Growth regulation of fibroblasts by thrombin,factor XIII and fibronectin

Summary The stimulation of fibroblast proliferation by thrombin and factor XIII is accompanied by an intracellular increase of cGMP. In contrast fibronectin inhibits the³H-thymidine uptake of fibroblasts. Pre-treatment of fibroblasts with neuraminidase eliminates the stimulating effect of thrombin completely and induces a shift of the optimum stimulating effect of factor XIII to higher concentrations. It is discussed that thrombin and factor XIII stimulate the proliferation of fibroblasts as growth hormones and regulate in combination with the inhibiting fibronectin the growth of fibroblasts in thrombus organization, would healing and in the arteriosclerotic vessel wall process.     

Immobilization of factor XIII on collagen membranes

Factor XIII from placenta was successfully grafted onto collagen membranes by the acyl-azide procedure. The transamidase activity retained on collagen membranes was determined by measuring the increase of fluorescence resulting from dansylcadaverine incorporation into casein. We studied the effect of different factors on the grafting: concentration and composition of the factor XIII preparation in the coupling solution and influence of the preactivation of factor XIII by thrombin. Stability studies have shown that the activity of factor XIII grafted on collagen membranes was almost constant over a period of 8 months. Sterilization by gamma-irradiation of factor XIII revealed a drastic loss of activity while the use of high-energized electron bombardment caused a reduced loss of activity. The potential of such a material for biomedical use is presently under investigation.     

Recombinant factor XIII supplemented clots resist lysis by plasmin and leucocyte elastase

We have examined the lysis of fibrin and plasma clots supplemented with supraphysiological concentrations of recombinant factor XIII(rFXIII) prior to clotting with thrombin. rFXIII supplements of up to 200 μg/mL reduced the lysis rate of fibrin clots by either plasmin or leucocyte elastase in a dose-dependent fashion. Plasma clots supplemented with rFXIII were larger and more resistant to plasmin lysis relative to controls. These effects were all greater when the clots were incubated for 20 h compared to 1 h.To determine whether these in vitro findings might have an in vivo correlate, rFXIII supplemented and unsupplemented fibrin clots labelled with ¹²⁵I-fibrin were implanted into the peritoneum of mice and lysis followed by the appearance of ¹²⁵I in the urine. Supplemented clots released significantly less ¹²⁵I over the first 10 days post-surgery. We conclude that supraphysiological concentrations of rFXIII reduce lysis to both plasmin and leucocyte elastase in vitro and that supplemented clots show a similar resistance in vivo.     

Linomide abolishes leukocyte adhesion and extravascular recruitment induced by tumor necrosis factor alpha in vivo

The immunomodulator Linomide (roquinimex) ameliorates the development of numerous inflammatory and immunological diseases, including sepsis, arthritis, and encephalomyelitis. However, the mechanism underlying this protective effect of Linomide remains unclear. In this study, we wanted to evaluate the effect of Linomide treatment on the different steps in the extravasation process of leukocytes stimulated by tumor necrosis factor alpha (TNF-alpha) in vivo. For this purpose, we used intravital microscopy in the mouse cremaster muscle microcirculation. We found that pretreatment with Linomide dose-dependently (3-300 mg/kg) reduced TNF-alpha-induced leukocyte adhesion and tissue recruitment. Notably, at 300 mg/kg response to TNF-alpha was nearly abolished, i.e. leukocyte adhesion was decreased by 83% and recruitment by 86%. In fact, the anti-inflammatory effect of this dose of Linomide corresponded in magnitude to the potency of 10 mg/kg of dexamethasone. Moreover, administration of Linomide did not alter the systemic leukocyte counts. On the other hand, 1-10 mg/kg of dexamethasone decreased the circulating number of mononuclear leukocytes by 77%. Taken together, our novel findings demonstrate that Linomide is a potent inhibitor of leukocyte adhesion and recruitment in cytokine-activated tissues. These data may help explain the documented protection provided by Linomide in inflammatory diseases characterized by cytokine and leukocyte accumulation.     

Fibrin cross-linking in congenital factor XIII deficiency.

Homozygous patients with factor XIII deficiency are devoid of immunologically identifiable A protein, the active enzymatic component. Quantitative studies of transamidase activity of the factor are available in only a few cases, and the fibrin cross-linking pattern is not well known. The present paper deals with the quantitative estimation of factor XIII transamidase activity (dansylcadaverine system), factor XIII molecular subunits, and the corresponding fibrin cross-linking pattern in seven homozygous patients with factor XIII deficiency. The results indicate that transamidase activity was present in all patients, and the range was 0.5-1.7%. The pattern of fibrin stabiisation showed an absence of cross-linking in two patients, the presence of gamma-gamma-dimers (traces) in four, and gamma-gamma-dimers plus incomplete alpha-polymers (traces) in one patient. In conclusion, the homozygous patients reported here were not completely devoid of functioning factor XIII.     

Preclinical safety and pharmacokinetics of recombinant human factor XIII

Factor XIII (FXIII) is a thrombin-activated protransglutaminase responsible for fibrin clot stabilization and longevity. Deficiency in FXIII is associated with diffuse bleeding and wound-healing disorders in humans. This report summarizes results from several studies conducted in adult cynomolgus monkeys (M. fascicularis) to evaluate the safety and pharmacokinetics of recombinant human factor XIII A(2) dimer (rFXIII). Intravenous slow bolus injection of rFXIII resulted in the expected formation of the heterotetramer rA(2)cnB(2), prolonged circulating half-life (5-7 days), and increased plasma transglutaminase activity. Recombinant FXIII was well tolerated as a single dose up to 20 mg/kg rFXIII (2840 U/kg), as repeated daily doses up to 6 mg/kg (852 U/kg) for 14 days, and as 3 repeated doses of 8 mg/kg (1136 U/kg) separated by 14 days. Overt toxicity occurred after a single intravenous injection of = 22.5 mg/kg rFXIII (3150 U/kg), or with 2 doses of = 12.5 mg/kg (1775 U/kg) administered within 72 hours. The rFXIII-mediated toxicity was expressed as an acute systemic occlusive coagulopathy. Evaluation of plasma samples from dosed animals demonstrated formation of cross-linked fibrin/fibrinogen oligomers and higher-order protein aggregates, which are hypothesized to be responsible for the observed vessel occlusion and associated embolic sequelae. These results demonstrate that rFXIII-mediated toxicity results from exaggerated pharmacological activity of the molecule at supraphysiological concentrations. The absence of observed toxicological effect with repeated intravenous doses up to 8 mg/kg (1136 U/kg) was used to support an initial clinical dose range of 0.014 to 0.35 mg/kg (2-50 U/kg).     

Scleredema of Buschke: remission with factor XIII treatment

We describe the case of a 55-year-old man with scleredema of Buschke of the torso complicated by insulin-dependent diabetes mellitus. Due to (i) the patient's poor general health status, (ii) the similarity between scleroderma and scleredema of Buschke, and (iii) the well known efficacy of factor XIII infusions in scleroderma, we attempted an intravenous treatment with factor XIII. This therapy resulted in marked increase of movements and in softening of the skin, together with ultrasonographic and histopathological improvements. In conclusion, to the best of our knowledge, this is the first case in which factor XIII has been successfully used for the treatment of scleredema of Buschke.     

Congenital factor XIII deficiency in the south of Tunisia

Factor XIII deficiency is a rare autosomal recessive congenital disorder of haemostasis characterised by a plasmatic factor XIII level less than 1% in homozygote and bleeding as of the youth. We report a study about ten patients with congenital factor XIII deficiency from seven south-Tunisian families, there are seven females. Umbilical bleeding was common and only two patients had intracranial bleeding. The standard screening tests are normal. Factor XIII activity was less than 1% in all patients. A sub-unit A deficit was detected for the ten patients. Out hemorrhagic context, five patients receive regular prophylactic transfusion with fresh frozen plasma.