Colin Downey

Loss of endothelial protein C receptors links coagulation and inflammation to parasite sequestration in cerebral malaria in African children.

Cerebral malaria (CM) is a major cause of mortality in African children and the mechanisms underlying its development, namely how malaria-infected erythrocytes (IEs) cause disease and why the brain is preferentially affected, remain unclear. Brain microhemorrhages in CM suggest a clotting disorder, but whether this phenomenon is important in pathogenesis is debated. We hypothesized that localized cerebral microvascular thrombosis in CM is caused by a decreased expression of the anticoagulant and protective receptors thrombomodulin (TM) and endothelial protein C receptor (EPCR) and that low constitutive expression of these regulatory molecules in the brain make it particularly vulnerable. Autopsies from Malawian children with CM showed cerebral fibrin clots and loss of EPCR, colocalized with sequestered IEs. Using a novel assay to examine endothelial phenotype ex vivo using subcutaneous microvessels, we demonstrated that loss of EPCR and TM at sites of IE cytoadherence is detectible in nonfatal CM. In contrast, although clotting factor activation was seen in the blood of CM patients, this was compensated and did not disseminate. Because of the pleiotropic nature of EPCR and TM, these data implicate disruption of the endothelial protective properties at vulnerable sites and particularly in the brain, linking coagulation and inflammation with IE sequestration.

Performance and prognostic importance of a new clinical and laboratory scoring system for identifying non-overt disseminated intravascular coagulation.

A template for diagnosing the non-overt phase of disseminated intravascular coagulation (DIC) has recently been proposed. However, validation of its performance and the proposal of a defining score are required. The aim was to assess feasibility of the non-overt DIC scoring template and its potential prognostic significance. Consecutive patients admitted to a university hospital intensive care unit were initially assessed over 2 months. Following this, a 12-month study examined the prognostic performance of the derived scores prospectively. Outcome parameters were overt DIC and 28-day mortality. The 2-month study, involving 66 patients and 919 time points, demonstrated practical feasibility and prognostic associations for mortality with scores of 5 and greater. The 12-month study involving 450 patients showed that the mortality rate was 29% (105 of 360) and 78% (70 of 90) for scores below 5 and scores of 5 or above, respectively. The mortality rate for overt DIC was also 78% (38 of 49). The non-overt DIC scoring template is workable and has prognostic relevance. A score of 5 and greater is recommended as diagnostic of non-overt DIC.

Microparticle-associated endothelial protein C receptor and the induction of cytoprotective and anti-inflammatory effects

The endothelial protein C receptor plays an important role within the protein C pathway in regulating coagulation and inflammation. This study provides information on the mechanisms by which activated protein C causes the release of the endothelial protein C receptor in microparticulate form from endothelial cells. See related perspective article on page 313. Background The endothelial protein C receptor plays an important role within the protein C pathway in regulating coagulation and inflammation. Recently, we described that endothelial protein C receptor can be released in vitro in microparticulate form from primary endothelial cells by exogenous activated protein C. Activated protein C bound to this endothelial protein C receptor retains anticoagulant activity and we hypothesize that this microparticulate endothelial protein C receptor-activated protein C complex can also cleave endothelial protease-activated receptor 1 to modulate inflammation and increase cell survival. Our main objective was, therefore, to study the effect that microparticle-associated endothelial protein C receptor-activated protein C has on endothelial function. Design and Methods Mini-arrays were used and probed with cDNA obtained from endothelial cells after treatment with microparticle-associated endothelial protein C receptor-activated protein C and results were confirmed by real time polymerase chain reaction. The functional relevance of changes at gene level were further analyzed by endothelial apoptosis and permeability assays, in the presence and absence of specific blockade of endothelial protein C receptor, protein C and protease-activated receptor 1. Results Gene profiling of endothelial cells stimulated by 40 nmol/L activated protein C on microparticles showed significant changes in anti-apoptotic and inflammatory pathways. This was accompanied by protease-activated receptor 1-dependent anti-apoptotic and barrier protective effects, the latter of which also involved sphingosine 1-phosphate receptor and vascular endothelial growth factor receptor-2/ kinase insert domain receptor. Protein C blockade reversed these effects showing specificity for activated protein C on microparticles. Furthermore, confocal microscopy and enzyme-linked immunosorbent assay of plasma obtained from septic patients during recombinant activated protein C treatment showed evidence of their presence in vivo. Conclusions Activated protein C on microparticle-associated endothelial protein C receptor release can induce protease-activated receptor 1-dependent endothelial effects. The mechanisms underlying barrier protection involve sphingosine 1-phosphate receptor and kinase insert domain receptor.

Early identification of sepsis and mortality risks through simple, rapid clot-waveform analysis. Implications of lipoprotein-complexed C reactive protein formation.

Abstract Objective. To determine if the rapid waveform profile of the activated partial thromboplastin time (aPTT) assay, which detects lipoprotein-complexed C reactive protein (LCCRP) formation, predicts sepsis and mortality in critically ill patients. Design. Observational, cohort study. Setting. General intensive therapy unit (ITU) of a tertiary care hospital. Patients and participants. A total of 1187 consecutive patients admitted to the ITU. Intervention. Activated partial thromboplastin time transmittance waveform analysis was performed within the first hour of admission to the ITU. The degree of change causing a biphasic waveform was quantified through the drop in light transmittance level. Measurements and results. Three hundred forty-six patients had a biphasic waveform on admission to the ITU with a mortality rate of 44% compared with 26% for those with normal waveforms. Logistic regression models showed direct correlation between the likelihood for sepsis and in-patient mortality with increasing waveform abnormalities. The mortality fraction was 0.3 with normal waveforms versus 0.6 when the light transmittance decreased by 30%. The odds ratio (OR) for mortality and sepsis were 4.5 and 11, respectively, from the most abnormal to normal aPTT waveforms. These were comparable with APACHE II scores and superior to those estimated by CRP for mortality (OR 2.3) / sepsis (OR 6.4) prediction. Conclusion. Waveform analysis within the first hour of ITU admission is a single, simple and rapid method of identifying the risks of mortality and sepsis. Its measure of LCCRP formation shows superior prediction over CRP alone and it warrants further assessment as a tool to triage and target prompt, appropriate treatment in the ITU.

Novel and diagnostically applicable information from optical waveform analysis of blood coagulation in disseminated intravascular coagulation

Transmittance waveform is the term applied to the optical profile generated from the process of clot formation on standard coagulation tests run on the MDA‐180, a new‐generation automated coagulation analyser. In patients with disseminated intravascular coagulation, a characteristically abnormal ‘biphasic change’ is seen on both the activated partial thromboplastin time and thrombin time waveforms. Increasing steepness of the initial slope on the waveform correlates with clinical deterioration and fulminant progression. Although the mechanism underlying the biphasic appearance remains to be elucidated, its identification provides the diagnostic laboratory with a simple, rapid and robust assay for disseminated intravascular coagulation that can help the clinician with urgent and appropriate therapeutic interventions.

Thrombin activatable fibrinolysis inhibitor and its fibrinolytic effect in normal pregnancy

We investigated changes in both thrombin activatable fibrinolysis inhibitor (TAFI) antigen levels and its functional effect on in vitro fibrinolysis in normal pregnancy. 152 pregnant women and 31 women in the immediate postpartum period were studied, with pregnancy divided into 6 windows at 4 weekly intervals. As TAFI influences and is in turn influenced by components of the protein C (PC) pathway, its measurements were correlated with levels of soluble thrombomodulin, PC, protein S (PS) and the overall phenotype of activated PC resistance (APCR). Compared with mean TAFI levels at booking gestation (6.6 +/- 1.2 microg/ml), levels peaked at 35-39 weeks gestation (9.6 +/- 2 microg/ml, p = 0.001), followed by a significant drop within 24 hours of delivery (7.2 +/- 1.1 microg/ml). In functional terms, the mean clot lysis time (CLT) (101 +/- 13 min at booking) also peaked at 35-39 weeks gestation (141 +/- 42 min, p = 0.007) and dropped after delivery (99 +/- 33 min), and was significantly correlated with gestational age (r = 0.410, p = 0.001) and could be abrogated in the presence of an inhibitor to TAFI activation. A significant negative correlation was found between TAFI levels and APCR (r = -0.478, p <0.001), APCRV (r = -0.598; p <0.001), PS (r = -0.490, P <0.001) and PC (r = -0.198, p = 0.02). In summary, there is a significant increase in TAFI levels, which translates into increased CLT during pregnancy. Furthermore, changes in TAFI contribute to the increasing APCR of pregnancy.

Assessment and clinical course of hypocalcemia in critical illness

IntroductionHypocalcemia is common in critically ill patients. However, its clinical course during the early days of admission and the role of calcium supplementation remain uncertain, and the assessment of calcium status is inconsistent. We aimed to establish the course of hypocalcemia during the early days of critical illness in relation to mortality and to assess the impact of calcium supplementation on calcium normalization and mortality.MethodsData were collected on 1,038 admissions to the critical care units of a tertiary care hospital. One gram of calcium gluconate was administered intravenously once daily to patients with adjusted calcium (AdjCa) <2.2 mmol/L. Demographic and outcome data were compared in normocalcemic (ionized calcium, iCa, 1.1-1.3 mmol/L) and mildly and severely hypocalcemic patients (iCa 0.9-1.1 mmol/L and <0.9 mmol/L, respectively). The change in iCa concentrations was monitored during the first four days of admission and comparisons between groups were made using Repeated Measures ANOVA. Comparisons of normalization and outcome were made between hypocalcemic patients who did and did not receive calcium replacement according to the local protocol. The suitability of AdjCa to predict low iCa was determined by analyzing sensitivity, specificity and receiver operating characteristic (ROC) curves. Multivariate logistic regression was performed to determine associations of other electrolyte derangements with hypocalcemia.Results55.2% of patients were hypocalcemic on admission; 6.2% severely so. Severely hypocalcemic patients required critical care for longer (P = 0.001) compared to normocalcemic or mildly hypocalcemic patients, but there was no difference in mortality between groups (P = 0.48). iCa levels normalized within four days in most, with no difference in normalization between those who died and survived (P = 0.35). Severely hypocalcemic patients who failed to normalize their iCa by day 4 had double the mortality (38% vs. 19%, P = 0.15). Neither iCa normalization nor survival were superior in hypocalcemic patients receiving supplementation on admission. AdjCa <2.2 mmol/L had a sensitivity of 78.2% and specificity of 63.3% for predicting iCa <1.1 mmol/L. Low magnesium, sodium and albumin were independently associated with hypocalcemia on admission.ConclusionsHypocalcemia usually normalizes within the first four days after admission to ICU and failure to normalize in severely hypocalcemic patients may be associated with increased mortality. Calcium replacement appears not to improve normalization or mortality. AdjCa is not a good surrogate of iCa in an ICU setting.

The clinical and functional relevance of microparticles induced by activated protein C treatment in sepsis

IntroductionActivated protein C (APC) induces release of microparticles (MP) from primary physiological cells, which are found in patients undergoing treatment with recombinant human APC (rhAPC) for severe sepsis. We hypothesised that APC on these circulating MPs activate endothelial protease-activated receptor 1 (PAR1) to induce anti-apoptotic and anti-inflammatory properties that can improve patient outcome.MethodsThis was an experimental study on clinical samples in an intensive care setting, and included patients with severe sepsis who fulfilled criteria for treatment with rhAPC. The number of CD13+ MPs from the patients were analysed to determine their origin. They were also quantified for endothelial protein C receptor (EPCR) and APC expression. Clinical relevance of these MPs were ascertained by comparing survival between the group receiving rhAPC (n = 25) and a control group of untreated patients (n = 25). MPs were also incubated with endothelial cells to analyse apoptotic gene expression, cytoprotection and anti-inflammatory effects.ResultsrhAPC treatment induced a significant increase in circulating MP-associated EPCR by flow cytometry (P < 0.05) and by quantitative ELISA (P < 0.005). APC expression also showed significant increases (P < 0.05). Numerically, CD13+ MPs were higher in rhAPC-treated survivors versus non-survivors. However, the number of non-survivors was low and this was not significantly different. APC on MPs was demonstrated to induce anti-apoptotic and endothelial barrier effects through the activation of endothelial PAR1.ConclusionsrhAPC treatment in patients with sepsis significantly increases circulating EPCR + MPs. These MPs were noted to express APC, which has specific anti-apoptotic and anti-inflammatory effects, with a non-significant correlative trend towards survival. This suggests that MPs could disseminate APC function and activate endothelial PAR1 at distal vascular sites.

Prothrombinase enhancement through quantitative and qualitative changes affecting very low density lipoprotein in complex with C-reactive protein

The biphasic waveform that can predict for disseminated intravascular coagulation (DIC) is due to the formation of a calcium-dependent complex between C reactive protein (CRP) and very low density lipoprotein (VLDL). As thrombin generation is pivotal to DIC, this aspect has been specifically investigated and the VLDL component has been found to increase prothrombinase activity via both quantitative and qualitative changes. The specific prothrombinase activity of VLDL from patients manifesting the biphasic waveform was 2.5 times that of normal individuals or critically ill patients without the biphasic waveform. This activity was due to an increase in anionic phospholipid surfaces that could be inhibited with excess annexin V and which was dependent on structurally intact apolipoprotein B. The qualitative change appeared to be due to a deficiency of phosphatidylethanolamine in VLDL from patients with the biphasic waveform. The functional consequence of this enhanced prothrombinase activity was an increased procoagulant effect in plasma. Using a modified activated partial thromboplastin time assay, the mean normal clot time decreased significantly when VLDL from patients with biphasic waveforms was substituted. These results indicate that VLDL derived from patients with the biphasic waveform can enhance thrombin procoagulant activity. As the CRP-VLDL complex exists in vivo, it could have a pathogenic role in disseminating the process of intravascular coagulation.

Laboratory evidence of disseminated intravascular coagulation is associated with a fatal outcome in children with cerebral malaria despite an absence of clinically evident thrombosis or bleeding

A procoagulant state is implicated in cerebral malaria (CM) pathogenesis, but whether disseminated intravascular coagulation (DIC) is present or associated with a fatal outcome is unclear.