Fibrin Stabilization Research Articles

Factor XIIIa Binding to Activated Platelets Is Mediated Through Activation of Glycoprotein IIb-IIIa

AbstractStabilization of a clot is dependent on fibrin cross-linking mediated by the transglutaminase, factor XIIIa (FXIIIa). In addition to fibrin stabilization, FXIIIa acts on a number of platelet-reactive proteins, including fibronectin and vitronectin, as well as the platelet proteins, glycoprotein (GP) IIb-IIIa, myosin, and actin. However, conditions inducing the platelet-activation dependent binding of FXIIIa have not been characterized nor have the sites mediating FXIIIa binding been identified. The generation of FXIIIa and consequent detection of FXIIIa on the platelet surface were compared with other thrombin-induced activation events; the rate at which FXIIIa bound to activated platelets was much slower than platelet degranulation or fibrin(ogen) binding. Whereas platelets could be rapidly induced to express a functional receptor for FXIIIa, the rate of FXIIIa binding to platelets is limited by the rate of conversion of FXIII to FXIIIa. Immunoprecipitation of radiolabeled platelets using polyclonal anti-FXIII A-chain antibody identified two proteins corresponding to GPIIb and GPIIIa. Preincubation of intact platelets with 7E3, a monoclonal antibody that blocks the fibrinogen binding site, or GRGDSP peptide inhibited FXIIIa binding by about 95% when measured by flow cytometry; FXIIIa binding to purified GPIIb-IIIa was also inhibited by 7E3. The binding of FXIIIa to purified GPIIb-IIIa was enhanced by the addition of fibrinogen, but not by that of fibronectin or thrombospondin, suggesting that FXIIIa also binds to fibrinogen associated with the complex. These observations suggest that activated platelets bearing FXIIIa may enhance stabilization of platelet-rich thrombi through surface-localized cross-linking events.

Factor XIIIa binding to activated platelets is mediated through activation of glycoprotein IIb-IIIa

Stabilization of a clot is dependent on fibrin cross-linking mediated by the transglutaminase, factor XIIIa (FXIIIa). In addition to fibrin stabilization, FXIIIa acts on a number of platelet-reactive proteins, including fibronectin and vitronectin, as well as the platelet proteins, glycoprotein (GP) IIb-IIIa, myosin, and actin. However, conditions inducing the platelet-activation dependent binding of FXIIIa have not been characterized nor have the sites mediating FXIIIa binding been identified. The generation of FXIIIa and consequent detection of FXIIIa on the platelet surface were compared with other thrombin- induced activation events; the rate at which FXIIIa bound to activated platelets was much slower than platelet degranulation or fibrin(ogen) binding. Whereas platelets could be rapidly induced to express a functional receptor for FXIIIa, the rate of FXIIIa binding to platelets is limited by the rate of conversion of FXIII to FXIIIa. Immunoprecipitation of radiolabeled platelets using polyclonal anti- FXIII A-chain antibody identified two proteins corresponding to GPIIb and GPIIIa. Preincubation of intact platelets with 7E3, a monoclonal antibody that blocks the fibrinogen binding site, or GRGDSP peptide inhibited FXIIIa binding by about 95% when measured by flow cytometry; FXIIIa binding to purified GPIIb-IIIa was also inhibited by 7E3. The binding of FXIIIa to purified GPIIb-IIIa was enhanced by the addition of fibrinogen, but not by that of fibronectin or thrombospondin, suggesting that FXIIIa also binds to fibrinogen associated with the complex. These observations suggest that activated platelets bearing FXIIIa may enhance stabilization of platelet-rich thrombi through surface-localized cross-linking events.

Ulcerative Colitis and Crohn’s Disease: Factor XIII, Inflammation and Haemostasis

An important role has been ascribed to plasma factor XIII (FXIII) in inflammation and wound healing. FXIII is necessary for fibrin stabilization and interacts with connective tissue and adhesive proteins and cells. In a prospective study, FXIII activity and parameters of coagulation, fibrinolysis and inflammation, were determined in patients with ulcerative colitis (UC; 13 active, 22 moderate) and Crohn's disease (CD; 36 active, 45 moderate). FXIII levels were lower in active than in moderate UC and CD, and were < 70% of normal values in 7/13 patients with active UC, and in 7/36 patients with active CD, although the median values did not fall below the normal range. FXIII was somewhat higher in active UC patients responding to therapy. The FXIII levels were widely scattered, and low values appear to be due to greatly enhanced turnover. A correlation between FXIII and the systemic levels of markers of activation of haemostasis and inflammation was lacking, but there was a correlation with the extent of bowel involvement. FXIII levels were lower in the patients with involvement beyond the sigmoid colon in CU (p = 0.0045), and both small and large bowel segments in CD (p = 0.0223) patients. This points to local consumption and/or loss of FXIII within the inflamed tissue, and provides an argument for FXIII substitution in the treatment of acute episodes of inflammatory bowel diseases.

Early cross-linked fibrin in human plasma contains alpha-polymers with intact fibrinopeptide A.

alpha-polymer formation, as opposed to gamma-chain dimerization has been considered a relatively late event in factor XIII-induced fibrin stabilization. Recently it has been shown, however, that plasma from healthy individuals and from patients with fibrinaemia contains small amounts of soluble fibrin/fibrinogen oligomers interlinked through dimerized gamma-chains as well as cross-linked alpha-chains. The present work was carried out to see if these early alpha-chain polymers also arise during coagulation of plasma in vitro. Plasma samples from healthy individuals, prepared by immediate centrifugation of blood collected without anticoagulant, were allowed to clot spontaneously for varying periods. The plasma clots were solubilized in SDS-urea-mercaptoethanol and samples were subjected to SDS-PAGE and Western blotting using polyclonal antibodies to human fibrinogen, or monoclonal antibodies specific either for A alpha/alpha-chains, for fibrinopeptide A-containing chains, for the N-terminus of the fibrin beta-chain or for the gamma-chains. Fibrin/fibrinogen oligomers were seen to form long before visible gelation of plasma. These oligomers were cross-linked through gamma-chain dimerization, but also through A alpha- or alpha-chain polymerization. The number and amount of alpha-polymers containing A alpha-chains increased immediately after clot formation, but these disappeared about 20 min later, due to complete removal of fibrinopeptide A (FPA) by thrombin. It is concluded that alpha-polymer formation is a very early event during plasma coagulation in vitro, and that both A alpha- and alpha-chains are involved.

Marshall-Marchetti-Krantz-Operation mit Fibrinklebung

Since broad experience concerning the stability of fibrin sealing is needed, it has become part of the routine program in orthopaedics and in traumatology. Because of these experiences, we decided to modify the standard Marshall-Marchetti-Krantz-Operation replacing all sutures on both sides of the urethra and the bladder neck by fibrin sealing. So far, 76 patients have been operated using to this new method. All patients had a urinary stress incontinence grade II-III, urodynamically verified and a pronounced urethro-cystocele. To judge the results of the operation the following check-ups were instituted: 1. Urodynamic control, 2. lateral cyst-urethrogram, 3. gynecological examination (anatomical control), 4. subjective, individual evaluation of the patient concerning the involuntary loss of urine. Our study shows that we achieved good surgical and functional results as can be demonstrated both clinically and by urodynamic check-up. The advantage of this method of operation using fibrin sealing is to be seen in the broad lifting up of the anterior vaginal wall without unphysiological fixation and at the same time avoiding all risks of sutures.

Alterations of haemostasis parameters with special reference to fibrin stabilization, factor XIII and fibronectin in patients with obliterative atherosclerosis

Intravascular and endoparietal fibrin deposition are thought to be involved in atherosclerotic process, especially when fibrin is stabilized by factor XIII of coagulation system. The study carried out on a group of 50 patients with obliterative atherosclerosis of the lower limbs revealed an increased plasma fibrin stabilizing activity apparently due to an increased F.XIII level. Furthermore, alterations in coagulation and fibrinolysis indicating hypercoagulable tendency were found. It is concluded, that the observed changes may contribute to the development of atherosclerotic process.

Early Alpha Chain Crosslinking in Human Fibrin Preparations

Purified fibrinogen preparations were clotted under crosslinking conditions and the evolution of alpha polymers was examined by Western blotting (SDS-PAGE). Monoclonal antibodies that bind to two localized regions within the COOH-terminal portion of the (A) alpha chains of fibrinogen (F-103, A alpha #259-276; F-102, A alpha #540-554) were used for immunodetection. Three crosslinked components (100K, 168K, 210K) that each exhibited coincident F-102 and F-103 immunoreactivities were evident as early as gamma dimer formation under the in vitro conditions employed. Initial events in the alpha chain crosslinking process appeared to include interactions between relatively intact chains and a variety of degraded ones that shared the structure A alpha #1-276, but differed in the extent to which regions between residues approximately #276 and approximately #539 were preserved. Degraded fibrinogen molecules whose A alpha chains all terminated before A alpha #540-554 were isolated from purified fibrinogen preparations by immunoaffinity chromatography on F-102 Sepharose. Immunoblotting data obtained for the crosslinking capacity of these degraded molecules indicated that early crosslinked components (95 K, 205 K) could form even in the absence of intact alpha chain partners. This crosslinking, moreover, could progress to the polymer stage. These findings demonstrate that some early crosslinking activity is localized exclusively within regions NH2-terminal to A alpha approximately #540-554 and suggest that fibrinogen molecules with partially degraded A alpha chains, which are likely to be circulating under many pathophysiologic conditions, can undergo fibrin stabilization through crosslinking despite a loss of at least 70 COOH-terminal A alpha chain residues.

Factor XIII: a marker of mono- and megakaryocytopoiesis.

Previous studies have shown that human platelets, megakaryocytes and peripheral blood monocytes contain the subunit a of plasma factor XIII (FXIII) which plays a key role in fibrin stabilization. To study the expression of FXIII subunit a during differentiation of the mono- and megakaryocytic cell lines, bone marrow smears were examined by immunomorphological methods. In addition to megakaryocytes, FXIII was detected in a large number of cells by a highly sensitive immunoperoxidase staining. Characterization of these cells was carried out by double immunofluorescent labelling in which the detection of FXIII subunit a was combined by the labelling of either Leu M3 monocyte/macrophage surface antigen or platelet factor 4 (PF4) a marker for megakaryocytic cells. On the course of differentiation from early precursors to mature megakaryocytes all phenotypic form of the megakaryocytic cell line expressed FXIII subunit a though there were considerable changes in its subcellular distribution pattern. Leu M3 positive cells of monocytopoiesis, i.e. monocytes and promonocytes and in all probability monoblasts as well, were also labelled for FXIII. On this basis FXIII subunit a could be considered as a common marker of megakaryo- and monocytopoiesis and its immunomorphological detection might provide a useful diagnostic tool for identifying normal and perhaps also malignant differentiation forms of these cell lines.

The transglutaminase in vascular cells and tissues could provide an alternate pathway for fibrin stabilization

A thrombin-independent transglutaminase (TG) has been identified in vascular cells and tissues from human, rabbit, rat, porcine, and bovine sources. The vascular TG had several properties that were similar but not identical to guinea pig liver TG. Both enzymes had similar chromatographic and electrophoretic properties, preferentially cross-linked the alpha-chains of fibrinogen, and reacted with polyclonal and monoclonal anti-guinea-pig liver TG antibodies. However, the TG from adult bovine aortic endothelial (ABAE) cells exhibited a novel Ca2+/Mg2+ dependence for enzymatic activity that was distinct from that of purified guinea pig liver TG. The mol wt of the vascular TG (79 +/- 3 kd) determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was slightly lower than the purified guinea pig liver TG (85 +/- 9 kd). The TG antigen was detected by immunohistochemical techniques in association with the endothelial and smooth muscle cells of arteries, veins, venules, and capillaries. The TG antigen also codistributed with the fibronectin antigen along the hepatic sinusoids. The ABAE cell TG cross-linked alpha 2-plasmin inhibitor to fibrinogen and caused the modified fibrinogen to be 40-fold more resistant to plasminolysis. A thrombin-independent TG in vascular cells of blood vessels could provide an alternate pathway to inhibit fibrinolysis and promote fibrin stabilization.

The Transglutaminase in Vascular Cells and Tissues Could Provide an Alternate Pathway for Fibrin Stabilization

Abstract A thrombin-independent transglutaminase (TG) has been identified in vascular cells and tissues from human, rabbit, rat, porcine, and bovine sources. The vascular TG had several properties that were similar but not identical to guinea pig liver TG. Both enzymes had similar chromatographic and electrophoretic properties, preferentially cross- linked the alpha-chains of fibrinogen, and reacted with polyclonal and monoclonal anti-guinea-pig liver TG antibodies. However, the TG from adult bovine aortic endothelial (ABAE) cells exhibited a novel Ca2+/Mg2+ dependence for enzymatic activity that was distinct from that of purified guinea pig liver TG. The mol wt of the vascular TG (79 +/- 3 kd) determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was slightly lower than the purified guinea pig liver TG (85 +/- 9 kd). The TG antigen was detected by immunohistochemical techniques in association with the endothelial and smooth muscle cells of arteries, veins, venules, and capillaries. The TG antigen also codistributed with the fibronectin antigen along the hepatic sinusoids. The ABAE cell TG cross-linked alpha 2-plasmin inhibitor to fibrinogen and caused the modified fibrinogen to be 40- fold more resistant to plasminolysis. A thrombin-independent TG in vascular cells of blood vessels could provide an alternate pathway to inhibit fibrinolysis and promote fibrin stabilization.

Effect of dextran 70 and saline on thrombus formation following arteriotomy and intimectomy in small arteries

Twenty-four arteries of rabbit ears, divided into three groups of eight, were prepared and 32P-platelets injected. Arteriotomy (7 mm) and intimectomy (5 mm) were performed and in vivo platelet accumulation followed for 2 hours. Group A comprised untreated control animals, group B was treated with 17 ml saline/kg bw, and group C with 1 g dextran and 17 ml saline/kg bw (Macrodex). Significant differences in platelet accumulation were observed only between the control and Macrodex groups at 105 and 120 minutes. In the control and saline groups four of eight vessels showed poor or no patency. All Macrodex vessels showed good patency. Control and saline vessels had large amounts of red thrombotic material, except for three saline cases with small amounts. After Macrodex treatment five of eight vessels were apparently clean, while the other three showed only small amounts of red thrombotic material. Dextran seems not to influence platelet function but rather to inhibit fibrin stabilization and probably increases fibrinolysis. Vascular patency was only endangered by the formation of solid fibrin-containing red thrombi.

Posthemorrhagic Hydranencephaly in the Fetal Period with Deficiency of Factor NIE (Fibrin Stabilizing Factor)

Posthemorrhagic Hydranencephaly in the Fetal Period with Deficiency of Factor NIE (Fibrin Stabilizing Factor)

Contribution of fibrin stabilization to clot strength. Supplementation of factor XIII-deficient plasma with the purified zymogen.

The contribution of fibrin stabilization to clot strength, measured as the static elastic modulus, was evaluated in human plasma by two independent procedures. In the first approach, amine inhibitors of fibrin stabilization were examined for their effects on the rigidity of normal plasma clots. It is a unique property of these inhibitors that they do not interfere with the reversible aggregation of fibrin molecules, i.e., do not delay clotting time, but selectively prevent only the formation of gamma-glutamyl-epsilon-lysine protein-to-protein linkages. Though the compounds tested were of different chemical structures and potencies, a fivefold reduction in clot strength was obtained in each instance. This value of 20% of normal seems to correspond to the rigidity of the Factor XIII-deficient plasma clot because, as demonstrated by the second approach, when a plasma specimen that genetically lacked the fibrin stabilizing factor was supplemented by the addition of measured amounts of the purified zymogen, a fivefold increase in clot strength could be achieved. The described procedure of evaluating Factor XIII in terms of correcting the elastic modulus of a deficient plasma clot is considered an important assay for the functional competence of purified preparations of the zymogen for the purpose of therapeutic application.

Determination of Factor XIII Activity and of Factor XIII Inhibitors Using an Ammonium-Sensitive Electrode

An assay for rapid factor XIII activity measurement has been developed based on the determination of the ammonium released during fibrin stabilization. Factor XIII was activated by thrombin and calcium. Ammonium was measured by an ammonium-sensitive electrode. It was demonstrated that the assay procedure yields accurate and precise results and that factor XIII-catalyzed fibrin stabilization can be measured kinetically. The amount of ammonium released during the first 90 min of fibrin stabilization was found to be 7.8 +/- 0.5 moles per mole fibrinogen, which is in agreement with the findings of other authors. In 15 normal subjects and in 15 patients suffering from diseases with suspected factor XIII deficiency there was a satisfactory correlation between the results obtained by the "ammonium-release-method', Bohn's method, and the immunological assay (r1 = 0.65; r2 = 0.70; p less than 0.01). In 3 of 5 patients with paraproteinemias the values of factor XIII activity determined by the ammonium-release-method were markedly lower than those estimated by the other methods. It could be shown that inhibitor mechanisms were responsible for these discrepancies.

Factors in fibrinolysis in the human vascular wall at different ages depending on the severity of atherosclerosis

Pieces of vessels from macroscopically unchanged regions of the aorta and inferior vena cava taken from subjects dying suddenly from various causes (70 persons) were investigated. The material was divided into three groups depending on age and the severity of the atherosclerotic changes in the aorta. When material was chosen for visual assessment of atherosclerotic changes in the intima of the aorta, special schemes of standard autopsied segments of the aorta suggested by Avtandilov [i] were used. The control group consisted of 20 young persons aged 17-29 years. Atherosclerotic changes in this group consisted of lipoidosis, accompanied in some cases by single fibrous plaques. The total area of aorta affected was 310%. Groups 2 and 3 consisted of elderly subjects (age 66.2  1.43 and 68.1  1.48 years respectively). In group 2 (26 subjects) the atherosclerotic changes were minimal for that age and, as in the control group, they were restricted to lipoidosis and atherosclerotic plaques. There were no complicated lesions in this group but the area involved was greater than in the young subjects, namely 10-30%. Group 3 consisted of 24 persons with severe morphological changes in the surface of the aorta, including complicated atherosclerotic formations (ulceration, hemorrhages, juxtamural thrombosis), and also areas of calcification. The total area of intima involved in this group exceeded 50%. In all cases the venous wall was free from any distinct morphological changes. Tissue extracts obtained from the vascular fragments were used in the ratio of 1:5 (i part of tissue material and 5 parts of isotonic solution). Activity of the vascular plasminogen activator and proactivator [8] was determined with the aid of kabikinase (a highly purified preparation of streptokinase marketed by the firm "Deutsche KABI Gmbh"); antiplasmin activity [2], the fibrin-stabilizi ng action [3], and also the clotting (thromboplastin) activity (from the substrate plasma recalcification time [4]) also were determined. In the last case, platelet-free bovine plasma was used. The results of the tests were subjected to statistical analysis by Student's nonparametric t test.

The influence of fibrin stabilization and fibrinolysis on the fibrin-adhesive system. A clinical study using radioactively marked fibrinogen as a tracer.

In many fields of surgery the use of highly concentrated human fibrinogen, which forms a fibrin clot when thrombin is added, is gaining increasing importance for tissue adhesion. A number of experimental and clinical reports on fibrin sealing have been published during recent years. The question underlying our present investigations is as follows: What happens to the tissue adhesive after its application to a wound in clinical use, i.e., how much time is required for complete resorption of the seal due to fibrinolytic processes? Skin graftings of the face (required to close defects caused by the excision of basal or spinal cell carcinomas) were performed by glueing rhomboid flaps or full thickness skin grafts to the wounds with the tissue adhesive to which 125I-marked fibrinogen has been added. Activity counts were recorded daily and CPM were plotted versus time to obtain summation curves. The physiological processes of fibrin stabilization and fibrinolysis are extensively discussed as both factors influence the tissue adhesive and thus have to be accounted for in clinical practice.

Fibrin oligomers of high molecular weight soluble in mca solutions: Implications on the time course of fibrin I formation

In gel filtration experiments, it was possible to demonstrate that the largest oligomers of fibrin still soluble in MCA solution contain at least 50 monomeric units. In the case of SDS-PAGE, it was also established that, after incubating fibrin s with F-XIIIa, the fibrin fraction, remaining soluble in MCA solution, has γ-chains which are largely dimeric. From these data we concluded that a critical minimal size is necessary to produce insoluble fibrin. On this basis, a quantitative approach has been attempted to describe the course of fibrin stabilization which finally yields a mathematical function connecting the amount of fibrini and the product of reaction time and enzyme concentration. The pattern of fibrini formation includes a latency phase which is followed by a relatively rapid increase in fibrini concentration. This phenomenon, already observed and interpreted by Loewy et al (6) in 1961, is easily explained by this function. Also, the influence of the critical minimal size of the oligomers, the substrate concentration, and the enzyme parameters (formation-constant and rate-constant) are discussed on the basis of this function.

Fibrinogen Marseille. A new case of congenital dysfibrinogenaemia.

A new case of congenital dysfibrinogenaemia was found in 2 members of the same family. The anomaly was characterized by an abnormal polymerization of fibrin monomers, whereas the release of fibrinopeptides by thrombin and fibrin stabilization by F XIII were normal. Investigation of the fibrinogen molecule did not lead to localizing the structural abnormality.

Studies on fibronectin in the skin. III. Indirect immunofluorescence studies in lichen planus

Fibronectin is a glycoprotein which is responsible for a varity of functions in the human organism, such as mediation of contact between cells and between cells and fibres, opsonic qualities, interaction in the stabilization of fibrin, etc. Fibronectin is an important constituent of the ground substance having a special affinity to collagen. In indirect immunofluorescence studies its presence has been abundantly demonstrated in normal human skin, in collagen-rich structures such as the basement membranes, the papillary and reticular dermis, and in the vascular and neural structures, demonstrable by its characteristic staining patterns. Fibronectin is not found in the epidermis. In lichen planus, the distribution in unaffected skin is identical with that in normal skin, whereas

Differences between Type I Autoimmune Inhibitors of Fibrin Stabilization in Two Patients with Severe Hemorrhagic Disorder

Inhibitors of fibrin stabilization of apparently autoimmune origin, found in two severely bleeding unrelated patients (W. G. and G. A.), were compared with regard to their biological target specificities, potencies and immunological characteristics. Both interfered only with the activation of fibrin stabilizing factor (coagulation Factor XIII) and, while totally preventing the conversion of this zymogen to the functional transamidating enzyme, fibrinoligase (Factor XIII(a)), they showed very little inhibition toward the enzyme itself. Thus, according to the classification of Lorand concerning biological specificities, both can be characterized as Type I inhibitors of fibrin stabilization. Potencies of the two inhibitors were quite similar when measured in conjunction with the plasma zymogen, but they differed remarkably in tests with platelet Factor 13. The inhibitor of patient W. G. prevented the activation of the zymogen from platelets, but that of G. A. had no effect on the platelet factor. It may therefore be concluded that the inhibitor of W. G. is directed exclusively against the a subunit which is a common constituent of plasma as well as platelet factors. The inhibitor of G. A., however, must be targeted against determinants uniquely characteristic for the ab ensemble of the plasma zymogen including the b subunit. On the basis of this difference in target specificity, the inhibitor of W. G. is designated as Type I-1 and that of G. A. as Type I-2. The inhibitors of both patients were isolated as immunoglobulins, and neutralization tests revealed that the antibody of W. G. comprised mainly heavy chains of the IgG1 and light chains of the kappa class. The antibody of G. A. proved to be considerably more heterogeneous and contained IgG1 and IgG3 heavy chains as well as kappa- and lambda-light chains. The finding that the antibody of W. G. inhibited conversion of platelet Factor 13 and also its thrombinmodified form, but had no effect on the thrombin and Ca(2+)-activated factor, is an indication that antigenic determinants existing both on the native zymogen and on its hydrolytically modified form become buried in the Ca(2+)-dependent step of activation. This is clear evidence for the occurrence of a significant conformational change in the protein structure attendant to the process of unmasking of its enzymic activity.