Development of a clot-targeted anticoagulant

The effectiveness of anticoagulant therapy is limited by the inability of many anticoagulant molecules to inhibit completely clot bound thrombin. Despite the development of direct, potent thrombin inhibitors such as hirudin, there remains significant opportunity to improve the treatment efficacy. Research in this area has provided evidence to indicate that the failure of existing anticoagulant therapies is due to the procoagulant nature of the clot itself. Clot-targeted anticoagulants offer the potential to: (1) simplify the administration methods; (2) reduce the quantity required for treatment, thus reducing the potential cost of treatment; (3) increase the local therapeutic concentration and (4) to prevent systemic side-effects. A model system has been developed for preparation of targeted anticoagulants using avidin and biotin for coupling. The Fab fragment of the monoclonal antibody DD3B6/22 which binds to the D-dimer component of cross-linked fibrin was prepared and conjugated to avidin. This conjugate was then reacted with the biotinylated specific active site thrombin inhibitor, biotin-PPACK (D-Phe-Pro-Arg-chloromethyl ketone). This targeted anticoagulant (TAC) molecule retained both the ability to bind D-dimer and inhibit thrombin. The reagent binds clots prepared in vitro, which correlates well with the good binding properties observed in an in vivo imaging trial carried out with DD3B6/22. The inhibition of clot procoagulant activity by the TAC reagent versus conventional inhibitors, biotin-PPACK, PPACK and hirudin was assessed in an in vitro assay developed in this study. Clots were pretreated with inhibitor and then thoroughly washed before the procoagulant activity was assayed. The TAC reagent was able to inhibit 90% of the procoagulant activity whereas the conventional inhibitors were only able to inhibit a maximum of 60%. This novel approach to targeting biological active molecules to the clot surface could be extended to include lytic agents or other anticoagulant molecules

Measurement of crosslinked fibrin degradation products. An immunoassay using monoclonal antibodies

We have prepared a monoclonal antibody which recognises an antigenic determinant on D-dimer, a specific fragment resulting from the degradation of crosslinked fibrin. This antibody has been used in the development of an enzyme-linked immunoassay for D dimer and related degradation products containing crosslinked gamma-gamma chains, to provide a simple assay of circulating crosslinked fibrin degradation products suitable for clinical use. Since these crosslinked fibrin degradation products are characteristic of fibrinolysis, as distinct from fibrinogenolysis, their measurement should aid in the diagnosis, evaluation and monitoring of thrombotic and thrombolytic states. In preliminary studies, low concentrations of crosslinked fibrin derivatives were detected in normal sera. High levels were found in 30/30 patients with disseminated intravascular coagulation and in the majority of patients having deep venous thrombosis or pulmonary embolism

Serial D-dimer levels in the assessment of tumor mass and clinical outcome in ovarian cancer

D-dimer (DD)-an end product of fibrinolysis-was measured in serum by enzyme immunoassay using a monoclonal antibody to the γγ crosslink in patients with ovarian cancer, before primary surgery and during chemotherapy for 12 months or more. Serial DD levels were found to have a high sensitivity for the detection of tumor in patients with subclinical disease (91%) as well as for predicting progression of disease (100%). As determined by a careful second-look laparotomy in patients with complete clinical remission the DD marker was highly predictive of tumors < 1 cm. The negative predictive value (82%) was higher than the positive predictive value (69%). However, there were 31% of the patients who showed a false-positive result; a close examination of the clinical data of these 9 patients failed to reveal an explanation for the positive DD levels. Despite the lack of specificity (50% in the present series), the findings support the use of DD in the assessment of patients with ovarian cancer, especially those with subclinical disease

Human pulmonary dirofilariasis: a review

The authors presented a detailed summary of the geographical distribution, clinical and pathological aspects of human pulmonary dirofilariasis. Although benign, this zoonosis, of which Dirofilaria immitis is the major etiological agent, represents a medical problem since it produces symptoms which may be confused with neoplasia and thus may subject patients to unnecessary thoracic surgery. Of 229 cases cited in the literature, only 17 were reported in Brazil, despite the existence of highly favorable conditions for the transmission of this infection in man. Thus it may well be that this parasitic infection remains underdiagnosed. Finally, the importance of a differential diagnosis between dirofilariasis and pulmonary neoplasia is emphasized in cases where there is a solitary subpleural nodule ("coin lesion") present. In addition, the development and improvement of modern immunological diagnostic techniques are essential to distinguish this benign disease from other pathological conditions and thus avoid unneccessary surgery. These techniques may reveal the true prevalence of this parasitic infection in our environment