Study On The Mechanism Of SLY-induced Platelets Aggregation And Platelets- Neutrophils Aggregation

Streptococcus suis(S. suis) as a new important zoonotic pathogens, erupted twice in Jiangsu and Sichuan and caused toxic shock syndrome, meningitis, bacteremia, threatened to people’s health seriously. Toxic shock syndrome in patients with clinical symptoms mainly for the high fever, shock, skin and mucosal bleeding, disseminated intravascular coagulation(DIC) etc., resulting in patients with multiple organ microcirculation dysfunction and failure, is the important reason that resulted in the deaths of patients. The anatomy of the dead with STSS found that multiple organ with micro thrombus, reduce blood clotting, implying blood coagulation system in patients with the disorder. Some virulence factors of S. suis interact with host blood system corresponding components and cause the patient blood coagulation dysfunction, but the virulence factors and the mechanism is still not clarified. Screening the corresponding virulence factors and to clarify the interaction mechanism, for a certain practical significance to the clinical therapy of S. suis STSS.As the main ingredient in human blood, platelet play a central role in the process of physiological hemostasis and blood coagulation, is also the promoter of thrombosis and constitute the main components of the micro thrombus. By contacting hemorrhage clinical manifestation and micro thrombus anatomic characteristics, we guess may be some virulence factors of S. suis interact with platelets, activated platelets. The activated platelet adheres to the vascular endothelial cell surface, gathered together each other to form the micro thrombus. Due to the formation of thrombus, consume large amounts of platelets in the blood circulation, results in the decrease of the function of blood coagulation, so the patient can bleeding, shock. In order to verify our conjecture, and find the virulence factor may interact with platelet, we design and carry out the experiment.Our laboratory staff stored 05ZYH33, at the same time structured a gene deletion strains ΔSLY, muramidase- released protein( MRP) gene deletion strains Δ MRP, adenosine synthetase(Ssads) gene deletion strains Δ Ssads, factor H binding protein gene deletion strains Δ Fhb and other mutant strains, provided materials for me in the subsequent experiment.We first used highly pathogenic S. suis wild strains 05 ZYH33 bacteria to interact with the platelet separated from whole blood of healthy people in this experiment and by detecting platelet activation markers of ATP and CD41 a, CD62 P, found the S. suis bacteria did not cause the activation of platelets, and were not statistically significant compared with negative control. After we used 05 ZYH33 culture supernatant of logarithmic metaphase or stability to interact with platelets, found the platelet activation index ATP,CD41 a and CD62 P will appear different degree of increase only interact with stability culture supernatant, compared with negative control no statistically significant, and the logarithmic culture supernatant cannot activate platelets. This suggested there are some ingredients in the S. suis supernatant can activate platelet, and it was secreted to the outside of the bacteria in bacteria plateau, and S. suis bacteria cannot directly activated platelets. At the same time, we used platelet aggregation analyzer found that only the S. suis plateau secretion can induce platelet aggregation, bacteria itself will not be able to induce platelet aggregation, platelet aggregation result is consistent with the results of platelet activation. This result further illustrates the toxin of S. suis in plateau secretion can activate platelet and induce the platelet aggregation.In order to further explore the virulence factors of S. suis activated platelets, we use the lab early build a variety of gene deletion strains of plateau culture supernatant to interact with platelet, such as Δ MRP, Δ SLY, to detect platelet ATP, CD41 a and CD62 P expression and the degree of platelet aggregation, found that only mutant strains Δ SLY cannot activate platelet and can’t induce platelet aggregation. SLY as a kind of exocrine toxin, does not exist in the surface of S. suis, and only in the plateau secrete. Mutant strains Δ SLY lacking of SLY genes do not secret SLY protein to the supernatant, these preliminary results confirmed the secretion of S. suis SLY was the virulence factors to activate platelet aggregation.We constructed recombinant hemolysin SLY through genetic engineering methods, in vitro expression and purification SLY. SLY can activate platelet and we confirmed SLY induced platelet aggregation from the macroscopic and microscopic by the platelet aggregation analyzer and electronic microscope. Fhb, MRP, Ssads known S. suis virulence factors are not able to cause platelet aggregation. Contact the results of the S. suis bacteria, logarithmic period and plateau cultural supernatant caused platelet aggregation further confirmed SLY is the only virulence factors of S. suis can induce platelet aggregation.We further explore the SLY activate platelet specific mechanism after finding the virulence factor. Because SLY can punch a hole in the cell membrane, we guess whether SLY can activate platelets in platelet surface pore forming way. First of all, Certain concentrations of cholesterol was put into 05ZYH33 plateau supernatant or SLY protein, combined with SLY protein and closed to the pore forming ability, platelets can’t be activated. At the same time, SLY, 353 amino acid mutation to lost the ability of pore forming(SLY(P353V)), to stimulate platelet, nor can activate platelet. In addition, the phospholipase C(PLC) inhibitors U73122 did not inhibit the platelet activation induced by SLY, which illustrated the SLY activated platelets protein not through G protein coupled receptors on the surface of the platelet but by form a hole in platelet surface. EGTA, conventional calcium chelator, was put into platelet rich plasma(PRP) can significantly inhibit 05ZYH33 stabilization and SLY protein induced platelet activation and aggregation, which illustrated the platelet aggregation induced by SLY need calcium ions in the plasma. In experiments of calcium ion internal flow, we found SLY cause platelet calcium ion internal flow, cholesterol closed the pore formation ability of SLY, cannot cause calcium ion internal flow, also won’t activate platelet. These results suggested SLY led to the internal flow of calcium ions by punching in platelet surface and activated platelets.Platelet surface molecular GPⅡb/Ⅲa subunit CD41 a increased only about 30% after activation, but the ability of GPⅡb/Ⅲa combined with fibrinogen(Fg) greatly increased. Eptifibatide closed off the Fg binding site of GPⅡb/Ⅲa, significantly reduced the platelet aggregation induced by SLY, which illustrated the SLY led to the GPⅡb/Ⅲa structure change by activating platelets and enhanced the affinity to Fg, then caused the platelet aggregation. Aspirin as a cyclo-oxygenase(COX) inhibitor, can also decrease the rate of the platelet aggregation induced by SLY. Evaluated two inhibitors, eptifibatide and aspirin, on platelet aggregation effect, we found that the GPⅡb/Ⅲa played a major role in platelet aggregation. Eptifibatide can slightly reduce amount of CD62 P expression at the same time, the GPⅡb/Ⅲa combined with Fg can put the extracellular signals into the intracellular, reduces the degree of platelet degranulation to a certain extent.SLY activated corresponding calmodulin by calcium internal flow induced platelet activation. Myosin light-chain kinase(MLCK) inhibitors ML- 7 and Rho protein inhibitors Y27632 was interacted with platelets, blocked the corresponding calmodulin activity, the results show that the ML- 7 inhibits platelet activation, and Y27632 has no effect on platelet activation. At the same time, only ML- 7 inhibited platelet CD62 P expression by using flow cytometry, which suggested the calcium ions into the platelet activated the MLCK, MLCK activated myosin light chain(MLC), MLC induced platelet degranulation, CD62 P was released to the platelet surface, rather than through the PLC- IP3 / DAG- MLCK and Rho- ROCK- MLCK pathways.CD62P, as a kind of integrin receptors combining with PSGL-1in the surface of the neutrophils(PMNs), induced the formation of platelet/neutrophils aggregation(PNA).The anti-CD62 P monoclonal antibody can significantly reduce the PNA formation rate, confirmed the PNA formation by CD62 P. PNA played a key role in the progress of neutrophilic exudate in endothelial cells to inflammation.The BALB/c mice were infected by 05ZYH33 wild strains and Δ SLY gene mutation, platelet volume increased and the number were reduced in whole blood 36 h later. Mice liver tissue was found pathological micro thrombus and coagulation necrosis in 05ZYH33 group, Δ SLY group not seen obvious abnormity in pathological section, indicated the SLY in vivo induced platelet activation and formed the micro thrombus.Summary, we found that the S. suis secretion SLY caused calcium ion internal flow by pore formation on platelet membrane, activated platelet MLCK and membrane surface GPⅡb/Ⅲa, platelet aggregation and PNA depend on GPⅡb/Ⅲa activation and high expression of CD62 P mediated. Platelet aggregation leads to the formation of thrombus, caused multiple organs damage and the consumption of platelets and the patient serious bleeding, and PNA will trigger inflammation and promote the formation of neutrophils migration, PNA will also be involved in the formation of thrombus. The various mechanisms led to the deaths of patients. We hope our research can provide theoretical basis for the treatment of patients with S. suis STSS.