Sami Guermazi

Protein S deficiency and antibodies to protein S in patients with Behçet's disease.

Thrombosis occurs in 20 to 30% of patients with Behçets disease (BD). Most of the reported hemostatic abnormalities are related to the inflammatory syndrome. We have assessed the activity of antithrombin III, protein C and protein S (PS), in 30 patients with BD and in 30 healthy controls. Thrombosis antecedents were found in 16 patients. Antithrombin III and protein C were within the normal range, however free PS and PSactivity were significantly decreased in patients as compared to control group. PS deficiency detected in eight patients, was associated to thrombosis in 6 of them. No correlation was found between free PS/total PS ratio and C4bBP levels. Antibodies to PS were screened by ELISA and were present in 6 patients, associated to PS deficiency in 4, and to thrombosis antecedents in 5 cases. PS deficiency was transient in two patients, associated to a persistent antiPS in one of them. These findings suggest that auto-immune acquired PS deficiency may be involved in the pathogenesis of thrombotic events in BD.

Further evidence for the presence of anti-protein S autoantibodies in patients with systemic lupus erythematosus

Acquired protein S (PS) deficiency in systemic lupus erythematosus (SLE) has been previously reported, but its mechanism and its possible thrombotic role have not been established. The aim of our study was to provide further evidence for auto-immune PS deficiency in 27 Tunisian SLE patients, using PS-specific enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance technology (SPR). PS deficiencies for PS activity, free PS or total PS, respectively, were found in 19, 18 and 12 patients. A significant correlation (r= −0.475,P< 0.016) was found between free/total PS ratio and C4bBP levels, suggesting a role of inflammation in free PS deficiency. Immunoglobulin IgG antibodies to PS were detected in four patients by both ELISA and SPR, in six patients only by ELISA, and in two patients only by SPR. Signals for anti-PS IgG by ELISA and SPR were, however, significantly correlated (r= 0.549,P= 0.003). These results suggest that an auto-immune mechanism could account for low PS activity in patients with SLE. Auto-antibodies to PS may form immune complexes, inducing increased clearance of PS or interfering with the protein C–protein S system.

Amino acid structure and characterization of a heterodimeric disintegrin from Vipera lebetina venom

A heterodimeric disintegrin designed as lebein was isolated from crude Vipera lebetina venom using gel filtration, anion and cation exchange chromatographies on FPLC. The amino acid sequence of each subunit determined by Edman degradation contains 64 residues with ten half-cystines and an RGD site at the C-terminal part of the molecule. The molecular mass of native lebein determined by mass spectrometry was found to be 14083.4 Da and those of alpha and beta subunits were 6992.05 and 7117.62, respectively. These value are in good agreement with those calculated from the sequences. This protein strongly inhibits ADP induced platelet aggregation on human platelet rich plasma with IC(50)=160 nM. Sequences of this protein subunits displayed significant sequence similarities with many other monomeric and dimeric disintegrins reported from snake venoms. We identified an amino acid residue (N) in the hairpin loop of both subunits (CNRARGDDMNDYC) which is different from all other reported motifs of disintegrins and this subtle difference may contribute to the distinct affinities and selectivities of this class of proteins.

FURTHER CHARACTERIZATION AND THROMBOLYTIC ACTIVITY IN A RAT MODEL OF A FIBRINOGENASE FROM VIPERA LEBETINA VENOM

Vipera lebetina fibrinogenase (VlF) was shown to render fibrinogen incoagulable and to solubilize fibrin. The fibrinogenolytic activity of this enzyme was found to be 33 mg fibrinogen/min/mg protein. The study of the specificity of this enzyme revealed that it has no effect on purified factor X, prothrombin and protein C and on the specific chromogenic substrates of their active form. Plasminogen was not activated by VlF but slightly degraded. We have also compared the effect of VlF and plasmin on fibrinogen and shown that these two enzymes have a different sites of cleavage. This enzyme inhibited human platelet aggregation on PRP initiated by ADP and collagen but was without effect on the aggregation of washed rabbit platelets using thrombin as agonist. Administration of VlF in rat did not show any necrosis or hemorrhage in treated rats organs. We therefore, examined the thrombolytic activity of VlF in a rat model of venous thrombosis. Thrombus was produced in the posterior vena cava by injection of human fibrinogen and thrombin. Injection of 5 mg/Kg body weight showed an evident flow restoration after one hour and measurement of the fibrinogen level a decrease of about 30% after 3 hrs. VlFs action is not dependent on plasminogen activators and may act synergistically with them, thereby providing an intriguing potential clinical application for dissolution of blood clots.

Procoagulant and platelet-aggregating properties of cerastocytin from Cerastes cerastes venom

Cerastocytin is a thrombin-like serine protease with potent platelet-proaggregating properties. It is able to activate factor XIII but is less active than thrombin on plasma coagulation. The aggregation induced by cerastocytin resembles that induced by thrombin, since rabbit washed platelets desensitized by a pretreatment with thrombin do not aggregate in the presence of cerastocytin. Furthermore, preincubation of platelets with monoclonal antibodies specific for glycoproteins GPIb or GPIIbIIIa blocks receptor sites for thrombin and fibrinogen, respectively, and prevents their aggregation induced by thrombin or cerastocytin. A monoclonal antibody, inhibitor of von Willebrand factor (VWF)-dependent agglutination, blocks the aggregation induced by cerastocytin. After activation with cerastocytin, washed rabbit platelets degranulate and secrete ATP and phospholipase A2. However, cerastocytin is less potent in inducing the release of phospholipase A2 than in inducing ATP secretion.

Anti-thrombomodulin antibodies and venous thrombosis.

Thrombomodulin (TM) is a cell surface endothelial glycoprotein having anticoagulant properties. It inhibits thrombin, and activates protein C, leading to the inhibition of activated factors V and VIII. TM autoantibodies could theoretically predispose to thrombosis. We have tested 83 unselected patients with deep venous thrombosis, 36 males and 47 females aged from 1 to 70 years [mean ± standard deviation (SD), 34.2 ± 14.5 years] for the presence of IgG anti-TM in their plasmas. Tests were performed by enzyme-linked immunosorbent assay (ELISA) using recombinant human TM kindly provided by PAION GmbH (Aachen, Germany). Results are expressed as the optical density (OD) differences between coated and un-coated wells. Plasmas from 83 normal volunteer donors were used to define the cut-off value as the mean of absorbance of the control group + 3 SDs. The median OD of normal controls group was 0.024 (mean, 0.034; SD, 0.066; range, −0.048 to 0.309). The median OD obtained with plasmas from patients was 0.048 (mean, 0.114; SD, 0.215; range, −0.039 to 1.312) and was significantly higher than that of the control group (P < 0.0001). Choosing a cut-off value of 0.232 (mean OD of the control group + 3 SDs), 11 patients are considered as positive for IgG autoantibodies to TM and three normal controls are weakly positive. Selected plasma with IgG anti-TM and purified IgG were further tested by dot blot using recombinant purified TM and were found positive. Purified IgG of positive plasmas inhibits protein C activation by TM and thrombin, suggesting that anti-TM antibodies have a procoagulant effect. Interestingly, in our study, anti-TM antibodies are found in three of six patients with Budd–Chiari syndrome and four of eight patients with cerebral venous thrombosis. In conclusion, thrombomodulin autoantibodies could be a new interesting marker of thrombophilia easily detected by ELISA.

Importance of preanalytical step in hemostasis

The preanalytical conditions are the major component to assure the reliability and validity of results in hemostasis. They extend from the requirement of analysis, patient preparation, blood collection and the conditions of transport, centrifugation and plasma storage until analysis. They are the most important sources of erroneous or inconclusive results; mainly the quality of the analytical stage has seen great progress of instruments and reagents. Control and standardization efforts of these preanalytical conditions are essential to ensure the quality of exploration in hemostasis.