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PubMed1994

Receptor dimerization determines the effects of growth hormone in primary rat adipocytes and cultured human IM-9 lymphocytes.

Binding of GH to its cell surface receptors is thought to result in the formation of a complex comprised of one molecule of hormone per two molecules of receptor. It has been proposed that this hormone-induced receptor dimerization is important for the mechanism of signal transduction. We have developed a mathematical model for quantitative evaluation of the biological responses associated with sequential receptor dimerization. Based on these predictions, we have investigated whether GH-induced receptor dimerization plays a role in two classical effects of GH, i.e. stimulation of lipogenesis in primary rat adipocytes and GH receptor down-regulation in cultured human IM-9 lymphocytes. Model predictions of biological responses linked to dimer formation yielded a bell-shaped pattern, with self-antagonism at high GH concentrations when monomeric GH-receptor complexes become predominant. The GH lipogenic bioactivity curve was indeed biphasic and first increased in a concentration-dependent manner between 10(-10)-10(-8) M GH (ED50, 0.5 nM), up to a maximum of 1.7-fold stimulation above basal. Then, the response decreased continuously above 5 x 10(-8) M GH, returning to basal levels around 10(-5) M GH. Incubation of IM-9 cells with wild-type human GH resulted in a dose-dependent loss of their surface receptors. In contrast, a human GH analog (G120R), mutated in the second binding surface of the hormone and, therefore, unable to induce GH receptor dimerization, failed to induce receptor down-regulation in the IM-9 cells. Furthermore, when added together with wild-type human GH, human GH(G120R) inhibited, in a concentration-dependent manner, the down-regulation induced by wild-type human GH. Taken together, these data support the hypothesis that receptor dimerization is critical for the stimulation by GH of both lipogenesis in primary rat adipocytes and receptor down-regulation in cultured human IM-9 lymphocytes.

Adipocytes↗
PubMed1992

Localization and regulation of the activin-A dimer in human placental cells.

Subunits of activin and inhibin and their mRNAs are present in human placental and decidual cells. However, evidence for the presence of intact activin dimers in the human placenta and their regulation has been lacking. Using a monoclonal antibody raised against the human activin-A dimer, we examined the cellular localization of immunoreactive activin-A dimer in human placentas of different gestational ages (8-41 weeks). In addition, we determined the effects of culture and various potential regulators on the cellular accumulation of immunoreactive activin-A dimer in trophoblast cells from human first trimester placentas. Activin-A dimer was found in both cyto- and syncytiotrophoblast cells of all gestational ages studied. Immunoreactive activin-A also was detected in placental Hofbauer cells in first and second trimester placentas as well as in cells of the placental membranes. Exposure of these cells to cAMP, GnRH, activin, inhibin, transforming growth factor-beta, dexamethasone, and interleukin-1 did not significantly change the intensity of immunostaining for activin-A dimer. These results together with previous data suggest that placental cells are a source of activin-A and that activin-A may be a paracrine and/or endocrine regulator of feto-maternal interactions during pregnancy.

Activins↗
PubMed2004

[Interpretation of increased D-dimer values].

BACKGROUND: The determination of D-dimer concentration is an essential part in the diagnostic procedure of thromboembolic diseases, e.g. deep vein thrombosis, pulmonary embolism. D-dimers are the products of fibrin hydrolysis with elevated levels in fibrinolytic processes. QUESTION: In the clinical practice problems exist in the interpretation of increased D-dimer concentrations, especially without thromboembolic disease. Before starting further expensive imaging diagnostics other reasons (i.e. pregnancy, neoplasma, systemic inflammatory disease, advanced arteriosclerosis) should be considered in differential diagnosis. CONCLUSION: The determination of the concentration of D-dimers is involved in the diagnostic strategy in thromboembolic diseases. However, this parameter is not suited for routine screening. Its high predictive-negative value is proved for the exclusion of thrombosis or pulmonary embolism in case of negative test result. Since a range of diseases and physiological conditions lead to increased D-dimer values, a positive D-dimer result does not verify the diagnosis of thromboembolism.

Antifibrinolytic Agents↗
PubMed2003

D-dimer in acute aortic dissection.

STUDY OBJECTIVE: Laboratory testing plays a minor role in the assessment of aortic dissection. Its main value is in the exclusion of other diseases. Following an incidental observation, we systematically investigated the relationship between elevated d-dimer levels and acute aortic dissection. DESIGN: We prospectively tested d-dimer levels in patients with suspected acute aortic dissection (10 patients). In addition, we investigated 14 patients who had received a confirmed diagnosis of thoracic aortic dissection during the previous 5 years, in whom d-dimer testing had been performed for differential diagnosis. Thirty-five patients with acute chest pain of other origin served as a control group. SETTING: Tertiary referral hospital. PATIENTS: Twelve patients had type A dissection (Stanford classification), and 12 patients had type B. MEASUREMENTS AND RESULTS: A d-dimer analysis was performed (Tina-quant assay; Roche Diagnostics; Mannheim, Germany) [normal limit of the assay, 0.5 micro g/mL]. The result of the d-dimer test was positive (ie, > 0.5 micro g/mL) in all patients (sensitivity of the test, 100%) with a mean value of 9.4 micro g/mL and a range of 0.63 to 54.7 micro g/mL. The degree of the elevation was correlated to the delay from the onset of symptoms to laboratory testing (mean, 12.6 h; range, 1 to 120 h) and showed a trend to the extent of the dissection, but not to the outcome (14 patients could be discharged; 10 patients died). CONCLUSIONS: Based on our observation, we suggest that testing for d-dimer should be part of the initial assessment of patients with chest pain, especially if aortic dissection is suspected. A negative test result makes the presence of the disease unlikely.

Acute Disease↗
PubMed2003

Biochemical characterization of beta2-adrenergic receptor dimers and oligomers.

G Protein-coupled receptor dimerization/oligomerization has been well established during the last several years. Studies have demonstrated the existence of dimers/digomers both in vitro and in living cells. However, a thorough characterization of the biochemical nature of receptor dimers and oligomers as well as their occurrence at the cell surface has not been properly addressed. In this study, we show that both beta2-adrenergic receptor (beta2AR) dimers and oligomers exist at the plasma membrane and that the detection of such species, following receptor solubilization and resolution by denaturing polyacrylamide gel electrophoresis (SDS-PAGE), does not result from the formation of spurious disulfide bonds during cell lysis. Moreover, our results indicate that the biochemical nature of beta2AR dimers is different from that of the oligomers. Although both complexes are partially resistant to SDS denaturation, disulfide bonding is absolutely required for the stability of beta2AR oligomers but not dimers in SDS-PAGE. Indeed, dimeric species can be detected even in the presence of high concentrations of reducing and alkylating agents. Although the different biochemical nature of the dimers and oligomers may be indicative of distinct biological roles in cells, additional studies will be required to further elucidate the biosynthesis and function of these receptor forms.

Blotting, Western↗
PubMed1997

Introduction of a proline residue into position 31 of the loop of the dimeric 4-alpha-helical protein ROP causes a drastic destabilization.

The exchange of an alanine with a proline residue in position 31 of the loop region of the dimeric 4-alpha-helical-bundle protein ROP causes a reduction in the alpha-helix content of 7% and a reduction in stability of about 40% compared to the wild type parameters. The Gibbs energy of unfolding by denaturants extrapolated linearly to zero denaturant concentration, delta G0D (buffer, 25 degrees C), has been determined to be 43 kJ (mol dimer)-1. The corresponding ROPwt value is 72 kJ (mol dimer)-1 (Steif et al., 1993). The extrapolated delta G0D values obtained from urea and GdmHCI un- and refolding studies are identical within error limits. Deconvolution of the stability values into enthalpy and entropy terms resulted in the following parameters. At T1/2 = 43 degrees C (Cprotein = 0.05 mg.ml-1) the ROP A31P mutant is characterized by delta Hv.H.0 = 272 kJ (mol dimer)-1, delta Cp = 7.2 kJ (mol dimer)-1 K-1, delta S0 = 762 J (mol dimer)-1 K-1. These parameters are only approximately 50% as large as the corresponding values of ROPwt. We assume that the significant reduction in stability reflects the absence of at least one hydrogen bond as well as deformation of the protein structure. This interpretation is supported by the reduction in the change in heat capacity observed for the A31P mutant relative to ROPwt, by the increased aggregation tendency of the mutant and by the reduced specific CD absorption at 222 nm. All results support the view that in the case of ROP protein the loop region plays a significant role in the maintenance of native structure and conformational stability.

Alanine↗
PubMed1981

Effects of pH and NaCl concentration on binding of covalently-linked insulin dimers to liver plasma membranes.

The effects of NaCl concentration and pH on binding to purified rat liver plasma membranes were compared for labelled insulin and two covalently-linked insulin dimers. Specific binding of both dimers and insulin increased as NaCl concentration increased from 0 to 1M in 0.05M Tris/HCl buffer. The initial rise in binding was much greater for dimers than for insulin. Specific binding of dimers was sensitive to pH changes in 0.029M barbital sodium acetate buffer, but this effect was more marked with insulin, which reached a sharp binding peak at pH 7.7 to 8.0. Both total and non-specific dimer binding were approximately 2-fold greater at pH 5.0 than at pH 8.0. Specific-binding fraction determinations of both dimers relative to insulin have been shown to be critically dependent on the experimental conditions of Na+/Cl- ion concentration and pH. These observations allow a better understanding of the nature of ligand-receptor interaction.

Animals↗
PubMed2003

Validation of an automated immunoturbidimetric assay for measurement of plasma D-dimer.

The performance characteristics and diagnostic accuracy of a new rapid automated quantitative immunoturbidimetric D-dimer assay, Auto-Dimer (Biopool, Umeå, Sweden) were evaluated in a population of 135 outpatients with suspected deep-vein thrombosis of a lower limb. Enzyme-linked immunosorbent assay was used as the reference method. The Auto-Dimer assay showed good reproducibility. The correlation between Auto-Dimer and enzyme-linked immunosorbent assay was high (r = 0.91, p < 0.05) and agreement in classifying patients above or below the cut-off was good (kappa coefficient 0.74, 95% CI 0.62-0.86). At a cut-off of 340 microg/l the sensitivity and specificity of Auto-Dimer were high (100% and 61%, respectively). These data show that Auto-Dimer is a reliable screening test for exclusion of deep-vein thrombosis. The assay could be included in prospective patient management studies in order to obtain further information on its clinical usefulness.

Automation↗
PubMed2005

The relationship between ambulation ability before surgery and the D-dimer value after total hip arthroplasty: the evaluation of ambulation ability by the timed "Up & Go" test.

We examined whether ambulatory ability before surgery might influence the post-operative D-dimer level after total hip arthroplasty (THA). One hundred two patients with hip osteoarthritis receiving THA were included in the current study. The patients were all female, and their ages ranged from 45 to 81 (average 65.0 +- 9.3 years). Age, operated side, body mass index (BMI), disease duration before surgery, pre-operative pain evaluated by visual analogue scale (VAS), total cholesterol value, maximal circumference of the lower leg of the operated side, and timed "Up & Go"test (TUG) before surgery, were retrospectively investigated to examine their relationship with D-dimer levels on post-operative day 7. Patients were divided into 2 groups according to the D-dimer value: over 10 microg/ml (Group D), and under (Group N). Patients in group D (N= 52)were older, had a higher BMI, and had less ambulatory ability than patients in group N (N= 50). As age showed a relationship with the D-dimer value on the 7th day and TUG results, patients in the 2 groups were further subdivided into 50's, 60's, and 70's age brackets. In the 50's bracket, patients in group D had higher BMI than patients in group N, but time for TUG was not significantly different. In the 60's and 70's bracket, patients in group D had less ambulatory ability than patients in group N, but the time for TUG was not directly correlated with the D-dimer value. The results suggest that pre-operative low ambulatory ability in patients with osteoarthritis over 60 years might influence the postoperative D-dimer after THA, indicating the potential risk for post-operative deep venous thrombosis.

Aged↗
PubMed2006

Family C 7TM receptor dimerization and activation.

The family C seven transmembrane (7TM) receptors constitutes a small and especially well characterized subfamily of the large 7TM receptor superfamily. Approximately 50% of current prescription drugs target 7TM receptors, this biologically important family represents the largest class of drug-targets today. It is well established that family C 7TM receptors form homo- or hetero-dimers on the cell surface of living cells. The large extra-cellular domains (ECD) have been crystallized as a dimer in the presence and absence of agonist. Upon agonist binding, the dimeric ECD undergoes large conformational changes that lead to receptor activation. Despite extensive studies of the receptor transmembrane domain, several key features, including the exact organization of the complete receptor dimer, the sequence of events leading to receptor activation, and the functional significance of dimerization, have yet to be fully defined. This review presents the biochemical support for family C 7TM receptor dimerization and discusses its importance for receptor biosynthesis, surface expression, ligand binding and activation, since lessons learnt here may well be applicable to the whole superfamily of 7TM receptors.

Animals↗
PubMed2003

Rational use of D-dimer measurement to exclude acute venous thromboembolic disease.

Clinical diagnosis of venous thromboembolic (VTE) disease is often inaccurate because signs and symptoms are nonspecific. Testing for the absence of D-dimer levels in the blood of patients with suspected deep venous thrombosis and pulmonary embolism can assist in ruling out these illnesses. Some highly sensitive D-dimer assays have sufficient specificity to assist in the exclusion of VTE disease. Numerous clinical management trials using D-dimer measurement in association with additional diagnostic tests have shown that it is safe to withhold anticoagulant therapy in selected patients with suspected VTE disease who have negative D-dimer assay results. Applying these diagnostic strategies can potentially decrease the need for radiological testing. The simplicity of measuring D-dimer levels creates the potential for misuse. For safe patient management, clinicians must understand the indications for and limitations of D-dimer measurement in the diagnosis of VTE disease.

Antifibrinolytic Agents↗
PubMed2003

The use of the rapid D-dimer test for the exclusion of acute venous thromboembolism in a regional hospital.

BACKGROUND: The performance of rapid D-dimer ELISA assay has been validated as a part of various diagnostic work-ups in tertiary care hospitals for the exclusion of acute thromboembolism in the medical emergency department. In order to measure the performance of this test outside of predetermined protocols and in a different medical setting, we retrospectively analysed a cohort of adult patients admitted to the emergency department of a regional hospital with a suspicion of acute venous thromboembolism. METHODS: All D-dimer assays performed during an 18-month period were retrieved. The patients' data were collected from hospital charts. Six-month follow-up was determined either by a written or telephone questionnaire or after contact with the patient's physician. The patients for whom this process was completed were included in the study and a retrospective diagnostic assessment was performed using a combination of clinical probability and objective testing. The diagnosis was then compared to the result of the initial D-dimer assay. RESULTS: During the study period 494 patients were included with 110 venous thromboembolic episodes. The sensitivity and negative predictive value of the D-dimer assay were respectively 94.5% (95% CI 88.4 to 97.7%) and 96.8% (95% CI 93.2 to 98.7%). CONCLUSIONS: The yield of the rapid D-dimer assay in this study is comparable to the results of management studies performed in tertiary centres. D-dimer ELISA assay can be used to exclude venous thromboembolism, particularly in cases with a low clinical probability, in the emergency department and for larger populations in various clinical settings, even in the absence of a formal diagnostic work-up. False negative results can occur, particularly in the presence of a high clinical probability of acute thromboembolism.

Acute Disease↗
PubMed1999

Clinical utility of a rapid whole-blood D-dimer assay in patients with cancer who present with suspected acute deep venous thrombosis.

BACKGROUND: Although D-dimer assays have high negative predictive values for the diagnosis of deep venous thrombosis, their accuracy in patients with cancer is uncertain. OBJECTIVE: To compare the clinical utility of a whole-blood D-dimer assay for the diagnosis of deep venous thrombosis in patients with and those without cancer. DESIGN: Retrospective analysis of three prospective studies. SETTING: Two tertiary care hospitals. PATIENTS: 1068 consecutive outpatients with suspected deep venous thrombosis. MEASUREMENTS: All patients underwent D-dimer testing and assessment with a priori diagnostic strategies that incorporated impedance plethysmography, compression ultrasonography, or contrast venography. Patients in whom deep venous thrombosis was not diagnosed initially were followed for 3 months for the development of thrombosis. Results of D-dimer testing were assessed according to the final diagnosis based on objective testing and clinical follow-up. Cancer status was identified at presentation. RESULTS: The prevalence of deep venous thrombosis was 48.8% in 121 patients with cancer and 14.6% in 947 patients without cancer. Although the sensitivity of the D-dimer assay was similar in patients with and those without cancer (86.4% [95% CI, 75.0% to 94.0%] and 82.6% [CI, 75.2% to 88.5%], respectively), the specificity was significantly lower in patients with cancer (48.4% [CI, 35.5% to 61.4%] and 82.2% [CI, 79.4% to 84.8%]), as was the negative predictive value (78.9% [CI, 62.7% to 90.4%] and 96.5% [CI, 94.9% to 97.8%]). In contrast, the likelihood ratios of a negative test result (0.28 [CI, 0.14 to 0.56] and 0.21 [CI, 0.15 to 0.31]) did not differ significantly. CONCLUSIONS: A negative D-dimer test result in patients with cancer does not reliably exclude deep venous thrombosis because the negative predictive value of the test is significantly lower in these patients than in patients without cancer.

Acute Disease↗
PubMed2003

A diagnostic strategy involving a quantitative latex D-dimer assay reliably excludes deep venous thrombosis.

BACKGROUND: Because clinical diagnosis is inaccurate, objective testing is usually considered necessary when patients present with suspected deep venous thrombosis (DVT). OBJECTIVE: To determine whether a negative result on a quantitative latex D dimer assay eliminates the need for further investigation in patients with a low or moderate pretest probability of DVT. DESIGN: Prospective cohort study. SETTING: Three tertiary care hospitals in Canada. PATIENTS: 556 consecutive outpatients with suspected first DVT. INTERVENTION: Patients were categorized as having a low, moderate, or high pretest probability of DVT and then underwent D-dimer testing. Patients with low or moderate pretest probability and a negative D-dimer result had no further diagnostic testing and received no anticoagulant therapy. Serial compression ultrasonography was performed in all other patients. Patients who did not receive a diagnosis of DVT were followed for symptomatic venous thromboembolism. MEASUREMENTS: Objectively confirmed symptomatic venous thromboembolic events during 3 months of follow-up. RESULTS: 283 patients (51%) had low or moderate pretest probability and a negative D-dimer result. One of these patients had DVT during follow-up (negative likelihood ratio, 0.05 [CI, 0.01 to 0.23]). The negative likelihood ratio of the d -dimer test in all patients was 0.03 (CI, 0.01 to 0.16). CONCLUSION: A negative result on a quantitative latex d -dimer assay safely eliminates the need for further testing in patients with low or moderate pretest probability of DVT.

Fibrin Fibrinogen Degradation Products↗
PubMed1998

[The levels of FDP, FDP-E and D-dimer in patients with rheumatoid arthritis].

We studied the relationship of FDP and D-dimer levels of rheumatoid arthritis (RA) patients with their activities of RA. We evaluated FDP/D-dimer levels of thirty-six RA patients. And we also evaluated FDP/D-dimer levels of 14 patients with systemic lupus erythematosus (SLE) and 12 patients with other rheumatic diseases as control. RA patients were divided into two groups according to their activities. Nineteen patients, who fulfilled at least three of four activities criteria, were classified as active group [RA (A) group], and other 17 patients were classified as not-active group [RA (B) group]. FDP and D-dimer levels of RA patients were higher than those of SLE or other patients group significantly (P < 0.01). Furthermore, in RA patients, high levels of FDP and D-dimer were observed in RA (A) group compared to RA (B) group. In some RA patients, decrease of FDP and D-dimer levels were observed according to their RA activities. FDP and D-dimer levels were not correlated with the level of rheumatoid factor (RF). These results show that the FDP and D-dimer levels were elevated according to RA activity in RA patients.

Adult↗
PubMed1999

D-dimer after delivery in uncomplicated pregnancies.

UNLABELLED: D-dimer is now widely used as a coagulation marker. During pregnancy the D-dimer level increases until term even in uncomplicated pregnancies. The aim of the study was to establish the D-dimer immediately after delivery in uncomplicated pregnancies. A rapid immunoturbidimetric assay for D-dimer determination was employed in 100 consecutive deliveries. D-dimer level increased significantly in all women after delivery (increase from 1 to more than 10 times over the normal range). CONCLUSION: An increase in fibrinolysis is associated with pregnancy and delivery, and D-dimer level must be interpreted only in association with other clinical, laboratory and instrumental methods when pathological conditions (e.g. pulmonary embolism, deep vein thrombosis or disseminated coagulation) are suspected.

Cesarean Section↗
PubMed2000

Derivation of a simple clinical model to categorize patients probability of pulmonary embolism: increasing the models utility with the SimpliRED D-dimer.

We have previously demonstrated that a clinical model can be safely used in a management strategy in patients with suspected pulmonary embolism (PE). We sought to simplify the clinical model and determine a scoring system, that when combined with D-dimer results, would safely exclude PE without the need for other tests, in a large proportion of patients. We used a randomly selected sample of 80% of the patients that participated in a prospective cohort study of patients with suspected PE to perform a logistic regression analysis on 40 clinical variables to create a simple clinical prediction rule. Cut points on the new rule were determined to create two scoring systems. In the first scoring system patients were classified as having low, moderate and high probability of PE with the proportions being similar to those determined in our original study. The second system was designed to create two categories, PE likely and unlikely. The goal in the latter was that PE unlikely patients with a negative D-dimer result would have PE in less than 2% of cases. The proportion of patients with PE in each category was determined overall and according to a positive or negative SimpliRED D-dimer result. After these determinations we applied the models to the remaining 20% of patients as a validation of the results. The following seven variables and assigned scores (in brackets) were included in the clinical prediction rule: Clinical symptoms of DVT (3.0), no alternative diagnosis (3.0), heart rate >100 (1.5), immobilization or surgery in the previous four weeks (1.5), previous DVT/PE (1.5), hemoptysis (1.0) and malignancy (1.0). Patients were considered low probability if the score was <2.0, moderate of the score was 2.0 to 6.0 and high if the score was over 6.0. Pulmonary embolism unlikely was assigned to patients with scores < or =4.0 and PE likely if the score was >4.0. 7.8% of patients with scores of less than or equal to 4 had PE but if the D-dimer was negative in these patients the rate of PE was only 2.2% (95% CI = 1.0% to 4.0%) in the derivation set and 1.7% in the validation set. Importantly this combination occurred in 46% of our study patients. A score of <2.0 and a negative D-dimer results in a PE rate of 1.5% (95% CI = 0.4% to 3.7%) in the derivation set and 2.7% (95% CI = 0.3% to 9.0%) in the validation set and only occurred in 29% of patients. The combination of a score < or =4.0 by our simple clinical prediction rule and a negative SimpliRED D-Dimer result may safely exclude PE in a large proportion of patients with suspected PE.

Algorithms↗
PubMed2000

[The usefulness of the association of clinical probability, rapid plasma measurement of D-dimer, compression echography of the lower limbs and echocardiography in the diagnosis of acute pulmonary embolism].

BACKGROUND: The aim of this study was to investigate the diagnostic utility of clinical probability, rapid plasma D-dimer assay, compression ultrasonography (CUS) and transthoracic echocardiography (TTE) in patients with suspected pulmonary embolism. METHODS: One hundred forty consecutive outpatients with suspected pulmonary embolism were enrolled in a prospective trial. We evaluated sensitivity, specificity, positive and negative predictive value of a combination of clinical probability, D-dimer, CUS and TTE using perfusion lung scan and pulmonary angiography as a combined gold standard for determining the presence or absence of pulmonary embolism. Clinical probability was assessed in accordance with the PIOPED criteria. The D-dimer (Nycocard) level was considered as abnormal > 0.3 mg/l, the CUS if incompressibility of the leg veins was showed, and the TTE if right ventricular dilation was present, in the absence of chronic pulmonary disease. The combination of these tests was considered consistent with the presence of pulmonary embolism if D-dimer plus CUS and/or TTE showed abnormal results. A pulmonary embolism was excluded if D-dimer and CUS showed normal findings or a low clinical probability was associated with normal findings of CUS and TTE. RESULTS: One hundred eleven patients were evaluated. Pulmonary embolism was present in 45/111 (40%) patients. The combination of tests showed positive findings in 39/39 patients with pulmonary embolism, negative findings in 47/50 without pulmonary embolism and non-diagnostic results in 22/111 (20%) patients (95% confidence interval--CI 12-28%). There were three false positive and no false negative results. Sensitivity and specificity were 100 and 94% respectively (95% CI 92-100% and 87-100%); positive and negative predictive values were 93 and 100% (95% CI 85-100% and 93-100%). None of these tests, separately, showed enough sensitivity and specificity. CONCLUSIONS: The combination of clinical probability, D-dimer, CUS and TTE was highly accurate to confirm or rule out pulmonary embolism.

Acute Disease↗