Umme Amara

Molecular Intercommunication between the Complement and Coagulation Systems

The complement system as well as the coagulation system has fundamental clinical implications in the context of life-threatening tissue injury and inflammation. Associations between both cascades have been proposed, but the precise molecular mechanisms remain unknown. The current study reports multiple links for various factors of the coagulation and fibrinolysis cascades with the central complement components C3 and C5 in vitro and ex vivo. Thrombin, human coagulation factors (F) XIa, Xa, and IXa, and plasmin were all found to effectively cleave C3 and C5. Mass spectrometric analyses identified the cleavage products as C3a and C5a, displaying identical molecular weights as the native anaphylatoxins C3a and C5a. Cleavage products also exhibited robust chemoattraction of human mast cells and neutrophils, respectively. Enzymatic activity for C3 cleavage by the investigated clotting and fibrinolysis factors is defined in the following order: FXa > plasmin > thrombin > FIXa > FXIa > control. Furthermore, FXa-induced cleavage of C3 was significantly suppressed in the presence of the selective FXa inhibitors fondaparinux and enoxaparin in a concentration-dependent manner. Addition of FXa to human serum or plasma activated complement ex vivo, represented by the generation of C3a, C5a, and the terminal complement complex, and decreased complement hemolytic serum activity that defines exact serum concentration that results in complement-mediated lysis of 50% of sensitized sheep erythrocytes. Furthermore, in plasma from patients with multiple injuries (n = 12), a very early appearance and correlation of coagulation (thrombin–antithrombin complexes) and the complement activation product C5a was found. The present data suggest that coagulation/fibrinolysis proteases may act as natural C3 and C5 convertases, generating biologically active anaphylatoxins, linking both cascades via multiple direct interactions in terms of a complex serine protease system.

Interaction Between the Coagulation and Complement System

The complement system as a main column of innate immunity and the coagulation system as a main column in hemostasis undergo massive activation early after injury. Interactions between the two cascades have often been proposed but the precise molecular pathways of this interplay are still in the dark. To elucidate the mechanisms involved, the effects of various coagulation factors on complement activation and generation of anaphylatoxins were investigated and summarized in the light of the latest literature. Own in vitro findings suggest, that the coagulation factors FXa, FXIa and plasmin may cleave both C5 and C3, and robustly generate C5a and C3a (as detected by immunoblotting and ELISA). The produced anaphylatoxins were found to be biologically active as shown by a dose-dependent chemotactic response of neutrophils and HMC-1 cells, respectively. Thrombin did not only cleave C5 (Huber-Lang et al. 2006) but also in vitro-generated C3a when incubated with native C3. The plasmin-induced cleavage activity could be dose-dependently blocked by the serine protease inhibitor aprotinin and leupeptine. These findings suggest that various serine proteases belonging to the coagulation system are able to activate the complement cascade independently of the established pathways. Moreover, functional C5a and C3a are generated, both of which are known to be crucially involved in the inflammatory response.

Alteration of complement hemolytic activity in different trauma and sepsis models.

Complement activation is involved in various diseases in which innate immunity plays a crucial role. However, its pathophysiological relevance is not clearly understood. Experimental models have been widely used to characterize the role of complement activation under different pathological conditions, such as hypoxemia, ischemia and reperfusion, tissue damage, and polymicrobial invasion. Screening of the complement status and function is, however, strongly dependent on the laboratory-specific techniques being used to sample and measure complement, making it difficult to compare the results found in different laboratories. Therefore, we evaluated complement function by measuring complement hemolytic activity (CH50) in various animal models of isolated ischemia reperfusion (I/R: kidney, liver, gut), hemorrhagic traumatic shock (HTS), endotoxic shock (LPS), and sepsis (CLP). Complement activation was less pronounced in isolated models of ischemia and reperfusion, whereas a strong complement response was observed early after HTS, CLP, and LPS. In summary, CH50 is a well-established, quick, and cost-effective screening method of complement function. However, because we obtained different results in clinically relevant animal models, further differentiation using specific complement factor analysis is necessary.

Pyrosequencing-based strategy for a successful SNP detection in two hypervariable regions: HV-I/HV-II of the human mitochondrial displacement loop

Forensic analysis of mitochondrial displacement loop (D‐loop) sequences using Sanger sequencing or SNP detection by minisequencing is well established. Pyrosequencing has become an important alternative because it enables high‐throughput analysis and the quantification of individual mitochondrial DNAs (mtDNAs) in samples originating from more than one individual. DNA typing of the mitochondrial D‐loop region is usually the method of choice if STR analysis fails because of trace amounts of DNA and/or extensive degradation. The main aim of the present work was to optimize the efficiency of pyrosequencing. To do this, 31 SNPs within the hypervariable regions I and II of the D‐loop of human mtDNA were simultaneously analyzed. As a novel approach, we applied two sets of amplification primers for the multiplexing assay. These went in combination with four sequencing primers for pyrosequencing. This method was compared with conventional sequencing of mtDNA from blood and biological trace materials.