Factor XIII A-Subunit V34L Variant Affects Thrombus Cross-Linking in a Murine Model of Thrombosis.

OBJECTIVE: Factor XIII (FXIII) cross-links fibrin upon activation by thrombin. Activation involves cleavage at residue 37 by thrombin, releasing an activation peptide. A common polymorphism (valine to leucine variant at residue 34, V34L), located in the activation peptide, has been associated with increased activation rates and paradoxically a protective effect in cardiovascular disease. There is, currently, no data available on the effects of V34L from in vivo models of thrombosis. We examined the effect of FXIII V34L on clot formation and cross-linking in vivo. APPROACH AND RESULTS: We generated a panel of full-length recombinant human FXIII-A2 variants with amino acid substitutions in the activation peptide to investigate the effect of these variants on activation rate, and we used wild-type, V34L, and alanine to glycine variant at residue 33 variants to study the effects of varying FXIII activation rate on thrombus formation in a murine model of FeCl3 injury. FXIII activation assay showed that residues 29, 30, 33, and 34 play a critical role in thrombin interaction. Full-length recombinant human FXIII-A2 V34L has significant effects on clot formation, structure, and lysis in vitro, using turbidity assay. This variant influenced fibrin cross-linking but not size of the thrombus in vivo. CONCLUSIONS: Mutations in the activation peptide of full-length recombinant FXIII regulate activation rates by thrombin, and V34L influences in vivo thrombus formation by increased cross-linking of the clot.

Congenital blood coagulation factor XIII deficiency and successful deliveries: a review of the literature.

Congenital deficiency of blood coagulation factor XIII is an uncommon, inherited disorder characterized by hemorrhagic diathesis, habitual abortions and defective wound healing. We analyzed 8 reported successful pregnancies in women with a congenital deficiency of A-subunit of factor XIII (XIIIA), in which the plasma level of maternal factor XIIIA and/or the precise replacement therapies are described. Because decidual bleeding usually begins from 5 to 6 weeks' gestation and, without replacement therapy, spontaneous abortion always occurs, we herein offer the following prenatal and peripartum management guidelines and observations: i) the level of plasma A-subunit of factor XIII antigen (XIIIA-Ag) or factor XIII activity (XIII-act) must be at least 2%-3%, and, if possible, higher than 10% to prevent decidual bleeding and miscarriage during the pregnancy; ii) factor XIIIA concentrate is better than fresh frozen plasma or cryoprecipitate for replacement therapy; iii) the administration of 250 international units (IU) every 7 days is sufficient to maintain the level of plasma XIIIA-Ag or XIII-act more than 10% in the early period of gestation (through 22 weeks' gestation); however, 500 IU every 7 days is indicated in the later period (from 23 weeks' gestation) to maintain that level; iv) during labor, the desired level of plasma XIIIA-Ag or XIII-act should be higher than 20%, and, if possible, higher than 30% in order to make ready for any risk of severe obstetrical hemorrhagic complications; thus a booster dose of 1000 IU is indicated before labor; v) no replacement therapy is necessary in the puerperium because it is usually uneventful without it.

Safety and pharmacokinetics of recombinant factor XIII-A2 administration in patients with congenital factor XIII deficiency.

Congenital factor XIII (FXIII) deficiency is associated with a tendency for severe bleeding, a risk for spontaneous abortion, and a high rate of spontaneous intracranial hemorrhage. This phase 1 escalating-dose study was developed to evaluate the safety and pharmacokinetics of a single administration of human recombinant FXIII-A2 (rFXIII-A2) homodimer in adults with congenital FXIII deficiency. Pharmacokinetics and activity of rXIII and changes in endogenous B subunit levels were assessed. Recombinant FXIII-A2 homodimer were complexed with endogenous FXIII-B subunits to form an FXIII-A2B2 heterotetramer with a half-life of 8.5 days, similar to that of endogenous FXIII. The median dose response was a 2.4% increase in FXIII activity based on unit per kilogram rFXIII administered. After the administration of rFXIII-A2, clot solubility normalized as measured by clot lysis in urea. Clot strength and resistance to fibrinolysis, as assessed by thromboelastography, also improved. Safety reviews were conducted before each dose escalation; no serious adverse events, including bleeding or thrombosis, were noted during the study. In addition, there was no evidence of the generation of specific antibodies to rFXIII or yeast proteins. Recombinant FXIII appears to be a safe and potentially effective alternative for FXIII replacement in patients with FXIII deficiency.

Gelatin-based biomimetic tissue adhesive. Potential for retinal reattachment.

An adhesive that cures under moist/wet conditions could facilitate surgical procedures for retinal reattachment. We are investigating an adhesive that mimics the factor XIIIa-mediated crosslinking of fibrin that occurs in the late stages of the blood coagulation cascade. Specifically, we use gelatin as the structural protein (in place of fibrin), and crosslink gelatin using a calcium-independent microbial transglutaminase (in place of the calcium-dependent transglutaminase factor XIIIa). Injection of gelatin and microbial transglutaminase (mTG) into the vitreous cavity of Sprague Dawley white rats did not elicit structural or cellular damage to the retina as evidenced from histological evaluation 2 weeks post-injection. Qualitative in vitro studies indicate that the gelatin-mTG adhesive binds to bovine retinal tissue under wet conditions. Quantitative lap-shear tests were performed with more robust bovine tissue from the choroid and sclera. The lap-shear strength of the biomimetic gelatin-mTG adhesive was independent of tissue-type and ranged from 15 to 45 kPa, which is comparable to the values reported for other soft-tissue adhesives. These studies suggest that the mTG-crosslinked gelatin may provide a simple, safe, and effective adhesive for ophthalmic applications.

[Mechanisms influencing cellular physiology as a concept of treatment for wound healing disturbances]. / Mechanismen zur Beeinflussung der Zellphysiologie als Wundheilungskonzept.

AIM: Recent knowledge about repair mechanisms in different types of tissue is the basic of actual therapeutic efforts. Center of several experimental and clinical approaches is the influencing of angiogenesis with an also distinct meaning concerning wound healing. Therefore, application of growth factors, gene transfer, and employment of genetically manipulated cells often aim at angiogenesis. Nevertheless, manipulation of angiogenesis also leads to secondary problems such as hyperpermeability followed by impairment of local wound milieu. Our study was done to identify mechanisms to protect from disturbances of endothelial barrier function. METHOD: In a first experimental investigation on cultured endothelial cells, the influence of plasma-transglutaminase (Factor XIII) to endothelial barrier function was studied. In a second step, the influence of Factor XIII on wound healing properties was investigated in patients with a chronic venous ulceration. RESULTS: Activated Factor XIII (FXIIIA*) led to a dose-dependent reduction of endothelial cell permeability of 30 % compared to control with a maximum effect using 1 to 5 U/mL. Clinical investigation revealed a nearly complete reduction of wound secretion. CONCLUSION: Experimental studies revealed that activated Factor XIII stabilizes endothelial barrier under basic conditions as well as under conditions of induced hyperpermeability. Clinical study revealed that Factor XIII also distinctly reduces wound secretion. Therefore, plasma-transglutaminase may offer a new therapeutic option to treat the local or generalized leakage-syndrome.