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Four missense mutations in the factor XIII A-subunit gene, Arg260Leu, Ala318Val, Thr398Asn and Gly210Arg, were previously reported by us in patients with severe factor XIII deficiency. The objective of our study was to discern the effect of all four mutations on the stability and intracellular localization of the factor XIII A-subunit by their expression in COS cells. In-vitro mutagenesis, transient expression of the mutants in COS cells and subsequent pulse-chase analyses were carried out. Intracellular localization of wild-type and mutant proteins was analyzed by immunohistochemistry using a monoclonal antibody against factor XIII A-subunit. Pulse-chase analyses of metabolically labeled proteins demonstrated rapid intracellular degradation of each mutant protein as compared with wild type. Immunocytochemical and immunofluorescence analyses disclosed that wild-type and all four mutant factor XIII A-subunit proteins were diffusely distributed within the cytoplasm but not in the endoplasmic reticulum of the COS-7 cells. The Arg260Leu, Ala318Val, Thr398Asn and Gly210Arg mutations in FXIII A-subunit cause rapid intracellular degradation of the corresponding mutated protein.

Factor XIII subunit A (FXIII A) is synthesized by megakaryocytes, and monocytes/macrophages. In addition, the liver has been reported as an extrahaematopoietic source of FXIII A. At present, the extent of contribution of either haematopoietic or extrahaematopoietic sources to the plasma FXIII A level is unknown. We studied the effect of bone marrow aplasia due to high-dose chemotherapy followed by autologous peripheral blood stem cell transplantation (ASCT) on plasma FXIII A activity and concentration in 20 patients with haematological or solid tumour malignancies. A multiple linear regression model was used to assess the effect of gender, age, malignancy and treatment types, platelet and monocyte counts, abnormal liver function tests, prothrombin time, and number of platelet transfusions on FXIII activity measured in plasma before and following ASCT. Significant correlation between platelet counts and FXIII A activity in plasma was observed (r = 0.51, P = 0.0001), which remained after the adjustment for the aforementioned parameters (multiple R = 0.52, P = 0.0001). In contrast, no significant correlation between FXIII A levels and monocyte counts was observed (r = 0.19), and this lack of correlation persisted after the adjustment. These results suggest that in the ASCT model, following myeloablation, platelets but not monocytes are the haematopoietic cells that contribute significantly to plasma FXIII A levels. In addition, extra-haematopoietic sources of FXIII synthesis may also contribute to FXIII levels in plasma.

Objective— Factor XIII (FXIII), a plasma transglutaminase that stabilizes fibrin clots at the final stages of blood coagulation by crosslinking fibrin monomers, is essential for embryo implantation and participates in tissue remodeling and wound healing, processes that involve angiogenesis. The aim of our study was to analyze the effect of FXIII on angiogenesis using in vitro and in vivo models and to examine the role of FXIII in the basic steps of angiogenesis, ie, migration, proliferation, and apoptosis/cell survival. Methods and Results— In the Matrigel tube formation model, only FXIIIa caused a dose-dependent enhancement of array formation. This proangiogenic effect was not associated with alterations in vascular endothelial growth factor (VEGF) protein levels nor VEGF or VEGFR2 mRNA levels. FXIIIa, but not nonactivated or transglutaminase-inactivated FXIII, significantly enhanced endothelial cell migration and proliferation and inhibited apoptosis. After treatment of HUVECs with FXIIIa, almost complete disappearance of mRNA of thrombospondin 1 (TSP-1) and a marked reduction in the secretion of TSP-1 protein were observed. A reduction in TSP-1 protein synthesis, although to a lesser extent, was observed on treatment of microvascular endothelial cells with FXIIIa. In a rabbit cornea model, injection of FXIIIa caused neovascularization associated with almost complete disappearance of TSP-1 in the cornea. Conclusions— These results show that FXIIIa exhibits a novel proangiogenic activity that is associated with downregulation of TSP-1 and also involves stimulation of endothelial cell proliferation and migration and inhibition of apoptosis. These findings might shed light on the mechanism by which FXIII mediates tissue repair and remodeling.

Abstract —In a previous report by Freedman et al ( J Clin Invest. 1996;97:979–987), plasma from 2 brothers with stroke or transient ischemic attack inactivated the antiplatelet effects of nitric oxide (NO), and this effect was found to be a consequence of a deficiency of plasma glutathione peroxidase (GSH-Px). In this study, we attempted to define the generalizability of this deficiency by studying NO-mediated antiplatelet effects in 7 families with familial childhood stroke. Seven families with familial childhood stroke that consecutively presented to a large referral center were included in the study. We monitored ADP-induced aggregation of normal gel-filtered platelets (GFP) in platelet-poor plasma (PPP) from normal individuals and from patients in the presence or absence of an NO donor (S-nitroso–glutathione). Surface P-selectin expression of normal GFP in patients’ PPP was analyzed by flow cytometry after incubation with a P-selectin–specific monoclonal antibody in the presence or absence of the NO donor. We also measured GSH-Px activity in plasmas from family members and normal controls using standard methods. In 6 of 7 families, NO failed to inhibit platelet P-selectin expression and platelet aggregation in PPP from the affected family members and some of their relatives. Of 4 families studied, 3 probands and their corresponding affected parent had 50% decrease in plasma GSH-Px activity. In some patients with childhood stroke, impaired metabolism of reactive oxygen species as a result of reduced GSH-Px activity results in NO insufficiency that affects normal platelet inhibitory mechanisms and predisposes to arterial thrombosis.

Haemostatic efficacy and pharmacokinetic analysis of solvent/detergent (S/D) treated, virus inactivated plasma (OctaplasTM, Germany) was evaluated in eight patients with hereditary factor VII, X and XI deficiency and in three patients with acquired coagulation disorders due to liver disease. The patients received the S/D plasma for treatment of haemarthrosis, menorrhagia or before surgical procedures. In all the patients the S/D plasma was sufficient to prevent or stop bleeding. Side effects included urticaria (one patient) and moderate anaphylactoid reaction (one patient). No evidence of plasma-born viral infections was observed up to 12 months after the treatment (95% confidence limits 0–22%). Calculated mean half-life of coagulation factors VII, X and XI was 4.36 h, 49.21 h and 44.5 h, respectively, similar to that observed with fresh-frozen plasma. Because of retained coagulation factor integrity and improved viral safety, S/D plasma could be considered a superior alternative to standard fresh-frozen plasma.

Three intragenic restriction fragment length polymorphisms (RFLPs) were used to study linkage and analyse the mode of inheritance in type I and type III von Willebrand's disease (vWD). In two families linkage was established between Sac I RFLPs and the inheritance of type I vWD. RFLP analysis of amniocyte DNA from a potentially affected foetus enabled us to establish a prenatal diagnosis of vWD in a third family with type I vWD. Linkage was also established in four families between the Sac I and two Taq I RFLPs and the inheritance of type III vWD. All type III probands were homozygotes and inherited the same mutant vWF allele from both parents. Heterozygous carriers from one type III family were phenotypically normal and could be detected only by linkage analysis, whereas carriers from the remaining three type III families were asymptomatic but had decreased values of vWF antigen and activity. RFLP-based linkage analysis of vWD alleles provides a way to improve the diagnostic precision, detect carriers, and may be useful for prenatal diagnosis of type III vWD.