Sensitive and selective detection of free FXIII activation peptide: a potential marker of acute thrombotic events.

Coagulation factor XIII (FXIII) stabilizes fibrin fibers and is therefore a major player in the maintenance of hemostasis. FXIII is activated by thrombin resulting in cleavage and release of the FXIII activation peptide (AP-FXIII). The objective of this study was to characterize the released AP-FXIII and determine specific features that may be used for its specific detection. We analyzed the structure of bound AP-FXIII within the FXIII A-subunit and interactions of AP-FXIII by hydrogen bonds with both FXIII A-subunit monomers. We optimized our previously developed AP-FXIII ELISA by using 2 monoclonal antibodies. We determined high binding affinities between the antibodies and free AP-FXIII and demonstrated specific binding by epitope mapping analyses with surface plasmon resonance and enzyme-linked immunosorbent assay. Because the structure of free AP-FXIII had been characterized so far by molecular modeling only, we performed structural analysis by nuclear magnetic resonance. Recombinant AP-FXIII was largely flexible both in plasma and water, differing significantly from the rigid structure in the bound state. We suggest that the recognized epitope is either occluded in the noncleaved form or possesses a structure that does not allow binding to the antibodies. On the basis of our findings, we propose AP-FXIII as a possible new marker for acute thrombotic events.

Pruritus of unknown origin and elevated total serum bile acid levels in patients without clinically apparent liver disease.

BACKGROUND AND AIM: Generalized pruritus of unknown origin (PUO) is a highly distressing condition that is unrelated to any underlying dermatologic or systemic disorder (e.g. cholestasis). Little is known about the potential contribution of elevated total serum bile acid (TSBA) levels to PUO. Our aim in the present study was to investigate the role of elevated TSBA levels in patients with PUO and the efficacy of ursodeoxycholic acid (UDCA) and cholestyramine therapy. METHODS: Retrospective study comprising 117 patients with chronic pruritic conditions (PUO, atopic disease, asteatotic eczema, latent cholestasis, etc.); 99 patients with available TSBA levels were included and compared with healthy controls. RESULTS: Elevated TSBA levels were detected more frequently in patients with chronic pruritic diseases than in the control population (28.28% vs 6%; P<0.001) with significantly higher pathological absolute levels (mean 17.45±34.46 µmol/L vs 6.02±4.73 µmol/L; P=0.001). Patients with PUO (n=18) showed the second-highest prevalence of pathological bile acid level elevation (83.3%; control population 6%; P<0.001), after patients with subclinical cholestasis and presented with particularly high TSBA serum values (mean 37.79±53.38 µmol/L; P<0.001). Cholestyramine (n=9) and UDCA (n=8) therapy were both effective in lowering TSBA levels and lead to substantial improvement of pruritus in patients with elevated TSBA levels. CONCLUSIONS: Total serum bile acid levels are elevated in a high proportion of patients with PUO. These results provide evidence of a potential involvement of subclinical cholestasis in the pathogenesis of PUO. We suggest that evaluation of TSBA levels should be included in the diagnostic work-up of patients with chronic unexplained pruritus.

Coagulation factor XIII activation peptide and subunit levels in patients with acute ischaemic stroke: a pilot study.

INTRODUCTION: We have recently shown that FXIII activation peptide (AP-FXIII) can be measured in plasma. The objective of this pilot study was to investigate for the first time if AP-FXIII can be detected in plasma from patients with acute ischaemic stroke. MATERIALS AND METHODS: We included 66 patients with acute ischaemic stroke admitted between 1 and 7 hours after the onset of clinical symptoms. We collected plasma samples upon admission and on the two following days and measured AP-FXIII and subunit levels by ELISA. Clinical stroke severity was assessed by NIHSS stroke score. RESULTS: AP-FXIII could be detected in 34 patients upon admission (range 0.2-26.3 ng/ml), on day 1 in 15 patients (0.2-10.4 ng/ml), and on day 2 in 11 patients (0.1-15.1 ng/ml. AP-FXIII was higher in patients with severe stroke. Lower AP-FXIII levels upon admission were associated with clinical improvement. FXIII-A and FXIII-B subunit levels decreased significantly from day 0 to day 1. CONCLUSIONS: For the first time, we detected AP-FXIII in patients upon an acute thrombotic event. The decrease in FXIII subunit levels during acute ischaemic stroke is evidence for ongoing coagulation activity and FXIII consumption. Our results suggest that FXIII activation and concomitant AP-FXIII release might be associated with an unfavourable short-term clinical outcome. Larger studies are needed to further investigate whether AP-FXIII might serve as a diagnostic and/or prognostic marker for acute thrombotic diseases.

Heteromeric complex formation of ASK2 and ASK1 regulates stress-induced signaling.

Apoptosis signal-regulating kinase 2 (ASK2) is an interaction partner of the highly related ASK1. Here, we describe a regulatory function of ASK2 in stress signaling-induced cleavage of caspase-3 and poly(ADP-ribose) polymerase (PARP). Increased cleavage of caspase-3 and PARP was demonstrated by overexpression as well as knockdown of ASK2 after stress-induction by serum-starvation. We show that ectopically expressed ASK2 homo-oligomerized while endogenous ASK2 and ASK1 formed hetero-oligomers, which decreased upon serum-starvation. Co-expression of ASK2 and ASK1 stabilized these two proteins and reduced starvation-induced caspase-3 activation and degradation of PARP. Analysis of the intracellular localization of ASK2 exhibited a similar localization compared with ASK1 in the nucleus, cytoplasm, and in mitochondria. We propose that ASK2 regulates stress-induced caspase-3 and PARP cleavage in a dose-dependent manner by heteromeric complex formation with ASK1.