ristocetin and Disseminated-Intravascular-Coagulation

Pathology data from the anthrax animal models show evidence of significant increases in vascular permeability coincident with hemostatic imbalances manifested by thrombocytopenia, transient leucopenia, and aggressive disseminated intravascular coagulation. In this study we hypothesized that anthrax infection modulates the activity of von Willebrand factor (VWF) and its endogenous regulator ADAMTS13, which play important roles in hemostasis and thrombosis, including interaction of endothelial cells with platelets. We previously demonstrated that purified anthrax neutral metalloproteases Npr599 and InhA are capable of cleaving a variety of host structural and regulatory proteins. Incubation of human plasma with these proteases at 37 degrees C in the presence of urea as a mild denaturant results in proteolysis of VWF. Also in these conditions, InhA directly cleaves plasma ADAMTS13 protein. Npr599 and InhA digest synthetic VWF substrate FRETS-VWF73. Amino acid sequencing of VWF fragments produced by InhA suggests that one of the cleavage sites of VWF is located at domain A2, the target domain of ADAMTS13. Proteolysis of VWF by InhA impairs its collagen binding activity (VWF:CBA) and ristocetin-induced platelet aggregation activity. In plasma from anthrax spore-challenged DBA/2 mice, VWF antigen levels increase up to 2-fold at day 3 post-infection with toxigenic Sterne 34F(2) strain, whereas VWF:CBA levels drop in a time-dependent manner, suggesting dysfunction of VWF instead of its quantitative deficiency. This conclusion is further supported by significant reduction in the amount of VWF circulating in blood in the ultra-large forms. In addition, Western blot analysis shows proteolytic depletion of ADAMTS13 from plasma of spore-challenged mice despite its increased expression in the liver. Our results suggest a new mechanism of anthrax coagulopathy affecting the levels and functional activities of both VWF and its natural regulator ADAMTS13. This mechanism may contribute to hemorrhage and thrombosis typical in anthrax.

Topics: ADAM Proteins; ADAMTS13 Protein; Animals; Anthrax; Anti-Bacterial Agents; Bacterial Proteins; Blood Platelets; Cell Communication; Collagen; Disease Models, Animal; Disseminated Intravascular Coagulation; Endothelial Cells; Hemostasis; Humans; Leukopenia; Metalloendopeptidases; Metalloproteases; Mice; Plasma; Platelet Aggregation; Protein Binding; Protein Structure, Tertiary; Ristocetin; Spores, Bacterial; Substrate Specificity; Thrombocytopenia; Thrombosis; Time Factors; Urea; von Willebrand Factor

Topics: Adenosine Diphosphate; Diagnostic Errors; Disseminated Intravascular Coagulation; Female; Humans; Obstetric Labor Complications; Platelet Aggregation; Platelet Function Tests; Pregnancy; Ristocetin; Time Factors

The specific detection of fibrin monomer and fibrin degradation products is of high importance in the laboratory diagnosis of intravascular clotting (disseminated intravascular coagulation, deep vein thrombosis). The methods proposed until now are partly time-consuming, needing special laboratories or insensitive and poorly specific. Applying ristomycin instead of ristocetin (another member of the vancomycin antibiotics) a new simple, specific and sensitive method has been elaborated and recommended for the laboratory diagnosis of intravascular coagulation and its differentiation from primary fibrinogenolysis. The results obtained from in vitro and animal experiments and from human studies are presented.

Topics: Animals; Chemical Precipitation; Disseminated Intravascular Coagulation; Female; Fibrin; Fibrin Fibrinogen Degradation Products; Humans; In Vitro Techniques; Male; Rabbits; Ristocetin; Streptokinase; Thrombin; Thrombophlebitis

The ristocetin precipitation test was designed as a simplified test to detect fibrin monomers and fibrinogen/fibrin degradation products (FPD/fdp). The ristocetin precipitation test is positive in plasma samples containing either fibrin monomer (greater than 5--10 microgram/ml) or early fdp (greater than 50--100 microgram/ml). The ristocetin precipitation test is negative in plasma with fibrinogen concentrations to 1,000 mg/dl or fibrinogen degradation products FDP) and late fdp to 400 microgram/ml. The ristocetin precipitation test is positive in plasmas collected from rabbits after the infusion of thrombin (2.7 u/kg) or thrombin and streptokinase (10,000 u/kg); the test is negative in plasmas from animals treated with streptokinase or saline solution alone. The ristocetin precipitation test is negative in normal human plasmas and plasmas from patients who have primary firbinogenolysis, but positive in plasmas from patients with disseminated intravascular coagulation. These results suggest that the restocetin precipitation test can be a useful test for the detection of plasma fibrin monomers and early fdp.

Topics: Animals; Chemical Precipitation; Disseminated Intravascular Coagulation; Fibrin; Fibrin Fibrinogen Degradation Products; Humans; Rabbits; Ristocetin