Control of Platelet Fibronectin Internalization and Cell Surface Retention by Fibrinogen: Lessons from Hypofibrinogemic Patients.

Abstract Although fibrinogen (Fg) has been considered essential for platelet aggregation, we previously demonstrated that thrombi still formed in Fg-deficient mice. In these animals, platelet fibronectin (Fn) content is increased >3 fold, suggesting that Fn may play a role in thrombosis and hemostasis. However, it is unclear whether Fg controls human platelet Fn internalization. We recently identified severely hypofibrinogenaemic patient born from a consanguineous marriage (Xu X et al. Thromb and Haemost2006; 95: 931–5). The patient and his father had respective plasma Fg levels of 1.7mg/dL (approximately 20 times less than the normal level) and 10.5 mg/dL. We examined the platelet Fn content of the patient, his parents, and healthy donors by immuno-electron microscopy (IEM), western blot, and flow cytometry (FACS) using a rabbit anti-human Fn-specific antibody (Sigma, USA). Significant increase of patient platelet Fn content was detected by all three methods, in which mean fluorescence intensity (MFI) of the patient’s platelet Fn by FACS was 3-fold higher than healthy donors. No concomitant alteration of platelet thrombospondin-1 and vitronectin was detected. These data are consistent with our observations in animal models. To examine the role of platelet Fn content in platelet aggregation, we isolated human platelets via sepharose 2B gel filtration. Cell surface Fn expression after thrombin stimulation was examined by FACS. Surprisingly, although platelet Fn was increased, cell surface Fn was not increased. Rather, surface Fn was approximately 6-fold less than that of his father and 10-fold less than healthy donors. Using ELISA, we observed that the patient’s platelet Fn was mainly released into the extracellular medium after thrombin treatment since the supernantant contained 2–3 fold higher levels of Fn than that of healthy donors (472.8 ± 13 ng/mL versus 213.5 ±4.7 ng/mL). Since fibrin has been reported as a receptor of Fn on platelet surface, we tested the hypothesis that high expression of platelet surface Fn in healthy donors was due to fibrin formation on the platelet surface after thrombin treatment. Gel filtered platelets from healthy donors were treated with either thrombin or thrombin receptor activation peptide (TRAP, at 100μM). The latter is able to induce platelet Fg release but not fibrin formation. We found that fibrin was indeed expressed on the platelet surface after thrombin but not TRAP treatment, which were detected by both FACS and IEM with a monoclonal anti-fibrin specific antibody (clone T2G1, Accurate Bilchemicals, USA). Consistent with these data, Fn was only significantly detected on the surface after thrombin but not TRAP treatment (MFI = 10.9 ± 2.5 versus 1.3 ± 0.3). To the best of our knowledge, this is first report that thrombin converts platelet-released Fg to fibrin on the platelet surface. Our data also suggested that Fg not only controls platelet Fn internalization but also regulates platelet surface Fn retention by the fibrin-Fn interaction.

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