Hsp70 Trap Assay for Detection of Misfolded Subproteome Related to Myelodysplastic Syndromes.

The onset and progression of numerous serious diseases (e.g., various types of malignancies, neurodegenerative diseases, and cardiac diseases) are, on a molecular level, associated with protein modifications and misfolding. Current methods for the detection of misfolded proteins are not able to detect the whole misfolded subproteome and, moreover, are rather laborious and time consuming. Herein, we report on a novel simple method for the detection of misfolded proteins employing a surface plasmon resonance (SPR) biosensor and heat shock protein 70 (Hsp70) that recognizes and traps misfolded proteins in a nucleotide-dependent manner. We use this method for the detection of misfolded proteins in blood plasma of patients with various subtypes of myelodysplastic syndromes (MDS) and healthy donors. Our results reveal significantly elevated levels of misfolded proteins in the two stages of MDS that are most affected by oxidative stress: low-risk (RARS) and intermediate-risk (RCMD) patients. This approach can be extended to a variety of diseases and provides unique insights into the thus far unexplored area of blood proteome.

The effect of the biological variability of samples on Coomassie blue dye based fast staining for SDS-PAGE in nonfixed gels.

Fast-staining protocols based on the use of Coomassie blue dye for SDS-PAGE separated proteins, represent a quick and simple solution for protein visualization. It has been shown however, that in some cases a phenomenon of missing spots or spot discoloration may be observed in the proteome pattern when the standard fast-staining protocol is used. In this work, it is demonstrated that this occurrence is affected by the biological variability of samples, and therefore, cannot be observed in all samples. Moreover, it is demonstrated that the phenomenon is manifested exclusively in nonfixed gels, and that including a fixation step into the fast-staining protocol prevented this phenomenon. In conclusion, it has been demonstrated that standard Coomassie blue dye based fast staining for SDS-PAGE resolved proteins is affected by the biological variability of samples in nonfixed gels.

No clinical evidence for performing trough plasma and intracellular imatinib concentrations monitoring in patients with chronic myelogenous leukaemia.

This multicentre study focused on monitoring imatinib mesylate (IMA) trough plasma (Ctrough ) and intracellular (IMA Cintrac ) concentrations in 228 chronic myelogenous leukaemia patients. The median of measured IMA Ctrough in our patient group was 905.8 ng ml (range: 27.7-4628.1 ng/ml). We found a correlation between IMA Ctrough and alpha 1-acid glycoprotein plasma concentrations (rS = 0.42; p < 0.001). All other analysed parameters revealed only weak (gender, dose of IMA per kg) or not significant (age, albumin, creatinine plasma concentration or body mass index) impact on measured IMA Ctrough. The IMA Ctrough decreased during the first 6 months and significantly increased later during treatment. The IMA Ctrough at the first month of therapy did not differ between patients with and without an optimal response at the 12th (p = 0.724) and 18th month (p = 0.135) of therapy. There were no significant differences in medians of IMA Ctrough between both groups measured during the first year of treatment. The IMA Cintrac during the first month were not different between patients with and without an optimal response at the 6th (p = 0.273) and the 12th month (p = 0.193) of therapy. Our data obtained from real life clinical practice did not find a benefit of routine and regular IMA Ctrough nor IMA Cintrac therapeutic drug monitoring in chronic myelogenous leukaemia patients or for subsequent adjustments of the IMA dose based on these results. Moreover, actual alpha 1-acid glycoprotein plasma concentration should be used for proper interpretation of IMA Ctrough results.

Fibrinopeptides A and B release in the process of surface fibrin formation.

Fibrinogen adsorption on a surface results in the modification of its functional characteristics. Our previous studies revealed that fibrinogen adsorbs onto surfaces essentially in 2 different orientations depending on its concentration in the solution: "side-on" at low concentrations and "end-on" at high concentrations. In the present study, we analyzed the thrombin-mediated release of fibrinopeptides A and B (FpA and FpB) from fibrinogen adsorbed in these orientations, as well as from surface-bound fibrinogen-fibrin complexes prepared by converting fibrinogen adsorbed in either orientation into fibrin and subsequently adding fibrinogen. The release of fibrinopeptides from surface-adsorbed fibrinogen and from surface-bound fibrinogen-fibrin complexes differed significantly compared with that from fibrinogen in solution. The release of FpB occurred without the delay (lag phase) characteristic of its release from fibrinogen in solution. The amount of FpB released from end-on adsorbed fibrinogen and from adsorbed fibrinogen-fibrin complexes was much higher than that of FpA. FpB is known as a potent chemoattractant, so its preferential release suggests a physiological purpose in the attraction of cells to the site of injury. The N-terminal portions of fibrin ß chains including residues Bß15-42, which are exposed after cleavage of FpB, have been implicated in many processes, including angiogenesis and inflammation.

Staining of proteins for 2D SDS-PAGE using Coomassie Blue--speed versus sensitivity?

Several new fast staining protocols for the visualization of proteins separated by SDS-PAGE utilizing Coomassie Blue staining (CBS) have been described in literature. The sensitivity of a newly designed staining protocol is usually estimated using 1D SDS-PAGE of serially diluted protein samples. However, this approach is not predictive and satisfactory for 2D SDS-PAGE capable of resolving hundreds or thousands of different proteins in a single analysis. In this work, a new fast staining protocol recently introduced by Dong et al. (PLoS One 2011, 6, e22394) was compared to colloidal CBS. The number of detectable spots in 2D SDS-PAGE of identical blood plasma samples in repeated runs was chosen as a sensitivity criterion. Further, the influence of gel boiling on the subsequent protein identification by MS was investigated. In spite of its advantages, the staining protocol according to Dong et al. (PLoS One 2011, 6, e22394) seems to be less sensitive than colloidal Coomassie staining when the number of detected spots is the evaluating criterion. No obvious influence of gel boiling on the protein identification was observed.

Plasma proteome changes associated with refractory anemia and refractory anemia with ringed sideroblasts in patients with myelodysplastic syndrome.

BACKGROUND: Refractory anemia and refractory anemia with ringed sideroblasts are two myelodysplastic syndrome (MDS) subgroups linked with anemia. MDS is a group of heterogeneous oncohematological bone marrow disorders characterized by ineffective hematopoiesis, blood cytopenias, and progression of the disease toward acute myeloid leukemia. The aim of this study was to search for plasma proteome changes in MDS patients with refractory anemia and refractory anemia with ringed sideroblasts. RESULTS: A total of 26 patient and healthy donor plasma samples were depleted of fourteen high-abundant plasma proteins, separated with 2D electrophoresis, and statistically processed with Progenesis SameSpots software. 55 significantly differing spots were observed and corresponded to 39 different proteins identified by nanoLC-MS/MS. Changes in the fragments of the inter-alpha-trypsin inhibitor heavy chain H4 protein were observed. Using mass spectrometry-based relative label-free quantification of tryptic peptides, there were differences in alpha-2-HS-glycoprotein peptides, while no differences were observed between the control and patient sample groups for retinol-binding protein 4 peptides. CONCLUSIONS: This study describes plasma proteome changes associated with MDS patients with refractory anemia and refractory anemia with ringed sideroblasts. Changes observed in the inter-alpha-trypsin inhibitor heavy chain H4 fragments were in agreement with our previous studies of other MDS subgroups: refractory cytopenia with multilineage dysplasia and refractory anemia with excess blasts subtype 1. Mass spectrometry-based relative quantification of retinol-binding protein 4 peptides has shown that there are differences in the modification of this protein between refractory anemia with excess blasts subtype 1 patients and MDS patients with refractory anemia and refractory anemia with ringed sideroblasts. Alpha-2-HS-glycoprotein seems to be a new potential MDS biomarker candidate.

Complete identification of proteins responsible for human blood plasma fouling on poly(ethylene glycol)-based surfaces.

The resistance of poly(ethylene glycol) (PEG) against protein adsorption is crucial and has been widely utilized in various biomedical applications. In this work, the complete protein composition of biofilms deposited on PEG-based surfaces from human blood plasma (BP) was identified for the first time using nanoLC-MS/MS, a powerful tool in protein analysis. The mass of deposited BP and the number of different proteins contained in the deposits on individual surfaces decreased in the order of self-assembling monolayers of oligo(ethylene glycol) alkanethiolates (SAM) > poly(ethylene glycol) end-grafted onto a SAM > poly(oligo(ethylene glycol) methacrylate) brushes prepared by surface initiated polymerization (poly(OEGMA)). The BP deposit on the poly(OEGMA) surface was composed only of apolipoprotein A-I, apolipoprotein B-100, complement C3, complement C4-A, complement C4-B, histidine-rich glycoprotein, Ig mu chain C region, fibrinogen (Fbg), and serum albumin (HSA). The total resistance of the surface to the Fbg and HSA adsorption from single protein solutions suggested that their deposition from BP was mediated by some of the other proteins. Current theories of protein resistance are not sufficient to explain the observed plasma fouling. The research focused on the identified proteins, and the experimental approach used in this work can provide the basis for the understanding and rational design of plasma-resistant surfaces.

The effect of reagents mimicking oxidative stress on fibrinogen function.

Fibrinogen is one of the plasma proteins most susceptible to oxidative modification. It has been suggested that modification of fibrinogen may cause thrombotic/bleeding complications associated with many pathophysiological states of organism. We exposed fibrinogen molecules to three different modification reagents-malondialdehyde, sodium hypochlorite, and peroxynitrite-that are presented to various degrees in different stages of oxidative stress. We studied the changes in fibrin network formation and platelet interactions with modified fibrinogens under flow conditions. The fastest modification of fibrinogen was caused by hypochlorite. Fibers from fibrinogen modified with either reagent were thinner in comparison with control fibers. We found that platelet dynamic adhesion was significantly lower on fibrinogen modified with malondialdehyde and significantly higher on fibrinogen modified either with hypochlorite or peroxynitrite reflecting different prothrombotic/antithrombotic properties of oxidatively modified fibrinogens. It seems that, in the complex reactions ongoing in living organisms at conditions of oxidation stress, hypochlorite modifies proteins (e.g., fibrinogen) faster and more preferentially than malondialdehyde. It suggests that the prothrombotic effects of prior fibrinogen modifications may outweigh the antithrombotic effect of malondialdehyde-modified fibrinogen in real living systems.

Plasma protein alterations in the refractory anemia with excess blasts subtype 1 subgroup of myelodysplastic syndrome.

BACKGROUND: Refractory anemia with excess blasts subtype 1 (RAEB-1) is a subgroup of myelodysplastic syndrome. It represents a heterogeneous group of oncohematological bone marrow diseases, which occur particularly in elderly patients. The aim of this proteomic study was to search for plasma protein alterations in RAEB-1 patients. RESULTS: A total of 24 plasma samples were depleted of fourteen high-abundant plasma proteins, analyzed with 2D SDS-PAGE, compared, and statistically processed with Progenesis SameSpots software. Proteins were identified by nanoLC-MS/MS. Retinol-binding protein 4 and leucine-rich alpha-2-glycoprotein were relatively quantified using mass spectrometry. 56 significantly differing spots were found; and in 52 spots 50 different proteins were successfully identified. Several plasma proteins that changed either in their level or modification have been described herein. The plasma level of retinol-binding protein 4 was decreased, while leucine-rich alpha-2-glycoprotein was modified in RAEB-1 patients. Changes in the inter-alpha-trypsin inhibitor heavy chain H4, altered protein fragmentation, or fragments modifications were observed. CONCLUSIONS: This study describes proteins, which change quantitatively or qualitatively in the plasma of RAEB-1 patients. It is the first report on qualitative changes in the leucine-rich alpha-2-glycoprotein in the RAEB-1 subgroup of myelodysplastic syndrome. Described changes in the composition or modification of inter-alpha-trypsin inhibitor heavy chain H4 fragments in RAEB-1 are in agreement with those changes observed in previous study of refractory cytopenia with multilineage dysplasia, and thus H4 fragments could be a marker specific for myelodysplastic syndrome.

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.

Surface plasmon resonance biosensor for the detection of VEGFR-1--a protein marker of myelodysplastic syndromes.

The surface plasmon resonance (SPR) biosensor system with dispersionless microfluidics for the direct and label-free detection of a soluble vascular endothelial growth factor receptor (sVEGFR-1) is described. The detection approach takes advantage of an affinity interaction between sVEGFR-1 and its ligand, vascular endothelial growth factor (VEGF-A), which is covalently immobilized on the surface of the SPR sensor. The ability of the immobilized VEGF-A to specifically bind the sVEGFR-1 receptor is demonstrated in a buffer. The detection of sVEGFR-1 in 2% human blood plasma is carried out by using the sequential injection approach. The detection limit of 25 ng/mL is achieved. In addition, we demonstrate that the functional surface of the sensor can be regenerated for repeated use.

Mass spectrometry, data re-analysis, and homology modelling predict posttranslational modifications of leucine-rich alpha-2-glycoprotein as a marker of myelodysplastic syndrome.

BACKGROUND: Leucine-rich alpha-2-glycoprotein (LRG) has been repeatedly proposed as a potential plasma biomarker for myelodysplastic syndrome (MDS). OBJECTIVE: The goal of our work was to establish the total LRG plasma level and LRG posttranslational modifications (PTMs) as a suitable MDS biomarker. METHODS: The total plasma LRG concentration was determined with ELISA, whilst the LRG-specific PTMs and their locations, were established using mass spectrometry and public mass spectrometry data re-analysis. Homology modelling and sequence analysis were used to establish the potential impact of PTMs on LRG functions via their impact on the LRG structure. RESULTS: While the results showed that the total LRG plasma concentration is not a suitable MDS marker, alterations within two LRG sites correlated with MDS diagnosis (p= 0.0011). Sequence analysis and the homology model suggest the influence of PTMs within the two LRG sites on the function of this protein. CONCLUSIONS: We report the presence of LRG proteoforms that correlate with diagnosis in the plasma of MDS patients. The combination of mass spectrometry, re-analysis of publicly available data, and homology modelling, represents an approach that can be used for any protein to predict clinically relevant protein sites for biomarker research despite the character of the PTMs being unknown.

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.

Incorporation of Fibrin, Platelets, and Red Blood Cells into a Coronary Thrombus in Time and Space.

We describe the internal structure, spatial organization and dynamic formation of coronary artery thrombi from ST-segment elevation myocardial infarction patients. Scanning electron microscopy (SEM) revealed significant differences among four groups of patients (<2 hours; 2-6 hours; 6-12 hours, and >12 hours) related to the time of ischemia. Coronary artery thrombi from patients presenting less than 2 hours after the infarction were almost entirely composed of platelets, with small amounts of fibrin and red blood cells. In contrast, thrombi from late presenters (>12 hours) consisted of mainly platelets at the distal end, where clotting was initiated, with almost no platelets at the proximal end, while the red blood cell content went from low at the initiating end to more than 90% at the proximal end. Furthermore, fibrin was present mainly on the outside of the thrombi and older thrombi contained thicker fibers. The red blood cells in late thrombi were compressed to a close-packed, tessellated array of polyhedral structures, called polyhedrocytes. Moreover, there was redistribution from the originally homogeneous composition to fibrin and platelets to the outside, with polyhedrocytes on the interior. The presence of polyhedrocytes and the redistribution of components are signs of in vivo clot contraction (or retraction). These results suggest why later thrombi are resistant to fibrinolytic agents and other treatment modalities, since the close-packed polyhedrocytes form a nearly impermeable seal. Furthermore, it is of particular clinical significance that these findings suggest specific disparate therapies that will be most effective at different stages of thrombus development.

Plasma proteome changes in cardiovascular disease patients: novel isoforms of apolipoprotein A1.

BACKGROUND: The aim of this proteomic study was to look for changes taking place in plasma proteomes of patients with acute myocardial infarction (AMI), unstable angina pectoris (UAP), and stable angina pectoris (SAP). METHODS: Depleted plasma proteins were separated by 2D SDS-PAGE (pI 4-7), and proteomes were compared using Progenesis SameSpots statistical software. Proteins were identified by nanoLC-MS/MS. Proteins were quantified using commercial kits. Apolipoprotein A1 was studied using 1D and 2D SDS-PAGE, together with western blotting. RESULTS: Reciprocal comparison revealed 46 unique, significantly different spots; proteins in 34 spots were successfully identified and corresponded to 38 different proteins. Discrete comparisons of patient groups showed 45, 41, and 8 significantly different spots when AMI, UAP, and SAP were compared with the control group. On the basis of our proteomic data, plasma levels of two of them, alpha-1 microglobulin and vitamin D-binding protein, were determined. The data, however, failed to prove the proteins to be suitable markers or risk factors in the studied groups. The plasma level and isoform representation of apolipoprotein A1 were also estimated. Using 1D and 2D SDS-PAGE, together with western blotting, we observed extra high-molecular weight apolipoprotein A1 fractions presented only in the patient groups, indicating that the novel high-molecular weight isoforms of apolipoprotein A1 may be potential new markers or possible risk factors of cardiovascular disease. CONCLUSION: The reported data show plasma proteome changes in patients with AMI, UAP, and SAP. We propose some apolipoprotein A1 fractions as a possible new disease-associated marker of cardiovascular disorders.

Plasma proteome changes associated with refractory cytopenia with multilineage dysplasia.

BACKGROUND: Refractory cytopenia with multilineage dysplasia (RCMD) is a subgroup of myelodysplastic syndrome (MDS), which belongs to oncohematological diseases, occurring particularly in elderly patients, and represents a heterogeneous group of bone marrow diseases. The goal of this study was to look for plasma proteins that changed quantitatively or qualitatively in RCMD patients. RESULTS: A total of 46 plasma samples were depleted, proteins were separated by 2D SDS-PAGE (pI 4-7), and proteomes were compared using Progenesis SameSpots statistical software. Proteins were identified by nanoLC-MS/MS. Sixty-one unique, significantly (p < 0.05, ANOVA) different spots were found; proteins in 59 spots were successfully identified and corresponded to 57 different proteins. Protein fragmentation was observed in several proteins: complement C4-A, complement C4-B, inter-alpha-trypsin inhibitor heavy chain H4, and endorepellin. CONCLUSIONS: This study describes proteins, which change quantitatively or qualitatively in RCMD patients, and represents the first report on significant alterations in C4-A and C4-B complement proteins and ITIH4 fragments in patients with MDS-RCMD.

Molecular Dynamic Simulations Suggest That Metabolite-Induced Post-Translational Modifications Alter the Behavior of the Fibrinogen Coiled-Coil Domain.

Fibrinogen is an abundant blood plasma protein that, inter alia, participates in blood coagulation. It polymerizes to form a fibrin clot that is among the major components of the thrombus. Fibrinogen reactions with various reactive metabolites may induce post-translational modifications (PTMs) into the protein structure that affect the architecture and properties of fibrin clots. We reviewed the previous literature to find the positions of PTMs of fibrinogen. For 7 out of 307 reported PTMs, we used molecular dynamics simulations to characterize their effect on the behavior of the fibrinogen coiled-coil domain. Interactions of the γ-coil with adjacent chains give rise to π-helices in Aα and Bß chains of even unmodified fibrinogen. The examined PTMs suppress fluctuations of the γ-coil, which may affect the fibrinolysis and stiffness of the fibrin fibers. Citrullination of AαR104 and oxidations of γP70 and γP76 to glutamic semialdehyde unfold the α-helical structure of Aα and Bß chains. Oxidation of γM78 to methionine sulfoxide induces the formation of an α-helix in the γ-coil region. Our findings suggest that certain PTMs alter the protein secondary structure. Thus, the altered protein structure may indicate the presence of PTMs in the molecule and consequently of certain metabolites within the system.

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.

Proteome changes in platelets activated by arachidonic acid, collagen, and thrombin.

BACKGROUND: Platelets are small anucleated blood particles that play a key role in the control of bleeding. Platelets need to be activated to perform their functions and participate in hemostasis. The process of activation is accompanied by vast protein reorganization and posttranslational modifications. The goal of this study was to identify changes in proteins in platelets activated by different agonists. Platelets were activated by three different agonists - arachidonic acid, collagen, and thrombin. 2D SDS-PAGE (pI 4-7) was used to separate platelet proteins. Proteomes of activated and resting platelets were compared with each other by Progenesis SameSpots statistical software; and proteins were identified by nanoLC-MS/MS. RESULTS: 190 spots were found to be significantly different. Of these, 180 spots were successfully identified and correspond to 144 different proteins. Five proteins were found that had not previously been identified in platelets: protein CDV3 homolog, protein ETHE1, protein LZIC, FGFR1 oncogene partner 2, and guanine nucleotide-binding protein subunit beta-5. Using spot expression profile analysis, we found two proteins (WD repeat-containing protein 1 and mitochondrial glycerol-3-phosphate dehydrogenase) that may be part of thrombin specific activation or signal transduction pathway(s). CONCLUSIONS: Our results, characterizing the differences within proteins in both activated (by various agonists) and resting platelets, can thus contribute to the basic knowledge of platelets and to the understanding of the function and development of new antiplatelet drugs.

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.