Synthetic Pentasaccharide, LMWH and UFH but not Hirudin inhibit factor VIIa generation and prothrombin activation during coagulation of human plasma.

G.T.Gerotziafas^1,2, L. Bara², M.F. Bloch¹, Z. Foka³,P.E. Makris³, M.M. Samama¹.

¹ Service d’Hématologie Biologique. Hôpital Hôtel-Dieu de Paris, France.

² Laboratoire de Thrombose Experimentale Faculté de Medecine, Broussais Hôtel -Dieu, Univ. P.&M. Curie-Paris VI, France.

³ Thrombosis & Haemostasis Unit, 1st Prop. Pathologic. Clinic, AHEPA University Hospital, Thessaloniki, Greece.

In the present study we compared the effect of UFH, LMWH (enoxaparin), synthetic pentasaccharide (PS) and hirudin on FVIIa generation and prothrombin consumption during in vitro clotting of normal platelet poor plasma. Coagulation was initiated after triggering extrinsic or intrinsic clotting system.

Materials & Methods. We measured FVIIa and residual prothrombin (factor II) levels in the following experimental systems: 1) Intrinsic system activation: Pseudo-serum (ψ-serum) was obtained as follows; pool - PPP was recalcified by adding an equal volume of CaCl₂ (0,025 M). After 60 min incubation in glass tubes at 37^oC, clot was discarded by winding it on a plastic spatula and the supernatant liquid (ψ-serum) was collected. 2) Extrinsic system activation: In 100 μl pool PPP we added 100 μl diluted thromboplastin (TP) (1/250) and after 3 min incubation, clotting was triggered by adding 100 μl of CaCl₂ solution (0,025 M). The procedure was performed in plastic tubes. Ψ-serum was prepared as described above. 3)Extrinsic and intrinsic system activation: In 100 μl pool PPP we added 100 μl diluted TP (1/250) and after 3 min incubation, clotting was triggered by adding 100 μl of CaCl₂ solution (0,025 M). The procedure was performed in glass tubes. Ψ-serum was prepared as described above. In plasma samples we added either buffer or increasing concentrations of UFH, or enoxaparin or PS or rHirudin. FVIIa was measured with a newly developed one stage clotting assay (Wildgoose et al, Blood 80-1, 25-28, 1992). Residual factor II was measured in ψ-serum using a FII deficient plasma FVIIa and FII were measured in PPP.

Results: FVIIa levels in ψ-serum (54,7±15, 258±18, 184±18 ng/ml, in extrinsic, intrinsic and intrinsic + thromboplastin ψ-serum) were significantly increased as compared to PPP (3 ng/ml). UFH, LMWH and PS inhibited factor VIIa generation and/or activity in both extrinsic and intrinsic system (70%-90% inhibition of FVIIa generation). The inhibitory effect of heparins and PS on FVIIa generation in intrinsic system ψ-serum, was not modified by the addition of diluted TP. The inhibitory effect of heparins and PS was concentration dependent. rHirudin had no effect on factor on FVII activation in any experimental system. UFH, LMWH and PS significantly inhibited prothrombin activation in both extrinsic and intrinsic experimental system. r-Hirudin did not prevent prothrombin activation. Interpretation : We demonstrate for first time that PS, LMWH and UFH significantly inhibit FVIIa generation and/or activity as well as prothrombin activation during clotting of human plasma after both extrinsic and intrinsic clotting system activation. This effect is independent of the presence of TF. On the contrary, r-hirudin (a pure and specific anti-IIa agent) has no effect neither on FVIIa generation and/or activity nor on prothrombin activation. Since FVIIa/TF has a pivotal role in thrombogenesis the clinical relevance of this activity of PS and heparins has to be evaluated.

Inhibition of factor VII activation by UFH, LMWH and Synthetic Pentasaccharide. Mechanism of action. Preliminary results.

G.T.Gerotziafas^1,2, L. Bara², M.F. Bloch¹, Z. Foka³, P.E. Makris³, M.M. Samama¹.

¹ Service d’Hématologie Biologique, Hôpital Hôtel-Dieu de Paris, France.

² Laboratoire de Thrombose Experimentale Faculté de Medecine, Broussais Hôtel -Dieu, Univ. P. & M. Curie-Paris VI, France.

³Thrombosis & Haemostasis Unit, 1st Prop. Pathologic. Clinic, AHEPA University Hospital, Thessaloniki, Greece.

Factor VII activation is an essential step in the process of blood coagulation and plays an important role in thrombogenesis. Heparins (UFH and LMWH) have a major role in the prevention and treatment of venous and arterial thrombosis. We have recently demonstrated that UFH, LMWHs and the synthetic pentasaccharide (PS) inhibit FVIIa generation during in vitro coagulation of whole blood or PPP. In the present study we investigated some aspects of the mechanism of this activity of heparins and PS on FVIIa generation. Materials & Methods. We measured FVIIa levels in PPP and in the following experimental system : Intrinsic system activation pseudo-serum (ψ-serum) was obtained as follows; PPP was recalcified by adding an equal volume of CaCl₂ (0,025 M). After 60 min incubation in glass tubes at 37^oC, clot was discarded by winding it on a plastic spatula and the supernatant liquid (ψ-serum) was collected. FVIIa levels were measured in ψ-serum prepared by normal pool PPP and factor VII, XII, XI, IX, VIII, X, V and II deficient plasmas. In plasma samples we added either buffer or UFH (0,4 IU/ml), or enoxaparin or PS (0,4 anti-Xa IU/ml) or recombinant Hirudin (2 μg/ml). Normal PPP was incubated with an anti-TFPI monoclonal antibody (moab) (American Diagnostics). After 60 min incubation, we added either buffer or UFH or Εnoxaparin or PS and ψ-serum was prepared as described. Factor VIIa was measured with a newly developed one stage clotting assay.

Results : UFH and enoxaparin abolished their inhibitory effect on FVIIa generation when ψ-sera were prepared from FIX, FXI, or FXII and to lesser degree from FX deficient plasmas. PS inhibited FVIIa generation in every studied plasma. rHirudin did not have any effect on FVIIa generation. Experiments with the anti-TFPI moab demonstrated that in ψ-serum treated with the moab, FVIIa levels were 3,6 folds higher than the control. The presence of the anti-TFPI moab did not modify the inhibitory effect of UFH, LMWH and PS which remained important (90-98 % inhibition).

Interpretation : After intrinsic system activation FVII is activated mainly by FIXa. UFH and LMWH intervent in FVIIa generation by inhibiting mainly the activation of FVII by the FIXa and by reducing the generation of FIXa (by inhibiting FXII and FXI). The inhibitory effect of UFH and LMWH on FVIIa generation was partially abolished when FX deficient plasma was used. Thus, the inhibition of FXa by heparins does not seem to play a major role in the inhibition of FVIIa generation in this experimental system. Moreover, the inhibitory effect on FVIIa generation did not pass via the inhibition of FIIa since rHirudin (a pure strong and specific anti-IIa agent) did not have any inhibitory effect on FVIIa generation. PS probably had a direct inhibitory effect on FVIIa generation and/or activity. UFH, LMWH and PS inhibit FVIIa generation independently of the presence of TFPI.

DETECTION OF THE FACTOR V LEIDEN USING SSCP.

Kouidou S ¹, Lambropoulos A², Foka Z, Gerotziafas G, Pithara E, Kotsis A², Makris PE.

Haem & Thromb Unit AHEPA, University Hospital, 1- Biochemistry Dep and 2- Biology Dep of University of Thessaloniki- GREECE.

The most common defect in patients with hereditary venous thrombosis is a missense mutation of G èto A at position 1691 in 10 exon of the factor V gene. This results in a mutant factor V (Factor V Leiden) where Arg at position 506 is substituted by a Gln resulting in . As a consequence, this mutant factor V exhibits an abnormal resistance to cleavage by activated protein C (APC). This defect is a well established genetic risk factor in venous thrombosis. Because of the high prevalence of factor V Leiden in normal population (2-7%), it would be reasonable to develop a rapid and easy to perform method for screening the genetic abnormality in population at risk.

A series of methods have been used to detect this common defect, employing PCR and, for most of them, endonuclease restriction. We have developed an alternative method to screen easily and rapidly a large number of samples. After performing PCR in 1μl of whole blood which had stayed frozen in -20^oC, a non isotopic single-strand conformation polymorphism (SSCP) analysis of PCR products to find out individuals which carry factor V Leiden was employed. Gel was run 18 ^oC, for 2 hours at 500 Volts. Under these conditions we were able to discriminate the homozygous and the heterozygous for the factor V Leiden mutation, as detected by means of PCR and endonuclease restriction with Mnl1. No difference was observed when the run was performed at 20^oC.

Abstract Categories: 92 or 7

A NEW POLYMORPHISM (NEW MUTATION ?) IN THE FACTOR V GENE OF A PATIENT CARRYING THE FACTOR V LEIDEN MUTATION.

Lambropoulos A, Kouidou S, Foka Z, Gerotziafas G, Kotsis A, Makris PE.

Haem & Thromb Unit AHEPA, University Hospital, 1- Biology Dep and 2- Biochemistry Dep of University of Thessaloniki- GREECE.

The proband is a man, 53 years old, of greek origin, who suffered deep venous thrombosis (DVT) one year ago. His family had also a history of DVT. In the proband, plasma levels of AT-III, protein C and S, Heparin cofactor II, fibrinogen and plasminogen, a₂-antiplasmin, and PAI were normal, there was no evidence of lupus anticoagulants, but the aPC- resistance test was as low as 1.3. He was screened for the factor V Leiden mutation by a PCR in 1 μl of whole blood using the primers FV3 and FV6. The final product (147bp) was submitted to two different procedures: a) RFLP using the restriction endonuclease Mnl1, and electrophoresis on a 5% Metaphore gel. While factor V Leiden heterozygous present 4 bands (25, 37, 81 and 118 bp), homozygous 2 bands (25 and 118 bp) and normals 3 bands (25, 37 and 81 bp), this proband presented three bands: 147, 25, and 118 bp which corresponded to one uncut allelic and to one bearing the Leiden mutation.

b) SSCP using electrophoresis of the PCR product which has submitted denaturation on a non-denaturing polyadrylamide gel at different temperatures. While at 18 and 20^o C there was no difference between the normal pattern and the proband, at 4^o C he exhibited a distinctly abnormal pattern with a slow moving band and lacking the very fast one. We can assume that there may be a new mutation in the region of the gene which is responsible for the Arg506 cleavage of factor Va by the activated protein C, which has to be identified by a DNA-sequence procedure.. The impact of this mutation in the factor V behaviour has to be determined by Western-blot analysis.

SSCP ANALYSIS IN AN ATTEMPT TO EXPLAIN THE POSSIBLE CAUSE OF APC-RESISTANCE IN PATIENTS NOT BEARING THE FACTOR V LEIDEN MUTATION

Foka Z, Kouidou S¹, Lambropoulos A², Gerotziafas G, Pithara E, Kotsis A², Makris PE.

Haem & Thromb Unit AHEPA, University Hospital, 1- Biochemistry Dep and 2- Biology Dep of University of Thessaloniki- GREECE.

The aim of the study was to try to explain the abnormal aPC resistance test in patients who did not harbour the factor V Leiden mutation. The question was if these patients corrected their ratio with the addition of plasma deficient in factor V and if they harboured another mutation in the region of 147 bp long around the 1691 nucleotide of the gene of factor V.

Materials: In a previous study we found that 33 out of 172 patients with unexplained thrombophilic tendency had an abnormal aPC-resistance test, but 11 patients among them didn’t bear the factor V Leiden mutation.. These 11 persons consested our material

Methods: We determined: a) aPC-r test after the dilution of the patients plasma with plasma deficient in protein C. b) PCR using the primers FV3 and FV6 in 1 ml of whole blood and reading the results of PCR with single strand conformation polymorphisms (SSCP). SSCP analysis is a simple method which provides a rapid and sensitive means for detecting sequence variations, as small as single base point mutations. The SSCP method relies upon the separation of single- stranded (denatured) DNA which have formed hairpin secondary structures, on a non- denaturing polyacrylamide gel. Different conformations exhibit different electrophoretic mobilities and the complementary strands migrate as separate bands on the gel, therefore, small differences in sequence can be detected. We ran the gel at 18, 20 and 4^oC.

Results: a) None of the patients corrected the aPC ratio after the addition of plasma deficient in factor V. b)None of the patients didn’t exhibit an abnormal pattern in SSCP electrophoresis. We can assume that mutations in the region around the 1691 nucleotide of the factor V gene can’t explain the abnormal resistance to protein C and we probably have to search for other mutations around the regions responsible for Arg 306 and Arg 679.

IN VITRO EFFECTS OF VITAMIN C ON FACTORS XII AND XI

- PRELIMINARY RESULTS

PE Makris¹, Z Foka¹, G Gerotziafas¹, E Pithara¹, S Karkabounas, A Evangelou.

Department of Physiology of University of Ioannina

1-Haemostasis and Thrombosis Unit A’ Propedeutic Medical Clinic, University of Thessaloniki, Greece.

The aim of the study was to check the effect of vitamin C on the factor XII and XI (as they are factors which can be activated by conformational changes -and the vitamin C is a compound which can act by the same way). The main hypothesis is if the vitamin C can activate these factors.

Material: and Methods We used crystallic powder of vitamin C, in dilution corresponding in the following molecular concentrations: M/2, M/10, M/100, M/1000. Each of these concentration was added to normal plasma; in parallel we added equal quantities of buffer in another series of normal plasma as a control group. The determinations were effectuated, after incubation for 30sec, 5min, 15min, 30min, and 60min at 37oC. In this experiment we tested the following parameters: FXII:C, FXI:C, Plasminogen, a2-antiplasmin, with a chromogenic substrate in the BCT of Behring and the TAT, F1+2, and PAP using the elisa technique from the Behring Co.

Results. Our results demonstrate that in the dilution of M/2 and M/10 the factors XII and XI disappeared and at the same time the a2-antiplasmin was significantly reduced. The levels of TAT, F1+2 and PAP were not affected. This effect is not time dependent and is observed only in high concentrations of vitamin C (in small concentrations of vitamin C - M/100 and M/1000- there is no effect on factors XII and XI).

ARTHROSCOPIC SYNOVECTOMY IN HAEMOPHILIACS.

P Gigis , F Athanasiadou, K Natsis, Charitou P, D Katriou, PE Makris.

Haemostasis and Thrombosis Unit A’ Medical Propedeutic Clinic,

B Paediatric Clinic of University of Thessaloniki,

TEFAA Thessaloniki- Greece.

The aim of this presentation is to show the surgical treatment of the permanent ancylosis of both the knee- joints of a young haemophiliac by arthroscopic synovectomy. This method was chosen because it is less Haemorragic and the use of video-camera permits the control over the possible complications.

Our case is a young haemophiliac 11 years old of Albanian origin who suffered from permanent ancylosis in flexion of 90o of both the knee- joints. The laboratory control revealed severe haemophilia A (FVIII:C = 0.45%) without the presence of inhibitor. The roendgenography and the magnetic resonance scan showed the presence of articular space and this fact permitted the execution of arthtroscopic synovectomy.

Operation procedure: The patient was covered with 100 IU/Kg BW of rFVIII (kogenate) every 8 hours. In parallel with synovectomy we reduced a part of articular cartilage. The haemorrhage was the usual for such operations.

The movement of the knee- joints was impressingly improved and the young patient is able to walk almost normally now after five years of crippling.