Search

BACKGROUND: Congenital disorders of glycosylation are rare inherited diseases affecting many different proteins. The lack of glycosylation notably affects the hemostatic system and leads to deficiencies of both procoagulant and anticoagulant factors. OBJECTIVE: To assess the hemostatic balance in patients with multiple coagulation disorders by using a thrombin generation assay. METHOD: We performed conventional coagulation assays and a thrombin generation assay on samples from patients with congenital disorder of glycosylation. The thrombin generation assay was performed before and after activation of the protein C system by the addition of soluble thrombomodulin. RESULTS: A total of 35 patients were included: 71% and 57% had low antithrombin and factor XI levels, respectively. Protein C and protein S levels were abnormally low in 29% and 26% of the patients, respectively, whereas only 11% displayed low factor IX levels. Under baseline conditions, the thrombin generation assay revealed a significantly higher endogenous thrombin potential and thrombin peak in patients, relative to controls. After spiking with thrombomodulin, we observed impaired involvement of the protein C system. Hence, 54% of patients displayed a hypercoagulant phenotype in vitro. All the patients with a history of stroke-like episodes or thrombosis displayed this hypercoagulant phenotype. CONCLUSION: A thrombin generation assay revealed a hypercoagulant in vitro phenotype under baseline condition; this was accentuated by impaired involvement of the protein C system. This procoagulant phenotype may thus reflect the risk of severe vascular complications. Further research will have to determine whether the thrombin generation assay is predictive of vascular events.

Heparin-induced thrombocytopenia (HIT) involves heparin-dependent antibodies which induce platelet activation. In the present study, we searched for a relationship between the polymorphism of the Fc receptor (FcgammaRIIa) and the development of HIT. In this purpose, all the donors were genotyped for their FcgammaRIIA and HIT patients were selected on the basis of at least one positive answer by 14C-serotonin release assay (SRA). The frequency distribution of the FcgammaRIIa polymorphism in the HIT patient group was similar to that observed in the healthy control group. Moreover, a statistical analysis taking into account our results and those of 3 previously published studies, suggested at most only a weak association between HIT and the FcgammaRIIa-131 polymorphism. Laboratory tests used to diagnose HIT rely on the activation of normal donor platelets but fail to detect every HIT positive patient. We determined the role of FcgammaRIIa-131 polymorphism on the reactivity of control platelets to HIT plasmas. When control platelet FcgammaRIIa-131 was of Arg/Arg form, only 47% of the HIT plasmas were positive by SRA, compared to 81% and 74% for His/His or His/Arg forms, respectively. We also compared the level of anti PF4/heparin antibodies in the HIT plasmas with the response obtained by SRA. The mean anti PF4/heparin antibodies level in HIT plasma was significantly lower in negative SRA than in positive tests when using control platelets from FcgammaRIIa-Arg/Arg 131 and heterozygous donors. Thus, the variability of control platelets to respond to HIT plasmas in the SRA test is related to both the FcgammaRIIa-131 polymorphism, and to the amount of anti PF4/heparin antibodies.

Synthetic peptides (TRAP or Thrombin Receptor Activating Peptide) corresponding to at least the first five aminoacids of the new N-terminal tail generated after thrombin proteolysis of its receptor are effective to mimic thrombin. We have studied two different TRAPs (SFLLR, and SFLLRN) in their effectiveness to induce the different platelet responses in comparison with thrombin. Using Indo-l/AM-labelled platelets, the maximum rise in cytoplasmic ionized calcium was lower with TRAPs than with thrombin. At threshold concentrations allowing maximal aggregation (50 microM SFLLR, 5 microM SFLLRN and 1 nM thrombin) the TRAPs-induced release reaction was about the same level as with thrombin, except when external calcium was removed by addition of 1 mM EDTA. In these conditions, the dense granule release induced by TRAPs was reduced by over 60%, that of lysosome release by 75%, compared to only 15% of reduction in the presence of thrombin. Thus calcium influx was more important for TRAPs-induced release than for thrombin-induced release. At strong concentrations giving maximal aggregation and release in the absence of secondary mediators (by pretreatment with ADP scavengers plus aspirin), SFLLRN mobilized less calcium, with a fast return towards the basal level and induced smaller lysosome release than did thrombin. The results further demonstrate the essential role of external calcium in triggering sustained and full platelet responses, and emphasize the major difference between TRAP and thrombin in mobilizing [Ca2+]i. Thus, apart from the proteolysis of the seven transmembrane receptor, another thrombin binding site or thrombin receptor interaction is required to obtain full and complete responses.