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.

Thrombosis & Haemostasis Unit, 1st Prop. Pathological Clinic AHEPA Hospital of Thessaloniki. Greece.

INTRODUCTION

Heparin induced thrombocytopenia (HIT), a severe complication of heparin treatment, appears 3 or more days after the start of heparin therapy. In some patients, thrombocytopenia may be associated with thromboembolic complications.

·      The syndrome is associated with platelet activation via the platelet membrane receptor FcγII

·      Platelet activation is triggered by an antibody which appears during the treatment (HIT-ab).

·      However, the nature of the antigen has not been well determined.

·      Recent studies, using ELISA assay or flow cytometry, have demonstrated that the complex heparin-platelet factor 4 (PF4) is the epitope of an antibody present in plasma, serum or purified IgG of patients with HIT.

However, it is not clear if this is the Ab that triggers platelet activation in the presence of heparin.

AIM OF THE STUDY

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

I. Study in purified system

·      Washed platelets (according to Mustard method)

·      Total IgG ,purified with affinity chromatography on protein G colon, from pool plasma obtained from 22 patients with HIT.

·      Total IgG purified from healthy donors and from patients treated with streptokinase as negative control.

·      IgG were used in different concentrations (150 to 1500 μg/ml)

II. Study in citrated PRP

PRP : control platelets (from healthy donors)

PPP from 6 unrelated HIT-patients

Control of platelet responsiveness (washed or PRP):

1.    to classic agonists : ADP, arachidonic acid, adrenaline, collagen and thrombin.

2.    to immunological agonists: monoclonal antibodies (moab) : PL2-49, LeoA1, AlB-6 and antistreptokinase antibodies.

The immunological type of platelet aggregation was controlled using the moab IV-3 (specific to FcγRII).

Platelet inhibitors : EDTA, iloprost, RGDS, aspirin, β-γ-methylen ATP.

To demonstrate the role of PF4 in heparin dependant platelet aggregation induced by the IgG-HIT we used a polyclonal anti-PF4 rabbit antibody (offered by Serbio lab.). Its specificity against purified and intraplatelet human PF4 was controlled withWestern blot analysis.

Antibodies reacting with complexes 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

 I. Study on the mechanism of platelet aggregation triggered by HIT-IgG and heparin, in purified system.

Lag time and aggregation velocity. Increasing concentrations of HIT-IgG and 1 IU/ml of unftactionated heparin (UFH)

Platelet activation depends on heparin concentration.

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 μg/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.

Positive reaction of HIT-IgG (A) and of a pooled-PPP HIT (B) on an ELISA with microwells coated by the complex heparin-PF4.

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 (A) or immunological agonists (B).

A.

B.

In a system made of normal PRP reconstituted with PPPs from 6 different patients and 1 IU/ml of heparin,

the anti-PF4 antibody completely inhibited the aggregation in 2 cases (table 1a),

it had a partial effect in 1 case (table 1b) and none in 3 cases (table 1c).

Table 1a : Ab anti - PF4 completely inhibited heparin dependant platelet aggregation induced by HIT-PPP.

Table 1b : Ab anti - PF4 partially inhibited heparin dependant platelet aggregation induced by HIT-PPP.

Table 1c : Ab anti - PF4 had no effect on heparin dependant platelet aggregation induced by HIT-PPP.

HIT-IgG and heparin activate platelets via the membrane receptor FcγII. 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.

·      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.