Comparative effects of two synthetic oligosaccharides on platelet activation induced by plasma from HIT patients

Authors


J-M. Herbert, Cardiovascular-Thrombosis Research Department, Sanofi-Synthélabo Research, 195 Route d'Espagne, 31036 Toulouse, France.
Tel.: +33 05 6116 2361; fax: +33 05 6116 2286; e-mail: jean-marc.herbert@sanofi-synthelabo.com

Abstract

Summary.  Heparin-induced thrombocytopenia (HIT) is a serious secondary event encountered in the clinical use of heparin. HIT results from the consumption of platelets that are immunologically activated by antibodies directed against complexes formed by platelet factor 4 (PF4) and sulfated polysaccharides that activate platelet aggregation, leading to paradoxical, life-threatening thrombosis. There is strong evidence that the ability of heparin and related compounds to induce HIT is closely linked to the structure of the polysaccharide, and particularly to its negative charge and to the length of the molecule. To test this hypothesis, we synthesized two sulfated oligosaccharides: SanOrg123781, a 16-mer, presenting two terminal charged domains separated by a 7-mer neutral linker, and SR121903, a highly sulfated 17-mer. Both of them displayed strong anti-factor (F) Xa and anti-FIIa activities but their affinities for PF4 were markedly different. SR121903 displaced PF4-bound heparin, whereas SanOrg123781 did not, underlining the importance of the charge of the molecule for the interaction with PF4. Platelet studies, in the presence of HIT serum, showed that SR121903 induced the secretion of platelet-dense granules (measured by the release of serotonin) whereas SanOrg123781 did not, a result in accordance with an absence of affinity of this molecule for PF4. These results were confirmed by measurements of platelet activation by flow cytometry (measured by annexin V binding, CD62 detection and activation of the GpIIb–IIIa complexes). In conclusion, we have demonstrated the importance of the charge of the polysaccharides in the HIT-induced platelet reactions measured by diverse methods, of which some are described for this purpose for the first time.

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