V. De Filippis

Interaction of the 268-282 region of glycoprotein Ibα with the heparin-binding site of thrombin inhibits the enzyme activation of factor VIII

Activation of factor VIII (FVIII) by thrombin plays a fundamental role in the amplification of the coagulation cascade and takes place through specific proteolytic cleavages at Arg(372), Arg(740) and Arg(1689). Full FVIII activation requires cleavage at Arg(372), a process involving the alpha-thrombin exosite-II; referred to as heparin-binding site (HBS). The present study was aimed at investigating the effect of glycoprotein Ibalpha (GpIbalpha; 1-282 fragment) binding to thrombin HBS on FVIII activation. Similar experiments were also performed using a synthetic peptide modelled on the 268-282 sequence of GpIbalpha, and sulphated successfully at all tyrosine residues present along its sequence, at positions 276, 278 and 279. Both GpIbalpha 1-282 and the sulphated GpIb 268-282 peptides induced a progressive decrease (up to 70%) in activated FVIII generation, assessed by coagulation and FXa-generation assays. Furthermore, SDS/PAGE and Western-blot experiments showed that the specific appearance of the 44 kDa A2 domain on cleavage of the FVIII Arg(372)-Ser(373) peptide bond was delayed significantly in the presence of either GpIbalpha 1-282 or GpIb 268-282 peptide. Moreover, the effect of the latter on thrombin-mediated hydrolysis of a peptide having the sequence 341-376 of FVIII was investigated using reverse-phase HPLC. The k (cat)/ K (m) values of the FVIII 341-376 peptide hydrolysis by thrombin decreased linearly as a function of the GpIbalpha 268-282 peptide concentration, according to a competitive inhibition effect. Taken together, these experiments suggest that the sulphated 268-282 region of GpIbalpha binds to thrombin HBS, and is responsible for the inhibition of the Arg(372)-Ser(373) bond cleavage and activation of FVIII.

β2 -Glycoprotein I binds to thrombin and selectively inhibits the enzyme procoagulant functions.

This work was aimed at characterizing the interaction of β2‐glycoprotein I (β2GPI), an abundant plasma protein of unknown function, with human thrombin, the final effector protease in the coagulation cascade.

Oxidized von Willebrand factor is efficiently cleaved by serine proteases from primary granules of leukocytes: divergence from ADAMTS-13

To cite this article: Lancellotti S, De Filippis V, Pozzi N, Oggianu L, Rutella S, Scaglione GL, Maset F, Peyvandi F, Mannucci PM, De Cristofaro R. Oxidized von Willebrand factor is efficiently cleaved by serine proteases from primary granules of leukocytes: divergence from ADAMTS‐13. J Thromb Haemost 2011; 9: 1620–7.

Elucidation of the disulfide-folding pathway of hirudin by a topology-based approach

A theoretical model for the folding of proteins containing disulfide bonds is introduced. The model exploits the knowledge of the native state to favor the progressive establishment of native interactions. At variance with traditional approaches based on native topology, not all native bonds are treated in the same way; in particular, a suitable energy term is introduced to account for the special strength of disulfide bonds, as well as their ability to undergo intramolecular reshuffling. The model thus possesses the minimal ingredients necessary to investigate the much debated issue of whether the refolding process occurs through partially structured intermediates with native or non‐native disulfide bonds. This strategy is applied to a context of particular interest, the refolding process of hirudin, a thrombin‐specific protease inhibitor, for which conflicting folding pathways have been proposed. We show that the only two parameters in the model (temperature and disulfide strength) can be tuned to reproduce well a set of experimental transitions between species with different number of formed disulfides. This model is then used to provide a characterization of the folding process and a detailed description of the species involved in the rate‐limiting step of hirudin refolding. Proteins 2003;53:000–000.

Impaired sperm function in infertile men relies on the membrane sterol pattern

Membrane cholesterol removal appears a key step for the gain of fertility potential during sperm maturation. However, the membrane sterol pattern in sperm cells from infertile patients, with impaired sperm parameters, has been poorly investigated. To elucidate a causative link between sperm membrane composition in male fertility, here we have investigated the levels of cholesterol and its oxidized derivatives 7β‐hydroxycholesterol and 7‐keto‐cholesterol in sixteen infertile patients with oligo‐asthenozoospermia and 16 normozoospermic (N) fertile subjects. Furthermore, ten of 16 N fertile subjects agreed to receive a defined testicular thermal challenge by adhering to a programme of sauna sessions for 1 month. Semen samples were obtained from each of the participants, and sperm parameters were assessed according to the World Health Organization criteria. Sperm levels of cholesterol, 7β‐hydroxycholesterol and 7‐keto‐cholesterol were quantified by ultra‐pressure liquid chromatography mass spectrometry. The results showed that oligo‐asthenozoospermia patients had a huge amount of cholesterol content compared with fertile subjects (12.40 ± 6.05 μg/106 cells vs. 0.45 ± 0.28 μg/106 cells, p < 0.001, N and oligo‐asthenozoospermia, respectively). Also, oxidized derivatives were significantly higher in oligo‐asthenozoospermia patients (7β‐hydroxycholesterol: 1.96 ± 1.03 ng/106 cells vs. 0.075 ± 0.05 ng/106 cells, p < 0.001 and 7‐keto‐cholesterol: 1.11 ± 0.72 ng/106 cells vs. 0.005 ± 0.003 ng/106 cells, p < 0.001). Moreover, sauna exposure, in parallel with a progressive worsening of sperm motility parameters, was associated with a reversible increase in sperm cholesterol after the third and fourth week of treatment, whilst 7β‐hydroxycholesterol and 7‐keto‐cholesterol levels showed an earlier enhancement starting from the second week. Our data show for the first time in humans a strong difference in the cholesterol and its oxidized derivatives of infertile and fertile subjects. These findings suggest a strict biochemical link relating testis function, sperm membrane status and male fertility potential.

The Core Domain of Hirudin from the Leech Hirudinaria manillensis

Hirudin is a small (∼ 7 kDa) disulfide-crosslinked polypeptide known as the most potent and specific inhibitor of thrombin, a serine protease that plays a key role in the coagulation cascade and pathology of thrombotic diseases (Tapparelli et al., 1993). We have previously shown that the N-terminal proteolytic fragment 1–47 of hirudin HM2 from Hirudinaria manillensis (Fig. 1) maintains inhibitory action towards thrombin (Vindigni et al., 1994). An analog of fragment 1–47 with a Tyr3 →Trp exchange (Y3W) was prepared by solid-phase chemical synthesis and shown to be ~5-fold more active than the Tyr3 natural fragment 1–47 in inhibiting thrombin (De Filippis et al., 1995). In this study, we report the results of experiments of chemical modification and peptide bond cleavage at the level of Trp3 of the synthetic Y3W peptide 1–47 with the aim to prepare modified and truncated species of the Y3 W peptide analog. Modification reactions were carried out also on fragment 1–41, prepared by V8-protease digestion of the synthetic Y3W peptide 1–47 at Glu41. All peptide derivatives prepared in the course of this work were isolated to homogeneity and assayed for biological activity. The results of this study revealed the critical role of the N-terminal portion of peptide 1–47 in the inhibition of thrombin, providing experimental support for the proposed mechanism of interaction between hirudin and thrombin advanced in previous studies.