Rainer Seitz

Diagnosis and classification of factor XIII deficiencies

H. P . K OHLER ,* A. IC H I NO SE , R . SE ITZ ,§ R . A . S . AR I ENS– and L . MUSZBEK ,** ON BEHALF OF THE FACTOR X I I I AND F IBR INOGEN SSC SUBCOMMITTEE OF THE ISTH *Laboratory for Hemostasis Research, Department of Hematology, University Hospital of Bern, Bern; Department of Internal Medicine, Spital Netz Bern Hospitals, Bern, Switzerland; Department of Molecular Patho-Biochemistry, Yamagata University School of Medicine, Yamagata, Japan; §Paul-Ehrlich-Institut, Langen, Germany; –Section on Mechanisms of Thrombosis, Leeds Institute for Health, Genetics and Therapeutics, University of Leeds, Leeds, UK; and **Clinical Research Center and Research Group of the Hungarian Academy of Sciences, University of Debrecen, Medical and Health Science Center, Debrecen, Hungary

International Registry on Factor XIII Deficiency: A basis formed mostly on European data

FXIII deficiency is known as one of the rarest blood coagulation disorders. In this study, the phenotypic and in part genotypic data of 104 FXIII-deficient patients recorded from 1993 - 2005 are presented. The most common bleeding symptoms were subcutaneous bleeding (57%) followed by delayed umbilical cord bleeding (56%), muscle hematoma (49%), hemorrhage after surgery (40%), hemarthrosis (36%), and intracerebral bleeding (34%). Prophylactic treatment was initiated in about 70% of all patients. FXIII-B subunit-deficient patients had a milder phenotype than patients with FXIII-A subunit deficiency. The most frequent mutation affecting the F13A gene was a splice site mutation in intron 5 (IVS5-1G>A). This mutation was found in eight (17%) of 46 analyzed families. The haplotype analysis of patients carrying the IVS5-1A allele was consistent with a founder effect. The international registry (http://www.f13-database.de) will provide clinicians and scientists working on FXIII deficiency with a helpful tool to improve patient care and direct future studies towards better understanding and treatment of the disease.

Activation of coagulation and fibrinolysis in patients with lung cancer: relation to tumour stage and prognosis

Activation of coagulation and fibrinolysis within tumour tissues is thought to be associated with tumour growth, angiogenesis, and metastasis. The plasma levels of markers of thrombin and plasmin generation are sensitive tools for monitoring activation of coagulation and fibrinolysis. We studied 47 patients with histologically confirmed lung cancer, 15 with small cell (SCLC) and 32 with non-small cell lung cancer (NSCLC). The plasma levels of the following markers were assessed: thrombin-antithrombin III complex (TAT), prothrombin activation fragment Fl + 2, plasmin-α2:-antiplasmin complex (PAP) and the split product from cross-linked fibrin, D-dimer. The first sample was obtained before receiving any specific antineoplastic treatment. The patients were followed thereafter until treatment was terminated. There was no difference in activation markers between patients with SCLC and NSCLC. Comparing patients with limited disease to those with extensive disease, there were significant differences in TAT (median 3.0 (1.9–9.8) vs 5.3 (1.8–35.6) μg/l, P = 0.021) and D-dimer (569 (135–1948) vs 1288 (120–2221) μg/I, P = 0.014). According to the response to subsequent treatment, those who achieved complete or partial tumour remission had significantly lower baseline levels samples than non-responders (TAT 2.9 (1.9–4.0) vs 4.7 (1.8–35.6) μg/l, P = 0.0047;D-dimer527(135–1149)w 1242 (120–2221) μg/l, P = 0.0013). Thus, the increase of TAT and D-dimer appears to be related to tumour spread. The results suggest that high levels of these markers might be a sign of unfavourable prognosis in patients with lung cancer. The possible predictive value and the relevance of these markers in supporting diagnostic and therapeutic decisions should be further evaluated.

Detection of a novel plasma serine protease during purification of vitamin K-dependent coagulation factors.

A novel serine protease (PHBSP) was purified from human plasma by two chromatographic steps with a final yield of 1.6 mg/l plasma. The protease consists of two disulfide‐bridged chains of about 50 and 30 kDa with the light chain containing the active site of the enzyme. NH2‐terminal sequence analysis revealed identity to the deduced amino acid sequence of HGFA‐like mRNA. The activity of PHBSP is strongly dependent on Ca2+ ions and is efficiently inhibited by α2‐antiplasmin and aprotinin. Possible functions of PHBSP in the hemostatic system are discussed.

Experience of mandatory nucleic acid test (NAT) screening across all blood organizations in Germany: NAT yield versus breakthrough transmissions.

BACKGROUND: Mandatory nucleic acid test (NAT) blood screening was introduced in Germany in 1999 for hepatitis C virus (HCV) RNA and in 2004 for human immunodeficiency virus Type 1 (HIV‐1) RNA. Minimal sensitivity limits of 5000 IU HCV RNA/mL and 10,000 IU HIV‐1 RNA/mL were defined for the individual donation facilitating testing of minipools (MPs). The NAT yield obtained from all blood organizations is summarized. Transfusion‐associated virus transmissions despite NAT screening (“breakthrough transmissions”) are analyzed.

Activation of proPHBSP, the zymogen of a plasma hyaluronan binding serine protease, by an intermolecular autocatalytic mechanism.

Abstract The hyaluronic acid binding serine protease (PHBSP), an enzyme with the ability to activate the coagulation factor FVII and the plasminogen activator precursors and to inactivate factor VIII and factor V, could be isolated from human plasma in the presence of 6m urea as a single-chain zymogen, whereas under native conditions only its activated two-chain form was obtained. The total yield of proenzyme (proPHBSP) was 5–6 mg/l, corresponding to a concentration of at least 80–100nm in plasma. Upon removal of urea, even in the absence of charged surfaces a rapid development of amidolytic activity was observed that correlated with the appearance of the two-chain enzyme. The highest activation rate was observed at pH 6. ProPHBSP processing was concentration-dependent following a second order kinetic and was accelerated by catalytic amounts of active PHBSP, indicating an intermolecular autocatalytic activation. Charged macromolecules like poly-L-lysine, heparin, and dextran sulfate strongly accelerated the autoactivation, suggesting that in vivo proPHBSP activation might be a surface-bound process. The intrinsic activity of the proenzyme was determined to be 0.25–0.3%, most likely due to traces of PHBSP. The presence of physiological concentrations of known plasma inhibitors of PHBSP, like α2 antiplasmin and C1 esterase inhibitor, but not antithrombin III/heparin, slowed down zymogen processing. Our in vitro data suggest that the autoactivation of proPHBSP during plasma fractionation is induced by the removal of inhibitors of PHBSP and is accelerated by charged surfaces of the chromatographic resins.

Identification of kallikrein and FXIa as impurities in therapeutic immunoglobulins: implications for the safety and control of intravenous blood products

Background and Objectives  The occurrence of thromboembolic events (TEEs) with intravenous immunoglobulin lots (IVIGs) raised the question of the causative agent for these adverse events. We investigated the predominant plasma proteases in 19 IVIG lots from five manufacturers including three lots associated with adverse events.

An update of the mutation profile of Factor 13 A and B genes

Mutational reports over the past two decades have accumulated an immense amount of literature for inherited Factor XIII deficiency. However, the genotype and phenotype correlations for inherited Factor XIII deficiency are complicated. While many studies clearly prove a cause and effect relationship for the reported mutations, others are lacking in this regard. The F13B gene remains an elusive component as far as inherited Factor XIII deficiencies are concerned. Also, an in-depth analysis into the heterozygous state of this deficiency is also lacking. In this review we have tried to analyze and present an exhaustive amount of mutational data from the past three decades. The source of our mutational data is our website dedicated to Factor XIII deficiencies (www.F13-database.de) as well as literature search done on the Pubmed (www.ncbi.nlm.nih.gov/pubmed).

Inhibition of bFGF/EGF-dependent endothelial cell proliferation by the hyaluronan-binding protease from human plasma

Recently we identified a plasma serine protease with a high affinity to glycosaminoglycans like heparin or hyaluronic acid, termed hyaluronan-binding protease (HABP). Since glycosaminoglycans are found on cell surfaces and in the extracellular matrix a physiological role of this plasma protease in a pericellular environment was postulated. Here we studied the influence of HABP on the regulation of endothelial cell growth. We found that HABP efficiently prevented the basic fibroblast growth factor/epidermal growth factor (bFGF/EGF)-dependent proliferation of human umbilical vein endothelial cells. Proteolytic cleavage of adhesion molecules was found to be involved, but was not solely responsible for the anti-proliferative activity. Pre-treatment of growth factor-supplemented cell culture medium with HABP indicated that no direct contact between the active protease and cells was required for growth inhibition. In vitro studies revealed a growth factor-directed activity of HABP, resulting in complexation and partial hydrolysis and, thus, inactivation of basic fibroblast growth factor, a potent mitogen for endothelial cells. Heparin and heparan sulfate fully protected bFGF from complexation and cleavage by HABP, although these glycosaminoglycans are known to enhance the proteolytic activity of HABP. This finding suggested that free circulating bFGF rather than bFGF bound to heparan sulfate proteoglycans would be a physiologic substrate. In conclusion, down-regulation of bFGF-dependent endothelial cell growth represents an important mechanism through which HABP could control cell growth in physiologic or pathologic processes like angiogenesis, wound healing or tumor development.

The Hyaluronan-Binding Serine Protease from Human Plasma Cleaves HMW and LMW Kininogen and Releases Bradykinin

Abstract The influence of the hyaluronanbinding protease (PHBSP), a plasma enzyme with FVII and prourokinase activating potency, on components of the contact phase (kallikrein/kinin) system was investigated. No activation or cleavage of the proenzymes involved in the contact phase system was observed. The procofactor high molecular weight kininogen (HK), however, was cleaved in vitro by PHBSP in the absence of any charged surface, releasing the activated cofactor and the vasoactive nonapeptide bradykinin. Glycosoaminoglycans strongly enhanced the reaction. The cleavage was comparable to that of plasma kallikrein, but clearly different from that of coagulation factor FXIa. Upon extended incubation with PHBSP, the light chain was further processed, partially removing about 60 amino acid residues from the Nterminus of domain D5 of the light chain. These cleavage site(s) were distinct from plasma kallikrein or FXIa cleavage sites. PHBSP and, more interestingly, also plasma kallikrein could cleave low molecular weight kininogen in vitro, indicating that domains D5H and D6H are no prerequisite for kininogen cleavage. PHBSP was also able to release bradykinin from HK in plasma where the procofactor circulates predominantly in complex with plasma kallikrein or FXI. In conclusion, PHBSP represents a novel kininogencleaving and bradykininreleasing enzyme in plasma that shares significant catalytic similarities with plasma kallikrein. Since they are structurally unrelated in their heavy chains (propeptide), their similar in vivo catalytic activities might be directed at distinct sites where PHBSP could induce processes that are related to the kallikrein/kinin system.