Sepsis-associated Thrombocytopenia Research Articles

Construction and verification of a nomogram of factors influencing the risk of death in patient with sepsis-associated thrombocytopenia

To construct a nomogram prediction model for predicting the risk of death in patients with sepsis-associated thrombocytopenia (SAT) in intensive care unit (ICU) for early indentification and active intervention. Clinical data of SAT patients admitted to ICU of the First Affiliated Hospital of Nanjing Medical University from December 2019 to August 2021 were retrospectively collected, including demographic data, laboratory indicators, etc. According to the prognosis at 28 days, the patients were divided into the death group and the survival group, and the differences of clinical variables between the two groups were compared. Multivariate Logistic regression analysis was performed to analyze the independent risk factors influencing mortality of patients within 28 days, then a nomogram predictive model was constructed and its performance was verified with internal data. Receiver operator characteristic curve (ROC curve) was used to evaluate the diagnostic effectiveness of the nomogram model, and the clinical applicability of this model was evaluated by clinical decision curve analysis (DCA). A total of 275 patients were included, with 95 deaths at 28 days and a 28-day mortality of 34.5%. Compared with the survival group, acute physiology and chronic health evaluation II (APACHE II), sequential organ failure assessment (SOFA), lactic acid (Lac), platelet distribution width (PDW) on day 5 of ICU admission, blood urea nitrogen (BUN), total bilirubin (TBIL), aspartate aminotransferase (AST), C-reactive protein (CRP) of patients in the death group were higher, activated partial thromboplastin time (APTT) and prothrombin time (PT) were longer, platelet count (PLT) on day 3 and day 5 of ICU admission, direct bilirubin (DBIL), fibrinogen (FIB) were lower, the history of chronic lung disease, mixed site infection, lung infection, bloodstream infection, Gram-negative bacterial infection and fungal infection accounted for a higher proportion, the history of diabetes mellitus, urinary tract infection and no pathogenic microorganisms cultured accounted for a lower proportion, and the proportion of the use of vasoactive drugs, mechanical ventilation (MV), continuous renal replacement therapy (CRRT), bleeding events and platelet transfusion were higher. Multivariate Logistic regression analysis showed that APACHE II score at the day of ICU admission [odds ratio (OR) = 1.417, 95% confidence interval (95%CI) was 1.153-1.743, P = 0.001], chronic lung disease (OR = 72.271, 95%CI was 4.475-1 167.126, P = 0.003), PLT on day 5 of ICU admission (OR = 0.954, 95%CI was 0.922-0.987, P = 0.007), vasoactive drug (OR = 622.943, 95%CI was 10.060-38 575.340, P = 0.002), MV (OR = 91.818, 95%CI was 3.973-2 121.966, P = 0.005) were independent risk factors of mortality in SAT patients. The above independent risk factors were used to build a nomogram prediction model, and the area under the curve (AUC), sensitivity and specificity were 0.979, 94.7% and 91.7%, respectively, suggesting that the model had good discrimination. The Hosmer-Lemeshow goodness of fit test showed a good calibration with P > 0.05. At the same time, DCA showed that the nomogram model had good clinical applicability. Patients with SAT has a higher risk of death. The nomogram model based on APACHE II score at the day of ICU admission, chronic lung disease, PLT on day 5 of ICU admission, the use of vasoactive drug and MV has good clinical significance for the prediction of 28-day mortality, and the discrimination and calibration are good, however, further verification is needed.

Myristica fragrans Extract Inhibits Platelet Desialylation and Activation to Ameliorate Sepsis-Associated Thrombocytopenia in a Murine CLP-Induced Sepsis Model.

Sepsis, characterized by an uncontrolled host inflammatory response to infections, remains a leading cause of death in critically ill patients worldwide. Sepsis-associated thrombocytopenia (SAT), a common disease in patients with sepsis, is an indicator of disease severity. Therefore, alleviating SAT is an important aspect of sepsis treatment; however, platelet transfusion is the only available treatment strategy for SAT. The pathogenesis of SAT involves increased platelet desialylation and activation. In this study, we investigated the effects of Myristica fragrans ethanol extract (MF) on sepsis and SAT. Desialylation and activation of platelets treated with sialidase and adenosine diphosphate (platelet agonist) were assessed using flow cytometry. The extract inhibited platelet desialylation and activation via inhibiting bacterial sialidase activity in washed platelets. Moreover, MF improved survival and reduced organ damage and inflammation in a mouse model of cecal ligation and puncture (CLP)-induced sepsis. It also prevented platelet desialylation and activation via inhibiting circulating sialidase activity, while maintaining platelet count. Inhibition of platelet desialylation reduces hepatic Ashwell-Morell receptor-mediated platelet clearance, thereby reducing hepatic JAK2/STAT3 phosphorylation and thrombopoietin mRNA expression. This study lays a foundation for the development of plant-derived therapeutics for sepsis and SAT and provides insights into sialidase-inhibition-based sepsis treatment strategies.

Sepsis and Thrombocytopenia: A Nowadays Problem.

Sepsis is a life-threatening organ failure produced by a dysregulated host response to infection that involves 15.6% of hospital mortality. The most common signs and symptoms of sepsis are hypotension, tachypnea, fever, and leukocytosis, whether suspected or confirmed. Including a major one, thrombocytopenia is a sign that is an independent predictor of poor outcomes in patients with sepsis, increasing their mortality rate and their length of stay in the intensive care unit (ICU). So far, the ongoing treatment for this problem is securing the airway, treating hypoxemia, and providing vascular access for hydration, antibiotic delivery, and vasopressors, if needed. This article has reviewed the different possible mechanisms found for sepsis-associated thrombocytopenia, going from the most acknowledged one as decreased platelet production to the potential aftermath of sepsis itself as disseminated intravascular coagulation (DIC). This article has also discussed the future treatment for patients suffering from thrombocytopenia and sepsis, going from phase I and II trials as GI antagonists to the well-known drug aspirin as a possible treatment for this problem.

Prediction Models for Sepsis-Associated Thrombocytopenia Risk in Intensive Care Units Based on a Machine Learning Algorithm.

Sepsis-associated thrombocytopenia (SAT) is a common complication in the intensive care unit (ICU), which significantly increases the mortality rate and leads to poor prognosis of diseases. Machine learning (ML) is widely used in disease prediction in critically ill patients. Here, we aimed to establish prediction models for platelet decrease and severe platelet decrease in ICU patients with sepsis based on four common ML algorithms and identify the best prediction model. The research subjects were 1,455 ICU sepsis patients admitted to Dongyang People's Hospital affiliated with Wenzhou Medical University from January 1, 2015, to October 31, 2019. Basic clinical demographic information, biochemical indicators, and clinical outcomes were recorded. The prediction models were based on four ML algorithms: random forest, neural network, gradient boosting machine, and Bayesian algorithms. Thrombocytopenia was found to occur in 732 patients (49.7%). The mechanical ventilation time and length of ICU stay were longer, and the mortality rate was higher for the thrombocytopenia group than for the non-thrombocytopenia group. The models were validated on an online international database (Medical Information Mart for Intensive Care III). The areas under the receiver operating characteristic curves (AUCs) of the four models for the prediction of thrombocytopenia were between 0.54 and 0.72. The AUCs of the models for the prediction of severe thrombocytopenia were between 0.70 and 0.77. The neural network and gradient boosting machine models effectively predicted the occurrence of SAT, and the Bayesian models had the best performance in predicting severe thrombocytopenia. Therefore, these models can be used to identify such high-risk patients at an early stage and guide individualized clinical treatment, to improve the prognosis of diseases.

Elevated angiotensin II induces platelet apoptosis through promoting oxidative stress in an AT1R-dependent manner during sepsis.

Thrombocytopenia is independently related with increased mortality in severe septic patients. Renin‐angiotensin system (RAS) is elevated in septic subjects; accumulating studies show that angiotensin II (Ang II) stimulate the intrinsic apoptosis pathway by promoting reactive oxygen species (ROS) production. However, the mechanisms underlying the relationship of platelet apoptosis and RAS system in sepsis have not been fully elucidated. The present study aimed to elucidate whether the RAS was involved in the pathogenesis of sepsis‐associated thrombocytopenia and explore the underlying mechanisms. We found that elevated plasma Ang II was associated with decreased platelet count in both patients with sepsis and experimental animals exposed to lipopolysaccharide (LPS). Besides, Ang II treatment induced platelet apoptosis in a concentration‐dependent manner in primary isolated platelets, which was blocked by angiotensin II type 1 receptor (AT1R) antagonist losartan, but not by angiotensin II type 2 receptor (AT2R) antagonist PD123319. Moreover, inhibiting AT1R by losartan attenuated LPS‐induced platelet apoptosis and alleviated sepsis‐associated thrombocytopenia. Furthermore, Ang II treatment induced oxidative stress level in a concentration‐dependent manner in primary isolated platelets, which was partially reversed by the AT1R antagonist losartan. The present study demonstrated that elevated Ang II directly stimulated platelet apoptosis through promoting oxidative stress in an AT1R‐dependent manner in sepsis‐associated thrombocytopenia. The results would helpful for understanding the role of RAS system in sepsis‐associated thrombocytopenia.

The role of recombinant human thrombopoietin in critically ill patients with sepsis-associated thrombocytopenia: a clinical study

To investigate the effect of recombinant human thrombogenin (rhTPO) on sepsis-associated thrombocytopenia. A prospective randomized controlled study was conducted. One hundred patients with sepsis-associated thrombocytopenia admitted to the department of critical care medicine of the First Affiliated Hospital of Zhengzhou University from August 2019 to October 2020 were enrolled. The enrolled patients were divided into rhTPO-using group (TPO group) and routine group (control group) by random number table method, with 50 cases in each group. Both groups were treated according to the guideline of Sepsis-3. In addition, TPO group received rhTPO 15 000 U, once daily for 7 days. Geneal information and acute physiology and chronic health evaluation II (APACHE II) were recorded. The levels of platelet count (PLT), blood coagulation function [prothrombin time (PT) and prothrombin activity (PTA)], myocardial enzyme indexes [troponin (Tn) and creatine kinase (CK)], liver and kidney function [aspartate aminotransferase (AST), total bilirubin (TBil) and creatinine (Cr)] and inflammatory biomarkers [procalcitonin (PCT) and C-reactive protein (CRP)] were recorded before treatment and 1, 3, 5 and 7 days after treatment. The infusion volume of blood components, duration of mechanical ventilation, length of stay in ICU, total length of hospitalization, total cost of hospitalization and 28-day outcome were recorded. According to whether the PLT was lower than 50×109/L, the patients in TPO group were divided into the TPO A group (PLT ≥ 50×109/L, 16 cases) and TPO B group (PLT < 50×109/L, 34 cases), and the absolute value of PLT increase, duration of mechanical ventilation, length of stay in ICU, total length of hospitalization, total cost of hospitalization and 28-day outcome of the two groups were compared. (1) In TPO and control groups, there were no statistically significant differences in gender, age, proportion of patients with primary infection site, APACHEII score, PLT, coagulation function, myocardial enzymes, liver and kidney function and inflammation indexes before treatment (all P > 0.05). (2) The PLT levels of the TPO group were significantly higher than those of the control group on the 5th and 7th day after treatment (×109/L: day 5, 63.94±44.01 vs. 49.85±29.26, day 7, 125.85±112.31 vs. 76.81±50.87, both P < 0.05), and there were no statistically significant differences in PT, PTA, Tn, CK, AST, TBil, Cr, PCT or CRP before and on the 1, 3, 5, 7 days after treatment between TPO and control groups (all P > 0.05). (3) The amount of platelet transfusion in the TPO group was lower than that in the control group [treatment amount: 0 (0, 0) vs 0 (0, 2.00), P = 0.001]. (4) There were no statistically significant differences in mechanical ventilation time, length of stay in ICU, total length of hospitalization, total cost of hospitalization or 28-day outcome between TPO and control groups (all P > 0.05). The mechanical ventilation time, ICU stay time and total hospitalization time of TPO A group were longer than those in TPO B group, but the differences were not statistically significant [mechanical ventilation time (hours): 131.00 (0, 311.00) vs. 50.00 (0, 192.00), ICU stay time (days): 14.44±8.57 vs. 11.73±9.24, total hospitalization time (days): 15.00 (6.00, 23.50) vs. 18.00 (8.00, 31.00), all P > 0.05]. The absolute value of PLT increase in TPO A group was higher than that of TPO B group, but the difference was not statistically significant [×109/L: 65.00 (16.50, 131.50) vs. 36.00 (18.00, 130.00), P > 0.05]. RhTPO can significantly increase the PLT of patients with sepsis-related thrombocytopenia, thereby reduce the amount of platelet transfusion, but it cannot shorten the length of ICU stay time and total hospitalization time, and it cannot reduce 28-day mortality.

Recombinant human thrombopoietin in critically ill patients with sepsis-associated thrombocytopenia: A clinical study

BackgroundSepsis causes varying degrees of thrombocytopenia that are closely related to the likelihood of patient mortality. This study analysed the effect of recombinant human thrombopoietin (rhTPO) on the platelet count in critically ill patients with sepsis-associated thrombocytopenia and provided a reference for its treatment. Material/methodsThe study was a retrospective analysis of the clinical data of patients. Patients were divided into an rhTPO group and control group according to rhTPO use during treatment. Demographical and clinical data (age, sex, history of hypertension, diabetes, platelet counts, mortality rate, etc.) of the patients were collected and analysed using statistical software; p < 0.05 was considered statistically significant. ResultsOf 213 patients, 84 constituted the rhTPO group and 129 constituted the control group. The increase in platelet counts was significantly higher in the rhTPO group than in the control group on the third day (43.01 ± 18.23 × 109/L vs. 36.31 ± 14.17 × 109/L, p = 0.003), fifth day (71.51 ± 39.59 × 109/L vs. 42.95 ± 20.48 × 109/L, p < 0.001) and seventh day (115.36 ± 69.41 × 109/L vs. 62.54 ± 42.70 × 109/L, p < 0.001). Further statistical analysis of the data of patients with platelet counts ≤30 × 109/L and >30 × 109/L and APACHE II scores >15 and ≤15 at the time of diagnosis showed that the increase in platelet counts in the rhTPO group was greater. There was no significant between-group difference in volume of platelet transfusions (rhTPO group 15.42 ± 17.20 vs. control group 10.93 ± 17.48, p = 0.068). The cost of ICU treatment in patients with rhTPO was higher (RMB 126,936.21 ± 86,548.27 vs. 101,685.28 ± 77,291.75, p = 0.027); however, the ICU stay time was shorter (9.20 ± 5.38 vs. 10.88 ± 6.82, p = 0.047). There was no significant difference in 28-day mortality (rhTPO group: 25.0% vs. control group: 34.1%, p = 0.158) between the two groups. ConclusionFor patients with severe thrombocytopenia or severe sepsis, rhTPO was efficacious in increasing their platelet counts, resulting in a shorter ICU stay time.

Research Advances in the Subtype of Sepsis-Associated Thrombocytopenia.

The incidence and mortality of sepsis in the intensive care unit (ICU) are extremely high. Thrombocytopenia, one of the most common laboratory abnormalities, is correlated with prognosis in sepsis. The pathophysiology of sepsis-associated thrombocytopenia (SAT) remains unclear and may be associated with several factors such as platelet activation due to vascular injury and pathogen, suppression of bone marrow, platelet-targeted antibodies and desialylation. This review summarized all these possible mechanisms in the 3 subtypes of SAT: increased platelet consumption, reduced platelet production and increased platelet destruction. Based on the clinically available platelet parameters, the evidence for identifying SAT subtypes and the recent progress in treatments according to these subtypes are proposed to provide new prospects for the management of SAT.

Macrophage Depletion Mitigates Platelet Aggregate Formation in Splenic Marginal Zone and Alleviates LPS-Associated Thrombocytopenia in Rats.

Sepsis is often accompanied with thrombocytopenia partly due to platelet sequestration in the lung and liver. The spleen can store up to one-third of circulating platelets and can also significantly affect platelet transfusion outcomes by accumulating platelets. However, in sepsis, it is not clear whether there are platelet changes in the spleen which could contribute to sepsis-associated thrombocytopenia and also influence platelet transfusion outcomes. By using confocal microscopy, we examined endogenous rat platelets and infused human platelets in the spleen of severe combined immune deficient Rag2 KO rats which were injected intraperitoneally with lipopolysaccharide (LPS). LPS-injected Rag2 KO rats developed sepsis as indicated by increased TNFa, IL-6, IL-1b, and IL-10 levels and thrombocytopenia. Large platelet aggregates were observed in the spleen with majority located in the marginal zone and closely associated with CD169+ macrophages. Depletion of macrophages by clodrosome resulted in reduction of LPS-induced cytokine generation and alleviated LPS-induced thrombocytopenia. Macrophage depletion also remarkedly diminished large platelet aggregate formation in the splenic marginal zone but had less effect on those in red pulp. Infusion of human platelets into LPS-injected rats failed to raise platelet counts in the peripheral blood. In LPS-injected rat spleen, human platelets interacted with aggregated rat platelets in the marginal zone. In contrast, human platelets infused into control rats were located outside of splenic marginal zone. This study provides morphological evidence of platelet aggregates in the splenic marginal zone in sepsis which can interact with infused platelets and thus can contribute to platelet infusion refractoriness in sepsis. It indicates that macrophages play an important role in LPS-associated thrombocytopenia. It also suggests that CD169+ macrophages support platelet aggregate formation in the splenic marginal zone.

The Use of High Dose Eltrombopag in the Management of Sepsis-Associated Thrombocytopenia in Critically Ill Patients.

IntroductionSepsis is a life-threatening condition, and sepsis-associated thrombocytopenia (SAT) is a common consequence of the disease where platelet count falls drastically within a very short time. Multiple key factors may cause platelet over-activation, destruction and reduction in platelet production during the sepsis. Eltrombopag is a thrombopoietin receptor agonist and is the second-line drug of choice in the treatment of chronic immune thrombocytopenia (ITP).Aim of the studyThe objective of this study was to observe the therapeutic outcome of high dose eltrombopag in SAT management in critically ill patients.Material and MethodsThis 6-month-long single group, observational study was conducted on seventeen ICU patients with SAT. Eltrombopag 100 mg/day in two divided doses was given to each patient. Platelet counts were monitored. A low platelet blood count returning to 150 K/μL or above, is taken as indicative of a successful reversal of a thrombocytopenia event.ResultsThe mean Apache II score of patients (n= 17) was 18.71 (p-value: >0.05). No eltrombopag-induced adverse event was observed among the patients during the study period. Thrombocytopenia events were reversed successfully in 64.71% of patients (11; n= 17) within eight days of eltrombopag therapy.ConclusionsThe therapeutic potentiality of high dose eltrombopag regime in the management of sepsis-associated thrombocytopenia was found clinically significant in over two-thirds of critically ill adult patients enrolled in the study. These data may point to a new strategy in the management of acute type of thrombocytopenia in septic patients.

Acquired Platelet GPVI Dysfunction As Possible Predictor for Early Sepsis Diagnosis and Poor Outcome

Introduction: Sepsis is a major contributor to the global disease burden with estimated 20 million cases per year. With every hour prior to begin of therapy mortality increases by over 7%. Hence, early diagnosis is indispensable for patient survival, though it is hampered by the lack of known pathognomonic symptoms. Sepsis diagnosis is set according to an increase of the sequential organ failure assessment (SOFA) score, reflecting organ dysfunction. Severe symptoms comprising dysfunctional hemostasis are disseminated intravascular coagulation and formation of massive edema due to loss of vascular integrity. Functional hemostasis and maintenance of vascular integrity depends particularly on the platelet surface receptor glycoprotein (GP)VI that dimerizes upon activation and transduces its signals via the associated immunoreceptor tyrosine activation motif (ITAM) of the Fc-gamma chain. Sepsis-associated thrombocytopenia has been extensively investigated and low platelet counts are implemented in the SOFA score.Platelet function during sepsis, however, remains ill-defined with few reports using ADP or thrombin receptor activating peptide (TRAP) and overall conflicting results. Objectives: We assessed platelet function in patients with sepsis III criteria in a single center study at three times during disease: (I) intensive care unit (ICU) admission day; (II) day five to seven at ICU; (III) day of ICU discharge. Methods: Fifteen patients were recruited from the ICU of the University Hospital Würzburg, Germany (m:f ratio 9:6; median age 70; mortality: 40%). Platelets in whole blood were stimulated with ADP, TRAP, or the GPVI agonist collagen-related peptide (CRP). Activation was detected flow cytometrically by P-selectin (CD62P) exposure and integrin GPIIb/IIIa activation (PAC-1 binding). Results were correlated with light transmission aggregometry (LTA) and immunoblotting of phospho-Syk and -LAT. Co-incubation experiments were performed with plasma, whole blood or patient-borne bacterial isolates comprising Gram-negative (E. coli, K. pneumoniae) or Gram-positive (E. faecalis) strains. Results: Platelet function was markedly reduced in all patients assessed by flow cytometry and LTA despite overall unaltered receptor expression. The defect was most prominent after GPVI stimulation with CRP(Mean Geo-MFI ± SD; Control: 9356 ± 3460; Sepsis: 1438 ± 2090; p&amp;lt;.0001). In 14/15 patients GPVI-dysfunction was already present at time (I). In contrast, only 7/15 patients were thrombocytopenic at this early time. We did not consistently find elevated GPVI ectodomain plasma levels, low platelet counts, increased mean platelet volume, or elevated reticulated platelet levels in our cohort at early time points and minor alterations only late during disease progression. We conclude that none of these is a bona fide biomarker, neither for early sepsis diagnosis nor as a prognostic marker. Sepsis platelets failed to transduce the GPVI signal to induce tyrosine phosphorylation of Syk kinase or LAT. ITIM receptor-based tyrosine phosphatases SHP1 and SHP2 were neither preactivated in resting platelets nor responsive to GPVI stimulation. Of note, platelet aggregation upon GPVI stimulation increased in those patients whose condition ameliorated. We found a strong correlation of restored platelet aggregation by LTA with survival and could distinguish survivors from non-survivors using this marker for stratification (%max aggregation ± SD; survivors: 25 ± 8%; non-survivors: 4 ± 3%; p&amp;lt;0.01). Responsiveness to CRP was unaltered when platelets of healthy donors were co-incubated with patient blood borne bacterial isolates. Platelet reactivity was also unaltered when they were pre-incubated in plasma of sepsis patients. However, pre-incubation in whole blood clearly diminished the response toward CRP,suggesting that the abrogated GPVI signaling is in part mediated by cellular components. Conclusion: Our results strongly imply that GPVI hypo-responsiveness could serve as a bona fide marker for early sepsis diagnosis and also bears potential to act as a prognostic indicator for therapy monitoring, independent from the source of infection. Acknowledgement: Financial support was granted by the Deutsche Forschungsgemeinschaft (374031971 - TRR 240). Disclosures No relevant conflicts of interest to declare.

Factor XII Activation Promotes Platelet Consumption in the Presence of Bacterial-Type Long-Chain Polyphosphate In Vitro and In Vivo.

Objective- Terminal complications of bacterial sepsis include development of disseminated intravascular consumptive coagulopathy. Bacterial constituents, including long-chain polyphosphates (polyP), have been shown to activate the contact pathway of coagulation in plasma. Recent work shows that activation of the contact pathway in flowing whole blood promotes thrombin generation and platelet activation and consumption distal to thrombus formation ex vivo and in vivo. Here, we sought to determine whether presence of long-chain polyP or bacteria in the bloodstream promotes platelet activation and consumption in a coagulation factor (F)XII-dependent manner. Approach and Results- Long-chain polyP promoted platelet P-selectin expression, microaggregate formation, and platelet consumption in flowing whole blood in a contact activation pathway-dependent manner. Moreover, long-chain polyP promoted local fibrin formation on collagen under shear flow in a FXI-dependent manner. Distal to the site of thrombus formation, platelet consumption was dramatically enhanced in the presence of long-chain polyP in the blood flow in a FXI- and FXII-dependent manner. In a murine model, long-chain polyP promoted platelet deposition and fibrin generation in lungs in a FXII-dependent manner. In a nonhuman primate model of bacterial sepsis, pre-treatment of animals with an antibody blocking FXI activation by FXIIa reduced lethal dose100 Staphylococcus aureus-induced platelet and fibrinogen consumption. Conclusions- This study demonstrates that bacterial-type long-chain polyP promotes platelet activation in a FXII-dependent manner in flowing blood, which may contribute to sepsis-associated thrombotic processes, consumptive coagulopathy, and thrombocytopenia.

Role of matrix metalloproteinases 2 and 9, toll-like receptor 4 and platelet-leukocyte aggregate formation in sepsis-associated thrombocytopenia.

BackgroundThe development of thrombocytopenia in sepsis is a poor prognostic indicator associated with a significantly increased mortality risk. Mechanisms underlying this phenomenon remain to be clearly elucidated. Matrix metalloproteinases (MMPs) are enzymes that regulate the turnover of the extra-cellular matrix. MMP-2 is recognised as a platelet agonist with MMP-9 proposed as an inhibitor of platelet activation. The existence of MMP-9 in platelets is a subject of debate. There is limited evidence thus far to suggest that toll-like receptor 4 (TLR-4) and platelet-leukocyte aggregate (PLA) formation may be implicated in the development of sepsis-associated thrombocytopenia.ObjectivesTo investigate whether MMP -2/-9, toll-like receptor 4 (TLR-4) or platelet-leukocyte aggregate (PLA) formation are implicated in a decline in platelet numbers during septic shock.MethodsThis was an observational study which recruited healthy controls, non-thrombocytopenic septic donors and thrombocytopenic septic donors. MMP-2, MMP-9 and TLR-4 platelet surface expression as well as PLA formation was examined using flow cytometry. In addition MMP-2 and MMP-9 were examined by gelatin zymography and enzyme-linked immunosorbent assay (ELISA) using a 3 compartment model (plasma, intraplatelet and platelet membrane).ResultsThere was no difference found in MMP-2, MMP-9 or TLR-4 levels between non-thrombocytopenic and thrombocytopenic septic donors. PLA formation was increased in thrombocytopenic patients. MMP-9 was detected in platelets using flow cytometry, gelatin zymography and ELISA techniques.ConclusionsPlatelet consumption into PLAs may account for the development of thrombocytopenia in septic shock. MMP-9 is found in platelets and it is upregulated during septic shock.

Abstract 037: Coagulation Factor XII Promotes Platelet Consumption in the Presence of Microbial Polyphosphate Under Shear Flow

Background: Terminal complications of bacterial sepsis include development of disseminated intravascular consumptive coagulopathy. Bacterial constituents, including long-chain polyphosphates (polyP), have been shown to activate the contact pathway of coagulation in plasma. Recent work shows that activation of the contact pathway in flowing whole blood can promote thrombin generation and platelet activation and consumption distal to thrombus formation ex vivo and in vivo . Aim: Determine whether presence of long-chain polyP in the bloodstream promotes platelet activation and consumption in a coagulation factor (F)XII-dependent manner. Methods/Results: The addition of long-chain polyP to human whole blood promoted platelet P-selectin expression, microaggregate formation and platelet consumption in the bloodstream under shear in a FXII-dependent manner. Moreover, long-chain polyP accelerated thrombus formation on immobilized collagen surfaces under shear flow in a thrombin generation-dependent manner. Distal to the site of thrombus formation, platelet consumption was dramatically enhanced in the presence of long-chain polyP in the bloodstream. Inhibiting contact activation of coagulation using established and novel agents reduced fibrin formation on collagen as well as platelet consumption in the bloodstream distal to the site of thrombus formation. In vivo , FXII deficiency was protective against long-chain polyP occlusive lung thrombus formation in mice. Lastly, in a non-human primate model of sepsis, pretreatment of animals with an antibody blocking FXI activation by FXIIa (14E11) diminished LD 100 S. aureus -induced platelet and fibrinogen consumption. Conclusions: This study demonstrates that bacterial-type long-chain polyP promotes FXII-mediated thrombin generation and platelet activation in the flowing blood and could contribute to sepsis-associated thrombotic processes, consumptive coagulopathy and thrombocytopenia.

Abstract 091: Coagulation Factor XII Promotes Platelet Consumption in the Presence of Microbial Polyphosphate Under Shear Flow

Background: Terminal complications of bacterial sepsis include development of disseminated intravascular consumptive coagulopathy. Bacterial constituents, including long-chain polyphosphates (polyP), have been shown to activate the contact pathway of coagulation in plasma. Recent work shows that activation of the contact pathway in flowing whole blood can promote thrombin generation and platelet activation and consumption distal to thrombus formation ex vivo and in vivo . Aim: Determine whether presence of long-chain polyP in the bloodstream promotes platelet activation and consumption in a coagulation factor (F)XII-dependent manner. Methods/Results: The addition of long-chain polyP to human whole blood promoted platelet P-selectin expression, microaggregate formation and platelet consumption in the bloodstream under shear in a FXII-dependent manner. Moreover, long-chain polyP accelerated thrombus formation on immobilized collagen surfaces under shear flow in a thrombin generation-dependent manner. Distal to the site of thrombus formation, platelet consumption was dramatically enhanced in the presence of long-chain polyP in the bloodstream. Inhibiting contact activation of coagulation using established and novel agents reduced fibrin formation on collagen as well as platelet consumption in the bloodstream distal to the site of thrombus formation. In vivo , FXII deficiency was protective against long-chain polyP occlusive lung thrombus formation in mice. Lastly, in a non-human primate model of sepsis, pretreatment of animals with an antibody blocking FXI activation by FXIIa (14E11) diminished LD 100 S. aureus -induced platelet and fibrinogen consumption. Conclusions: This study demonstrates that bacterial-type long-chain polyP promotes FXII-mediated thrombin generation and platelet activation in the flowing blood and could contribute to sepsis-associated thrombotic processes, consumptive coagulopathy and thrombocytopenia.

Abstract 342: Coagulation Factor XII Promotes Platelet Consumption in the Presence of Microbial Polyphosphate Under Shear Flow

Background: Terminal complications of bacterial sepsis include development of consumptive coagulopathy referred to as disseminated intravascular coagulation. Bacterial constituents, including long-chain polyphosphates (polyP), have been shown to activate the contact pathway of coagulation in plasma. Recent work shows that activation of the contact pathway is capable of promoting thrombin generation and platelet activation and consumption in whole blood distal to thrombus formation under shear ex vivo and in vivo . Aim: Test the hypothesis that the presence of long-chain polyP in the bloodstream promotes platelet activation and consumption in a coagulation factor (F)XII-dependent manner. Methods and Results: Presence of long-chain polyP in whole blood promoted platelet aggregation on immobilized collagen surfaces under shear flow. Long-chain polyP enhanced fibrin formation and shortened clotting times of plasma and whole blood. The addition of long-chain polyP promoted platelet P-selectin expression, microaggregate formation and platelet consumption in the bloodstream under shear in a FXII-dependent manner. Moreover, long-chain polyP accelerated thrombus formation on immobilized collagen surfaces under shear flow. Distal to the sites of thrombus formation, platelet consumption was dramatically enhanced in the presence of long-chain polyP in the bloodstream. Inhibiting contact activation of the coagulation pathway reduced fibrin formation on collagen as well as platelet consumption in the bloodstream distal to the site of thrombus formation. Conclusions: This study demonstrates that bacterial-type long-chain polyP promotes FXII-mediated thrombin generation and platelet activation in the flowing blood and could exaggerate sepsis-associated thrombotic processes, consumptive coagulopathy and thrombocytopenia.

Phosphatidylserine-Mediated Platelet Clearance By Endothelium Decreases Platelet Aggregates and Procoagulant Activity in Sepsis

Introduction: Disorders of coagulation are common in sepsis, with disseminated intravascular coagulation (DIC) occurring in approximately 35 % of severe cases, contributing to microvascular dysfunction and death. Intensive platelet activation in sepsis facilitates platelet aggregation, leading to the formation of microthrombi and platelet depletion. This results in the development of DIC and sepsis-associated thrombocytopenia. Therefore, platelets must be cleared locally and quickly in the early phase of activation. Previous studies mainly focused on the clearance of activated cold-stored and aging platelets as well as platelets in immune-mediated thrombocytopenia. However, platelet activation and their clearance in sepsis are poorly understood. Platelets can form aggregates with leukocytes resulting in leukocyte death, the release of extracellular traps (ETs), increased endothelial permeability, and aggravated thrombosis. This study explored an alternate pathway for platelet disposal mediated by endothelial cells (ECs) through phosphatidylserine (PS) and examined the effect of platelet clearance on procoagulant activity (PCA) in sepsis.Methods: The subjects were septic patients (n=48) and healthy controls (n=48). Platelet engulfment by ECs was observed by electron microscopy, immunofluorescence, or immunochemistry both in vitro and in animal models. The PCA of platelets was measured by clotting time, purified coagulation complex assays, and fibrin formation.Results: Platelets in septic patients demonstrated increased levels of surface activation markers and apoptotic vesicle formation, and also formed aggregates with leukocytes. Activated platelets adhered to and were ultimately digested by ECs in vivo and in vitro. Blocking PS on platelets or integrin on ECs attenuated platelet clearance, resulting in increased platelet count in a mouse model of sepsis (p<0.05). Furthermore, platelet removal by ECs resulted in a corresponding decrease in platelet-leukocyte complex formation and markedly reduced generation of factor Xa and thrombin on platelets (p<0.01). Pretreatment with lactadherin increased phagocytosis of platelets by approximately 2-fold, diminished PCA by 70%, prolonged coagulation time, and attenuated fibrin formation by 50%. A large decline in PS exposure on platelets, associated platelet PCA, and PLA formation is seen in patients in remission, which could be attributed to the elimination of abnormal platelets.Conclusions: Our results suggest that PS-mediated clearance of activated platelets by the endothelium results in an anti-inflammatory, anticoagulant, and antithrombotic effect that contributes to maintaining platelet homeostasis during acute inflammation. Antiplatelet treatment has been suggested as a novel strategy in sepsis, and we speculate that promoting efficient removal of activated and apoptotic platelets could further improve patient outcomes. Therefore, clearance of activated platelets earlier in the disease process could hasten recovery of homeostasis in circulation by eliminating catalytic platforms for the coagulation pathway, protecting blood cells from excessive activation, and restoring their normal function. Endothelium, at least in part, contributes to platelet disposal and may further improve the hypercoagulable status in inflammation. It is noteworthy that PS-mediated and lactadherin-strengthened platelet engulfment may modify coagulopathy, and thus provide a new modality for treatment of septic clotting disorders. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Sepsis-associated thrombocytopenia

•Incidence and significance of sepsis-associated thrombocytopenia (SAT). •Analysis of studies examining potential mechanisms of SAT. •Discussion of other causes of thrombocytopenia in ICU. •Summary of ongoing research in this area.

Evaluating the safety and efficacy of recombinant human thrombopoietin among severe sepsis patients with thrombocytopenia: study protocol for a randomized controlled trial.

BackgroundSepsis is still a major health problem that causes high mortality in all populations. Organ dysfunction including sepsis-associated thrombocytopenia is prevalent among sepsis patients, resulting in increasing mortality rates. Considering the clinical role of platelets, thrombocytopenia in sepsis has led to a large spend in research activity and clinical trials in this area, yet there is no consensus upon which treatment should be administered. As a result, platelet transfusion is often indicated to resolve low platelet counts, leading to an increasing risk of the multiple risks transfusion brings, such as infectious or immune system complications. Given the role of thrombopoietin in stimulating proliferation and differentiation of megakaryocytes, our previous study investigated the potential benefits of recombinant human thrombopoietin in severe sepsis patients with thrombocytopenia. However, there are several limitations in the study, which may have led to bias in our conclusion. Thus, we are conducting this study in order to evaluate the safety and efficacy of recombinant human thrombopoietin in a large, varied population.Methods/DesignThe study is designed as a randomized, open-label, placebo-controlled, multi-center study in tertiary academic centers for evaluating the safety and efficacy of recombinant human thrombopoietin over placebo. An established total of 708 patients with sepsis and thrombocytopenia will undergo prospective random assignment to recombinant human thrombopoietin or placebo (a 1:1 ratio). The primary endpoint is 7-day all-cause mortality and 28-day all-cause mortality.DiscussionTo our knowledge, this is the first study to evaluate the safety and efficacy of recombinant human thrombopoietin among severe sepsis patients with thrombocytopenia in a varied population. With our study, the level of evidence for the treatment of these patients will be significantly raised.Trial registrationClinicalTrials.gov: NCT02094248. Registration date: 23 March 2014.Electronic supplementary materialThe online version of this article (doi:10.1186/s13063-015-0746-6) contains supplementary material, which is available to authorized users.