Role, Laboratory Assessment and Clinical Relevance of Fibrin, Factor XIII and Endogenous Fibrinolysis in Arterial and Venous Thrombosis.

Diseases such as myocardial infarction, ischaemic stroke, peripheral vascular disease and venous thromboembolism are major contributors to morbidity and mortality. Procoagulant, anticoagulant and fibrinolytic pathways are finely regulated in healthy individuals and dysregulated procoagulant, anticoagulant and fibrinolytic pathways lead to arterial and venous thrombosis. In this review article, we discuss the (patho)physiological role and laboratory assessment of fibrin, factor XIII and endogenous fibrinolysis, which are key players in the terminal phase of the coagulation cascade and fibrinolysis. Finally, we present the most up-to-date evidence for their involvement in various disease states and assessment of cardiovascular risk.

[Risk assessment of thrombotic events in patients with schizophrenia and schizoaffective disorder in the acute state: the 'fibrinodynamics' technology]. / Otsenka riska tromboticheskikh narushenii u bol'nykh shizofreniei i shizoaffektivnymi rasstroistvami v ostrom periode bolezni s ispol'zovaniem tekhnologii 'fibrinodinamika'.

AIM: To assess the risk of thrombotic events in patients with schizophrenia and schizoaffective disorder based on 'fibrinodynamics' technology. MATERIAL AND METHODS: A group of 76 women, including 38 with paranoid schizophrenia (F20.0), 18 with schizoaffective disorder (F25.1) in the acute stage, and 20 healthy controls, participated in the study. The technology includes the study of coagulation and fibrinolysis, Karmin author software, and calculation of peak time and hemostasis potential of spontaneous clots. Growth and lysis of fibrin clots were studied in plasma purified from platelets. All preanalytic procedures were conducted within 30 minutes after blood sampling. Blood serum was studied separately using the neuroimmunological test. Dynamic of brightness profiles of the clots was determined and a number of parameters (peak time and hemostasis potential of spontaneous clots) were calculated using the Karmin software. RESULTS: In patients with schizophrenia, the dynamic brightness profile of the clots has two peaks: the first peak is formed as a result of the growth and lysis of the clot initiated by the activator, the second peak is due to the growth and lysis of spontaneous clots in the volume of the measuring cuvette far from the activator. In healthy donors, the second peak under experimental conditions is absent. In the group of schizophrenic patients, a strong negative correlation is observed between the peak time of the second peak and the activity of leukocyte elastase (Spearman R = -0.75, p<0.0001), i.e. the greater the activity of elastase, the earlier the maximum of the second peak is formed and vice versa. In the control group, there is no such correlation. Evaluation of the potential of hemostasis of spontaneous clots showed that in 42% of schizophrenic patients this parameter is shifted above the norm, which indicates an increased risk of thrombosis of small brain arteries in these patients. CONCLUSION: The developed technology of 'fibrinodynamics' has a good potential for introduction into personalized medicine to identify increased risks of thrombosis of small cerebral vessels in patients with acute schizophrenia leading to the development of cognitive disorders and to control the normalization of hemostasis with antiplatelet or anticoagulant drugs.

An automated method for fibrin clot permeability assessment.

The fibrin clot permeability coefficient (Ks) is a useful measure of porosity of the fibrin network, which is determined by a number of genetic and environmental factors. Currently available methods to evaluate Ks are time-consuming, require constant supervision and provide only one parameter. We present an automated method in which drops are weighed individually, buffer is dosed by the pump and well defined clot washing is controlled by the software. The presence of a straight association between drop mass and their dripping time allows to shorten the measurement time twice. In 40 healthy individuals, Ks, the number of drops required to reach the plateau (DTP), the time to achieve the plateau (TTP) and the DTP/TTP ratio (DTR) were calculated. There was a positive association between Ks (r = 0.69, P < 0.0001) evaluated by using the manual [median of 4.17 (3.60-5.18) ·10⁻9 cm²) and the automated method [median of 4.35 (3.74-5.38) ·10⁻9 cm²]. The correlation was stronger (r = 0.85, P < 0.001) in clots with DTP of 7 or less (n = 12). DTP was associated with total homocysteine (tHcy) (r = 0.35, P < 0.05) and activated partial thromboplastin time (APTT) (r = -0.34, P < 0.05), TTP with Ks (r = -0.55, P < 0.01 for the manual method and r = -0.44, P < 0.01 for the automated method) and DTP (r = 0.75, P < 0.0001), and DTR with Ks (r = 0.70, P < 0.0001 for the manual method and r = 0.76, P < 0.0001 for the automated method), fibrinogen (r = -0.58, P < 0.0001) and C-reactive protein (CRP) (r = -0.47, P < 0.01). The automated method might be a suitable tool for research and clinical use and may offer more additional parameters describing fibrin clot structure.

[Simultaneous quantification of soluble fibrin and D-dimer in blood plasma for the assessment of the threat of thrombosis].

Soluble fibrin and D-dimer are the most specific markers of blood coagulation cascade and the threat of thrombosis. Two immunoassay test systems were designed using the fibrin-specific and D-dimer-specific monoclonal antibodies. The clinical trials of the test systems were carried out in Ukraine. The high informative value of soluble fibrin quantification as a prognostic indicator of the threat of thrombosis associated with hip replacement and endoprosthetics of the abdominal aorta was shown. Independent D-dimer quantification is less informative. Simultaneous quantification of soluble fibrin and D-dimer before operation and at different time intervals after it is required for the prediction of postoperative thrombotic complications and monitoring the efficiency of antithrombotic therapy. Only in this case it is possible to get information about the state of the balance between blood coagulation and fibrinolytic systems, and determine the degree of the threat of thrombosis.

In vivo assessment of intraplaque and endothelial fibrin in ApoE(-/-) mice by molecular MRI.

OBJECTIVE: Molecular magnetic resonance imaging (MRI) has emerged as a promising non-invasive modality to characterize atherosclerotic vessel wall changes on a morphological and molecular level. Intraplaque and endothelial fibrin has recently been recognized to play an important role in the progression of atherosclerosis. This study aimed to investigate the feasibility of intraplaque and endothelial fibrin detection using a fibrin-targeted contrast-agent, FTCA (EPIX Pharmaceuticals, Lexington, MA), in a mouse model of atherosclerosis. METHODS: Male apolipoproteinE-knockout mice (ApoE(-/-)) were fed a high fat diet (HFD) for one to three months. MRI of the brachiocephalic artery was performed prior to and 90 min after the administration of FTCA (n=8 per group). Contrast to noise ratios (CNR) and longitudinal relaxation rates (R1) of plaques were determined and compared to ex vivo fibrin density measurements on immunohistological sections stained with a fibrin-specific antibody and gadolinium concentrations measured by inductively coupled mass spectroscopy (ICP-MS). RESULTS: Molecular MRI after FTCA administration demonstrated a significant increase (p<0.05) in contrast agent uptake in brachiocephalic artery plaques. In vivo CNR measurements were in good agreement with ex vivo fibrin density measurements on immunohistochemistry (y=2.4x+11.3, R(2)=0.82) and ICP-MS (y=0.95x+7.1, R(2)=0.70). Late stage atherosclerotic plaques displayed the strongest increase in CNR, R1, ex vivo fibrin staining and gadolinium concentration (p<0.05). CONCLUSION: This study demonstrated the feasibility of intraplaque and endothelial fibrin imaging using FTCA. Direct in vivo fibrin detection and quantification could be useful for characterization and staging of coronary and carotid atherosclerotic lesions, which may aid diagnosis and intervention.

Periodontal wound healing with and without platelet-rich plasma: histologic observations and assessment of flap tensile strength.

BACKGROUND: Platelet-rich plasma (PRP) has been promoted as a surgical adjunct to enhance hard and soft tissue wound healing. Although anecdotally reported to be of value, the results of controlled studies examining the added effects of PRP on surgical procedures have been mixed. The purpose of this study was to test the effect of PRP on flap strength at various post-surgical time points in a minipig animal model. METHODS: Twelve Yucatan minipigs provided four sites per animal. PRP was prepared from each animal at the time of surgery. Following reflection of a mucoperiosteal flap in each quadrant, subgingival plaque and calculus were removed. Each surgical site was irrigated with sterile saline; prior to suturing, one randomly selected test quadrant in each arch was treated with PRP. Four animals were euthanized at day 14, and two animals were euthanized at 2, 7, 10, and 28 days. The flap strength in each quadrant was tested by attaching to a loop of 3-0 silk suture through the tissue; the force required to separate the flap from the tooth/bone interface was recorded for each site. A separate portion of each flap site was prepared for descriptive histologic examination, including inflammation, hemorrhage, and new bone growth. RESULTS: Flap strength was significantly less on day 2 compared to later time points, and there were no significant differences between the test and control groups. No histologic differences in healing between test and control sites were seen at any time point. CONCLUSIONS: PRP did not seem to contribute to greater flap strength at any post-surgical time point, nor was it associated with any histologic differences in wound healing in this Yucatan minipig model. The time points chosen for observation post-surgery, as well as the variability in the PRP platelet count, may have contributed to the lack of positive findings in this study.

[Assessment of selected haemostatic parameters in obese patients]. / Ocena wybranych parametrów ukladu hemostazy u osób otylych.

BACKGROUND: Epidemiologic data show significantly higher frequency of thromboembolic incidents in obesity than in normal weight persons. Disorders of haemostasis seem to play a crucial role in their development. In the literature there are only few papers assessing coagulation and fibrynolitic parameters in obese subjects. AIM OF THE STUDY: Assessment of protein C (PC), antithrombin (AT) and alpha2 antiplasmin (alpha2AP) activity and thrombomodulin (TM) concentration in the blood plasma of obese person (BMI > 30 kg/m(2)). MATERIALS AND METHODS: The study involved 32 patients (22 women and 10 men, mean age 39.7 +/- 15.3 years) and 20 healthy volunteers matched correctly according to sex and age who constituted a control group. In the examined subjects activity of PC, AT, alpha2 AP were assessed by means of the colorimetric methods and TM concentration in the blood plasma using ELISA method. RESULTS: No statistically significant differences in activities of PC, AT, alpha2 AP and TM concentrations between the patients and the control group were found. However the tendency to higher activities of PC and concentrations of TM were noticed in the obese patients. Assessing tested parameters according to sex, statistically significant differences were found in AT activity between the male patients and healthy men. Statistically lower values, but still in the normal range, were found in the obese men. Comparing the groups of women, significantly lower concentrations of TM were found in the obese ones. CONCLUSIONS: It seems that changed values of PC, AT, alpha2 AP activities are not responsible for increased risk of thromboembolic events in obese persons. Increased TM concentration in obese, may indirectly indicate endothelium damage.

Hemostatic markers and the sepsis-related organ failure assessment score in patients with disseminated intravascular coagulation in an intensive care unit.

We investigated the correlation between disseminated intravascular coagulation (DIC) score and hemostatic parameters and sepsis-related organ failure assessment (SOFA) score with clinical outcome of patients with DIC in an intensive care unit (ICU). The SOFA score was markedly elevated in patients with DIC relative to patients without DIC and significantly higher in non-survivors than in survivors. Abnormalities in almost all hemostatic parameters were significant in patients with DIC, but there was no significant difference in almost all hemostatic parameters between survivors and non-survivors. However, plasma antithrombin (AT) levels were significantly lower in non-survivors than in survivors. Soluble fibrin (SF) and tissue type plasminogen activator (tPA)-plasminogen activator inhibitor-I (PAI-I) complex correlated significantly with the SOFA score, whereas AT levels correlated significantly and negatively with the SOFA score. We conclude that the SOFA score is useful for predicting outcome in DIC patients in the ICU, and that hemostatic parameters, especially plasma AT levels, are also useful markers for organ failure and clinical outcome.

The use of coagulation activation markers (soluble fibrin polymer, TpP, prothrombin fragment 1.2, thrombin-antithrombin, and D-dimer) in the assessment of hypercoagulability in patients with inherited and acquired prothrombotic disorders.

A total of 260 consecutive patients, referred for hypercoagulable assessment, was included in this study. Four coagulation activation markers were utilized to assess these patients [enzyme-linked immunosorbent assays for soluble fibrin polymer (TpP), prothrombin fragment 1.2, thrombin-antithrombin complex, and D-dimer]. The mean levels of the activation markers directly correlated with the number of hypercoagulable abnormalities. The percentage of patients with increased TpP levels for each group was lower than the other activation markers. The findings indicate that activation markers reflect the number of underlying thrombophilic abnormalities. Our data suggest that there is a utility in performing a panel of coagulation activation markers to assess the thrombotic risk. The measurement of soluble fibrin polymer may be more reflective of an impending vascular event.

Immunohistological assessment of fibrin deposition and thrombus formation in canine mammary neoplasia.

A commercially available monoclonal antibody against human fibrin was used to detect fibrin in canine formalin-fixed, paraffin wax-embedded tissue by applying a slightly modified alkaline phosphatase anti-alkaline phosphatase (APAAP) technique. Twenty-eight mammary tumours from six bitches were examined for the presence of fibrin. Thrombi and extravascular fibrin deposits were detected in 15 tumours (12 complex adenocarcinomas, one adenocarcinoma, two solid carcinomas), and a single thrombus was detected in one adenoma; 12 tumours (three adenomas, one complex adenoma, four complex adenocarcinomas and four adenocarcinomas) did not show any staining reaction.

Zinc and liver cirrhosis: biochemical and histopathologic assessment.

Experimental liver cirrhosis was produced by administration of thioacetamide. Cirrhotic animals were divided into two groups: one group was given zinc sulphate and the second kept as cirrhotic control. Zinc-treated animals showed a restoration of normal hepatic and plasma zinc and copper levels. Similarly, plasma levels of aspartate aminotransferase, alanine aminotransferase, gamma-glutamyl aminotransferase, and total bilirubin decreased significantly. Light microscopic studies showed that most of the hepatocytes appeared normal in zinc-treated as compared with untreated cirrhotic animals. The amount of fibrin, reticulin, and collagen, which was high in the cirrhotic livers, decreased following zinc treatment. Staining with periodic acid Schiff's reagent showed the ability of hepatocytes to store glycogen after zinc treatment. These results revealed that zinc may have some beneficial effect in the treatment of liver cirrhosis.

Mechanism-oriented assessment of isotretinoin in chronic or subacute cutaneous lupus erythematosus.

Eight of ten patients with chronic or subacute cutaneous lupus erythematosus completed 16 weeks of oral isotretinoin therapy (80 mg/day). All eight patients noted an excellent clinical response without significant side effects. (Two patients did not return to initial two-week follow-up.) Peripheral blood B- and T-cell counts were unaffected by therapy. Therapy was associated with resolution of routine histopathologic abnormalities, conversion of abnormal lesional direct immunofluorescence microscopy to normal, normalization of the epidermis on electron microscopy, and reduction of all T cells near the dermoepidermal junction without change in ratio of T-helper/inducer cells to T-suppressor/cytotoxic cells. Isotretinoin is a clinically effective short-term therapy for chronic or possibly for subacute cutaneous lupus erythematosus. The primary mechanism of action remains unestablished.

Quantitative assessment of soluble fibrin in plasma by affinity chromatography--a comparative study with desAA-fibrin, desAABB-fibrin and fibrinogen.

A quantitative determination of soluble fibrin in plasma was carried out by affinity chromatography. For this purpose, desAA-fibrin and fibrinogen immobilized on Sepharose 4B were used at the stationary side whereas batroxobin-induced 125I-desAA-fibrin or thrombin-induced 125I-desAABB-fibrin mixed with plasma containing 131I-fibrinogen represented the fluid phase. The binding characteristics of these mixtures to the immobilized proteins were compared at 20 degrees C and 37 degrees C. Complete binding of both types of fibrin to the immobilized desAA-fibrin was always seen at 20 degrees C as well as at 37 degrees C. However, binding of soluble fibrin was accompanied by substantial binding of fibrinogen that was more pronounced at 20 degrees C. Striking differences depending on the temperature at which the affinity chromatography was carried out, were documented for the fibrinogen-fibrin interaction. At 20 degrees C more than 90% of the applied desAA-fibrin was bound to the immobilized fibrinogen whereas at 37 degrees C only a mean of 17% were retained at the fibrinogen-Sepharose column. An opposite finding with regard to the tested temperature was made with the desAABB-fibrin. Nearly complete binding to insolubilized fibrinogen was found at 37 degrees C (95%) but only 58% of the desAABB-fibrin were bound at 20 degrees C. The binding patterns did not change when the experiments were performed in the presence of calcium ions. The opposite behaviour of the two types of soluble fibrin to immobilized fibrinogen at the different temperatures, together with the substantial binding of fibrinogen in the presence of soluble fibrin to insolubilized fibrin in every setting tested, devaluates affinity chromatography as a tool in the quantitative assessment of soluble fibrin in patient's plasma.

Quantitative assessment of the effects of platelet depletion in the autologous phase of nephrotoxic serum nephritis.

The effects of platelet depletion with antibody have been studied in two models of the autologous phase of nephrotoxic nephritis in the rabbit. In the 'telescoped' model (animals pre-immunized to sheep IgG injected with sheep nephrotoxic antibody), platelet depletion did not alter intraglomerular fibrin deposition or evidence of glomerular damage, but did significantly reduce proteinuria during the first 3 days of the 5 day experiment. In the 'passive' model (animals injected with hyperimmune rabbit antiserum to sheep IgG 48 hr after sheep nephrotoxic antibody and killed 3 hr later), platelet depletion was associated with significantly fewer intraglomerular polymorphonuclear leucocytes (PMN), but again did not alter intraglomerular fibrin deposition. The results indicate that platelets are involved in the initiation of glomerular PMN localization in the autologous phase, but that fibrin-induced glomerular injury is platelet-independent.

[Thrombolytic treatment with urokinase (author's transl)]. / Thrombolytische Therapie mit Urokinase

Compared with streptokinase, thrombolytic treatment with urokinase has the advantages of being better tolerated and of practically unlimited applicability. Its disadvantage is the high cost. A good lytic action can be obtained with a dosage of 150,000 Ploug Units/12 hours for a duration of lysis of 8-14 days combined with heparin, the therapy being monitored by determination of the products of fibrinolysis. This dosage is not possible if the time factor plays a decisive role in the success of the treatment, e.g. in myocardial infarction. Urokinase is indicated when streptokinase cannot be used, or if continuation of the streptokinase therapy is necessary because of extensive thromboses.