen are involved in the clot formation. Scanning electron microscopy revealed thicker fibres, which were significantly different from the normal control. CONCLUSION: A case of dysfibrinogenaemia, found by routine coagulation testing, was genetically identified as a novel fibrinogen variant (gamma Y262C) that has been named Liberec.

A novel fibrinogen variant--Praha I: hypofibrinogenemia associated with gamma Gly351Ser substitution.

OBJECTIVES: A 25-yr-old man from Prague had abnormal bleeding after several surgical operations with low fibrinogen level and hypofibrinogenemia was suspected. PATIENTS AND METHODS: The patient, 25 yr-old male had a low fibrinogen concentration as determined by the thrombin time and immunoturbidimetrical method. His 48-yr-old mother presented with normal coagulation tests, normal fibrinogen level and reported no history of bleeding. To identify the genetic mutation responsible for this hypofibrinogen, genomic DNA extracted from the blood was analyzed. Fibrin polymerization measurement, kinetics of fibrinopeptide release, fibrinogen clottability measurement, mass spectroscopy, and scanning electron microscopy were performed. RESULTS: DNA sequencing showed heterogeneous fibrinogen gammaG351S mutation in the propositus. The mutant chain was found not to be expressed to the circulation by matrix-assisted laser desorption/ionization time of flight mass spectrometry. Scanning electron micrographs of the patient's fibrin clot as well as kinetics of fibrinopeptide release and fibrin polymerization were found to be normal. CONCLUSION: A case of hypofibrinogenemia gammaG351S was found by routine coagulation testing and was genetically identified.

The adhesion of blood platelets on fibrinogen surface: comparison of two biochemical microplate assays.

The biocompatibility of materials is frequently assessed by blood platelet adhesion, since platelet adhesion plays a considerable role in blood interaction with artificial surfaces. Blood platelets adhesion is an essential event in haemostatic and thrombotic processes. The aim of this study was to simultaneously compare simple biochemical assays widely used for evaluation of platelet static adhesion based on the determination of enzymatic activity of either lactate dehydrogenase (LDH) or acid phosphatase (ACP) in lysates of adhered platelets. Adhesion of platelets from platelet-rich plasma and washed platelets activated by either ADP or thrombin on surfaces covered with fibrinogen and well defined fibrin was studied. The results demonstrated that the amounts of adhered platelets estimated by the LDH method were significantly lower as compared with the amount obtained by ACP method. LDH but not ACP release from platelets during adhesion was shown to take place. It suggests that the LDH method should be used rather as an assay of platelet integrity. The ACP method is much more suitable for quantitative determination of platelet adhesion especially in the development and evaluation of haemocompatibility of new biomaterials.