Comparison of two functional assays for the determination of activated protein C resistance (APCR). First study in Greece.

G.T. Gerotziafas*, E Pithara **, P.E. Makris**, MM Samama *.

*Service d’Hematologie Biologique, Hopital Hotel-Dieu de Paris, France.

**Thrombosis and Haemostasis Unit, AHEPA University Hospital, Thessaloniki Greece.

A new coagulation disorder related to familial thrombophilia has been recently described. This disorder is characterised by an abnormally low prolongation of APTT when APC is added in plasma. Several studies demonstrated a high prevalence of APCR among thrombophilic patients. Therefore, the screening of APCR is of paramount importance. So far, the screening of APCR has been performed with assays based on APTT prolongation. However, these methods are influenced by heparin treatment, and alterations of coagulation factors. Recently, Vasse et al evaluated the use of an assay based on PT prolongation when APC is added. All APCR screening tests reported in the literature have been performed using automated instruments. In this study, we present an evaluation of two APCR screening tests based on APTT and PT prolongation respectively. Because automated coagulometers may not be readily available to all hospital or private laboratories, we studied the effectiveness of APCR screening methods when performed manually.

Materials and Methods: Blood samples were obtained from 10 healthy male volunteers (mean age 365,2, control group) and 42 thrombophilic patients (mean age 4010,5). All patients suffer more than one DVT or SVT episode and they had a same familial history. Blood was anticoagulated in buffered sodium -citrate and PPP was prepared. All samples were frozen at -40^oC until they were analysed. They had normal levels of ATIII, PC, PS, HC-II, Plg and the lupus anticoagulant tests were negative. The following assays for anticoagulant APC response were performed: a) APTT prolongation based method: 100 ìl of PPP were mixed with 100 ìl of CK-prest reagent (Diagnostica-Stago, France) and incubated for 3 minutes at 37^oC before adding 100 ìl of a solution containing 25 mM CaCl₂ with or without APC (Kindly offered by Serbio -France) at a final concentration of 16 nM, to trigger coagulation. b) Prothrombin Time prolongation based method: 50 ìl of PPP were pre-incubated for 3 minutes at 37^oC and then mixed with 100 ìl clotting solution of Neoplastine (Diagnostica-Stago, France) diluted 1:60 in 12 mM CaCl₂ in presence or no of 16 nM APC. Results are expressed as a ratio: clotting time of test with APC/clotting time of test without APC. Intra-assay quality control was studied. Both tests were repeated 9 times in the same sample (reproducibility- test). Results: 1-Concerning the reproducibility -test. The CV ratios was 5% (APTT baqsed test) and 4% (PT based test). 2-The mean value of APCR ratio in the control group was 2,180,33 and 2,370,55 (respectively on APTT and PT based methods). The two tests had a significant correlation. All ratios lower than mean value -2SD (<1,52 and <1,30 for APTT and PT based assays, c.i. 95%) are defined as pathological. 3-Among patient population 11 subjects (or 26,1%) were no-responders to added APC in both tests. 4 patients were no responders only with APTT based test. 5 other patients were good responders with APTT based test but they had an abnormal response to added APC with PT based test.

Conclusions: APCR screening tests based on APTT or PT prolongation after APC addition, may be performed manually since the C.V. for the two methods of APCR is accepted (<7%). The combination of the two tests (APTT and PT based tests) may be useful to discriminate real APCR patients, since levels of FVIII, vWF, FVII and contact system activation influence the ratios of APTT and PT based tests. The prevalence of APCR in our series of thrombophilic patients is 26%. Molecular biologic confirmation will be performed.

A Prof. MM SAMAMA.

De ma part et aussi de la part de ma famille et de Grigoris nous vous shouetons BONNE ANNEE , BONNE CHANCHE, et a bientot.

Makris Pantelis.

L' abstract pour le facteur VII arrivera dans quelques heures.

Notre No de FAX est 19-3031-909751.

The role of PF4 in in vitro platelet activation induced by the antibodies from patients with Heparin Induced Thrombocytopenia.

G.T Gerotziafas, C. Lecrubier, P.E. Makris, M.M. Samama, T. Lecompte.

Service d’ Hematologie Biologique. Hopital Hotel-Dieu de Paris, France.

Thromb. & Haem. Unit, A’ Prop. Path. Clinic AHEPA Hospital of Thessaloniki. Greece.

Heparin induced thrombocytopenia (HIT) may be a severe complication of heparin treatment. The syndrome is associated with platelet activation via the FcãRII caused by an antibody which appears during the treatment (HIT-ab). However, the nature of the antigen has not been well determined. A number of studies using ELISA or Flux Cytometry methods has recently demonstrated that the complex heparin-Platelet Factor 4 (PF4) is the epitope of an antibody included in plasma, serum or purified IgG of patients with HIT. However, it is not clear yet if the antibody against the complex heparin-PF4 is responsible for platelet activation in the presence of heparin. In this study we clarify the role of PF4 as a necessary component for in vitro platelet aggregation triggered by IgG or platelet poor plasma (PPP) from patients with HIT. We also studied some aspects of the mechanism of platelet aggregation induced by the antigenic complex (heparin-PF4) and the HIT responsible antibody.

Materials and Methods: As control platelets we used either washed (according to Mustard method) or citrated PRP obtained from healthy donors. Platelet responsiveness (washed or PRP) to classic agonists (ADP, arachidonic acid, adrenaline, collagen and thrombin) and immunological agonists (monoclonal antibodies (moab): PL2-49, LeoA1, AlB-6 and antistreptokinase antibodies) was controlled. The immunological type of platelet aggregation was controlled using the moab IV-3 (specific to FcãRII). Moreover, we used the following platelet inhibitors: EDTA, iloprost, RGDS, aspirin, â-ã-methylen ATP. Total IgG were purified with affinity chromatography on protein G colon, from pool plasma obtained from 22 patients with HIT (Kelton et al). Moreover, we used total IgG purified from healthy donors and from patients treated with streptokinase as negative control. IgG were used in different concentrations (150 to 1500 ìg/ml). A polyclonal anti-PF4 rabbit antibody was offered by Serbio laboratories. Its specificity against purified and intraplatelet human PF4 was controlled withWestern blot analysis. Antibodies reacting with the complex heparin-PF4 were detected using the kit ELISA with microwells coated by the complex heparin-PF4 (offered by Dr. Amiral). Unfractionated heparin was use in different concentrations (0.001 to 10 IU/ml). Results: In purified system, the intensity of platelet aggregation depends on the HIT-IgG and heparin concentration. However, heparin in high concentrations has an inhibitory effect. The optimum conditions in terms of maximum aggregation velocity and minimum aggregation lag time were obtained with 500 ìg/ml HIT-IgG and 0.1 to 1 IU/ml of heparin. Total HIT-IgG include an antibody against the complex heparin-PF4 as it was revealed by a positive reaction on the ELISA. The F(ab’)₂ of the antibody anti-PF4 inhibits platelet aggregation induced either by HIT-IgG or by the pool PPP of HIT patients in the presence of heparin. The inhibitory effect of anti-PF4 antibody was specific for the aggregation induced by HIT related antibody and heparin, since its presence did not modify platelet aggregation triggered by classic or immunological agonists. In a system composed with PRP from healthy volunteers reconstituted with PPPs from 6 different patients and 1 IU/ml of heparin, the anti-PF4 antibody completely inhibited the aggregation in 2 cases, it had a partial effect in 1 case and no effect in 3 cases. HIT-IgG and heparin activate platelets via the membrane receptor FcãII. The aggregation was completely abolished by the IV-3 moab. This aggregation depends on the presence of Ca⁺⁺(it was inhibited by EDTA) and on the binding of Fg to GP IIb-IIIa (inhibitory effect of the peptide RGDS) Platelet activation and aggregation was inhibited by c-AMP augmentation and depends on Arachidonic acid and ADP pathways. Conclusions: The present study demonstrates that PF4 is a necessary component of the antigenic complex epitope of the antibody responsible to HIT. The formation of the complex heparin-PF4 is essential for the in vitro platelet activation by the antibodies responsible to HIT. However, PF4 probably is not the only cofactor of heparin in the formation of the antigenic complex. The variable results in individualised experiments permits to postulate that in some cases a component other than PF4 may be the cofactor of heparin. Furthermore, our results demonstrate that there is an optimal ratio of heparin and PF4 concentrations in order to obtain maximal platelet aggregation.

0033142348061 öá÷

LMWHs inhibit in vitro FVII activation.

The role of FVII and FVIIa is curcial in the coagulation process. Both UFH and LMWH induce TFPI release and inhibit TF/FVIIa comlex.

A method for the measurment of factor VIIa has been recently described.

In the present study, we studied the effect of LMWHs on in vitro FVII activation during whole blood coagulation.

Materials and Methods.

We measured FVIIa levels in citrated PPP and serum obtained from whole blood, with a newly developed one stage clotting assay (as described by Wildgoose et al, Blood 80-1, 25-28, 1992). This assay has a high specificity and sensitivity to FVIIa, using recombinant thromboplastin (residues 1-218) truncated to interact only with FVIIa (kindly offered by Pr.Y.Nemerson). Results were exressed as ng/ml. Standard curves were constructed with varying concentrations (1.5 to 300 ng/ml) of purified recombinant FVIIa (kindly offered by U.Hedner). To eliminate the possibility of heparin interfering with the factor VIIa clotting assay, we compared the standard curves obtained by measuring clotting time, as a function of FVIIa concentration in the presence of increasing concentrations of LMWH (0.2, 0.4, 0,8, and 1.6 IU/ml of Lovenox and Fragmin.) to the standard one.

Whole blood from two healthy volonteers was collected glass tubes and clotted in the presence or absence of increasing concentrations of Lovenox or Fragmin (corresponding to 0.2, 0.4, 0.8 and 1.6 anti-Xa plasma IU/ml). Serum was obtained after incubation at 37^oC for 4h of whole blood collected in a glass tube without any other anticoagulant than LMWH. Serum was collected after 20 min centrifugation at 2000 g and stored at -20 ^oC.

The effect of LMWHs circulating

Blood was collected from 3 healthy volonteers and FVIIa levels were measure in serum. One week later the same subjects were treated by Lovenox (40 mg two injections per day ). Four hours after the 3rd injection blood was collectred and serum FVIIa levels were measured as described above.

Results

LMWHs at concentrations equal or inferior to 1 anti-Xa IU/ml have no effect on the measurement of FVIIa.

FVIIa levels in serum were significantly increased as compared to PPP. However, LMWHs added in vitro in concentrations superior than 1 anti-Xa IU/ml significantly inhibited FVIIa generation in serum of both individuals. Both studied LMWHs exerted a similar effect.

FVIIa levels in serum of three controls before and after LMWH treatment were 6.57 ± 2.54 and 1.43 ± 0.6 IU/ml (p<0.001 piared t-test).

The role of PF4 in in vitro platelet activation induced by the antibodies from patients with Heparin Induced Thrombocytopenia.

G.T Gerotziafas*, C. Lecrubier*, P.E. Makris#, M.M. Samama*, T. Lecompte**.

*Service d’ Hematologie Biologique. Hopital Hotel-Dieu de Paris, France.

**Centre Regional de Transfusion et d’Hematologie, Nancy, France.

#Thromb. & Haem. Unit, AHEPA Hospital of Thessaloniki. Greece.

Heparin induced thrombocytopenia (HIT) may be a severe complication of heparin treatment. The syndrome is associated with platelet activation via the FcãRII caused by an antibody which appears during the treatment. However, the nature of the antigen has not been well determined. Recent studies using ELISA or Flux Cytometry methods have demonstrated that the complex heparin-Platelet Factor 4 (PF4) is the epitope of an antibody included in plasma, serum or purified IgG of patients with HIT. However, it is not clear yet if the antibody against the complex heparin-PF4 is responsible for platelet activation in the presence of heparin. In this study we clarify if the presence of the complex heparin-PF4 is necessary for in vitro platelet aggregation triggered by IgG or platelet poor plasma (PPP) from patients with HIT. We also studied some aspects of the mechanism of platelet aggregation induced by the antigenic complex (heparin-PF4) and the HIT responsible antibody. Materials and Methods: As control platelets we used either washed (according to Mustard method) or citrated PRP obtained from healthy donors. Platelet responsiveness to classic (ADP, arachidonic acid, adrenaline, collagen and thrombin) and immunological agonists (monoclonal antibodies (moab): PL2-49, LeoA1, AlB-6) was controlled. The immunological type of platelet activation was controlled using the moab IV-3 (specific to FcãRII). Moreover, we used the following platelet inhibitors: EDTA, iloprost, RGDS, aspirin, â-ã-methylen ATP. Total IgG were purified with affinity chromatography on protein G colon, from pool plasma obtained from 22 patients with HIT. Moreover, we used total IgG purified from healthy donors and from patients treated with streptokinase as negative control. IgG were used in different concentrations (150 to 1500 ìg/ml). A polyclonal anti-PF4 rabbit antibody was offered by Serbio laboratories. Its specificity against purified and intraplatelet human PF4 was controlled with Western blot analysis. Antibodies reacting with the complex heparin-PF4 were detected using the kit ELISA with microwells coated by the complex heparin-PF4 (offered by Dr. Amiral, Serbio lab. France). Unfractionated heparin was used in different concentrations (0.001 to 10 IU/ml). Results: In purified system, the intensity of platelet aggregation depends on HIT-IgG and heparin concentration. Heparin in high concentrations has an inhibitory effect. The maximum aggregation velocity and minimum aggregation lag time were obtained with 500 ìg/ml HIT-IgG and 0.1 to 1 IU/ml of heparin. Total HIT-IgG include an antibody against the complex heparin-PF4 as it was revealed by a positive reaction on the ELISA. Pre-incubation of washed platelets or control PRP with the F(ab’)₂ of the antibody anti-PF4 inhibits platelet aggregation induced either by HIT-IgG (or by the pool PPP of HIT patients) in the presence of heparin. The effect of the anti-PF4 antibody is concentration dependant and specific to the aggregation induced by the HIT responsible antibody and heparin, since its presence did not modify platelet aggregation triggered by classic or immunological agonists. Aggregation induced by HIT-IgG or HIT-PPP and heparin (in washed platelet or PRP) was completely abolished by the IV-3 moab (activation via the FcãRII). This aggregation depends on the presence of Ca⁺⁺(it was inhibited by EDTA) and on the binding of Fg to GP IIb-IIIa (inhibitory effect of the peptide RGDS) Platelet activation and aggregation was inhibited by c-AMP augmentation and depends on Arachidonic acid and ADP pathways. Conclusion: The present study demonstrates that PF4 is a necessary component of the antigenic complex-epitope of the antibody responsible to HIT. The formation of the complex heparin-PF4 is essential for in vitro platelet activation by the HIT responsible antibodies. Preincubation with an anti-PF4 antibody results to inhibition of platelet aggregation induced by HIT-IgG (or PPP) and heparin.