Which coagulation factors are serine proteases

At different time points, samples were collected from the supernatant and the bacterial load was determined. As seen in Figure 3 B, fXIII-induced cross-linking significantly reduced the release of bacteria from the clot, suggesting that they are immobilized and killed within the clot. These results show that S pyogenes bacteria are covalently woven into a fibrin network by the action of fXIII and therefore their dissemination from the clot is prevented.

In vitro data suggest that coagulation is part of the early innate immune response, which in a concerted action triggers the immobilization and killing of S pyogenes inside a clot. We therefore hypothesized that the prevention of bacterial dissemination and their clearance may dampen the inflammatory response at the site of infection.

To investigate this, we took advantage of a skin infection model that was established with KTL3, another S pyogenes strain of the M1 serotype. Twenty-four hours after infection, mice were killed and the skin from the local focus of infection was surgically removed and stained for histopathologic analysis. Biopsies were also stained with Giemsa to detect infiltrating cells.

Analysis of tissue samples from wild-type animals showed that bacteria were found in clustered patches and some neutrophils had been recruited Figure 4 E. Infected animals show signs of inflammation white arrows and tissue damage black arrows. Data are presented as mean values of plasma samples obtained from 3 or 5 noninfected and 9 infected animals obtained from 3 independent experiments. At higher magnification it appears that bacteria are an integral part of the fibrin network from infected wild-type mice Figure 4 G inset.

To assess the contribution of immobilization of bacteria to their dissemination, clotting times of the intrinsic pathway of coagulation aPTTs were measured.

Increased aPTTs are a sign of a systemic response to the infection. The PT remained unaltered after 24 hours of infection in both groups of mice data not shown. These results demonstrate that the lack of fXIII leads to an increased inflammatory response at the infectious site combined with an induction of systemic reactions. To determine whether the results obtained from the animal studies also apply to the clinical situation, biopsies from patients with necrotizing fasciitis caused by S pyogenes were analyzed by immunohistology and electron microscopy.

Figure 5 depicts massive tissue necrosis at the site of infection, and subsequent immunodetection in serial tissue sections showed positive staining for the M1 protein and fXIII at these sites.

This suggests an influx of plasma to the infected focus, and indeed cross-linking activity at the same location was recorded Figure 5 top lane. As controls, biopsies from healthy persons were used, in which no signal was seen when subjected to the same experimental protocol Figure 5 bottom lane.

In addition, we found a striking colocalization of the 2 antibodies Figure 6 A , suggesting bacterial cross-linking at the infected site. When the biopsies were analyzed by scanning electron microscopy, massive bleeding at the infected site was recorded data not shown , and bacteria were found to be clustered and entrapped inside the fibrin network Figure 6 B.

Figure 6 C shows immunostaining at the bacterial surface in regions in contact with fibrin fibers. These findings are in agreement with the in vitro and in vivo experiments, and illustrate that immobilization of bacteria and generation of antimicrobial activity is seen within the fibrin network in patients with severe and invasive infections with S pyogenes. Immunohistochemical analysis of human biopsies. Tissue biopsies were obtained from patients with necrotizing fasciitis caused by S pyogenes top and from healthy volunteers bottom.

Stainings without primary antibodies were negative data not shown. Confocal microscopy revealed colocalization of both antibodies, seen at higher magnification in the inset figure. Cell nuclei are stained in blue with DAPI. B Scanning electron microscopy showing bacteria entrapped in the fibrin network arrows in a biopsy from a patient with streptococcal necrotizing fasciitis.

D Transmission electron microscopy shows dead bacteria inside the fibrin network in a biopsy from a patient with streptococcal necrotizing fasciitis. The scale bar indicates 0. To determine whether treatment with fXIII is able to prevent bacterial spreading in an animal model of infection, wild-type mice were subcutaneously infected with S pyogenes.

Three hours after challenge, half of the mice were treated with FibrogamminP, a human plasma fXIII concentrate that was injected into the site of infection. Infected animals were killed after 24 hours of infection, and bacterial loads in the blood, liver, and spleen were determined. As depicted in Figure 7 , FibrogamminP treatment resulted in decreased bacterial loads in the blood, liver, and spleen of the treated mice, suggesting that fXIII dampens the systemic dissemination of S pyogenes in the infected animals.

Mice received a subcutaneous injection of S pyogenes and were treated with FibrogamminP 3 hours after infection. Nontreated mice served as controls. Twenty-four hours after infection, mice were killed and bacterial loads in the blood A , liver B , and spleen C were determined.

Data are presented as means of 10 mice per group and were obtained from 3 independent experiments. The results presented in this study support the concept that fXIII has an important role in the early immune response to bacterial infections. We have shown that fXIII triggers an immobilization of bacteria within the fibrin network at the local focus of infection, which is combined with an induction of plasma-derived antimicrobial activity and subsequent bacterial killing.

The 2 mechanisms work in concert and may together diminish early bacterial dissemination and down-regulate the inflammatory response. Sensing the first signs of inflammation and rapid elimination of an invading microorganism are key features of the early immune response to infection.

In particular, potential ports of microbial entry are at great risk and therefore need special protection. Therefore, the immune system has developed mechanisms that allow an efficient clearance of inhaled eg, with the help of mannose-binding lectin 24 or swallowed eg, by the action of intestinal mucins 25 pathogens.

Wounds present another port of entry and are associated with a great risk of promoting infections and allowing microorganisms to enter the circulatory system. To prevent their dissemination and eventual systemic complications, it is of great importance that the host's defense system is activated as soon as wound sealing begins.

It therefore appears likely that coagulation plays an important role in these very early processes. However, the extent and underlying mechanisms of this contribution to immunity are poorly understood. In the present study, we show for the first time that, in addition to its proinflammatory role, coagulation plays an active role in the containment and elimination of bacteria in infections caused by S pyogenes. Our data support a model based on 2 separate mechanisms involving a fXIII-triggered covalent immobilization of microorganisms inside the fibrin network and the generation of antimicrobial activity.

Apart from bacteria, 26 fungi 27 and viruses 28 have also been reported to interact with the contact system, supporting the notion that contact activation is subject to the principles of pattern recognition. In addition to the generation of antimicrobial peptides due to activation of the intrinsic pathway of coagulation, processing of thrombin has recently been shown to release host defense peptides with a broad specificity.

The in vivo data presented herein show that the lack of fXIII evokes pathologic inflammatory reactions, which is illustrated by a massive neutrophil influx to the site of infection and subsequent tissue destruction as seen in the infected mice.

The inability to immobilize bacteria in a fibrin network leads to a dramatic increase of the intrinsic-driven clotting time in these animals, which is an indication of more severe systemic infection in the knockout compared with wild-type mice. For example, it has been reported for Yersinia pestis that mutant strains lacking the plasminogen activator Pla failed to cause an otherwise systemic infection when tested in an subcutaneous murine infection model.

Human plasma fXIII is fully active in mice, 23 and as a proof of concept we administered the human protein in a murine infection model. When wild-type mice were treated with human plasma fXIII, dissemination of S pyogenes was significantly reduced compared with nontreated mice. Recent findings showing that fXIII also cross-links surface proteins from other bacterial species, such as E coli and S aureus bacteria, 9 imply that the mechanism described herein is an important part of the early immune response.

Our results underline the importance of fXIII in the early defense against S pyogenes and suggest that fXIII is an interesting target for the development of novel antimicrobial therapies.

Clotting has been previously implicated in immunity in invertebrate models, where its immune function is more visible because of the lack of redundancy with adaptive effector mechanisms. One of the best-studied examples is the clotting system of the horseshoe crab, which is triggered by minute amounts of bacterial elicitors such as lipopolysaccharide. This leads to the production of antimicrobial activity and communication with other effector systems.

We show here that blood clotting, which has so far been mostly been studied in the context of its physiologic hemostatic function, plays a key role in immunity both as an effector mechanism and by communicating with other branches of the immune system. This leads to fast and efficient instant immune protection, which keeps infections localized and leaves additional time for other effector mechanisms to be activated. The publication costs of this article were defrayed in part by page charge payment.

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Contribution: T. The remaining authors declare no competing financial interests. Herwald med. Sign In or Create an Account. Sign In. Serpins are Ser ine P rotease In hibitor s. The serpins inhibit the action of their respective serine protease by mimicking the three-dimensional structure of the normal substrate of the protease.

Their abundance reflects their importance: putting a stop to proteolytic activity when the need for it is over. A number of inherited human diseases are caused by a deficiency of a particular serpin.

The deficiency usually results from a mutation in the gene encoding the serpin. Alphaantitrypsin inactivates the elastase secreted by neutrophils. When the lungs become inflamed, neutrophils secrete elastase as a defensive measure. However, it is important to inactivate this elastase as soon as its job is done.

That is the function of alphaantitrypsin. People with an inherited deficiency of alphaantitrypsin they are homozygous for a point mutation in its gene are prone to emphysema. Publication types Research Support, U.

Gov't, P. Substances Protein C Thromboplastin Endopeptidases.