The structure of recombinant human cellular factor XIII zymogen was solved in its monoclinic crystal form and refined to an R-factor of 18.3% (R free = 23.6%) for all data between 40.0 and 2.1 A î resolution. Two non-proline cis peptide bonds were detected. One is between Arg QIH and Tyr QII close to the active site cysteine residue (Cys QIR ) and the other is between Gln RPS and Phe RPT at the dimerization interface. The structure and the role of these cis peptides are discussed in the light of their possible importance for factor XIII function.z 1998 Federation of European Biochemical Societies.

SummaryBlood coagulation factor XIII (FXIII) promotes cross-linking of fibrin during blood coagulation; impaired clot stabilization in human genetic deficiency is associated with marked pathologies of major clinical impact, including bleeding symptoms and deficient wound healing. To investigate the role of FXIII we employed homologous recombination to generate a targeted deletion of the inferred exon 7 of the FXIII-A gene. FXIII transglutaminase activity in plasma was reduced to about 50% in mice heterozygous for the mutant allele, and was abolished in homozygous null mice. Plasma fibrin γ-dimerization was also indetectable in the homozygous deficient animals, confirming the absence of activatable FXIII. Homozygous mutant mice were fertile, although reproduction was impaired. Bleeding episodes, hematothorax, hematoperitoneum and subcutaneous hemorrhage in mutant mice were associated with reduced survival. Arrest of tail-tip bleeding in FXIII-A deficient mice was markedly and significantly delayed; replacement of mutant mice with human plasma FXIII (Fibrogammin® P) restored bleeding time to within the normal range. Thrombelastography (TEG) experiments demonstrated impaired clot stabilization in FXIII-A mutant mice, replacement with human FXIII led to dose-dependent TEG normalization. The mutant mice thus reiterate some key features of the human genetic disorder: they will be valuable in assessing the role of FXIII in other associated pathologies and the development of new therapies.

von Willebrand disease (vWD) is a bleeding disorder that results from defects in the quality or quantity of von Willebrand factor (vWF), a glycoprotein essential for normal thrombus formation. vWF circulates in plasma as multimers in sizes ranging up to 20,000 kd. The high molecular weight vWF (HMWvWF) multimers are most essential for primary hemostasis, whereas the lower molecular weight multimers are less functionally active. For many patients, the treatment of choice is factor replacement with a vWF/FVIII concentrate, preferably one with a high content of HMWvWF multimers. Given that the commercially available vWF/FVIII concentrates seem to differ substantially in their biochemical properties as well as in their clinical efficacy, we did a comparative study with 12 vWF/FVIII concentrates to investigate content and activities of FVIII and vWF, as well as the content of HMWvWF multimers. The content of HMWvWF multimers varied considerably among the 12 concentrates. The specific vWF activities, as assessed by ristocetin cofactor activity (vWF:RCo) and collagen-binding activity (vWF:CB), correlated well with the HMWvWF content of the products. Of the products tested, Haemate P/Humate-P had the highest content of HMWvWF multimers (with a multimer pattern closest to that of normal human plasma), the highest specific vWF activities, and the highest values of vWF:RCo and vWF:CB per unit of FVIII:coagulant (C). The goal of bleeding prophylaxis and treatment in type 2, severe type 1, and type 3 vWD patients is to normalize vWF activities (vWF:RCo and vWF:CB) and FVIII:C preferentially by vWF/FVIII concentrates containing the high vWF multimers and a high vWF:RCo/FVIII ratio to achieve normal primary and secondary hemostasis. Based on the present study of a comparative analysis of currently available vWF/FVIII concentrates, a classification of vWF/FVIII products is proposed.

The effect of factor XIII on endothelial barrier function was studied in a model of cultured monolayers of porcine aortic endothelial cells and saline-perfused rat hearts. The thrombin-activated plasma factor XIII (1 U/ml) reduced albumin permeability of endothelial monolayers within 20 min by 30 ± 7% (basal value of 5.9 ± 0.4 × 10−6 cm/s), whereas the nonactivated plasma factor XIII had no effect. Reduction of permeability to the same extent, i.e., by 34 ± 9% could be obtained with the thrombin-activated A subunit of factor XIII (1 U/ml), whereas the iodoacetamide-inactivated A subunit as well as the B subunit had no effect on permeability. Endothelial monolayers exposed to the activated factor XIII A exhibited immunoreactive deposition of itself at interfaces of adjacent cells; however, these were not found on exposure to nonactivated factor XIII A or factor XIII B. Hyperpermeability induced by metabolic inhibition (1 mM potassium cyanide plus 1 mM 2-deoxy-d-glucose) was prevented in the presence of the activated factor XIII A. Likewise, the increase in myocardial water content in ischemic-reperfused rat hearts was prevented in its presence. This study shows that activated factor XIII reduces endothelial permeability. It can prevent the loss of endothelial barrier function under conditions of energy depletion. Its effect seems related to a modification of the paracellular passageways in endothelial monolayers.