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Summary. Background and objective: Several studies have suggested that thrombin-activatable fibrinolysis inhibitor (TAFI) levels are associated with the risk of arterial thrombosis, but results have been contradictory. We studied functional TAFI levels and TAFI gene polymorphisms in 124 patients with a recent ischemic stroke and 125 age-and sex-matched controls to establish the role of TAFI in ischemic stroke. Methods and results: Functional TAFI levels, defined as TAFI-related retardation (RT), the difference in clot lysis time (LT) in the absence or presence of a specific activated TAFI inhibitor (potato carboxypeptidase inhibitor [PCI]), were higher in patients than controls (19.5 ± 4.2 vs. 17.7 ± 3.7 min, P < 0.005). Clot LTs in the presence of PCI, which were independent of TAFI, were also increased in ischemic stroke patients. This indicates that in these patients fibrinolysis is impaired not only by high TAFI levels, but also by other mechanisms. Individuals with functional TAFI levels in the highest quartile had an increased risk of ischemic stroke compared with the lowest quartile [odds ratio (OR) 4.0, 95% confidence interval (CI): 1.6-9.8]. In an unselected group of 36 of the 125 stroke patients functional TAFI levels were also measured at 3 months, and were persistently high. This indicates that increased functional TAFI levels after stroke are not caused by an acute phase reaction. No difference was found between patients and controls with respect to TAFI genotype distribution. Conclusions: Increased functional TAFI levels, resulting in decreased fibrinolysis, are associated with an increased risk of ischemic stroke.

Intracellular polysaccharide fractions were isolated from calcifying B‐type cells of Emiliania huxleyi and separated by electrophoretic fractionation. In all fractions, the polysaccharide was immunologically related to the polysaccharide of (extracellular) B‐type coccoliths (CP‐B) and not to polysaccharides of A‐type coccoliths (CP‐A). Most polysaccharide fractions also contained protein material. The fraction with the largest proportion of protein was used to raise antibodies. The resulting antiserum, α‐BP, contained antibodies against both CP‐B‐ and protein‐epitopes. The antibodies specific for polysaccharide‐epitopes reacted with intracellular polysaccharide fractions of B‐type cells only. In contrast, the antibodies specific for protein‐epitopes reacted with the intracellular fractions of B‐type as well as A‐type cells. With immunolocalization, the presence of protein antigen in a layer surrounding both types of cells was demonstrated. A cDNA library of E. huxleyi was screened with α‐BP, and a gene called gpa was isolated. The open reading frame of gpa was found to encode a protein (GPA) of 36,608 D, containing, inter alia, 24% acidic residues (18% glutamic acid and 6% aspartic acid), 12% proline, and 23% alanine. GPA has two repeats, one containing a sequence resembling the Ca2+‐binding loop of EF‐hands. Overproduction of GPA in a prokaryotic system yielded a dimeric product capable of binding Ca2+. The possible role of GPA in the formation of coccoliths in E. huxleyi is discussed.

Pseudomonas putida GB-1-002 catalyzes the oxidation of Mn2+. Nucleotide sequence analysis of the transposon insertion site of a nonoxidizing mutant revealed a gene (designatedcumA) encoding a protein homologous to multicopper oxidases. Addition of Cu2+ increased the Mn2+-oxidizing activity of the P. putidawild type by a factor of approximately 5. The growth rates of the wild type and the mutant were not affected by added Cu2+. A second open reading frame (designatedcumB) is located downstream from cumA. BothcumA and cumB probably are part of a single operon. The translation product of cumB was homologous (level of identity, 45%) to that of orf74 ofBradyrhizobium japonicum. A mutation in orf74resulted in an extended lag phase and lower cell densities. Similar growth-related observations were made for the cumAmutant, suggesting that the cumA mutation may have a polar effect on cumB. This was confirmed by site-specific gene replacement in cumB. The cumB mutation did not affect the Mn2+-oxidizing ability of the organism but resulted in decreased growth. In summary, our data indicate that the multicopper oxidase CumA is involved in the oxidation of Mn2+ and that CumB is required for optimal growth of P. putida GB-1-002.

SummaryDecrease of fibrinolytic potential is considered to be a risk factor for arterial thrombosis. The recently described thrombinactivatable fibrinolysis inhibitor (TAFI) attenuates fibrinolysis by cleaving of the C-terminal lysine residues from fibrin, thereby inhibiting tPA mediated plasminogen activation. The role of plasma TAFI antigen (Ag) levels and gene polymorphisms in arterial thrombosis is still not elucidated. In this prospective study, the association between plasma TAFI Ag levels and the TAFI gene polymorphisms, Ala147Thr, Thr325Ile and -438A/G, with refractory unstable angina pectoris (UAP) was determined. The study population consisted of 209 patients with UAP of whom 76 were refractory and 133 non-refractory to medical treatment. In the same study population the contribution of these polymorphisms to plasma TAFI Ag levels was determined.Plasma TAFI Ag levels were significantly higher in non-refractory patients compared to refractory patients (geometric mean 114.4 and 105.6 U/dl respectively, p=0.042). Plasma TAFI Ag levels in the lowest quartile resulted in a 2.6 fold (95% confidence interval 1.2-5.9) increased risk for refractory UAP compared to plasma TAFI Ag levels in the upper quartile. The three studied TAFI polymorphisms had an independent and additive effect on plasma TAFI Ag levels. However, no significant association between the individual TAFI polymorphisms and refractiveness was observed.In conclusion, in this study population plasma TAFI Ag levels are significantly correlated with refractiveness in patients with UAP. Furthermore, all three polymorphisms contribute independently to plasma TAFI Ag levels, but not to refractiveness.Part of this paper was originally presented at the joint meetings of the 16th International Congress of the International Society of Fibrinolysis and Proteolysis (ISFP) and the 17th International Fibrinogen Workshop of the International Fibrinogen Research Society (IFRS) held in Munich, Germany, September, 2002.