For all their help and patience with me in the UMMC laboratory, I would like to thank Dr. to thank. Finally, I would like to thank all my professors in the Chemistry and Biochemistry Department for all the ways they prepared me to complete a project like this and to pursue my ambitions after college. Nathan Hammer, for taking an interest in my success even in freshman year and for always giving me wise counsel and pushing me to be the best I could be, both in the classroom and in future endeavors.
In theory, since platelets are destroyed in sepsis, PMP levels in plasma may increase and thus play an important physiological role in the cascade deregulation of the coagulation mechanism characteristic of sepsis. Data collected and analyzed to date in the context of this thesis (79 of 182 sepsis patients), a correlation between PMP level and sepsis severity was observed, but in an opposite manner than expected.
Sepsis
Thrombosis in Sepsis
DIC, which simultaneously causes adverse coagulation and bleeding, leads to increased platelet destruction.14 The simultaneous occurrence of aberrant coagulation in some organs and bleeding in others significantly complicates sepsis treatment, as therapeutic measures can help. If DIC becomes severe, deep vein thrombosis in multiple organs will lead to multiple organ dysfunction syndrome (MODS), the predominant sign of death in sepsis cases.14.
Sepsis Diagnosis
Critical care physicians often use the Sequential Organ Failure Assessment (SOFA) score, a simple and objective score that can calculate both the number and severity of organ dysfunction in six organ systems (respiratory, coagulation, hepatic, cardiovascular , renal, and neurological).16 For the coagulation system score, a low circulating platelet count, also known as thrombocytopenia, corresponds to a high SOFA score, indicating a greater likelihood of organ dysfunction. Once sepsis-induced abnormalities such as hypotension can no longer be improved by fluid administration, the patient's condition may be diagnosed as septic shock.17 Because most sepsis-related deaths result from DIC and subsequent MODS, it may improve of current methods or finding new ways to earlier recognize platelet abnormalities and organ dysfunction is an important step in increasing successful treatment of sepsis.
Platelet-Derived Microparticles and Their Possible Role in Sepsis
- Platelet-Derived Microparticle Overview and Biochemistry
- Physiological Function
- PMP Protocols and Flow Cytometry
- Pathophysiological Role of Platelets in Sepsis
- Possible Prognostic Value of PMPs
Important biological molecules found in PMPs include specific membrane adhesion proteins such as P-selectin and integrins, tissue factor (TF) and other functional effectors that can regulate a variety of processes - aggregation, adhesion, expression of molecules, proliferation of cell proliferation, apoptosis, endothelial migration—cytokines and growth factors.6 However, of greatest interest to the present study, the externalization of phosphatidylserine (PS), which occurs with platelet activation and/or platelet apoptosis, provides an efficient platform for the aggregation of blood coagulation enzymes such as thrombin via the thrombin activation complex. Thrombin converts fibrinogen to fibrin, culminating in the activation of the coagulation cascade.6 The presence of. The coagulation mechanism occurs in three steps: adhesion to extracellular molecules, activation of chemical messengers and receptors and a cellular conformational change, and aggregation of cells through receptor bridges.8 In some cases, harmful clotting can be induced in healthy blood. .
Severe tissue damage can result in failure of all organ systems and even death. The analogous biochemical composition of PMPs suggests that PMP may also play a role in the coagulation cascade—normal and aberrant—however, the extent of their effect is not well understood. To achieve this separation, researchers can exploit the difference in size between PMPs and platelets through a series of centrifugations.9 Once isolated, research on PMPs can go in a number of directions; however, for this study, quantification of PMPs is the next step.
Exposure of phosphatidylserine helps to identify procoagulant PMPs because it is expressed only on the outer surface of the plasma membrane in activated or apoptotic platelets.6-7 To avoid false positive detection by flow cytometry, a. Flow cytometry works by passing cells individually through a laser and recording the different wavelengths of scattered light emitted by the cell (Figure 1.1).12 The. Sepsis reduces the hemostatic function of circulating platelets, preserves adhesion molecule expression and secretion ability, and modulates growth factor production, suggesting that sepsis alters the hemostatic function of platelets and increases.
As platelets are increasingly broken down and consumed, it seems intuitive that severe sepsis could cause an increase in PMPs, which could worsen the deregulation of the clotting system, leading to a vicious cycle. Because sepsis deregulates the coagulation system, resulting in increased platelet breakdown through apoptosis and macrophage consumption, we believe that PMP concentrations in blood plasma may increase as they are released from platelets. To test our hypothesis that sepsis causes an increase in the number of PMPs and the idea that PMPs are a new, useful parameter of diagnostic and therapeutic value in severe sepsis, we first quantified and compared PMP concentrations in patients with severe sepsis with the degree of disease severity and a number of other sepsis-related clinical factors, including adverse clinical outcomes and factors including previous medical treatments.
Methods
- Definitions
- Demographic and Clinical Factors
- Methods of Isolation
- Preparation for Quantification by Flow Cytometry
- PMP Quantification by Flow Cytometry
- Statistical Methods
Each enrolled patient or the patient's legally authorized next of kin previously provided written informed consent. Isolated MPs were fluorescently stained with specific antibodies to quantify the level of PMPs in the plasma of each patient. 50 µL of suspended MPs were added to three tubes with no antibody, control antibody, and triple positive antibody mixture added in the order of flow cytometer acquisition.
100 µL of AccuCheck Beads (Life . Technologies, Frederick, MD) was added and mixed thoroughly by hand just prior to acquisition on the flow cytometer. Identical samples were similarly incubated with isotype control IgG antibodies to test for false positives. The total number of beads counted was taken from graphs produced by the flow cytometer as in Figure 3.3.
Despite log transformation, PMP distributions remained non-parametric and therefore two-sample Wilcoxan rank-sum (Mann-Whitney) tests were performed with PMP levels to test for statistically significant differences between patients meeting the primary of death in hospital versus survival. Differences in PMP levels among a variety of clinical factors, including source of infection (pneumonia, UTI, surgical wound), past medical history (diabetes, liver disease, chronic renal failure, congestive heart failure, hypertension, malignancy, transplantation), clinical presentation (lactate elevation, spontaneous bacterial peritonitis) and gender were similarly tested. Linear regression was used to assess for statistically significant associations between PMP level and both age and platelet count.
A Kruskal-Wallis equality-of-populations rank test was performed on the patients' SOFA coagulation scores, a measure of how sick someone is, to determine the association between PMPs and disease severity, as assessed by organ failure. Mann-Whitney tests were then performed with PMP based on the SOFA coagulation score of the patient to further test for statistically significant correlations with in-hospital death or survival. Logistic regression was used to see if both PMP and platelet counts were independent predictors of death.
Results and Discussion
- PMP Concentrations Compared to In-Hospital Death or Survival
- The Association of PMPs with SOFA Coagulation
- PMP Concentrations Compared to Other Clinical Factors
- Discussion
- Conclusion
Having established a correlation between the number of PMP and mortality, we wanted to further explain this idea to see if the concentration of PMP could indicate not only the death or survival of the patient, but also the specific degree of severity of the disease. For the coagulation system, the degree of dysfunction is determined by evaluating the number of platelets (Table 4.2). At a P < .05 (Table 4.3), the results indicate that there is an association between SOFA coagulation score and PMPs and, therefore, between platelet count and PMP.
A logistic regression was performed with PMP and platelet count to determine whether these two measures represent independent predictors of death or whether they are collinear, suggesting that they may represent alternative measures of the same underlying phenomenon (Table 4.4). Analyzing the 79 patients so far, these two factors do not appear to be independent predictors (Table 4.4), meaning that they may be two different measures of the same underlying physiological process. The statistical distribution of data from our enrolled patients provided an initial indication that PMP concentrations tend to appear predominantly at lower levels in severe sepsis (Figure 4.1).
One of the main focuses of this study was to determine whether PMP concentration could be a useful parameter of prognostic value in cases of severe sepsis and whether the PMP count could be used to assess the probability of a patient's outcome to decide. To further test the parallel between PMP and platelet count, we performed a statistical analysis on PMP count separated by the patient's SOFA coagulation score, a quantitative measure of sepsis severity, which is scored based on patient's platelet count. This analysis showed that PMP and platelet count are not independent factors, but appear to cancel each other out.
Thus, while PMPs do represent a prognostic marker, the same data appear to be available via a common clinical, easy-to-perform test in platelet count. Of particular note, if completion of the remaining samples shows that PMPs are an independent predictor of mortality after controlling for initial platelet count, it may be that after platelet count is considered, PMPs do contribute to mortality, all things being equal. Previous research has attributed low platelet counts in septic patients to cytokine abundance, endothelial damage and bone marrow suppression.14 Since cytokine abundance and endothelial damage should contribute to an increase in PMPs, bone marrow suppression may occur at a sufficient level. to overcome those effects, yielding PMP and platelet counts that mirror each other as we observed in our results; however, further research will be needed to confirm this.
PMP concentration does correlate with the degree of sepsis severity and patient survival; however, platelet count provides a much more convenient parameter for critical care physicians in determining the severity of. Analysis of the remaining patients enrolled in this study will be necessary to confirm the results established in this thesis.
